Cell Transplantation, Vol. 22, pp. 571–617, 2013 Printed in the USA. All rights reserved. Copyright 2013 Cognizant Comm. Corp.
0963-6897/13 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368912X655208 E-ISSN 1555-3892 www.cognizantcommunication.com
Review Systematic Review of Induced Pluripotent Stem Cell Technology as a Potential Clinical Therapy for Spinal Cord Injury Anne S. Kramer,* Alan R. Harvey,* Giles W. Plant,† and Stuart I. Hodgetts* *Spinal Cord Repair Laboratory, School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, Western Australia †Stanford Partnership for Spinal Cord Injury and Repair, Stanford Institute for Neuro-Innovation and Translational Neurosciences, Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA
Transplantation therapies aimed at repairing neurodegenerative and neuropathological conditions of the central nervous system (CNS) have utilized and tested a variety of cell candidates, each with its own unique set of advantages and disadvantages. The use and popularity of each cell type is guided by a number of factors including the nature of the experimental model, neuroprotection capacity, the ability to promote plasticity and guided axonal growth, and the cells’ myelination capability. The promise of stem cells, with their reported ability to give rise to neuronal lineages to replace lost endogenous cells and myelin, integrate into host tissue, restore functional connectivity, and provide trophic support to enhance and direct intrinsic regenerative ability, has been seen as a most encouraging step forward. The advent of the induced pluripotent stem cell (iPSC), which represents the ability to “reprogram” somatic cells into a pluripotent state, hails the arrival of a new cell transplantation candidate for potential clinical application in therapies designed to promote repair and/or regeneration of the CNS. Since the initial development of iPSC technology, these cells have been extensively characterized in vitro and in a number of pathological conditions and were originally reported to be equivalent to embryonic stem cells (ESCs). This review highlights emerging evidence that suggests iPSCs are not necessarily indistinguishable from ESCs and may occupy a different “state” of pluripotency with differences in gene expression, methylation patterns, and genomic aberrations, which may reflect incomplete reprogramming and may therefore impact on the regenerative potential of these donor cells in therapies. It also highlights the limitations of current technologies used to generate these cells. Moreover, we provide a systematic review of the state of play with regard to the use of iPSCs in the treatment of neurodegenerative and neuropathological conditions. The importance of balancing the promise of this transplantation candidate in the light of these emerging properties is crucial as the potential application in the clinical setting approaches. The first of three sections in this review discusses (A) the pathophysiology of spinal cord injury (SCI) and how stem cell therapies can positively alter the pathology in experimental SCI. Part B summarizes (i) the available technologies to deliver transgenes to generate iPSCs and (ii) recent data comparing iPSCs to ESCs in terms of characteristics and molecular composition. Lastly, in (C) we evaluate iPSC-based therapies as a candidate to treat SCI on the basis of their neurite induction capability compared to embryonic stem cells and provide a summary of available in vivo data of iPSCs used in SCI and other disease models. Key words: Stem cell therapy; Spinal cord; Induced pluripotency; Trophic support; Replacement; Regeneration
Pathophysiology of Spinal Cord Injury and Stem Cell Therapies Pathophysiology of Spinal Cord Injury (SCI) Most human spinal cord injuries (SCI) are contusions resulting in dislocation of vertebral columns after an initial traumatic event. These injuries are diffuse and result in
central cavitation surrounded by spared white matter. SCI follows a biphasic pattern referred to as “primary” and “secondary” phases. SCI involves extensive interaction of various immune cells, resident CNS cells, noncellular components (such as adhesion molecules, cytokines, and chemokines) to mount inflammatory, immune, and scar
Online prepub date: August 27, 2012. Address correspondence to Stuart I. Hodgetts, Spinal Cord Repair Laboratory, School of Anatomy, Physiology and Human Biology, The University of Western Australia, 35 Stirling Highway, Perth, 6009, Western Australia. Tel: +61-8-6488-8642; Fax: +61-8-6488-1051; E-mail:
[email protected]
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tissue responses resulting in extensive tissue destruction, cyst formation, scar tissue formation, Wallerian degeneration, and Schwannosis (45). Most details on the pathophysiology of SCI have been obtained from experimental SCI in animal models as well as stereotyped responses from traumatic brain injury. Despite extensive data detailing the events of experimental SCI [for reviews, see refs. (96,108, 168,248,249,297,306,318,324,333)] the exact course of events in human SCI is less clear. There seem to be basic similarities, but there is evidence that fundamental differences may exist, such as the extent and importance of the astrocytic response and demyelination (270). The primary phase consists of the mechanical compression of tissues and nerves, which causes membranes to rupture (326). The extent of axonal death rises exponentially with the initial force of impact (109) and the sparing of fibers is reflected in locomotor deficits (168,331). Complete spinal cord transection is rare, and most incomplete or contusion human injuries spare a rim of mostly demyelinated peripheral axons (27,43,382). Generalized immediate spinal cord swelling is usually the first visible sign of injury, disrupting normal blood flow and causing hypoxia, usually accompanied by hemorrhage (41,46,96,193,303). Mechanical factors are also in part responsible for damage to the blood–brain barrier (BBB), which increases permeability, facilitates peripheral cell invasion into the lesion and contributes to swelling and inflammatory events. The primary phase of events responsible for the imme diate wave of cell death is followed by a secondary phase, which results in widespread and prolonged tissue destruction mediated by excitotoxic, oxidative, inflammatory, and immune events. These are interlinked events, poorly defined especially in humans, and animal models seem to have species and strain specific differences as well as differences depending on the type of injury (47,96,111, 177,298,342). Together, these factors are typically far more damaging than that of the primary phase and can affect surrounding cells up to several millimeters from the lesion epicentre in rodents and several centimeters in humans. Excitotoxicity and Oxidative Damage. Ionic imbalances of key ions (such as sodium, potassium, and calcium) and subsequent excessive depolarization are a product of ischemia, hypoxia, and hemorrhage (288,314). This contributes to excitotoxicity (excessive stimulation by neurotransmitters such as glutamate), formation of reactive oxidative species (ROS), and lipid peroxidation (55,175,219,365). These events cause loss of osmotic balance, disruption of cell membranes, altered mitochondrial and metabolic activity, leading to widespread cell death and axonal degeneration (377). Inflammatory and Immune Responses. The damaged BBB allows influx of leukocytes, triggering an inflammatory response (170). Microglia and macrophages are elevated almost immediately post-SCI, peak at about 8 h and
remain elevated for weeks postinjury (96,205). These cells slowly clear debris by phagocytosis, participate in scar tissue formation, cause neurotoxic “bystander damage,” and secrete various neuroprotective factors. Neutrophil infiltration peaks at about 24 h post-SCI (96,111), returns to normal levels within the first week postinjury, and causes neuronal and glial toxicity by release of cytokines, ROS, and neurotoxic enzymes. Lymphocytes are elevated by 3–4 days post-SCI and peak by 7 days. Cytokines (including tumor necrosis factor-a, interleukins-1b and -6) are released from inflammatory cells (as well as neuronal and glial cells), peak within 1–6 h and decline by 1 week postinjury (96). These cytokines exacerbate inflammation and BBB permeability, promote apoptosis and necrosis of neurons and oligodendrocytes, reduce axonal growth, and may impair locomotor recovery (194). Cavity/Cyst Formation. Contusion injuries are characterized by formation of cystic structures, which arise from the clearance of debris by macrophages and progressive loss of neural tissue (19,111,270). A spared rim of white matter usually persists, but cysts represent a physical barrier that inhibits neurite regeneration. The cysts are delineated by the formation of scar tissue, which also eventually becomes inhibitory. Scar Tissue Formation. Scar tissue is now widely accepted to be both a physical as well as a chemical barrier for axonal growth and regeneration, at least in experimental SCI (108,306,320). Its composition as well as spatiotemporal distribution is extremely dynamic and malleable within the first few weeks postinjury (186,364) at which point the composition stabilizes and does not change (“chronic injury”). Scar tissue is mainly composed of macrophages, microglial cells, pericytes, oligodendrocytes precursor cells (OPCs), oligodendrocytes, meningeal cells, and macrophages, each of which express/secrete several growth inhibitory molecules. The key molecular components include extracellular matrix components (especially collagens), sulfate proteoglycans (including chondroitin sulfate, heparin sulfate, and keratin sulfate), tenascins, semaphorins, and ephrins [for reviews, see refs. (13,81,108,110,123,186)]. Inhibitory molecules within the glial scar are important contributors to abortive neuronal regeneration and may cause collapse of axonal growth cones (9,86,87,165,250,290); thus, the reduction of scar tissue is a commonly used indicator of morphological improvements in experimental SCI. However, it is equally well accepted that cells participating in scar tissue formation, especially astrocytes, also have a neuroprotective role by secreting growth promoting and/or neuroprotective factors (36,306,333,388). Overall, axonal growth after SCI depends on (i) molecular and spatiotemporal factors that balance inhibitory and growth promoting influences (165), (ii) the subtype of axon that is involved (e.g., serotonergic
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fibers rarely grow into scar tissue) (88,139,140,155,309), and (iii) the intrinsic growth state of the mature neuron. Wallerian Degeneration and Demyelination. Degeneration of the axon and myelin sheath distal to the injury after axonal disconnection with the cell body is followed by slow removal of neural tissue debris by macrophages and microglia cells (4,20,102,270,304,418). This is a protrac ted process in humans, beginning 3–6 days postinjury and becoming more extensive after 12–15 days postinjury (48,102). After 1 year postinjury, there is almost a complete loss of axons in affected tracts (48). Oligodendrocytes undergo apoptosis following SCI in both experimental and human SCI (83,102,112,247). Rapid loss of the oligodendrocyte population leads to demyelination (in expe rimental animal models within 1.5–4 h postinjury) and loss of remyelination (48,369). The resulting extensive myelin debris renders the injury site an effective inhibitor to axonal regrowth. Furthermore, demyelination directly contributes to dysfunction of axons and action potential transmission (264). Yet, the extent and importance of demyelination in human SCI is unclear. Primary demyelination, which is the destruction of myelin while still obtaining intact axons, seems to be uncommon in humans (47,168,270), but demyelination associated with Wallerian degeneration is more common (4). Several authors have reported a gradual loss of myelin, but not to the same degree in all patients (48,125). Current Stem Cell Therapies in Spinal Cord Injury Cellular transplantation approaches are aimed at targeting the pathophysiology of SCI, in particular to replace lost endogenous neuronal and/or glial cells, provide a more
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favorable growth environment to mask or neutralize inhibitory molecules, and enhance and direct any intrinsic regenerative ability that neurons may possess (17,229,233,317) (Fig. 1). Traditional cell transplants commonly used in experimental SCI are neural stem/progenitor cells, glial precursors, olfactory ensheathing glial cells, mesenchymal stem cells, and Schwann cells, each of which can produce some morphological or behavioral benefits (for review, see ref. 367). The delayed loss of myelinating oligodendrocytes provides another problem that can realistically benefit from cellular transplants. Replacing lost endogenous oligoden drocytes with donor oligodendrocyte precursor cells (OPCs) or a mixed transplant of neural stem/precursor cells can limit damage and potentially restore function by replacing myelinating oligodendrocytes. Specifically, studies have found transplantation of OPCs results in differentiation to mature oligodendrocytes, which can increase myelination up to 50%, and correlates with improvements in loco motor function (33,52,80,106,157,176,254,328). However, obtaining large quantities of purified human OPCs or neural stem/progenitor cells (which can give rise to OPCs) is problematic. Embryonic stem cell (ESC) lines that can be differentiated towards neural or glial lineages are a useful option because they can yield very large quantities of donor OPCs, which cannot be easily obtained from adult stem cell sources (120). Yet, embryonic cell lines are not genetically identical to the patient and as such donors must be carefully immunologically matched to recipients, along with appropriate immunosuppression therapies that are known to have adverse long term effects (224). Furthermore, the use of ESCs is associated with ethical controversy
Figure 1. Stem cell transplants in SCI. The aim of stem cell transplants is to attenuate the pathology of SCI by replacing lost endogenous cells and functions, thereby influencing the local milieu and microenvironment to improve neuronal plasticity and promote functional recovery.
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(137,159,310,313). Somatic cell nuclear transfer (SCNT) and cell fusions (144,158,251,300,308,401) are alternative sources of pluripotent stem cells, but these are technically challenging, inefficient, costly, rely on donor oocytes (and as such also introduce ethical issues), and although it can produce cells with cloned nuclear DNA, the mitochondrial DNA remains of maternal origin which may be sufficient to elicit immune rejection (156). The discovery that somatic adult cells could be dedifferentiated (or “reprogrammed”) back into an ESC-like state (termed induced pluripotent stem cells or “iPSCs”) has been an important milestone in clinical regenerative research (362,410). iPSCs offer potential solutions to the various problems described above by providing an almost unlimited source of OPCs or other desirable cells from adult tissue, human iPSCs can be obtained by simple noninvasive procedures and delivered as an autograft, which is likely to reduce host immune responses (98,291,356). Furthermore, iPSCs have now been shown to reliably differentiate into definite neural lineages (see “Can iPSCs Form Functional Neurons? Neurite Induction Compared to ESCs”). This makes iPSC technology a feasible new alternative for cell-based therapy for many CNS conditions including SCI. iPSC technology offers solutions to the ethical issues invariably associated with the use of human ESCs (although iPSCs are themselves not free of ethical controversies) (44,84,231,336), as well as offering autologous transplant possibilities that may attenuate rejection problems. Generation and Characteristics of Induced Pluripotent Stem Cells What Are Induced Pluripotent Stem Cells? An Introduction to the Technology iPSCs are generated by introducing “reprogramming” factors involved in maintenance of pluripotency and selfrenewal (39,67,163,182,223,360,362). Originally, four transcription factors [octamer-binding transcription factor 3/4 (oct3/4), sex-determining region Y box 2 (sox2), Krüppel-like factor (klf-4), and c-myc] were used to establish the first iPSCs from mouse embryonic fibroblasts (MEFs) (362). These initial attempts yielded partially reprogrammed iPSCs (compared to the current standard; see “Criteria of Bona Fide iPSCs: Measuring Pluripotency Experimentally”) as they could not give rise to live chimeric mice and thus did not demonstrate authentic pluripotency. The field quickly advanced and soon improved protocols with variations of reprogramming factors (importantly, the oncogene c-myc was shown to be dispensable) (260,385), selection processes of iPSC clones [more appropriate selection markers such as nanog and oct3/4 (232,279,386) instead of F-box protein 15 (fbx15) (362) or no selection at all (26)] began to yield high-quality fully pluripotent
iPSCs with silenced transgenes and the ability to form live organisms by both chimera formation and tetraploid complementation, the most stringent experimental assessments of pluripotency (see “Criteria of Bona Fide iPSCs: Measuring Pluripotency Experimentally”). This has now been achieved with a wide range of species including rats (208,213), humans (361,410), pigs (103,104,395), sheep (218), horse (259), nonhuman primates (59,89,422), and endangered wildlife species (24). The underlying molecular mechanisms of pluripotency and reprogramming are complex interactions between transcription factors, epigenetic regulators, signaling pathways, and microRNA interactions (39,64,65,158,223,228,253, 295,360,394,400), which are not detailed in this review. The reprogramming factors act to induce pluripotency synergistically, requiring a delicate balance of factors for a sustained period of time (~10–12 days) to reprogram the cells (54,286,344,362). It is unclear why only small proportions of cells exposed to reprogramming factors become fully programmed (typically 0.0001–2.0%, depending on the delivery method used—which albeit low, is still more efficient than other methods to create pluripotent stem cells). Several models have been proposed to explain these low efficiencies, including a stochastic model (correct levels of expression of the factors occur by chance in small amounts and potent resistant forces such as epigenetic landscape, tumor suppressors, or senescence inductors oppose pluripotency) and an elite model (stating as yet unidentifiable cells or stem cells present within the culture at low frequencies are able to be reprogrammed or overcome barriers to pluripotency) (130,228,295,399), although given enough time all cells eventually become amenable to reprogramming (for review, see ref. 295). Nevertheless, upon correct exposure of reprogramming factors to cells, the sequential activation of endogenous pluripotent markers follows, marking the onset of pluripotency; alkaline phosphatase, stage-specific embryonic antigen (SSEA)-1, followed by oct3/4, and nanog. The transcriptional network governing pluripotency appears to be conserved across species, because human or mouse transgenes can be used to reprogram other species, and is not restricted by the germ layer of origin of the parental donor cell. The resultant iPSC cells share the same phenotypic markers and characteristics as ESCs— they are pluripotent, self-renewing, and rapidly proliferate in culture—features that links these cells to tumorigenicity (see “Tumorigenicity of iPSCs”). Originally, iPSCs were deemed almost indistinguishable from ESCs in terms of genetic and epigenetic composition, but with recent reports it is now increasingly apparent that iPSCs are neither identical to ESCs nor identical to other iPSC lines and may still bear a “memory” of their parental donor cell (see “Molecular Analysis of iPSCs”). The full implications of these differences are unknown at this time.
(Transient expression, DNA based vector)
Adenovirus (281,345,427)
(Excisable system, DNA-based vector)
PiggyBac transposon (167,379,392,411,412)
(Excisable system used in conjunction with viral or plasmid delivery)
Cre-Lox (61,167,285,338,339)
Retroviral/Lentiviral delivery (2,92,151,183,232, 273,279,340,350,361, 362,386)
Method
• Medium to high efficiency of reprogramming • Technically simple procedure—most common method employed • Only single transfection necessary • Polycistronic vector available (lentivirus) • Tetracycline-inducible transgene expression available (lentivirus) • Can be transgene free by excision (cre-lox) • Successfully used with both human and murine cell lines • Omission of c-myc possible while still maintaining acceptable efficiency levels • Most widely tested method; wide range of cell types tested using both feeder-dependent & feeder-independent methods • Excision of transgenes with cre-lox removes genomic integration of transgenes • Very high excision rates can be achieved using optimal levels of cre-expression • Polycistronic vector available • Tetracycline-inducible transgene expression available • Omission of c-myc possible while still maintaining acceptable efficiency levels • Successfully used with both human and murine cell lines • Can be used with viral or episomal plasmid vectors • Successfully tested on feeder-free culture • High efficiency of reprogramming • Excision of transgenes with transposon removes genomic integration of transgenes • Traceless removal (no residual sequence) after excision of transposon • Only single transfection necessary • No viral infection necessary • Polycistronic vector available • Tetracycline-inducible transgene expression available • Low risk of permanent genomic integration sites • Polycistronic vector available • Tetracycline-inducible transgene expression available • Successfully used with both human and murine cell lines • Successfully achieved with omission of c-myc
Advantages
Table 1. Arsenal of Current Technologies Available to Produce iPSCs
(continued)
• Very low efficiency of reprogramming • Absence of integration must be experimentally determined. • Requires retransfection as expression is lost after cell division; repeated delivery may be challenging for nonadherent cells • Not tested on feeder-free culture • Viral infection of cells • Low clinical translation potential due to impractical large scale generation
• Lower excision rate compared to cre-lox system • Possibility of reintegration following excision. May require multiple rounds of excision. Requires identification of correctly excised clones—time consuming & labor intensive • Transposition can result in chromosomal rearrangement around excision site • Not attempted with human cells or without c-myc • Not tested on feeder-free culture.
• Not a traceless excision—cre-mediated excision leaves entire long term repeat containing residual loxP sites at random location. These may disrupt function of endogenous genes • May require several rounds of recloning to ensure only correctly excised clones are propagated (note that absence of integration should be experimentally determined to ensure excision successful) • Low clinical translation potential due to safety concerns
• Permanent genomic integration • Random integration of virus may cause insertional mutagenesis and unpredictable gene dysfunction • Reactivation of transgenes may promote oncogenesis • Residual exogenous factor expression may alter the molecular characteristics of iPSCs lines. Some evidence that this also may affect functionality • Viral infection of cells • Retrovirus limited to dividing cells only
Disadvantages
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(Transient expression, protein based vector)
Recombinant protein delivery (179,424)
(Transient expression ligated to plasmid, DNA based vector)
Bacteriophage integrase (171,406)
(Transient expression, DNA based vector)
Plasmids, episomal plasmids & minicircle (69,72,121,162, 199,278,280, 281,409)
(Transient expression, RNA based vector)
Sendai virus (15,115,327)
Method
• Low risk of permanent genomic integration • No viral vector necessary. Can be delivered with nanotechnology (199) • Plasmid backbone can be excised to reduce amount of bacterial DNA and improve transfection efficiency (minicircle vector) (162) • Polycistronic vector available • Technically simple procedure • Successfully achieved with omission of c-myc • Successfully used with both human and murine cell lines • Medium efficiency of reprogramming. • Single integration site, at known location—mostly intergenic regions—which does not seem to disrupt gene function • Polycistronic vector available • Tetracycline-inducible transgene expression available • Only single transfection necessary • No viral vector necessary • Successfully used with both human and murine cell lines • No risk of permanent integration site; DNA free—safe method • Direct delivery of reprogramming proteins into cells—does not rely on transcription • No viral or bacterial vector necessary • Successfully used with both human and murine cell lines • Tested on feeder-free culture
• Medium to high efficiency of reprogramming • No permanent genomic integration sites • DNA free (does not go through DNA phase to replicate) • Successfully used with both human and murine cell lines
Advantages
Table 1. Arsenal of Current Technologies Available to Produce iPSCs (Continued)
• Very low efficiency of reprogramming • Slow & inefficient transduction • Proteins difficult to purify in large amounts (essential for higher efficiency) • Requires multiple applications (short half-life of proteins), which must be carefully timed; repeated delivery may be challenging for nonadherent cell • Transient nature leads to higher number of incomplete iPSCs (“partially reprogrammed”) • Overall technically complex and labor intensive • Not tested without c-myc
• Permanent genomic integration • May incorporate into introns • Exposure to integrase may result in chromosomal rearrangement • Not attempted without c-myc • Not tested on feeder-free culture
• Viral infection of cells • Continual cytoplasmic replication—requires removal of vector. Can be done by temperature sensitive vector or antibody removal • Not attempted without c-myc • Not tested on feeder-free culture • Neither polycistronic vector nor tetracycline-inducible transgene expression used • Very low efficiency of reprogramming • Absence of genomic integration must be experimentally determined— labor intensive. Random integration sites may not be easily identifiable • Vectors lost with cell division • May require addition of extra transcription factors for successful and more efficient reprogramming • Not tested on feeder-free culture
Disadvantages
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• Low efficiency of reprogramming • Not attempted with human cells • Not tested on feeder-free culture • Requires adjunct p53 knockout on construct, for fully reprogrammed iPSCs
• Depending on delivery method used, shares disadvantages of retroviral or plasmid delivery • Not tested on feeder-free culture
• Addition of miRNA increases efficiency of normal retroviral transduction • Polycistronic vector possible • miR-302 cluster sufficient to induce reprogramming—does not require use of traditional transcription factors (OSKM)— reduces tumor concerns. Can be delivered by (i) retroviral vector—very high efficiency (10%), faster reprogramming than any other published method (8) and (ii) Plasmid vector (214) • Successfully used with both human and murine cell lines • Vector offers extremely large size capacity for transgenes • Polycistronic vector possible • No viral vector necessary • Transgene free clones can be selected for (chromosome con struct lost at low frequencies), but requires clonal expansion
• High efficiency of reprogramming • No risk of permanent integration sites, DNA free • No viral vector necessary • Successfully used with human cell lines
• Reprogramming factors are in ESC-derived proteins of actively proliferating cells which is undefined • Protein extract may differ depending on different ESC lines which may affect reprogramming efficiency • Unknown which proteins or combination of proteins, if any, cause reprogramming • Not attempted with human cells • Not tested on feeder-free culture • Requires daily readministration—labor intensive, repeated delivery may be challenging for nonadherent cells • May require soluble interferon inhibitor • Has not been tested without omission of c-myc or feeder-free culture
• High efficiency of reprogramming • No genomic integration detected • Only requires single treatment with extract—does not seem to require normal exposure time to reprogramming factors • Does not require any additional small molecules or chemicals to aid to reprogramming • No viral or bacterial vector necessary
References are not an exhaustive list especially for viral-integrating methods [for reviews see refs. (65,128)]. Efficiency of reprogramming can be enhanced with: (i) incorporation of additional reprogramming factors (e.g. lin28, nanog, glis1) (421), (ii) downregulation/knockout of tumor suppressor related genes (e.g. p53, p16, p15, p19, INK4) (16,147,174,206,240,280,374,421), (iii) modification of cell-signalling (e.g. Wnt Pathway) (242), (iv) treatment with chemicals and small molecules (e.g. Valcroic acid, kenpaullone, thiazovivin, SB431542, PD0325901, vitamin C, Ps48, lithium, butyrate) (103, 151,211,215,227,232,235,330,378,428), (v) addition of anti-apoptotic agents (e.g. SV40) (289), (vi) silencing of apoptotic inducers (e.g. Puma) (195), (vii) selection process for fully pluripotent stem cells [e.g. drug selection for nanog, oct-4 (232,279,386) or reporter system with miRNAs (94)] and (viii) hypoxic conditions (374,408). Combining several of these “enhancers” can reduce the time needed for reprogramming (332). Note: rate of iPSC formation (not efficiency) can be increased by cell division, as this results in increased cumulative probability for the stochastic events to occur earlier in time (130). ESC, embryonic stem cell; iPSC, induced pluripotent stem cell; miRNA, micro RNA; OSKM, oct4, sox2, klf-4, c-myc.
Artificial chromosome construct (142)
(Transient expression, mRNA based vector) miRNA cluster constructs (8,166,212,214, 293,349)
mRNA delivery (381,398)
(Transient expression, undefined vector)
Protein extract delivery (76)
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Generation of iPSCs: Methods of Transgene Delivery Originally iPSCs were produced by delivering sox-2, oct3/4, klf-4, and c-myc transcription factors by retroviral transduction, resulting in permanent genomic integration (362). Since then, much research has focused on improving the reprogramming method (232,252,260, 279,361,385,386,410). From a clinical perspective, the ideal method would reliably generate safe iPSC clones at a high rate (i.e., efficient reprogramming) without inclusion of oncogenes or requiring permanent genome integration, and can be derived xeno-free, is a technically simple and cost-effective process (Table 1), and cells can be produced in a time frame optimal for therapeutic use. Choice of Transcription Factors. It is possible to vary the number and combinations of the transcription factors used, which may be critical for clinical applications. At least one transcription factor (typically oct4) is usually required, although several studies have used synthetic messenger RNA (mRNA) (381), proteins (76,179,424), or more recently microRNAs (miRNA) that circumvent the use of these transcription factors entirely (8,214). Omission of the oncogene c-myc is desirable to reduce tumor concerns but has been linked to significantly lower reprogramming efficiency (but notably promoting growth of fewer noniPSC colonies or “partially reprogrammed” iPSCs) (260) and may affect development potential (10). Generally, methods employing fewer transcription factors are less efficient compared to the traditional four factors, at least for fibroblasts. C-myc can be substituted with n-myc or l-myc (26,261,280). l-myc has been found to specifically enhance the number of fully reprogrammed colonies (more so in humans than in mice), whereas c-myc enhances the numbers of both fully reprogrammed iPSCs as well as partially reprogrammed clones. As such, the overall effect of l-myc is to increase numbers of iPSC clones, without increasing tumorigenicity (261). Yet, l-myc is not typically used in the majority of iPSC studies. Sox-2 can be substituted by sox-3, 7, 15, 17, or 18 (260) or chemicals (153), and klf-4 can be replaced by 18 other factors but at much lower reprogramming efficiencies (230,260). Other transcription factors such as nanog, Lin28, glioma-associated oncogene similar 1 (Glis1), B lymphoma Mo-MLV insertion region 1 (Bmi1), and T Box 3 (tbx3) can also be successfully used (127,230,257,410). No direct substitution for oct3/4 has been found to date (230,360). Permanent Genomic Integration: Retroviral and Lenti viral Delivery. Viral integration methods with retrovirus or lentivirus are the most technically simple, common, and reliable method, reprogramming cells into iPSCs at high efficiency and providing sustained levels and durations of factor expression required for successful reprogramming. Viral delivery has proved effective with a wide variety of
different donor species and cell types, tissue culture platforms, and omission of oncogenes (both c-myc and klf-4). Yet safety concerns associated with permanent genomic integration and modification make this method of transgene delivery unlikely to be used in a clinical setting. A typical retroviral or lentiviral transfection will leave up to 5–20 integration sites in the genome (241,362,390). Random integration of virus may cause insertional mutagenesis and unpredictable genetic dysfunction. Residual exogenous factors may also be reactivated, linking to high rates of teratoma formation in vivo (145,279), and alterations in molecular characteristics of iPSCs, inhibition of complete iPSC maturation, and potential differences of development capacity (339,410). Changes in vector design can help reduce these risks by reducing the number of integrations and controlling the expression and activation of the transgenes. Using polycistronic vectors (single cassette carrying all the transgenes under the control of a single promoter) can reduce the number of integration sites down to one (53,61,338–340), ensure that cells will receive all transgenes at the same time to promote more efficient reprogramming, and reduce numbers of partially reprogrammed iPSCs (121,281,339,340). Alternatively, phage integrase can be used for site-specific integration (406). The temporal timing of expression/activation of the reprogramming factors can be controlled using tetracycline-inducible promoters, which can significantly enhance reprogramming efficiency (40,128,384). MicroRNA (miRNA) can also be used to generate iPSCs (8,166,212,214,293,349). Several miRNA clusters have been found to be important regulators of the cell cycle and self-renewal and are specific to pluripotent stem cells (380). Recently, several clusters have been identified to be important in iPSC reprogramming, and delivery of these clusters in addition to the reprogramming factors (oct3/4, sox-2, klf-4 with and without c-myc) was found to considerably enhance the reprogramming efficiency (166,212,214,293,349). Notably, the miR-302 cluster has been found to be sufficient to induce reprogramming alone at high efficiency, without requiring any additional reprogramming factors (8,214). Excision Systems: Cre-Lox and Transposons. To over come problems associated with permanent genomic insertions, excisable systems to remove the transgenes can be used while still maintaining high reprogramming efficiencies. These include the cre-lox excision system and Transposons PiggyBac system, both of which use enzymes to remove the inserted transgenes. The cre-lox system was initially used as a means to investigate iPSC quality and pluripotency after removal of the transgenes (61,167,338,339). Following removal, iPSCs remain pluripotent and may exhibit improved development potential compared to their viral integrated counterpart (339). However, residual lox-P sites following excision of transgenes with cre-recombinase may still
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Figure 2. Stepwise considerations for using iPSC-derived cells for transplantations aimed at repairing SCI and other neurodegenerative and neuropathological conditions of the CNS.
pose a safety risk by potentially disrupting exogenous gene function. PiggyBac transposon allows the traceless excision of transgenes (167,392,411), but transposition can also result in chromosomal rearrangement around the excision site. Both excision systems may require clonal expansion of
iPSC colonies to ensure only successfully excised clones are propagated. Transient Transgene Expression Systems. Transient expression of the transgenes (categorized into DNA-based and DNA-free vectors) can overcome problems associated
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with genomic integrations of imperfect excision systems but are more difficult to achieve, as successful reprogramming requires sustained level of the transcription factors for 10–12 days. This may necessitate carefully timed multiple transfections, according to the half-life of the vector. Too low a concentration of transgene levels will promote incomplete reprogramming of iPSCs and promote differentiation, and too high a concentration can be toxic to target cells. DNA vectors [e.g., adenovirus (281,345,427), plasmids/ episomal plasmids (121,199,278,280,281,409), and mini circles (162)] confer very low efficiency of reprogramming, require multiple transfections due to vector dilution with cell division, and bear the risk of genomic integration. As such, iPSC clones derived by these methods still need to be extensively characterized for lack of integration. DNAfree vectors [including Sendai virus (15,115,327), mRNA (381,398), and protein delivery (76,179,424)] bear no risk of genomic integration. Sendai virus is an RNA-based vector that can reprogram donor cells to iPSCs at medium to high efficiency. Yet Sendai virus continually replicates in the cytoplasm where it poses a safety risk and must be removed (15,327). mRNA delivery is a highly efficient, albeit labor-intensive, method (381,398). Protein delivery using purified recombinant proteins ligated to small delivery molecules delivered directly into the cells is very inefficient, is technically complex, requires multiple carefully timed reapplications, but represents a safe delivery option without viral vectors (179,424). Protein delivery using undefined combinations of unpurified combinations
of protein extract, isolated from ESCs (76), is much more efficient and reportedly does not require the normal duration of reprogramming factors; a single application is sufficient for successful reprogramming. Generation of iPSCs: General Considerations of Delivery Approaches It is unlikely that methods involving genomic integrations will be routinely used in a clinical setting, but the lowefficiency and labor-intensive approach of most transient methods also represent an important barrier (see Fig. 2). Permanently integrated transgenes may not be problematic in a research setting, but it should be kept in mind that transgene-free iPSCs may more closely resemble ESCs (381). Regardless of approach, it is important that each new method is tested on both human and nonhuman donor cells. Murine cells are generally reprogrammed with greater ease and efficiency than human cells and require less exposure to transcription factors. Furthermore, human donor cells cannot be subjected to the most stringent pluripotency tests (tetraploid complementation and chimera formation), and each new technology must prove that it is able to establish bona fide iPSCs according to the strictest pluripotency criterion (see “Criteria of Bona Fide iPSCs: Measuring Pluripotency Experimentally”). Different donor cell types should also be tested, as the efficiency of reprogramming also depends on the donor cell type. Keratinocytes have been shown to have one of the highest efficiency to date (1,2). Progenitor cells, especially adipose progenitor cells or neuronal progenitor cells, are also easily reprogrammed
Table 2. iPSC Lines Should Be Validated for Pluripotency by These Standard Criteria Criteria Morphology Expression of pluripotent markers Telomerase & alkaline phosphatase activity In vitro differentiation Teratoma formation
Viable chimera formation Viable organism production: tetraploid complementation
Comment
Key References
High ratio of nucleus to cytoplasm, prominent nuclei, distinct nuclear architecture and distinct chromatin structure. Nanog, oct3/4, SSEA-1,3,4, sox-2, Tra-1-60/81 (human). Note: microarray for global gene expression desirable. Must be positive.
(361,362,410)
Give rise to all three germ layers. Commonly assessed by embryoid body formation and subsequent differentiation. Subcutaneous injection into immunocompromised hosts (usually NOD-SCID mice) must give rise to teratomas (consist of all three germ layers). Form chimeras when directly injected into recipient blastocyst. Generate viable offspring that are germ line competent.
(361,362,410)
(361,362,410) (361,362,410)
(361,362,410)
(279) (30,169,420)
Morphology and pluripotent marker expression is often used as a selection criterion to selectively propagate fully pluripotent iPSCs from surrounding nonreprogrammed cells (362,414). iPSCs express the same surface antigens as ESCs. Notably, no single marker can be reliably used to distinguish bona fide iPSCs from heterogeneous populations of partially reprogrammed cells (60). Chan et al. (60) found that markers commonly used such as nanog, oct4, TERT, SSEA-4, and GDF3 were not sufficient enough to correctly identify fully reprogrammed cells, as even partially reprogrammed cells could exhibit these features of pluripotency. Instead, the most reliable marker combination to identify fully reprogrammed cells were (1) retroviral silencing, (2) expression of Tra-1-60, DNMT3B, and Rex1, and (3) transient dim staining with Hoechst. The most stringent test of pluripotency is live organism production by tetraploid complementation and chimera formation. For obvious ethical reasons, these last two criteria cannot be employed for human cells (but see ref. 160, for chimera testing for human ESCs).
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Table 3. A Comparison of Pluripotent-Related Features of iPSCs and ESCs Feature Gene expression miRNA & lincRNA DNA methylation/ histones
Cell cycle Imprinted genes
Karyotype/ nuclear mutations
Mitochondria (function)
Mitochondria (mutations) Metabolome
Telomeres
Comment Very similar globally, but some differences in gene expression including key pluripotency-related genes (73,180). Higher variability compared to ESCs, may be dependent on location of cell within colony (263). Although majority of miRNA clusters are highly similar in ESC and iPSCs, several differences in miRNA cluster patterns found between iPSCs and ESCs with some clusters more highly expressed in ESCs and others being iPSC specific (73,266,343,389). Several lincRNAs have been found to be enhanced in iPSCs (222). iPSCs have methylation pattern typical of pluripotent stem cells with few consistent differences in several studies (73,124,135,216,315), but some differential methylated regions apparent in iPSC lines (90,95,135,216,296,315): –– Found in H3K9 histone (135). –– Frequently in CG regions, often hypermethylated and include genes involved in developmental process (95,216,296,315) and can be transmitted through differentiation (216). –– Aberrant methylation of some imprinted genes (see below). Similar kinetics to ESCs (shorter G1 phase, with accelerated histone locus body assembly) (119). Some iPSC lines have wrongly imprinted genes (78,294,343,366). –– Dlk1-Dio3 cluster in mouse iPSCs (78,343), but see contrary for human iPSCs (346). iPSCs that have abnormal Dlk1-Dio3 expression have poor development potential (chimera formation) (343), but appear to have normal in vitro differentiation potential (78). –– NNAT (366), H19 and KCNQOT1 (294). Note: Imprinting diseases can be modelled with iPSCs (57,404). Most iPSCs & ESCs maintain normal karyotype in culture (14,225,243,289,362,410). Recurring mutations of both include trisomy 8,12,17,X (14,22,122,197,244,359). Differences between iPSCs and ESCs include: –– Trisomy 17 not yet detected in iPSCs (197,244,246). –– Early and late passage differences: iPSCs have more chromosomal abnormalities and CNVs at early passage, while ESC acquire mutations at late passage (14,152,197,246). –– iPSCs have more gain mutations (ESCs have more delete mutations) (197). Other studies have not reported differences in frequencies of aberration (359) or differences between early and late passage iPSCs (197). Reprogramming induces mitochondrial remodelling to an immature phenotype: become morphologically, functionally, and similarly distributed as in ESCs (11,301,302). –– Change to an immature phenotype (round shape and underdeveloped cristae) (301,414). –– Reduction in mitochondrial numbers, ROS expression, and mtDNA content, indicating reduced mitochondrial biosynthesis (11,301). –– Superoxide levels were found to be lower in two iPSC clones compared to ESCs levels suggesting variation between iPSC lines (11). –– Upon differentiating iPSCs in vitro, mitochondria increase in numbers again to similar levels of the parental donor cells. In addition, mitochondria revert back to an elongated shape with more developed cristae (301) and show mitochondrial/oxidative stress pathway upregulation (11,301). iPSCs harbor mtDNA mutations (homo and heteroplasmic) (302). –– Mainly point mutations. –– Shared in frequency across different iPSC clones. Although iPSCs share similar metabolic signature as ESCs, some metabolites are significantly upregulated in iPSCs (284). –– Differences are not affected by type of donor cell. –– Late passage iPSCs are metabolically closer to ESCs compared to early passage. Reprogramming causes elongation of telomeres, increased activity of telomerase at a comparable rate to ESCs, and epigenetic changes associated with telomeres (150,239,361,407,410). –– Changes in telomeres occur in cells from embryonic or aged donors, whether iPSCs were generated with or without c-myc (239), or from fibroblasts from dyskeratosis congenital patients that suffer a disorder of telomere maintenance (5). –– Extent of telomere elongation following iPSC reprogramming is debated. Several reports have found that elongated and telomerase activity is comparable to ESCs (150,239,361,407,410), while Vaziri et al. (375) found the contrary. –– Several studies have shown that telomere length differs across different iPSC lines (245,351,375,407), the length of which is correlated with pluripotent potential (150).
iPSCs used for above data have passed all criteria (species appropriate) outlined in Table 2, (i.e., bona fide iPSCs). CNVs, copy number variation; Dlk1Dio3, d-like homologue 1-type 3 deiodinase; H19, imprinted maternally expressed long noncoding RNA transcript; KCNQOT1, imprinted paternally expressed long noncoding RNA transcript; lincRNA, long intergenic noncoding RNA; miRNA, microRNA; mtDNA, mitochondrial DNA; NNAT, neuronatin; ROS, reactive oxygen species.
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(and can efficiently reprogrammed with just oct4) whereas adult fibroblasts have a much lower reprogramming rate (183,329,353). Finally, the efficiency of reprogramming also depends on the culture platform used; the majority of studies focusing on iPSC reprogramming methods use a feeder-based system, which is generally more efficient than feeder-independent methods. Feeder-free and xenofree derivation of iPSCs are important for potential clinical applications (312,316) and may reduce variability found among different iPSC lines (79). Characteristics of iPSCs: A Comparison to ESCs Criteria of Bona Fide iPSCs: Measuring Pluripotency Experimentally. Once iPSCs have been generated, pluri potency must be assessed. Pluripotent ESCs have a unique morphology, genetic, and epigenetic signature compared to somatic cells (116). As pluripotent cells, iPSCs should be similar to ESCs. Various assays are available to validate iPSCs for pluripotency—all of which should be employed consistently—at least in nonhuman iPSCs (Table 2). To satisfy these criteria is important as inconsistencies in reprogramming can result, especially in transient transgene delivery methods. These “partially reprogrammed” iPSC lines are not fully pluripotent and exist in an undefined state, expressing some pluripotent markers but not others, and have limited differentiation potential (60). Importantly, lines derived from the same parental donor cell, using the same derivation method, can show differences in functional (differentiation) potential (30,169,343). From this, it is becoming increasingly clear that the pluripotent state can be dynamic and varied, thus a common standard to assess pluripotency should be used for assessment of new cell lines and which is becoming more critical as more technologies to create iPSCs are being developed (337). Molecular Analysis of iPSCs. Initial reports of iPSCs found them to be very similar to ESCs in terms of DNA methylation, gene expression, and other features related to pluripotency (361,362), but more detailed molecular analysis has suggested a distinct genetic signature that distinguishes iPSCs from ESCs (73,74,118,236) [but see refs. (124,267) for contrary]. Analysis of pluripotent-related gene expression, DNA methylation, cell cycle dynamics, microRNA, imprinted genes, and genomic stability has shown that iPSCs are indeed very similar to ESCs but also show some key differences (Table 3). Incomplete reprogramming or somatic memory may contribute to differences (18,118, 184,204,237,296). Interestingly, extended culture attenuates these differences, perhaps as iPSCs achieve a more fully pluripotent state, rendering late passage iPSCs transcriptionally closer to ESCs compared to early passage iPSCs (73,74,284,296,352). Exactly when this late stage is achieved seems to depend on the donor cell type (296).
Parental cell origin or method of reprogramming does not seem to contribute to potential differences. Recent reports show that iPSCs seem to have increased genomic instability compared to ESCs (122). It is likely that the reprogramming process itself may be largely responsible for this (152,216,246), in addition to viral integration, amplification of exogenous transgenes, insertional mutagenesis (122,305,359), and incomplete reprogramming (somatic memory) (95,296). These aberrations may affect differentiation potential and increase tumor concerns. The reprogramming process may also compromise mitochondrial integrity (302). Importantly, a study analyzing mitochondrial DNA found that all iPSC clones analyzed maintained correct mitochondrial function and energy metabolism as ESCs regardless of low or high mutational load (302). iPSC-Based Therapies as a Candidate for SCI Repair Can iPSCs Form Functional Neurons? Neurite Induction Compared to ESCs Although iPSCs share highly similar expression of genes related to pluripotency and development, there is evidence that iPSCs may occupy a distinct pluripotent “state” from ESCs (see “Molecular Analysis of iPSCs”). As such it is important to test that iPSCs have the same capacity as ESCs to generate a whole spectrum of regionspecific neural progenitors and functional neuronal subtypes for SCI therapies (and other CNS disorders). Since the initial isolation of ESCs, many neural induction protocols have been developed in attempts to yield functional neurons. Neural progenitors can be patterned along rostrocaudal and dorsoventral axes using specific morphogens such as sonic hedgehog, retinoic acid, fibroblast growth factor 8, or Wnt (for review, see ref. 129) in order to obtain region-specific neuronal subtypes such as spinal motor neurons, midbrain dopaminergic neurons, and forebrain glutamatergic and GABAergic neurons. Directed neural differentiation approaches in vitro generally involve either embryoid body (EB) formation, adherent monolayerdefined culture conditions, coculture with stromal feeder cells, or by default differentiation (348). Several optimizations in protocols have been shown to increase the yield of neural induction and include dual small body size–dual mothers against decapentaplegic (SMAD) inhibition with noggin and small molecule SB431532 (35,58,425), dual inhibition of activin/nodal and bone morphogenetic protein (BMP) signaling pathway (178) or sulfate reduction by chlorate treatment (323). Many studies have reported that iPSCs can be directed towards a neural lineage applying the same protocols (same developmental signals at the same concentration) that have been developed for ESCs (Table 4).
Fbs (371) • “Safe iPSC”* ––1´ iPSC, RV (OSKM) • 1´ “unsafe iPSC”* • 1´ ESC *Safety determined whether iPSC-derived NS formed tumors in the NOD-SCI mice brain in Miura et al. (255). “safe”, did not form tumors.
• 36´ iPSC • Generated by 11 different methods • Tested effects of (1) donor tissue of origin: (2) presence or absence of c-myc (3) presence or absence of drug selection for nanog or fbx15 • 3´ ESC
• NS (bIII+) – iPSC 4.9% – ESC ~9% • EB neural induction
• Presence or absence of c-myc or drug selection did not influence differentiation potential, nor did reprogramming method • Tissue of origin influenced differentiation potential and teratoma formation • iPSC derived from TTF had highest incidence of undifferentiated cells following differentiation into NS, and highest teratoma incidence following transplantation into NOD-SCID mice brain. Assessed up until 45 weeks. • Note that 3´ hepatocyte-iPSC and 1´ gastric epithelial-iPSC failed to differentiate into NS • iPSCs had similar differentiation efficiency into NS as ESCs • iPSC clones similar in in vivo outcome • “Unsafe iPSC clones” formed tumors at 4 weeks • “Safe iPSC clones” did not form tumors
• 1´ iPSC, RV (OSKM) • 1´ ESC
• NPC/Neurons (Ta1 promoter drug selection) – iPSC 12.5% – ESC 13.1% • EB neural induction • NS (bIII+/nanog–) • Efficiency depended on tissue of origin • EB neural induction
Fbs (A) (405)
Fbs (E), TTF (A), hepatocyte & gastric epithelial cell (255)
• Oct4 transgene reactivated in iPSC-derived neurons • iPSC neurons generally of GABAergic features (similar to ESC-derived neurons) • No difference in differentiation efficiency of iPSC or ESCs • Transplanted purified differentiated population into hind limb of nude mice to assess teratoma formation: no tumors evident at 5 weeks
• 1´ iPSC, RV (OSKM)
• NSC (bIII+): 61.5% • EB neural induction
Fbs (282)
• No comparison between different iPSC lines • Teratoma formation NR
• 3´ iPSC, RV (OSKM) • 1´ drug selected for nanog • 1´ drug selected for oct4 • 1´ no drug selection
• NPCs – Efficiency NR • EB neural induction with fibronectin
Results
Fbs (E + A) (387)
Cell Line(s) Details
Efficiency & Protocol
Donor (Age)
Table 4A. General Neural Induction in iPSCs—Mouse Donor Cells
(continued)
In vitro • Neuronal markers (IHC, PCR) • Electrophysiology In vivo • Transplanted into contused spinal cord. Differentiated into neurons, astrocytes and oligodendrocytes (IHC) (both “safe” and “unsafe clone”)
• Neuronal markers (IHC) • In vivo – transplanted into NODSCID mice brain, differentiated into neurons, astrocytes and oligodendrocytes (IHC)
• Neuronal markers (IHC)
• Neuronal markers (IHC) • In vivo: transplanted into fetal mouse brain. Differentiated into GABAergic, glutamatergic & catecholaminergic phenotype (IHC) and functional synapses (electrophysiology) • Neuronal markers (IHC, PCR) • Glial markers (IHC)
Verification of Phenotype
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• Neural lineage (CD24+) – iPSC 83% – ESC ~85% • EB neural induction
• Neural lineage (Efficiency NR) • EB neural induction • NPC Nestin+ - iPSC 54% - ESC 63% bIII+ - iPSC 36.3% - ESC 29% • EB neural induction • NSC (Efficiency NR) • Adherent monolayer culture condition
• Neural induction • Efficiency reported as relative fluorescence levels • Chick dorsal root ganglion conditioned medium (1–20% range)
• Neural lineage bIII+ - iPSC 63% - ESC 70% Nestin+ - iPSC 22% - ESC 18% • Stromal feeder cell line coculture
Fbs (E) (189)
Meningeal membrane (N) (397) Fbs (E) (322)
Fbs (E) (185)
Fbs (269)
Fbs(E) (201)
Efficiency & Protocol
Donor (Age)
• 1´ iPSC, RV (OSK) • 1´ ESC
• 1´ iPSC, RV (OSKM) with nanog drug selection • 1´ ESC
• 1´ iPSC, LV (OSKM)
• 1´ iPSC, RV (OSKM)
• 1´ iPSC, RV (OSKM)
• 4´ iPSC, RV (OSKN) • 1´ ESC • Assessed neural differentiation at early and late passage
Cell Line(s) Details
• Transduced NSCs with therapeutic gene • Transplanted into mouse brain with glioma. NSCs homed to tumor site & reduced gliosis • Teratoma formation NR • Whether changes were statistically significant was not reported • iPSC appeared to differ to ESC ––Proliferation fate (faster compared to ESC) ––Colony formation (not positive for nanog, inside of iPSC colony, unlike ESC) ––Cell fate after exposure to conditioned medium (ESC differentiated preferentially into motor neurons (Lim-3+) with neurite outgrowth, while iPSC differentiated mainly into sensory neurons (Brn-3), as well as large amount of cells not bIII+, with no neurite outgrowth) • No difference between neural differentiation efficiency between iPSC or ESC • In vitro coculture with cochlea: iPSC neural precursors extended neurite towards cochlea
• Neuronal markers (IHC, PCR) • Electrophysiology
• Early passage iPSC clones were deemed as not fully reprogrammed with significant lower expression of pluripotency markers as late passage iPSC or ESC • Early passage iPSC had poor neural differentiation capacity (~30%), and electrophysiological data showed weak currents and action potentials of only ~23% of cells • Late passage iPSC had a comparable differentiation capacity to ESC, and proper functioning neurons (electrophysiology) • iPSC can differentiate into all neuronal (Sox1, 3/Pax6, Nestin, bIII+), astrocytic (GFAP+) and oligodendrocyte lineages (O4+) • No difference in differentiation efficiency between ESC & iPSC lines • Exposure to RA increases aneuploidy (2´), micronuclei occurrence (2´) and decreases surviving expression (half) in iPSCs and ESCs
• Neuronal markers (IHC) • In vivo: transplanted into cochlea – survived and differentiated towards neural lineages (IHC)
• Motor & sensory neuronal markers (IHC)
• Neuronal markers (IHC, PCR)
• Neuronal markers (IHC)
• Neuronal markers (IHC) • Glial markers (IHC)
Verification of Phenotype
Results
Table 4A. General Neural Induction in iPSCs—Mouse Donor Cells (Continued)
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• Neural lineage (Pax6+): Efficiency ESC >80% (efficiency of iPSC NR) • Adherent monolayer culture condition with dual SMAD inhibition (noggin & SB431542)
Fbs (F) (58)
• 2× iPSC, LV (OSKM) • 1× ESC
• NE (Pax6+) (90% all groups except 1 iPSC line yielded 0%) • EB neural induction
Fbs (F, A) (413)
• Region-specific NEs can be produced with the same protocol as for ESC. • iPSC Line-to-line variation in efficiency –– LV-iPSC similar efficiency to ESC –– 2× RV-iPSC lines required longer time, but achieved similar efficiency at end –– 1× iPSC line failed to yield NE cells • Generated glutamatergic neurons (~60–65% all groups, except 1 line – 0%) • Generated DA neurons (90% • EB neural induction + dual inhibition of activin/ nodal and BMP signaling pathway
Fbs (E, N) & MSCs (A) (178)
• 3× iPSC RV (OSKM) • 6× ESC
Cell Line(s) Details
Results
• Significant line-to-line variability towards neural induction (both ESC and iPSC) (EB protocol only) – suggests innate intrinsic differences in both ESC & iPSC • Neural induction efficiency can be increased by dual inhibition of activin/nodal & BMP signaling pathway (by small molecules SB431542 & DM) regardless of intrinsic neural differentiation propensity • Dual inhibition of activin/nodal & BMP pathways reduces capacity to differentiate towards other nonneural lineages • iPSC line-to-line variation: variable NE differentiation effi• NE (Pax6+) • 5× iPSC, LV (OSNL) ciency – does not depend on viral integration of transgene, • 5× iPSC, episomal plasmid, –– iPSC: 15–87% residual transgene expression or type of donor cell (OSKMNL) (no permanent –– ESC: 91–97% • iPSC follow same temporal progression as ESC transgene integration) • EB neural induction • 2× iPSC, RV, (OSKM) • 5× ESC • NPC (Pax6+) • Similar differentiation efficiency of iPSC and ESC • 1× iPSC, LV (OSKMNL) • Addition of DM significantly increased neuronal yield • iPSC 70.4% • 2× ESC • Noggin or SB431542 alone resulted in poor neuronal yield • ESC 88.7% • Found that dual addition of DM + SB431542 did not yield • Adherent monolayer significantly higher amounts of neurons compared with DM culture condition with only, nor did Noggin + SB431542 Activin/BMP inhibition • Lines derived with DM protocol could terminally differenti(by DM, SB431542 and/or ate into neurons (bIII+) Noggin) • iPSC line-to-line variation: different neuronal differentiation • NPC – efficiency NR • 1× iPSC, RV (OSKM) bias (variable proportions of GABA and excitatory markers) • Spin EB neural induction • 1× iPSC line, dox-inducible (VGLUT1+: GABA+ ratio: 9:1 iPSC line #1, 3:7 iPSC line LV, (OSKML) #2) • 2× ESC • Neural rosette formation delayed in IPSC by 2–3 days compared to ESC • Did not compare ESC to iPSC efficiency • Neural lineage (efficiency • 2× iPSC, RV (OSKM) • Neural induction efficiency reported as relative mRNA measured as relative • 2× ESC (mouse) expression. Values were normalized in reference to normal mRNA expression of select EB protocol expression of mRNA (=1) neural markers – arbitrary • Chlorate treatment increase neural marker expression value) >3× (ESC) (significant) and ~1× expression of bIII+ (as • EB neural induction with sulfate reduction (chlorate mature neuronal marker) treatment) • Chlorate treatment increased neural marker expression ≤5× (iPSC) (no significance reported) and ~2× expression of bIII+ (as mature neuronal marker)
Efficiency/Protocol
Donor (Age)
Table 4B. General Neural Induction in iPSCs—Human Donor Cells (Continued)
• Neuronal markers [(IHC, PCR (mRNA), WB)]
• Neuronal markers (IHC, PCR) • Electrophysiology – formed functional synapses
• Neuronal markers (IHC, WB)
• Neuronal markers (IHC, PCR) • Electrophysiology • Glial markers (IHC)
• Neuronal markers (IHC, PCR)
Verification of Phenotype
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• mDA (TH ) ~5% (% of total bIII+ neurons) • EB neural induction, with fibronectin)
• mDA (TH+/aldh1a1+) iPSC 6.5% • EB neural induction • mDA (TH+) iPSC 2.5–4.9% (ESC NR) • Stromal feeder cell line coculture
• mDA (TH+) iPSC ~30% • EB neural induction, xeno-free
• mDA (TH+) ~35–45% (all groups) • Stromal feeder cell line coculture
• mDA (TH+) >60% (all groups) • EB neural induction with LMX1A overexpression
Human Fbs (F) (49) Macaque Fbs (89)
Human Fbs (F) & MSC (A) (358)
Human Fbs, Smooth muscle (N, A) (307)
Human Fbs (A) & keratinocytes (juvenile) (321)
+
Efficiency & Protocol
Mouse Fbs (E + A) (387)
Species, Donor (Age)
• 1× iPSC, RV (OSKM) • 1× iPSC, RV (OSKM) – from Fanconi anemia patient • 2× ESC lines
• 2× iPSC, RV (OSKM) • Compared to ESC from previous study using same protocol (358) with efficiency of 35.5% (TH+) • 3× iPSC, LV (OSNL) • 1× iPSC, LV (OSLNL) • 2× iPSC, RV (OSKM) • 2× iPSC, protein based (OSKM) • 2× ESC
Cell Line(s) Details • 3× iPSC, RV (OSKM) • 1× drug selected for nanog • 1× drug selected for oct4 • 1× no drug selection • 1× iPSC, RV (OSNL) • 1× ESC • 4× iPSC, RV (OSKM) • 1× ESC
Results
• iPSC could differentiate into NSCs and DA neurons at a comparable rate to ESC (from previous study) • Molecular analysis of gene expression of iPSC and ESC and their derived cells – are similar in composition • No teratoma formation (3 months) • iPSC-derived neurons could be efficiently transduced with baculovirus (potential for gene therapy) • Variable neural induction efficiency (50–90%) between iPSC lines (but no significance reported). No difference of mDA yield between groups reported. • No reported differences between different iPSC generated with different reprogramming factors • iPSC – protein-based most similar in cellular/ morphological characteristics to ESC. –– iPSC-LV derived NPCs had residual exogenous transgene expression, which was increased in response to cAMP treatment, and neural differentiation –– iPSC-LV, RV-derived NPCs exhibited early senescence • Teratoma formation following NPC transplantation • No reported difference between iPSC and ESC differentiation efficiency • Using LMX1A overexpression yielded 10-fold higher mDA numbers (all groups) compared to normal EB neural induction protocol • Teratoma formation NR
• Line-to-line variability in iPSC differentiation efficiency (not significant) • No teratoma formation (6 months)
• Yield of mDA similar in iPSC & ESC • Teratoma formation reported (6 weeks)
• No comparison between different iPSC lines • Teratoma formation reported when transplanted unpurified mDA population. Transplanting purified mDA did not result in teratomas after 2 months
Table 4C. Specific Neuronal Subtype Induction—Midbrain Dopaminergic Neurons
In vitro: • Neuronal/DA markers (IHC, PCR) • Synaptic markers (IHC) • DA release assay • Electrophysiology In vivo: Transplanted mDA precursors into adult mouse striatum. Survived to 4 month & differentiated into DA neurons (IHC)
In vitro: • Neuronal/DA markers (IHC, PCR) • DA release/uptake assay • Electrophysiology • In vivo: transplanted NPC’s into rat model of PD. Survived & differentiated; expressed DA markers (IHC), reduced disease phenotype (reduced motor asymmetry – behavioral)
• In vitro: Neuronal/ DA markers (IHC, PCR) • In vivo: Transplanted partially differentiated iPSC into rat model of PD. Differentiated into mDA (IHC, PCR) In vitro: Neuronal/DA markers (IHC, PCR) In vivo: • Transplanted into rat; graft survived & integrated (IHC) • Transplanted into rat model of PD. Graft survived (IHC), formed synapses (IHC) and reduced disease phenotype (reduced motor asymmetry – behavioral). • In vitro: Neuronal/ DA markers (IHC, PCR, microarray) • In vivo: transplanted into rat model of PD. Survived & expressed DA markers (IHC), and reduced disease phenotype (reduced motor asymmetry – behavioral)
In vitro: Neuronal/DA markers (IHC) In vivo: • Transplanted into mouse model of PD. Attenuated disease phenotype [reduced motor asymmetry (behavioral) & increased mDA neurons (IHC)] • Formed synaptic connections (electrophysiology).
Verification of Phenotype
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Human (Fbs) (138)
Human Fbs (N, A) (149)
Human (172)
Species, Donor (Age)
• Motor neurons (islet+) –– iPSC 33% –– ESC 28% (% of total bIII+ neurons) • EB neural induction or adherent monolayer culture condition • Motor neurons Olig2+ –– iPSC 60–70% –– ESC 41% HB9+ –– iPSC 10–23% –– ESC 30–50% (% of total bIII+ neurons) • EB neural induction • Motor neurons (CHAT+/HB9+) –– iPSC 50–72% –– ESC 50–62% • EB neural induction, then delivery of neurogenin, islet-1 & LIM/homeobox protein 3 (by AV transduction) (“N.I.L”)
Efficiency & Protocol
Cell Line(s) Details
• 3× iPSC, RV (OSKM) • 2× ESC
• 3× iPSC, LV (OSNL) • 1× iPSC, episomal plasmid (OSKMNL) • 1× ESC
• 1× iPSC – method of generation not specified • 1× ESC
Table 4D. Specific Neuronal Subtype Induction—Motor Neurons
• Protocol with N.I.L transduction shortened protocol to 11 days. No significant difference in differentiation efficiency between iPSC and ESC reported.
• EB neural induction yielded motor neurons at a too low efficiency • Adherent culture conditions yielded high numbers of motor neurons • Lower yield of neural specific EB from iPSC (~13%) compared to ESC counterpart (~33%) • Motor neuron generation comparable to ESC • Attained appropriate rostrocaudal identity (similar to ESC derived cells) • Proportion of OLIG2+ progenitors was higher in iPSC compared to ESC, but proportion of H9+ progenitors was lower. • No difference in differentiation efficiency between different iPSC lines
Results
• Motor neuron markers (IHC, PCR) • NMJ formation (IHC) • Electrophysiology
• Motor neuron/synaptic markers (IHC) • Electrophysiology • Coculture with muscle: neurite connections & contraction of myotubules
• Neuronal/motor neuron markers (IHC) • Electrophysiology
Verification of Phenotype
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• NPC (Efficiency NR) • EB neural induction
Macaque Fbs (E) – HD (59) Human Fbs (A) – SZ (292)
• Glutamate neurons – Efficiency NR • EB neural induction – direct neural differentiation by WNT3A
• Striatal neurons (DARPP-32+) ~10% (all groups) • EB neural induction
Human Fbs (A) – HD (416)
iPSC, RV (OSKM) • 4´ iPSC, RTT patients • 5´ iPSC, healthy donors • 1´ ESC
• GABAergic neurons (GABA+) iPSC ~17–20% (both groups) (% of total MAP2+ neurons) • EB neural induction
No details about iPSC generation specified • 1´ iPSC, HD patient • 1´ iPSC, healthy donor • 1´ ESC • 1´ iPSC, RV (OSK) from macaque model of HD iPSC, RV (OSKM) • 2´ iPSC, SZ patients • 1´ iPSC, SZ patient with 22q11.2 deletion • 2´ iPSC, healthy donors
iPSC, LV (OSKM) • 3´ iPSC, FL patient • 1´ iPSC, healthy donor • 1´ ESC
iPSC, RV (OSKM) • 5´ IPSC, RTT patients, mutations in different domains (WT-RTTaffected X chromosome inactive, monoallelicRTT and biallelic-RTT) • 3´ iPSC, healthy donors • 2´ ESC • 3´ iPSC, RV (OSKM), from RTT patient
• Evaluated ability to form (1) neural stem/progenitor cells (Nestin+) (2) neurons (bIII+) and (3) astrocytes (GFAP+) • Nestin: ~35–42% (all conditions) • bIII+ –– ESC/ WT-RTT: ~55% –– Mutant-RTT-iPSC: ~22% • EB neural induction • Neurons – Efficiency NR • EB neural induction
• Generated NCS – Efficiency NR • Stromal feeder cell line coculture method
Cell Line(s) Details
Efficiency
Human Fbs (A) – FD (202)
Human Fbs (A) – RTT (70) Human Fbs (A) – RTT (238)
Human Fbs (A) – RTT (181)
Donor (Age) & Disease
Table 4E. Neural Induction—iPSC Derived From Donors With Neurological Disease
• Disease phenotype retained in differentiated NPCs (increase of mutant huntingtin transcript & formation of intranuclear inclusions) • No comparison between differentiation efficiency given • Disease phenotype evident in differentiated cells (protein expression relevant to SZ pathogenesis. • 22q11.2 iPSC line had delayed reduction of nanog and oct4.
• Differentiated neurons had reduced soma size compared to neurons derived from WT-RTT iPSC (inactive X chromosome) • Differentiation efficiency was not compared to ESC, but was similar in both WT & RTT-iPSC. • Disease phenotype evident in differentiated cells (fewer synapses, defects in glutamate transport, reduced soma, reduced spine intensity, altered Ca2+ signaling & electrophysiological defects. Drug treatment (with IGF1) could attenuate disease phenotype (increase synapse number) • Note: no difference in neuronal survival/proliferation in RTT versus WT-iPSC. • No difference in differentiation efficiency between groups (data not shown) • Disease phenotype retained in differentiated cells: FL- iPSC – NCS had defects in IKBKAP splicing, decreased gene expression associated with peripheral neurogenesis & differentiation, and decreased migration • No difference between differentiation efficiency reported • Disease phenotype evident in differentiated cells [altered signaling pathway (ERK), increased cellular toxicity (caspase activity)]
• No difference in (1) EB formation (2) NSC formation in any condition • Mono & bi-allelic RTT-iPSC lines had significant lower neuron differentiation (bIII+ expression) compared to healthy iPSC, and WT-RTT iPSC lines, as well as lower Pax6+ levels • No difference in astrocyte differentiation (GFAP+)
Results/Disease Phenotype Retained?
(continued)
• Neuronal/glutamatergic markers (IHC, PCR, microarray) • Electrophysiology
• Neuronal markers (IHC)
• Neuronal markers (IHC)
• Neuronal markers (IHC, PCR, microarray)
• Neuronal/ glutamatergic markers (IHC, PCR) • Synaptic markers (IHC) • Electrophysiology
• Neuronal markers (IHC)
• Neuronal markers (IHC, PCR) • Astrocyte marker (IHC)
Verification of Phenotype
iPSC TECHNOLOGY FOR SCI 589
Human (A) Fbs – PD (82)
Human Fbs (A) – PD (325)
Human Fbs (A) – PD (91)
Human Fbs (A) – PD (268)
Human Fbs (A) – PD (338)
Human Fbs (A) – SZ (42)
Donor (Age) & Disease Cell Line(s) Details
Results/Disease Phenotype Retained?
iPSC, dox-inducible LV (OSKML) • No difference in differentiation efficiency between groups • 5´ iPSC lines, SZ patient • 30% neurons (bIII+) GABAergic • 3´ iPSC lines, healthy donor • 90% (all) • Neurons (NeuN/ bIII+) >90% (all) • Oligodendrocytes (O4+) 56% (all) • Astrocytes (GFAP+) 91.7% (all) • Stromal feeder cell line coculture
• Motor Neurons (HB9) 20% • EB neural induction • Motor neurons (HB9+) –– iPSC 10% –– ESC 11% • EB neural induction as well as with SMAD inhibition (58)
Human Fbs (A) – PD (234)
Human Fbs (A) – ALS (93)
Human Fbs (A) – ALS (35)
• mDA (TH+) –– iPSC 3–9% –– ESC 5% • Stromal feeder cell line coculture
Human Fbs (A) – PD (132)
• No overall difference in differentiation efficiency between groups. Suggests no presence/absence of c-myc or presence/absence of exogenous transgenes does not interfere with differentiation in vitro • No difference in vivo properties between any group: migration pattern, differentiation or proliferation • No teratoma formation at 12 weeks
• Residual transgene expression (sox2, klf-4)
• iPSC produced motor neurons at a comparable rate to ESC • No difference in variation depending on health status of donor, age, level of residual transgene expression, or 3 or 4 factors • Donor sex and “donor identity”* were associated with variation in differentiation potential (female improved potential). *donor identity – genetic background. • 3/16 iPSC lines had to be treated by modified protocol (SMAD inhibition for early neutralization) to achieve differentiation • Efficiency of differentiation of each line was reproducible by independent laboratory suggesting that these are intrinsic characteristics
• 1× iPSC, RV, (OSKM), ALS patient (82 years) • 16× iPSC, RV – Different variables: age, sex, health status, donor identity, 4 factors (OSKM) or 3 (OSK) • 6× ESC controls
• No overall difference in differentiation efficiency between groups – Suggests neither donor type or transgene expression influences efficiency. Note that 1× iPSC line showed enhanced neural differentiation efficiency • No teratoma formation (3 months) • Disease phenotype not evident in PD-iPSC-derived neurons (no a-synuclein inclusion bodies)
• iPSC, dox-inducible LV. Compared (1) presence or absence of c-myc, (2) excision or nonexcision of transgenes (cre-lox) • 1× iPSC (OSKM) • 2× iPSC (OSK) • 1× iPSC (OSK – excised transgenes • 1× ESC
• iPSC, dox-inducible LV (OSK) • 2× iPSC: transgene free (excision), PD patient • 2× iPSC: no excision; contains exogenous transgenes, PD patient • 1× iPSC, healthy donor • 1× ESC
(continued)
• Neuronal/motor neuron/synaptic markers (IHC, PCR) • Electrophysiology
In vitro: Neuronal/DA markers (IHC) In vivo: • Transplanted into unlesioned rat; survived & differentiated into several neuronal subtypes (IHC) • Transplanted into rodent model of PD. Graft survived & expressed Neuronal/DA markers (IHC). Behavioral improvement (reduced motor asymmetry). No improvement in more complex motor function. • In vitro: neuronal lineage specific markers (IHC) • In vivo: (1) transplanted NPC’s into subventricular zone of rat. Found migration pattern (via rostral migratory stream to olfactory bulb) similar in all groups, as well as differentiation into neurons, astrocytes and oligodendrocytes; (2) transplanted oligodendrocytes. Migration pattern (white matter pathway along corpus callosum & internal capsule) same among all groups. • Neuronal/motor neuron markers (IHC) • Glial markers (IHC)
iPSC TECHNOLOGY FOR SCI 591
• iPSC, RV (OSKM) • 2× iPSC, X-ALD childhood cerebral (severe) • 1× iPSC, adrenomye loneuropathy (AMN) • 1× iPSC, healthy donor • 1× ESC • 5× iPSC, RV (OSKM), SMA donor (type 1 with homozygous deletion of SMN-1 gene)
• 3× iPSC, RV (OSKM), PWS patient (paternal deletions in 15q11-q13) • 2× ESC • 2× iPSC, RV (OSKML), AngS patient (maternal deletions of 15q11-q13) • 1× iPSC, RV (OSKML), PWS patient (paternal deletions of 15q11-q13) • 1× iPSC, healthy donor • 1× ESC
• OPCs (PDGFR+) ~73–91% (all groups) • Oligodendrocytes – NR • EB neural induction (with DM & SB431542)
• Motor neurons (HB9+) ~7–10% (all groups) • EB neural induction (with DM & SB431542)
• Neuronal lineage (bIII+/ MAP2+) • Efficiency NR • EB neural induction
• Neuronal lineage (bIII+/ MAP2+) • Efficiency NR • EB neural induction
Human Fbs (A) – X-ALD (161)
Human Fbs (A) – SMA (62)
Human Fbs (A) – PWS (404)
Human Fbs – PWS & AngS (57)
iPSC, LV (OSNL) • 2× iPSC, ALS patient • 1× iPSC, healthy donor
Cell Line(s) Details
• Motor neurons (ChAT+) 4 weeks; 6 week time point: –– ALS-iPSC ~9.5; ~4.3% –– WT-iPSC ~12; ~22 (% of total bIII+ neurons) • Chopping technique with neural selection medium (355)
Efficiency
Human Fbs (child) – ALS (100)
Donor (Age) & Disease Results/Disease Phenotype Retained?
• No difference in differentiation efficiency between iPSCs and ESC. • Disease phenotype evident in differentiated cells (decreased capacity to form motor neurons, and abnormal neurite outgrowth). Disease phenotype could be rescued with SMN expression (transduction) – increased neurite outgrowth. • iPSCs could differentiate towards neuronal lineage, and give rise to neurons and astrocytes (s100b) • Disease phenotype retained in iPSCs (genomic imprint not deleted) (reduced expression of disease associated small nucleolar RNA HBII 85/SNORD116) • iPSCs could differentiate towards neuronal lineage but with variable efficiencies (data not shown) – only selected 1 AngS line & 1 normal iPSC line for further analysis. • Functional neurons could be obtained • Disease phenotype retained in iPSC & differentiated cells (genomic imprint not deleted, reduction in UBE3A for AngS line)
• No difference in differentiation efficiency between groups at 4 weeks. Further culture to 6 weeks showed significant fewer motor neurons in ALS-iPSC-derived neurons compared to WT. • Disease phenotype evident in differentiated cells (reduced SMN (survival motor neuron transcript), size/number of motor neurons (at 6 weeks), defective synapse formation). Valproic acid & tobramycin: attenuated disease phenotype (increase SMN) • No difference in differentiation efficiency between groups to OPCs or oligodendrocytes (MBP+) • Disease phenotype evident in differentiated oligodendrocytes cells (increased VLCFA) (very long chain fatty acid). This could be reversed by drug treatment with lovastatin or 4-phenylbutyrate.
Table 4E. Neural Induction—iPSC Derived From Donors With Neurological Disease (Continued)
• Neuronal/synapse markers (IHC) • Astrocyte markers (IHC) • Electrophysiology
• Neuronal markers (IHC) • Astrocyte markers (IHC)
• Motor neuron marker (IHC)
• Oligodendrocyte markers (IHC)
• Neuronal/motor neuron/synapse markers (IHC)
Verification of Phenotype
592 kramer ET AL.
• Oligodendrocytes (O4+) 43.5% • EB neural induction with A2B5+ immunopanning selection • Oligodendrocytes (MBP+) 18% • EB neural induction
Mouse Fbs (E) (274)
• Oligodendrocytes (O4+) 10 markers) (majority >90%) • EB neural induction (with EGF)
Human Fbs (A) (275)
Human Fbs (A) (299)
Mouse Fbs (E) (85)
• OPC (A2B5+) –– iPSC 14.1% –– ESC 12.6% • Oligodendrocytes (O4+) –– iPSC 2.3% –– ESC 24% • EB neural induction
Efficiency & Protocol
Mouse Fbs (E) (368)
Species, Donor (Age)
Table 4F. Induction Towards Oligodendrocyte Lineage
• Differentiation into NPC similar in iPSC and ESC (95.8% and 96% respectively) • Differentiation into OPCs similar in iPSC and ESC (14.1% and 12.6% respectively) • Differentiation into oligodendrocytes from OPCs very low in iPSC compared to ESC – intracellular factors in iPSCs inhibiting differentiation? • Higher efficiency of oligodendrocyte differentiation with A2B5 immunopanning, as compared to previous study (368)
• 1× iPSC, RV (OSKM) with nanog drug selection • 1× ESC
• No difference between 3 or 4 factor iPSC lines • PDGF protocol did not yield any oligodendrocytes • EGF yielded 90% • EB neural induction with patterning of NPCs
Efficiency & Protocol
Human Fbs (192)
Species, Donor (Age)
Table 4G. Induction Towards Astrocytic Lineage
• iPSC could be efficiently differentiated into astrocytes via NSS floating method. • No teratoma formation at 8 weeks
• No difference in differentiation efficiency between groups – one pluripotent cell can produce 2.8 × 1012 immature astrocytes • Produced region specific immature astrocytes by patterning NE cells. • Minimal contamination of immune cells or neuronal cells in resultant differentiated population. • Teratoma formation NR • Line to line variation of differentiation efficiency between iPSC lines
Results
• Astrocyte marker (IHC, PCR) • In vivo: transplanted into rat model of SCI. Graft survived at 8 weeks. No improvement of injury phenotype (locomotion, tissue sparing, myelination) instead increased sensitivity to mechanical stimuli
• Astrocyte markers (IHC, PCR) • In vitro: transwell migration assay
In vitro: • Astrocyte markers (IHC, PCR, WB) • Functional: Electrophysiology, glutamate uptake, promotes synaptogenesis, propagation of Ca2+ In vivo: Transplanted into mouse brain: retained regional identity
Verification of Phenotype
594 kramer ET AL.
• 1× iPSC, RV (OSKM)
• Photoreceptors (Pax6−/RHO+) (iPSC: 1–3%) • EB neural induction with DKK-1, IGLF-1 & noggin
Verification of Phenotype
In vitro: • Photoreceptor markers (IHC, PCR, WB) • Calcium imaging: functional membrane properties In vivo: • Transplanted into retinal degenerative mice (Rho–/–). Integrated with host – synapse formation (IHC), bipolar cell marker colocalization (IHC), and improvement in disease phenotype (ERG)
In vitro: • Rod photoreceptor markers (IHC, PCR) • Evidence of rod outer segment generation (IHC, morphology) In vivo: transplanted into swine with chemically ablated rods. No improvement of function (ERG), but evidence of rod integration (IHC) In vitro: • RPC markers (IHC, PCR) • Further differentiation into RPC lineages (IHC, PCR) In vivo: transplanted RPC into healthy mice. Note that few transplanted cells incorporated. Few expressed RHO+, and did not have outer segment (indicated immature phenotype) • RPC markers (IHC, PCR) • Further differentiated into RPC lineages (IHC, PCR)
“Efficiency & Protocol” lists the desired cell type that iPSCs were differentiated into, and the method of neural induction. The efficiency of differentiated cells obtained is reported as total number of cells unless otherwise stated. Typically only a few select markers are chosen to calculate differentiation efficiencies (chosen marker indicated in brackets following cell type), but cells are often more thoroughly phenotyped using additional markers by different techniques (“Verification of Phenotype”). “Cell line(s) details” documents number of iPSC lines used (and control ESC lines if applicable), as well as type of transgenes and method of transgene delivery used to generate iPSCs. Note that only studies which have included enough detail about the neural differentiation procedure are included. (A), adult; (E), embryonic; (F), fetal; (N), newborn; AngS, Angelman’s syndrome; AV, adenovirus; ALS, amyotrophic lateral sclerosis; bIII, bIII tubulin; BMSC, bone marrow stromal cell; DKK1, Dickkopf-1; DM, dorsomorphin; EB, embryoid body; ERG, electroretinography; Fbs, fibroblasts; FBS, foetal bovine serum; FD, familial dysautonomia; HD, Huntington’s disease; IHC, immunohistochemistry; IGF-1, insulin like growth factor-1; L, Lin28; LV, lentivirus/lentiviral; MAP2, microtubule associated protein 2; mDA, midbrain dopamine/dopaminergic neurons; MSC, mesenchymal stem cell; N, nanog; NCS, neural crest cell; NE, neuroepithelial cell; N.I.L, neurogenin; islet-1; LIM/homeobox protein 3; NMJ, neuromuscular junction; NPC, neural precursor cell; NR, not reported; NS, neurosphere; NSS, neural stem sphere; OPC, oligodendrocyte precursor cell; OSK, oct3/4, sox2, klf-4; OSKM, oct3/4, sox2, klf-4, c-myc; OSKMNL, oct3/4, sox2, klf-4, nanog, lin-28; PD, Parkinson’s disease; PWS, Prader-Willi Syndrome; RA, all trans retinoic acid; RHO, rhodopsin; RPC, retinal progenitor cell; RTT, Rett’s disease; RV, retrovirus/retroviral; SMA, spinal muscular atrophy; SMN, survival motor neuron; SSEA-1, stage specific embryonic antigen-1; SZ, schizophrenia/schizophrenic; TTF, tail tip fibroblast; WB, western blotting; WT, wild type; X-ALD, adrenoleukodystrophy.
Mouse Fbs (A) (372)
• No difference in differentiation efficiency between mouse or human iPSCs and comparable to ESC differentiation • Protocol with addition of DKK1 & lefty-A yielded more RPCs compared to other protocols • iPSCs could further differentiated into retinal ganglion cells, rod & cone photoreceptors (as well as into retinal pigment epithelium) • iPSCs could differentiate into mature photoreceptors, but about 30% of the population remained undifferentiated • Transplantation of –– undifferentiated population caused high rate of tumor formation –– 1 round of purification (anti-SSEA1 selection) lower rate of tumor –– 2 rounds of anti-SSEA1 selection: no tumors at 16 weeks
• 1× iPSCs, RV (OSKM) with nanog drug selection • 2× iPSCs, RV (OSKM) • 1× iPSC, RV (OSK) • 1× ESC (mouse)
• RPCs (Pax6+/Rx+) (~18–19% all groups) • EB neural induction with either –– No additional compounds –– Addition of DKK1 & Lefty-A –– Addition of DKK1, Lefty-A, FBS & Activin
Mouse Fbs (E) & Human Fbs (141)
• iPSCs could differentiated into RPCs and further differentiated into retinal ganglion cells, rod & cone photoreceptors • No tumor formation (4 weeks)
• RPCs (Pax6+/Rx+) (~21%) • EB + retinal conditioned medium.
Mouse Fbs (E) (287)
• 1× iPSC, RV (OSKM) with nanog drug selection
Results • iPSC could differentiate into rod photoreceptors
Cell Line(s) Details • 1× iPSC, LV (OSKM)
Efficiency & Protocol
• Rod photoreceptors (RHO+) (1–7%) • EB floating culture, with DKK1, noggin & ILGF-1, then plated on Matrigel (increased RHO+ cells with outer segment) (196)
Swine Fbs (F) (426)
Species, Donor (Age)
Table 4H. Induction Towards Retinal Neurons
iPSC TECHNOLOGY FOR SCI 595
(136)
(271)
(371)
Reference
iPSC generation • Donor cells: MEF • RV (OSKM) nanog drug selected Labeling: transduced with PKH26 Red fluorescent cell linker Differentiated into astrocytes (GFAP+) (neural stem sphere floating method)
iPSC generation • Donor cells: MEF or mouse TTF • RV (OSK with and without c-myc) • Nanog drug selection (safe) or no drug selection (unsafe) Labeling: transduced with CBRluc and mRFP Differentiated into secondary NS (“SNS”) (EB neural induction) iPSC generation • Donor cells: human Fbs • RV (OSKM) Labeling: transduced with Venus fluorescent protein Differentiated into NS (EB neural induction)
Cell Line Details
Injury model • Sprague–Dawley rats (adult, female) • Moderate contusion injury (200 kd, IH impactor) • Cell injections 3 or 7 days post-SCI (1 × 105 into lesion epicentre) • Immunosuppressed with cyclosporine A Injections @3 days post-SCI 1. SCI + iPSC-astrocytes (n = 20) 2. SCI + DMEM (n = 10) Injections @7 days post-SCI 3. SCI + iPSC-astrocytes (n = 9) 4. SCI + DMEM (n = 7) Time points • Histology: 8 weeks post-SCI • Behavior: weekly up to 8 weeks post-SCI
Injury model • NOD-SCID mice (adult, female) • Contusion (60 kd – IH impactor), T10 • Cell injections 9 days postinjury (5 ´ 105 into lesion epicentre) Groups 1. SCI + PBS (n = 29) 2. SCI + iPSC-NS (n = 31) Time points • Histology: 56 days post-SCI (n = 10), 112 days post-SCI (n = 18) • Behavior: weekly up to 56 or 112 days post-SCI
Injury model • C57BL/6J mice (adult, female) • Contusion (60 kd – IH impactor), T10 • Cell injection 9 days post-SCI (5 ´ 105 cells into lesion epicentre) Groups: note that not all groups/controls were used for all experiments 1. SCI + iPSC-SNS (38C2) (n = 19) 2. SCI + iPSC-SNS (335D1) (n = 9) 3. SCI + “unsafe” iPSC-SNS (256H18) (n = 9)‡ 4. SCI + “uns afe” iPSC-SNS (256H13) (n = 9)‡ 5. SCI + iPSC-PNS (primary) (n = 13) 6. SCI + ESC-SNS (n = 15) 7. SCI + fbs (n = 13) 8. SCI + PBS (n = 12) Time points • Histology: 35 or 42 days post-SCI • Behavior: weekly, up to 42 days post-SCI
Regime: SCI + Groups
Table 5. iPSC-Based Therapies in Experimental SCI
Histological profile: some improvements in iPSC grafted animals compared to PBS control • 18% iPSC-SNS (38C2) graft survival (Bioluminescence) at 35 days post-SCI, some cells had migrated as far as 4 mm from epicentre (rostral-caudal). Differentiation into neurons (Hu+), astrocytes (GFAP+), and oligodendrocytes (p-GST+) reported • iPSC-SNS (38C2) graft promoted myelination (LFB staining) compared to PBS control. Note: RFP+ cells colocalized with myelin basic protein (MBP), iPSC-SNS also transplanted into MBP-null shiverer mice and MBP+ were found together suggesting graft-derived oligodendrocytes are remyelinating • iPSC-SNS (38C2) graft promoted increased axonal growth (5HT) compared to PBS control. • iPSC-SNS (38C2) graft showed no tumor formation/nanog+ cells. Transplantation of “unsafe” iPSC-SNS (256H18) showed tumor formation, with nanog+ cells in all animals. Second “unsafe” (256H13) clone, only one animal showed tumor formation iPSC graft improved histological profile up to 112 days post-SCI • Graft survived (IHC) & differentiated into neurons (NeuN+, bIII+), astrocytes (GFAP+), and oligodendrocytes (APC+), with some left undifferentiated (Nestin+) • Neuronal subtypes mainly GABAergic • Synapses between host and donor cell observed (IHC) • Graft promoted angiogenesis (increase in VEGF & PECAM-1) • Graft promoted increased axonal regrowth (NF, 5HT, GAP43) • No change in CGRP+ (dorsal column) axons • Graft promoted tissue sparing Graft promoted myelination (LFB staining) • No tumor formation up to 112 days. No sig. difference in histological profile in iPSC-grafted animals compared to controls • 4.7% graft survived (IHC) at 8 weeks post-SCI • No change in myelin (LFB staining) (3 day injection regime) • No change in CGRP+ (dorsal column) axons (3 day injection regime) • No change in astrocytes (GFAP+) area (3 day injection regime) Functional recovery comparable to ESCSNS. • BMS experiment 1: compared groups 1,5,6,7,8 – iPSC-SNS & ESC-SNS slightly higher than PBS, Fbs & iPSC-PNS at day 42 • BMS experiment 2 : compared groups 2,3,4,5,8 – iPSC-SNS, ESC-SNS, and “unsafe” iPSC-SNS (256H13) scored slightly higher than PBS and “unsafe” iPSC-SNS (256H18) at day 42 (attributed this to tumor formation)
No improvement in motor function at 8 weeks post-SCI (both 3 & 7 day injection) • BBB-LS • Inclined Plane Test • SCANET movement Increased sensitivity to sensory stimuli in iPSC-astrocyte groups (in 3 day injection regime only) • Mechanical withdrawal (plantar aesthesiometer) – decreased threshold in iPSCastrocyte group • Thermal stimuli (nociceptive thresholds) – decreased threshold in iPSC-astrocyte group compared to unlesioned rats
Graft slightly improved motor recovery compared to PBS control • BMS (improvement since day 28 and up until day 112) • Rotarod (increased time) • Treadmill (DigiGait) (increased stride length) • Electrophysiology: motor evoked potential improvement
Histological Data
Behavioral Data
596 kramer ET AL.
iPSC generation • Donor cells: human fibroblasts • RV (OKS) Labeling: transduced with luciferase and GFP Differentiated into neuroepitheliallike stem cells (NES) (monolayer) iPSC generation • Donor cells: human fbs • RV (OSKM) • Undifferentiated Injury model • Nude rats (adult, female) • Moderate contusion (200 kd – IH impactor), T10 • Cell injections 7 days postinjury (2 injections each 2.5 ´ 105, 0.2 mm rostral & caudal from lesion epicentre) Groups 1. SCI + undifferentiated iPSC (n = 7) 2. SCI + human fbs (n = 7) 3. SCI + media (n = 9) Time points • Histology: Day 35 post-SCI • Behavioral: weekly, up to 4 weeks post-SCI
Injury model • NOD-SCID mice (adult, female) • Contusion (70 kd – IH impactor), T9 • Cell injection 7 days post-SCI (10 ´ 105 cells into lesion epicentre) Groups (numbers unknown) 1. SCI + iPSC-NES 2. SCI + human spinal cord NSC 3. SCI + culture medium Time points • Histology: 12 weeks post-SCI • MEP: 12 weeks post-SCI • Behavior: 8 weeks post-SCI • Cell ablation: 7 weeks post-SCI No sig. improvement in motor function in iPSC grafted animals. • BBB-LS: no sustained difference at day 35 (Note: day 28, significantly higher score for iPSC-grafted animals) • Gait parameters (RatWalk) – Stride length: no sig. difference at day 35. (Note: day 28, fbs had reduced front stride length) – Base of support*: no difference at day 35. (Note: day 28 significant reduction in front base of support in grafted animals (iPSC & fbs) – Placement of feet†: no difference at day 28 or 35.
Significant difference in functional recovery compared to media-only control, but no difference to NSC control. • BMS (no difference between NSC and iPSC-NES group, but significant improvement over medium only control group). Following cell ablation (Diphtheria toxin), no difference in BMS (trend for reduction in score only) • Electrophysiology (only compared iPSC-NES to medium only group): increased amplitude & decreased latency in iPSC-NES group No sig. difference in histological profile in iPSC-grafted animals compared to controls • ECM deposition: no sig. difference –– Collagen: trend for increased deposition in grafted animals (3–4-fold) (iPSC & fbs) –– Laminin: trend for increased deposition in grafted animals (2-fold) (iPSC & fbs) • Astrocytes (GFAP+): trend for decreased astrocytes in grafted animals (3-fold) (iPSC & fbs). Note that prominent GFAP+ processes were observed to invade lesion epicentre of grafted animals but not in media control. • Tissue sparing: no sig. difference • Axonal regrowth/sparing: no significant difference. Note that neurons (bIII+) were found to penetrate lesion epicentre in grafted animals only (iPSC & fbs) but not media-only animals –– bIII+: trend for increase (7-fold) for iPSC animals –– RT97+: trend for increase (3-fold) for grafted animals (iPSC & fbs) –– 5HT+ (descending): trend for increase (4-fold) for fb-grafted animals –– CGRP+ (dorsal columns): no change • No change in macrophage (ED1+) numbers across groups • No change in Schwann cell number recruitment/ phenotype across groups. Majority of Schwann cells were S100+/p75– • No evidence of tumor formation (any group)
Histological profile: (did not include SCI+NSC control group in analysis). • Graft survived (20%) (IHC) and differentiated into 75% neurons (Tuj1+), 20% astrocytes (GFAP +) and adult-bone marrow derived MSCs) Yes
Yes – 3/7 studies
(28,56, 198,265, 335,403, 417)
No
Yes
No
(198,210, 354) (131)
Yes
No
(396)
N/A – Healthy animals only (graft differentiated into desired phenotype) Yes
No
(25)
Yes
(154)
Yes
No
(7)
Liver disease Cirrhosis (M)
Hu, M,
Cardiac infarction Infarction in immuno competent & compromised hosts (M, R, RhM)
Hu, M, R, C
Differentiated graft (iPSCs into cardiac lineages), undifferentiated iPSC & comparison to adult derived MSCs
Ischemia (noncerebral) Hind limb (M) Sickle cell anemia (M)
Hu, M, C M
Hemophilia A (M)
M
Bone regeneration (M)
M
Differentiated grafts (iPSC-MSCs), (iPSC-EC) Autologous, differentiated graft & ex vivo gene therapy (iPSC-HP) Differentiated graft (iPSC-endothelial cells) Differentiated graft, transplanted as scaffold (iPSC-osteoblast)
Tracheal defect Surgical tracheal lesions (R) Diabetes Type 1 & 2 (M)
M
M
Pre-treated iPSCs with chondrocyte induction medium. Transplanted as scaffold Differentiated graft (iPSC, insulin-producing pancreatic b-like cells)
(85,299)
C, canine; EC, endothelial cell; HP, hematopoietic precursor; HSC, hematopoietic stem cell; Hu, human; M, mouse; MCA, middle cerebral artery; MSCs, mesenchymal stem cells; NES, neuroepithelial stem cell; NPC, neural precursor cell; OPC, oligodendrocyte precursor cell; R, rat; RhM, rhesus monkey; SNS, secondary neurospheres.
600 kramer ET AL.
results from these very limited number of studies suggest that iPSC-based therapy in SCI warrants more extensive and thorough testing. Ideally, research in this area should be conducted using comparable regimes in mouse and rat models, as outlined in the recommendations and guidelines developed by the International Campaign for Cures of Spinal Cord Injury Paralysis (ICCP) (107). As with pluripotent ESCs, several iPSC in vivo studies have reported high rates of teratoma formation (12,21,49– 51,63,66,255,272,307,319,357,371,372,383,402,405), a risk that seems to be increased with (i) c-myc or klf-4 reactivation (279), (ii) higher dosage of cells delivered (272,335,403) (also see ref. 417), (iii) changes in local environment; necrotic tissue such as the infarcted myocar dium seems to promote tumor expansion (402) (importantly, in SCI necrotic tissue is abundant), and (iv) the amount of undifferentiated cells present in the graft (85,255, 371,372). This latter point can be easily addressed by purifying the population by flow cytometry (e.g., negative selection for SSEA-1+ cells), which can prevent tumor for mation (372,387). But other studies have reported no evi dence of tumor formation in various animal models assessed at time points ranging from as early as 5 weeks to as late as 1 year (85,89,132,136,234,358,376,405). Of note, although one of the major advantages of iPSCs is potential patient-specific cells for treatment of disease, only very few studies to date have used autologous iPSCs for transplantation (131,198), which is important as recent evidence suggests that iPSCs may evoke an immune response in syngeneic recipients (419) (see “Immunoge nicity of Pluripotent Stem Cells”). Issues With Using iPSC-Based Therapies Tumorigenicity of iPSCs. Pluripotent stem cells and tumors share characteristics such as proliferative selfrenewal, lack of contact inhibition and telomerase activity, and they share expression of many common genes (29,75,117,341,393,415), and it has been shown that both pluripotent stem cells and their ESC derivatives can form teratomas in vivo (see “iPSCs in Spinal Cord Injury and Other In Vivo Models”). Teratoma formation is thought to be strongly linked to the number of undifferentiated cells in the graft (21,272). Furthermore, in vitro culture may inadvertently select for more tumorigenic ESCs/iPSCs, because clones that are fast growing and less likely to spontaneously differentiate are more likely to be preferentially propagated. These characteristics make the pluripotent stem cell likely to be more tumorigenic in vivo (187). Recent reports have indicated that iPSCs have increases risks of tumorigenicity compared to ESCs, as reprogramming factors used to induce pluripotency are oncogenic (c-myc and klf-4) (279), or linked to tumor pathways, and differences between iPSCs and ESCs in gene expression, epigenetics, and genomic instability, which have been
linked to increased tumorigenicity [for reviews, see refs. (23,187,191,258,276,391)]. Indeed, injection of iPSCs into nonobese diabetic/severe combined immunodeficient/interleukin 2g-null (NOD/SCID/IL-2–/–) mice (with both adaptive and innate immune deficiencies) resulted in aggressive teratomas with higher frequency and reduced time of onset compared to ESCs (126). Tumor formation risk can however be reduced with several strategies (for review, see ref. 187). Terminal differentiation of ESCs/iPSCs reduces the risk of teratoma formation, but differentiation alone cannot completely eliminate pluripotent stem cells from the population (113). Sorting by flow cytometry, for example, with negative selection of SSEA-1, which should be performed at least twice (372), antibody depletion of SSEA-5 (363), or possibly by natural killer (NK) cell targeting of pluripotent stem cells (97). The methods of obtaining iPSCs can also be modified to reduce the risk of tumors; the use of l-myc instead of c-myc (see “Generation of iPSCs: General Considerations of Delivery Approaches”), the use of nonintegrating transgene delivery systems to prevent tumorigenesis from reactivation of transgenes and/ or the integration of a suicide gene (50,68,200,423). Rigorous analysis of the cell line to select the lines with the least propensity of tumor formation for transplantation may also be beneficial (11,255,371). Lastly, regulation of glycogen synthase kinase 3b (Gsk3b) pathway may also be beneficial in reducing the tumor formation, at least in ESCs (209). Immunogenicity of Pluripotent Stem Cells. ESCs have demonstrated immune-privileged properties such as low levels of human leukocyte antigen (HLA) class I, absence of HLA class II, reduced NK ligands, as well as several in vivo demonstrations of immune evasion by transforming growth factor-b2 and serpin-6 expression, inducing T-cell apoptosis and T-regulatory-mediated suppression, in syngeneic and allogeneic transplants (3,31,98,99,188,207), even across a full major histocompatibility complex (MHC) barrier (226). But it is now becoming increasingly clear that ESCs are not truly immune privileged with several studies reporting that ESCs are capable of eliciting strong immune responses from both T-cells and NK cells (in syngeneic, allogeneic and xenogeneic recipients) (38,97,98,190,357). Inducing tolerance can be done with relative ease (in comparison to adult derived tissue), for example, by costi mulatory or coreceptor blockage (37,105,226,311). In contrast, the immunogenicity of iPSCs has not been extensively investigated so far. To date only four seminal reports on the immunogenicity of iPSCs have been published (89,97,291,419) (Table 7), and there are too few data to draw definite conclusions on iPSC immunogenicity. However, it is becoming clear that differences between iPSC and ESC immunogenicity may exist and it should
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Table 7. Summary of Reports Investigating Immunogenicity of iPSCs Reference (97)
(291)
(89) (419)
Comment • iPSCs/ESCs highly susceptible to killing by NK & LAK cells (in vitro assay). • In vivo: NKs target both pluripotent stem cells when activated with TLR-3 agonist poly (I:C) (teratoma frequency halved). • Engraftment of iPSCs/ESCs could be enhanced with short term immunosuppression (costimulatory blockade of cytotoxic T-lymphocyte associated antigen-4/anti-CD40 ligand/antilymphocyte function associated antigen 1) of both undifferentiated & differentiated cells (in allogeneic & xenogeneic recipients). • iPSCs have similar immunological properties as ESC (low MHC I and no MHC II). • iPSCs/ESCs increased MHC I upon interferon-g but unlike ESCs, iPSCs had no increase in CD16. • Syngeneic iPSC graft can cause a T-cell response (immune rejection, reduced tumor formation) compared to ESCs. • Immune rejection may be due to overexpression of genes of iPSC teratomas, (Zg16, Hormad1 & Cyp3A11). Note: Overexpression of these antigens was found in iPSC clones derived from both retroviral or nonintegrating methods (episomal vectors and recombinant proteins).
LAK, lymphocyte activated killer cell; NK, natural killer (cell).
not be simply assumed that an iPSC autograft will not be rejected by the host immune system. Immunoregulation by Donor Cells. The potential immunoregulatory capacity of many donor stem cell populations may influence many factors posttransplantation, including donor cell survival, successful engraftment (and subsequent transdifferentiation capability?) within the required site, as well as induction of endogenous (host) stem cell and/or repair mechanisms. Crosstalk between implanted donor stem cells and recipient immune cells (220) may therefore have a key role in determining the success of stem cell-mediated tissue regeneration (32). Mechanisms may involve inhibition of specific immune cell proliferation, activation, induction of tolerance (370), and/or suppression of maturation and function of specific subsets of populations (6). It is likely that donor iPSC transplantation may also involve interplay between these potential immunoregulatory factors in the host. Tracking the Fate of Donor iPSCs. Accurate assessment of the fate of donor cells in transplantation therapies often requires the use of markers to monitor the fate of grafted cells and any influence of/interaction with these cells on the host. Many types of protocols that have been used have their advantages and disadvantages, including immunohistochemistry (IHC), retrograde-labeling, electron microscopy (EM), and fluorescence in situ hybridization (FISH) (133). Technical limitations can also be compounded by factors such as immunogenicity of the marker of choice leading to specific immune responses (347) and ambiguity in assessment due to (transient?) loss of long-term expression of the marker in surviving cells (146,203,256), leakage of marker and subsequent uptake by host cells, toxicity from chronic exposure of markers, change in cellular distribution over time, and uptake/redistribution by scavenging host microglia/macrophages. Other markers may circumvent these factors
by the use of species-specific markers, markers associated with a specific gene promoter or adenoviral (AdV), adeno-associated viral (AAV), retroviral (RV), and lentiviral (LV) vectors for the introduction of foreign genes encoding either the bacterial marker enzyme b-galactosidase/LacZ or green fluorescent protein (GFP), fluorescent (usually rhodamine or fluorescein) dye-coated latex microspheres/beads/nanospheres, and combined analysis with other assays such as IHC, retrograde-labeling, or EM, FISH, or real-time quantitative PCR using donor-specific sequences [e.g., Y1 DNA of male cells transplanted into female hosts (34,134), as well as the use of thymidine, bromodeoxyuridine (BrdU), and ethynyl deoxyuridine (EDU) analogues (133)]. Conclusion iPSCs are a novel source of neural stem cells for SCI therapy that do not share the disadvantages of adult or embryonic-derived cells. To date, only a few studies have investigated iPSC-derived grafts in SCI models. The disparity in overall results allows no clear conclusions to be drawn as to how iPSC-based therapy compares to cells derived from other sources. It is important to be mindful of certain factors (e.g., choice of donor cell or method of transgene delivery) that may affect iPSC differentiation propensity towards neural lineage and may ultimately also affect in vivo outcome. As such, future in vivo studies should carefully consider (and provide sufficient details) on their method of choice for iPSC generation and characterization to ensure that only authentic iPSCs are used. At present, most in vivo studies to not provide sufficient details regarding the iPSCs used for transplantation, which can limit the conclusions that can be drawn from these reports Despite the promise iPSC technology holds in providing an unlimited source of desired donor cells, current technologies appear to be too inefficient, costly, and
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time-consuming, especially for safer nonviral, non-DNAintegrating methods, to be useful in a clinical setting anytime soon. Protocols must be continually developed to address these issues. Furthermore, after establishment of a new patient-specific cell line, it must be ensured that (i) the cell line is appropriate for the establishment of the necessary cells (line to line variability in establishment of tissue) and (ii) the donor population is devoid of undifferentiated cells and does not form tumors in vivo. At present, these processes can take several months to complete, which is not ideal in SCI since acute transplants (and other noncellular therapies) are vastly superior in functional and morphological improvements compared to chronic injury regimes, which tend to show greatly diminished, if any, improvements (148). Our understanding of how to direct differentiation towards a potentially heterogeneous population of donor cells that incorporate more stringent safety features also needs to be improved. Lastly, from preliminary investigations into the immunogenicity of iPSCs, it is becoming apparent that iPSC may be targeted by the immune system, requiring immune suppression/tolerance induction regimes. For these reasons, it is more likely that—with the current technologies and knowledge—an iPSC bank with ready-made cells produced under the most stringent GMP conditions, appropriately evaluated and characterized, will be more feasible than creating new patient-specific lines for the urgent treatment of SCI and other types of neurotrauma. References 1. Aasen, T.; Izpisua Belmonte, J. C. Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells. Nat. Protoc. 5:371–382; 2010. 2. Aasen, T.; Raya, A.; Barrero, M. J.; Garreta, E.; Consiglio, A.; Gonzalez, F.; Vassena, R.; Bilic, J.; Pekarik, V.; Tiscornia, G.; Edel, M.; Boue, S.; Izpisua Belmonte, J. C. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat. Biotechnol. 26:1276–1284; 2008. 3. Abdullah, Z.; Saric, T.; Kashkar, H.; Baschuk, N.; Yazdanpanah, B.; Fleischmann, B. K.; Hescheler, J.; Kronke, M.; Utermohlen, O. Serpin-6 expression protects embryonic stem cells from lysis by antigen-specific CTL. J. Immunol. 178:3390–3399; 2007. 4. Abe, Y.; Yamamoto, T.; Sugiyama, Y.; Watanabe, T.; Saito, N.; Kayama, H.; Kumagai, T. Apoptotic cells associated with wallerian degeneration after experimental spinal cord injury: A possible mechanism of oligodendroglial death. J. Neurotrauma 16:945–952; 1999. 5. Agarwal, S.; Loh, Y.-H.; McLoughlin, E. M.; Huang, J.; Park, I.-H.; Miller, J. D.; Huo, H.; Okuka, M.; dos Reis, R. M.; Loewer, S.; Ng, H.-H.; Keefe, D. L.; Goldman, F. D.; Klingelhutz, A. J.; Liu, L.; Daley, G. Q. Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients. Nature 464:292–298; 2010.
6. Akesson, E.; Wolmer-Solberg, N.; Cederarv, M.; Falci, S.; Odeberg, J. Human neural stem cells and astrocytes, but not neurons, suppress an allogeneic lymphocyte response. Stem Cell Res. 2:56–67; 2009. 7. Alipio, Z.; Liao, W.; Roemer, E. J.; Waner, M.; Fink, L. M.; Ward, D. C.; Ma, Y. Reversal of hyperglycemia in diabetic mouse models using induced-pluripotent stem (iPS)derived pancreatic b-like cells. Proc. Natl. Acad. Sci. USA 107:13426–13431; 2010. 8. Anokye-Danso, F.; Trivedi, C. M.; Juhr, D.; Gupta, M.; Cui, Z.; Tian, Y.; Zhang, Y.; Yang, W.; Gruber, P. J.; Epstein, J. A.; Morrisey, E. E. Highly efficient miRNAmediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 8:376–388; 2011. 9. Apostolova, I.; Irintchev, A.; Schachner, M. Tenascin-R restricts posttraumatic remodeling of motoneuron innervation and functional recovery after spinal cord injury in adult mice. J. Neurosci. 26:7849–7859; 2006. 10. Araki, R.; Hoki, Y.; Uda, M.; Nakamura, M.; Jincho, Y.; Tamura, C.; Sunayama, M.; Ando, S.; Sugiura, M.; Yoshida, M. A.; Kasama, Y.; Abe, M. Crucial role of c-myc in the generation of induced pluripotent stem cells. Stem Cells 29:1362–1370; 2011. 11. Armstrong, L.; Tilgner, K.; Saretzki, G.; Atkinson, S.; Stojkovic, M.; Moreno, R.; Przyborski, S.; Lako, M. Human induced pluripotent stem cell lines show stress defense mechanisms and mitochondrial regulation similar to those of human embryonic stem cells. Stem Cells 28:661–673; 2010. 12. Arnhold, S.; Klein, H.; Semkova, I.; Addicks, K.; Schra ermeyer, U. Neurally selected embryonic stem cells induce tumor formation after long-term survival following engraftment into the subretinal space. Invest. Opthalmol. Vis. Sci. 45:4251–4255; 2004. 13. Asher, R. A.; Morgenstern, D. A.; Moon, L. D. F.; Fawcett, J. W. Chondroitin sulphate proteoglycans: Inhibitory components of the glial scar. Prog. Brain Res. 132:611–619; 2001. 14. Baker, D. E. C.; Harrison, N. J.; Maltby, E.; Smith, K.; Moore, H. D.; Shaw, P. J.; Heath, P. R.; Holden, H.; Andrews, P. W. Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat. Biotechnol. 25:207–215; 2007. 15. Ban, H.; Nishishita, N.; Fusaki, N.; Tabata, T.; Saeki, K.; Shikamura, M.; Takada, N.; Inoue, M.; Hasegawa, M.; Kawamata, S.; Nishikawa, S.-I. Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proc. Natl. Acad. Sci. USA 108:14234–14239; 2011. 16. Banito, A.; Rashid, S. T.; Acosta, J. C.; Li, S.; Pereira, C. F.; Geti, I.; Pinho, S.; Silva, J. C.; Azuara, V.; Walsh, M.; Vallier, L.; Gil, J. Senescence impairs successful reprogramming to pluripotent stem cells. Genes Dev. 23:2134– 2139; 2009. 17. Bareyre, F.; Kerschensteiner, M.; Raineteau, O.; Metten leiter, T.; Weinmann, O.; Schwab, M. The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats. Nat. Neurosci. 7:269–277; 2004. 18. Bar-Nur, O.; Russ, H. A.; Efrat, S.; Benvenisty, N. Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet b-cells. Cell Stem Cell 9:17–23; 2011.
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19. Basso, D.; Beattie, M.; Bresnahan, J. Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Exp. Neurol. 139:244–256; 1996. 20. Becerra, J. L.; Puckett, W. R.; Hiester, E. D.; Quencer, R. M.; Marcillo, A. E.; Post, M. J. D.; Bunge, R. P. MR-Pathological comparisons of Wallerian degeneration in spinal cord injury. Am. J. Neuroradiol. 16:125–133; 1995. 21. Behfar, A.; Perez-Terzic, C.; Faustino, R. S.; Arrell, D. K.; Hodgson, D. M.; Yamada, S.; Puceat, M.; Niederlander, N.; Alekseev, A. E.; Zingman, L. V.; Terzic, A. Cardiopoietic programming of embryonic stem cells for tumor-free heart repair. J. Exp. Med. 204:405–420; 2007. 22. Ben-David, U.; Benvenisty, N. High prevalence of evolutionarily conserved and species-specific genomic aberrations in mouse pluripotent stem cells. Stem Cells 30:612–622; 2012. 23. Ben-David, U.; Benvenisty, N. The tumorigenicity of human embryonic and induced pluripotent stem cells. Nat. Rev. Cancer 11:268–277; 2011. 24. Ben-Nun, I. F.; Montague, S. C.; Houck, M. L.; Tran, H. T.; Garitaonandia, I.; Leonardo, T. R.; Wang, Y.-C.; Charter, S. J.; Laurent, L. C.; Ryder, O. A.; Loring, J. F. Induced pluripotent stem cells from highly endangered species. Nat. Methods 8:829–831; 2011. 25. Bilousova, G.; Jun, D. H.; King, K. B.; De Langhe, S.; Chick, W. S.; Torchia, E. C.; Chow, K. S.; Klemm, D. J.; Roop, D. R.; Majka, S. M. Osteoblasts derived from induced pluripotent stem cells form calcified structures in scaffolds both in vitro and in vivo. Stem Cells 29:206– 216; 2011. 26. Blelloch, R.; Venere, M.; Yen, J.; Ramalho-Santos, M. Generation of induced pluripotent stem cells in the absence of drug selection. Cell Stem Cell 1:245–247; 2007. 27. Blight, A. Cellular morphology of chronic spinal cord injury in the cat: Analysis of myelinated axons by linesampling. Neurosci. 10:521–543; 1983. 28. Blin, G.; Nury, D.; Stefanovic, S.; Neri, T.; Guillevic, O.; Brinon, B.; Bellamy, V.; Ruecker-Martin, C.; Barbry, P.; Bel, A.; Bruneval, P.; Cowan, C.; Pouly, J.; Mitalipov, S.; Gouadon, E.; Binder, P.; Hagege, A.; Desnos, M.; Renaud, J.-F.; Menasche, P.; Puceat, M. A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates. J. Clin. Invest. 120:1125– 1139; 2010. 29. Blum, B.; Benvenisty, N. The tumorigenicity of diploid and aneuploid human pluripotent stem cells. Cell Cycle 8:3822–3830; 2009. 30. Boland, M. J.; Hazen, J. L.; Nazor, K. L.; Rodriguez, A. R.; Gifford, W.; Martin, G.; Kupriyanov, S.; Baldwin, K. K. Adult mice generated from induced pluripotent stem cells. Nature 461:91–96; 2009. 31. Bonde, S.; Zavazava, N. Immunogenicity and engraftment of mouse embryonic stem cells in allogeneic recipients. Stem Cells 24:2192–2201; 2006. 32. Bonnamain, V.; Neveu, I.; Naveilhan, P. Neural stem/ progenitor cells as a promising candidate for regenerative therapy of the central nervous system. Front. Cell. Neurosci. 6:1–8; 2012. 33. Bonner, J.; Blesch, A.; Neuhuber, B.; Fischer, I. Promoting direcftional axon growth from neural progenitors grafterd into the injured spinal cord. J. Neurosci. Res. 88:1182– 1192; 2009.
603
34. Bosio, E.; Lee-Pullen, T. F.; Fragall, C. T.; Beilharz, M. W.; Bennett, A. L.; Grounds, M. D.; Hodgetts, S. I.; Sammels, L. M. A comparison between real-time quantitative PCR and DNA hybridization for quantitation of male DNA following myoblast transplantation. Cell Transplant. 13:817–821; 2004. 35. Boulting, G. L.; Kiskinis, E.; Croft, G. F.; Amoroso, M. W.; Oakley, D. H.; Wainger, B. J.; Williams, D. J.; Kahler, D. J.; Yamaki, M.; Davidow, L.; Rodolfa, C. T.; Dimos, J. T.; Mikkilineni, S.; MacDermott, A. B.; Woolf, C. J.; Henderson, C. E.; Wichterle, H.; Eggan, K. A functionally characterized test set of human induced pluripotent stem cells. Nat. Biotechnol. 29:279–286; 2011. 36. Bouzier-Sore, A. K.; Merle, M.; Magistretti, P. J.; Pellerin, L. Feeding active neurons: (Re)emergence of a nursing role for astrocytes. J. Physiol. Paris 96:273–282; 2002. 37. Boyd, A. S.; Fairchild, P. J. Approaches for immunological tolerance induction to stem cell-derived cell replacement therapies. Expert Rev. Clin. Immunol. 6:435–448; 2010. 38. Boyd, A. S.; Rodrigues, N. P.; Lui, K. O.; Fu, X.; Xu, Y. Concise review: Immune recognition of induced pluripotent stem cells. Stem Cells 30:797–803; 2012. 39. Boyer, L. A.; Lee, T. I.; Cole, M. F.; Johnstone, S. E.; Levine, S. S.; Zucker, J. R.; Guenther, M. G.; Kumar, R. M.; Murray, H. L.; Jenner, R. G.; Gifford, D. K.; Melton, D. A.; Jaenisch, R.; Young, R. A. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122:947–956; 2005. 40. Brambrink, T.; Foreman, R.; Welstead, G. G.; Lengner, C. J.; Wernig, M.; Suh, H.; Jaenisch, R. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2:151–159; 2008. 41. Bramlett, H. M.; Dietrich, W. D. Progressive damage after brain and spinal cord injury: Pathomechanisms and treatment strategies. Prog. Brain Res. 161:125–141; 2007. 42. Brennand, K. J.; Simone, A.; Jou, J.; Gelboin-Burkhart, C.; Tran, N.; Sangar, S.; Li, Y.; Mu, Y.; Chen, G.; Yu, D.; McCarthy, S.; Sebat, J.; Gage, F. H. Modelling schizophrenia using human induced pluripotent stem cells. Nature 473:221–227; 2011. 43. Bresnahan, J. C. An electron-microscopic analysis of axonal alterations following blunt contusion of the spinal cord of the rhesus monkey (Macaca mulatta). J. Neurol. Sci. 37:59–82; 1978. 44. Brown, M. T. Moral complicity in induced pluripotent stem cell research. Kennedy Inst. Ethics J. 19:1–22; 2009. 45. Bruce, J. H.; Norenberg, M. D.; Kraydieh, S.; Puckett, W.; Marcillo, A.; Dietrich, D. Schwannosis: Role of gliosis and proteoglycan in human spinal cord injury. J. Neurotrauma 17:781–788; 2000. 46. Bullock, R.; Maxwell, W. L.; Graham, D. I.; Teasdale, G. M.; Adams, J. H. Glial swelling following human cerebral contusion: An ultrastructural study. J. Neurol. Neurosurg. Psychiatry 54:427–434; 1991. 47. Bunge, R. P.; Puckett, W.; Becerra, J. L.; Marcillo, A.; Quencer, R. M. Observations on the pathology of human spinal cord injury: A review and classification of 22 new cases with details from a case of chronic cord compression with extensive focal demyelination. Adv. Neurol. 59:75–89; 1993. 48. Buss, A.; Brook, G. A.; Kakulas, B.; Martin, D.; Franzen, R.; Schoenen, J.; Noth, J.; Schmitt, A. B. Gradual loss of myelin and formation of an astrocytic scar during Wallerian
604 kramer ET AL.
degeneration in the human spinal cord. Brain 127:34–44; 2004. 49. Cai, J.; Yang, M.; Poremsky, E.; Kidd, S.; Schneider, J. S.; Iacovitti, L. Dopaminergic neurons derived from human induced pluripotent stem cells survive and integrate into 6-OHDA-lesioned rats. Stem Cells Dev. 19:1017–1023; 2010. 50. Cao, F.; Drukker, M.; Lin, S.; Sheikh, A. Y.; Xie, X.; Li, Z.; Connolly, A. J.; Weissman, I. L.; Wu, J. C. Molecular imaging of embryonic stem cell misbehavior and suicide gene ablation. Cloning Stem Cells 9:107–117; 2007. 51. Cao, F.; Lin, S.; Xie, X. Y.; Ray, P.; Patel, M.; Zhang, X. Z.; Drukker, M.; Dylla, S. J.; Connolly, A. J.; Chen, X. Y.; Weissman, I. L.; Gambhir, S. S.; Wu, J. C. In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery. Circulation 113:1005– 1014; 2006. 52. Cao, Q.; Howard, R.; Dennison, J.; Whittemore, S. Diffe rentiation of engrafted neuronal-restricted precursor cells is inhibited in the traumatically injured spinal cord. Exp. Neurol. 177:349–359; 2002. 53. Carey, B. W.; Markoulaki, S.; Hanna, J.; Saha, K.; Gao, Q.; Mitalipova, M.; Jaenisch, R. Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc. Natl. Acad. Sci. USA 106:157–162; 2009. 54. Carey, B. W.; Markoulaki, S.; Hanna, J. H.; Faddah, D. A.; Buganim, Y.; Kim, J.; Ganz, K.; Steine, E. J.; Cassady, J. P.; Creyghton, M. P.; Welstead, G. G.; Gao, Q.; Jaenisch, R. Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells. Cell Stem Cell 9:588–598; 2011. 55. Carlson, S. L.; Parrish, M. E.; Springer, J. E.; Doty, K.; Dossett, L. Acute inflammatory response in spinal cord following impact injury. Exp. Neurol. 151:77–88; 1998. 56. Carpenter, L.; Carr, C.; Yang, C. T.; Stuckey, D. J.; Clarke, K.; Watt, S. M. Efficient differentiation of human induced pluripotent stem cells generates cardiac cells that provide protection following myocardial infarction in the rat. Stem Cells Dev. 21:977–986; 2012. 57. Chamberlain, S. J.; Chen, P. F.; Ng, K. Y.; Bourgois-Rocha, F.; Lemtiri-Chlieh, F.; Levine, E. S.; Lalande, M. Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader-Willi syndromes. Proc. Natl. Acad. Sci. USA 107:17668–17673; 2010. 58. Chambers, S. M.; Fasano, C. A.; Papapetrou, E. P.; Tomishima, M.; Sadelain, M.; Studer, L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27:275– 280; 2009. 59. Chan, A. W. S.; Cheng, P.-H.; Neumann, A.; Yang, J.-J. Reprogramming Huntington monkey skin cells into pluripotent stem cells. Cell. Reprogram. 12:509–517; 2010. 60. Chan, E. M.; Ratanasirintrawoot, S.; Park, I.-H.; Manos, P. D.; Loh, Y.-H.; Huo, H.; Miller, J. D.; Hartung, O.; Rho, J.; Ince, T. A.; Daley, G. Q.; Schlaeger, T. M. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat. Biotechnol. 27:1033– 1037; 2009. 61. Chang, C.-W.; Lai, Y.-S.; Pawlik, K. M.; Liu, K.; Sun, C.-W.; Li, C.; Schoeb, T. R.; Townes, T. M. Polcystronic lentiviral vector for “hit and run” reprogramming of adult skin fibroblasts to induced pluripotent stem cells. Stem Cells 27:1042–1049; 2009.
62. Chang, T.; Zheng, W.; Tsark, W.; Bates, S.; Huang, H.; Lin, R. J.; Yee, J. K. Brief report: Phenotypic rescue of induced pluripotent stem cell-derived motorneurons of a spinal muscular atrophy patient. Stem Cells 29:2090–2093; 2011. 63. Chaudhry, G. R.; Fecek, C.; Lai, M. M.; Wu, W. C.; Chang, M.; Vasquez, A.; Pasierb, M.; Trese, M. T. Fate of embryonic stem cell derivatives implanted into the vitreous of a slow retinal degenerative mouse model. Stem Cells Dev. 18:247–258; 2009. 64. Chen, L.; Daley, G. Q. Molecular basis of pluripotency. Hum. Mol. Genet. 17:R23–R27; 2008. 65. Chen, L.; Liu, L. Current progress and prospects of induced pluripotent stem cells. Sci. China C. Life Sci. 52:622–636; 2009. 66. Chen, S.-J.; Chang, C.-M.; Tsai, S.-K.; Chang, Y.-L.; Chou, S.-J.; Huang, S.-S.; Tai, L.-K.; Chen, Y.-C.; Ku, H.-H.; Li, H.-Y.; Chiou, S.-H. Functional improvement of focal cerebral ischemia injury by subdural transplantation of induced pluripotent stem cells with fibrin glue. Stem Cells Dev. 19:1757–1767; 2010. 67. Chen, X.; Xu, H.; Yuan, P.; Fang, F.; Huss, M.; Vega, V. B.; Wong, E.; Orlov, Y. L.; Zhang, W.; Jiang, J.; Loh, Y.-H.; Yeo, H. C.; Yeo, Z. X.; Narang, V.; Govindarajan, K. R.; Leong, B.; Shahab, A.; Ruan, Y.; Bourque, G.; Sung, W.-K.; Clarke, N. D.; Wei, C.-L.; Ng, H.-H. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133:1106–1117; 2008. 68. Cheng, F.; Ke, Q.; Chen, F.; Cai, B.; Gao, Y.; Ye, C.; Wang, D.; Zhang, L.; Lahn, B. T.; Li, W.; Xiang, A. P. Protecting against wayward human induced pluripotent stem cells with a suicide gene. Biomaterials 33:3195–3204; 2012. 69. Cheng, L.; Hansen, N. F.; Zhao, L.; Du, Y.; Zou, C.; Donovan, F. X.; Chou, B.-K.; Zhou, G.; Li, S.; Dowey, S. N.; Ye, Z.; Chandrasekharappa, S. C.; Yang, H.; Mullikin, J. C.; Liu, P. P.; Progra, N. C. S. Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression. Cell Stem Cell 10:337–344; 2012. 70. Cheung, A. Y. L.; Horvath, L. M.; Grafodatskaya, D.; Pasceri, P.; Weksberg, R.; Hotta, A.; Carrel, L.; Ellis, J. Isolation of MECP2-null Rett Syndrome patient hiPS cells and isogenic controls through X-chromosome inactivation. Hum. Mol. Genet. 20:2103–2115; 2011. 71. Chiang, C.-H.; Chang, C.-C.; Huang, H.-C.; Chen, Y.-J.; Tsai, P.-H.; Jeng, S.-Y.; Hung, S.-I.; Hsieh, J.-H.; Huang, H.-S.; Chiou, S.-H.; Lee, F.-Y.; Lee, S.-D. Investigation of hepatoprotective activity of induced pluripotent stem cells in the mouse model of liver injury. J. Biomed. Biotechnol. 2011:219060; 2011. 72. Chiang, C. H.; Su, Y.; Wen, Z.; Yoritomo, N.; Ross, C. A.; Margolis, R. L.; Song, H.; Ming, G. I. Integration-free induced pluripotent stem cells derived from schizophrenia patients with a DISC1 mutation. Mol. Psych. 16:358–360; 2011. 73. Chin, M. H.; Mason, M. J.; Xie, W.; Volinia, S.; Singer, M.; Peterson, C.; Ambartsumyan, G.; Aimiuwu, O.; Richter, L.; Zhang, J.; Khvorostov, I.; Ott, V.; Grunstein, M.; Lavon, N.; Benvenisty, N.; Croce, C. M.; Clark, A. T.; Baxter, T.; Pyle, A. D.; Teitell, M. A.; Pelegrini, M.; Plath, K.; Lowry, W. E. Induced pluripotent stem cells and embryonig stem cells are distinguised by gene expression signatures. Cell Stem Cell 5:111–123; 2009.
iPSC TECHNOLOGY FOR SCI
74. Chin, M. H.; Pellegrini, M.; Plath, K.; Lowry, W. E. Molecular analyses of human induced pluripotent stem cells and embryonic stem cells. Cell Stem Cell 7:263–269; 2010. 75. Chiou, S.-H.; Yu, C.-C.; Huang, C.-Y.; Lin, S.-C.; Liu, C.-J.; Tsai, T.-H.; Chou, S.-H.; Chien, C.-S.; Ku, H.-H.; Lo, J.-F. Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinoma. Clin. Cancer Res. 14:4085–4095; 2008. 76. Cho, H.-J.; Lee, C.-S.; Kwon, Y.-W.; Paek, J. S.; Lee, S.-H.; Hur, J.; Lee, E. J.; Roh, T.-Y.; Chu, I.-S.; Leem, S.-H.; Kim, Y.; Kang, H.-J.; Park, Y.-B.; Kim, H.-S. Induction of pluripotent stem cells from adult somatic cells by protein-based reprogramming without genetic manipulation. Blood 116:386–395; 2010. 77. Choi, S. M.; Kim, Y.; Liu, H.; Chaudhari, P.; Ye, Z.; Jang, Y.-Y. Liver engraftment potential of hepatic cells derived from patient-specific induced pluripotent stem cells. Cell Cycle 10:2423–2427; 2011. 78. Christodoulou, C.; Longmire, T. A.; Shen, S. S.; Bourdon, A.; Sommer, C. A.; Gadue, P.; Spira, A.; Gouon-Evans, V.; Murphy, G. J.; Mostoslavsky, G.; Kotton, D. N. Mouse ES and iPS cells can form similar definitive endoderm despite differences in imprinted genes. J. Clin. Invest. 121:2313– 2325; 2011. 79. Chung, H. C. Y.; Lin, R. C. Y.; Logan, G. J.; Alexander, I. E.; Sachdev, P. S.; Sidhu, K. S. Human induced pluripotent stem cells derived under feeder-free conditions display unique cell cycle and DNA replication gene profiles. Stem Cells Dev. 21:206–216; 2012. 80. Cloutier, F.; Siegenthaler, M.; Nistor, G.; Keirstead, H. Transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into rat spinal cord injuries does not cause harm. Regen. Med. 1:469–479; 2006. 81. Condic, M.; Letourneau, P. Ligand-induced changes in integrin expression regulate neuronal adhesion and neurite outgrowth. Nature 389:852–856; 1997. 82. Cooper, O.; Hargus, G.; Deleidi, M.; Blak, A.; Osborn, T.; Marlow, E.; Lee, K.; Levy, A.; Perez-Torres, E.; Yow, A.; Isacson, O. Differentiation of human ES and Parkinson’s disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid. Mol. Cell. Neurosci. 45:258–266; 2010. 83. 83 Crowe, M. J.; Bresnahan, J. C.; Shuman, S. L.; Masters, J. N.; Beattie, M. S. Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nat. Med. 3:73–76; 1997. 84. Cyranoski, D. Stem cells: 5 things to know before jumping on the iPS bandwagon. Nature 452:406–408; 2008. 85. Czepiel, M.; Balasubramaniyan, V.; Schaafsma, W.; Stancic, M.; Mikkers, H.; Huisman, C.; Boddeke, E.; Copray, S. Differentiation of Induced Pluripotent Stem Cells Into Functional Oligodendrocytes. Glia 59:882–892; 2011. 86. Davies, S. J.; Goucher, D. R.; Doller, C.; Silver, J. Robust regeneration of adult sensory axons in degenerating white matter of the adult rat spinal cord. J. Neurosci. 19:5810– 5822; 1999. 87. Davies, S. J. A.; Fitch, M. T.; Memberg, S. P.; Hall, A. K.; Raisman, G.; Silver, J. Regeneration of adult axons in white matter tracts of the central nervous system. Nature 390:680–683; 1997. 88. de Castro, R.; Tajrishi, R.; Claros, J.; Stallcup, W.; Castro, D. Differential responses of spinal axons to transection:
605
Influence of the NG2 proteoglycan. Exp. Neurol. 192:299– 309; 2005. 89. Deleidi, M.; Hargus, G.; Hallett, P.; Osborn, T.; Isacson, O. Development of histocompatible primate-induced pluripotent stem cells for neural transplantation. Stem Cells 29:1052–1063; 2011. 90. Deng, J.; Shoemaker, R.; Xie, B.; Gore, A.; LeProust, E. M.; Antosiewicz-Bourget, J.; Egli, D.; Maherali, N.; Park, I.-H.; Yu, J.; Daley, G. Q.; Eggan, K.; Hochedlinger, K.; Thomson, J.; Wang, W.; Gao, Y.; Zhang, K. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat. Biotechnol. 27:353–360; 2009. 91. Devine, M. J.; Ryten, M.; Vodicka, P.; Thomson, A. J.; Burdon, T.; Houlden, H.; Cavaleri, F.; Nagano, M.; Drummond, N. J.; Taanman, J.-W.; Schapira, A. H.; Gwinn, K.; Hardy, J.; Lewis, P. A.; Kunath, T. Parkinson’s disease induced pluripotent stem cells with triplication of the a-synuclein locus. Nat. Commun. 2:440; 2011. 92. Dick, E.; Matsa, E.; Young, L. E.; Darling, D.; Denning, C. Faster generation of hiPSCs by coupling high-titer lentivirus and column-based positive selection. Nat. Protoc. 6:701–714; 2011. 93. Dimos, J.; Rodolfa, K.; Niakan, K.; Weisenthal, L.; Mitsumoto, H.; Chung, W. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321:1218–1221; 2008. 94. Di Stefano, B.; Maffioletti, S. M.; Gentner, B.; Ungaro, F.; Schira, G.; Naldini, L.; Broccoli, V. A microRNAbased system for selecting and maintaining the pluripotent state in human induced pluripotent stem cells. Stem Cells 29:1684–1695; 2011. 95. Doi, A.; Park, I.-H.; Wen, B.; Murakami, P.; Aryee, M. J.; Irizarry, R.; Herb, B.; Ladd-Acosta, C.; Rho, J.; Loewer, S.; Miller, J.; Schlaeger, T.; Daley, G. Q.; Feinberg, A. P. Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts. Nat. Genet. 41:1350–1353; 2009. 96. Donnelly, D. J.; Popovich, P. G. Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury. Exp. Neurol. 209:378– 388; 2008. 97. Dressel, R.; Nolte, J.; Elsner, L.; Novota, P.; Guan, K.; Streckfuss-Boemeke, K.; Hasenfuss, G.; Jaenisch, R.; Engel, W. Pluripotent stem cells are highly susceptible targets for syngeneic, allogeneic, and xenogeneic natural killer cells. FASEB J. 24:2164–2177; 2010. 98. Drukker, M.; Katchman, H.; Katz, G.; Even-Tov Friedman, S.; Shezen, E.; Hornstein, E.; Mandelboim, O.; Reisner, Y.; Benvenisty, N. Human embryonic stem cells and their differentiated derivates are less susceptible to immune rejection than adult cells. Stem Cells 24:221–229; 2006. 99. Drukker, M.; Katz, G.; Urbach, A.; Schuldiner, M.; Markel, G.; Itskovitz-Eldor, J.; Reubinoff, B.; Mandelboim, O.; Benvenisty, N. Characterization of the expression of MHC proteins in human embryonic stem cells. Proc. Natl. Acad. Sci. USA 99:9864–9869; 2002. 100. Ebert, A. D.; Yu, J.; Rose, F. F.; Mattis, V. B.; Lorson, C. L.; Thomson, J. A.; Svendsen, C. N. Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457:277–280; 2009. 101. Emdad, L.; D’Souza, S.; Kothari, H.; Qadeer, Z.; Germano, I. Efficient differentiation of human embryonic
606 kramer ET AL.
and induced pluripotent stem cells into functional astrocytes. Stem Cells Dev. 21:404–410; 2012. 102. Emery, E.; Aldana, P.; Bunge, M. B.; Puckett, W.; Srinivasan, A.; Keane, R. W.; Bethea, J.; Levi, A. D. O. Apoptosis after traumatic human spinal cord injury. J. Neurosurg. 89:911–920; 1998. 103. Esteban, M. A.; Wang, T.; Qin, B.; Yang, J.; Qin, D.; Cai, J.; Li, W.; Weng, Z.; Chen, J.; Ni, S.; Chen, K.; Li, Y.; Liu, X.; Xu, J.; Zhang, S.; Li, F.; He, W.; Labuda, K.; Song, Y.; Peterbauer, A.; Wolbank, S.; Redl, H.; Zhong, M.; Cai, D.; Zeng, L.; Pei, D. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell 6:71–79; 2010. 104. Esteban, M. A.; Xu, J.; Yang, J.; Peng, M.; Qin, D.; Li, W.; Jiang, Z.; Chen, J.; Den, K.; Zhong, M.; Cai, J.; Lai, L.; Pei, D. Generation of induced pluripotent stem cell lines from tibetan miniature pig. J. Biol. Chem. 284:17634– 17640; 2009. 105. Fairchild, P. J. The challenge of immunogenicity in the quest for induced pluripotency. Nat. Rev. Immunol. 10:868–875; 2010. 106. Faulkner, J.; Keirstead, H. Human embryonic stem cellderived oligodendrocyte progenitors for the treatment of spinal cord injury. Transpl. Immunol. 15:131–142; 2005. 107. Fawcett, J.; Curt, A.; Steeves, J.; Coleman, W.; Tuszynski, M.; Lammertse, D. Guidelines for the conduct of clinical trials for spinal cord injury as developed by the ICCP panel: Spontaneous recovery after spinal cord injury and statistical power needed for therapeutic clinical trials. Spinal Cord 45:190–205; 2007. 108. Fawcett, J. W.; Asher, R. A. The glial scar and central nervous system repair. Brain Res. Bull. 49:377–391; 1999. 109. Fehlings, M. G.; Tator, C. H. The relationships among the severity of spinal cord injury, residual neurological function, axon counts, and counts of retrogradely labeled neurons after experimental spinal cord injury. Exp. Neurol. 132:220–228; 1995. 110. Fitch, M. T.; Silver, J. CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure. Exp. Neurol. 209:294–301; 2008. 111. Fleming, J. C.; Norenberg, M. D.; Ramsay, D. A.; Dekaban, G. A.; Marcillo, A. E.; Saenz, A. D.; PasqualeStyles, M.; Dietrich, W. D.; Weaver, L. C. The cellular inflammatory response in human spinal cords after injury. Brain 129:3249–3269; 2006. 112. Frei, E.; Klusman, I.; Schnell, L.; Schwab, M. E. Reactions of olidodendrocytes to spinal cord injury: Cell survival and myelin repair. Exp. Neurol. 163:373–380; 2000. 113. Fu, W.; Wang, S. J.; Zhou, G. D.; Liu, W.; Cao, Y.; Zhang, W. J. Residual undifferentiated cells during differentiation of induced pluripotent stem cells in vitro and in vivo. Stem Cells Dev. 21:521–529; 2012. 114. Fujimoto, Y.; Abematsu, M.; Falk, A.; Tsujimura, K.; Sanosaka, T.; Juliandi, B.; Semi, K.; Namihira, M.; Komiya, S.; Smith, A.; Nakashima, K. Treatment of a mouse model of spinal cord injury by transplantation of human induced pluripotent stem cell-derived long-term self-renewing neuroepithelial-like stem cells. Stem Cells 6:1163–1173; 2012. 115. Fusaki, N.; Ban, H.; Nishiyama, A.; Saeki, K.; Hasegawa, M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci. 85:348–362; 2009.
116. Gan, Q.; Yoshida, T.; McDonald, O. G.; Owens, G. K. Concise review: Epigenetic mechanisms contribute to pluripotency and cell lineage determination of embryonic stem cells. Stem Cells 25:2–9; 2007. 117. Ghosh, Z.; Huang, M.; Hu, S.; Wilson, K. D.; Dey, D.; Wu, J. C. Dissecting the oncogenic and tumorigenic potential of differentiated human induced pluripotent stem cells and human embryonic stem cells. Cancer Res. 71:5030–5039; 2011. 118. Ghosh, Z.; Wilson, K.; Wu, Y.; Hu, S.; Quertermous, T.; Wu, J. Persistent donor cell gene expression among human induced pluripotent stem cells contributes to differences with human embryonic stem cells. PLoS One 5:e8975; 2010. 119. Ghule, P. N.; Medina, R.; Lengner, C. J.; Mandeville, M.; Qiao, M.; Dominski, Z.; Lian, J. B.; Stein, J. L.; Van Wijnen, A. J.; Stein, G. S. Reprogramming the pluripotent cell cycle: Restoration of an abbreviated G1 phase in human induced pluripotent stem (iPS) cells. J. Cell. Physiol. 226:1149–1156; 2011. 120. Goh, E. L. K.; Ma, D. K.; Ming, G. L.; Song, H. J. Adult neural stem cells and repair of the adult central nervous system. J. Hematother. Stem Cell Res. 12:671–679; 2003. 121. Gonzalez, F.; Barragan Monasterio, M.; Tiscornia, G.; Montserrat Pulido, N.; Vassena, R.; Batlle Morera, L.; Rodriguez Piza, I.; Belmonte, J. C. I. Generation of mouseinduced pluripotent stem cells by transient expression of a single nonviral polycistronic vector. Proc. Natl. Acad. Sci. USA 106:8918–8922; 2009. 122. Gore, A.; Li, Z.; Fung, H.-L.; Young, J. E.; Agarwal, S.; Antosiewicz-Bourget, J.; Canto, I.; Giorgetti, A.; Israel, M. A.; Kiskinis, E.; Lee, J.-H.; Loh, Y.-H.; Manos, P. D.; Montserrat, N.; Panopoulos, A. D.; Ruiz, S.; Wilbert, M. L.; Yu, J.; Kirkness, E. F.; Belmonte, J. C. I.; Rossi, D. J.; Thomson, J. A.; Eggan, K.; Daley, G. Q.; Goldstein, L. S. B.; Zhang, K. Somatic coding mutations in human induced pluripotent stem cells. Nature 471:63–67; 2011. 123. Goritz, C.; Dias, D. O.; Tomilin, N.; Barbacid, M.; Shupliakov, O.; Frisen, J. A pericyte origin of spinal cord scar tissue. Science 333:238–242; 2011. 124. Guenther, M. G.; Frampton, G. M.; Soldner, F.; Hockemeyer, D.; Mitalipova, M.; Jaenisch, R.; Young, R. A. Chromatin structure and gene expression programs of human embryonic and induced pluripotent stem cells. Cell Stem Cell 7:249–257; 2010. 125. Guest, J. D.; Hiester, E. D.; Bunge, R. P. Demyelination and Schwann cell responses adjacent to injury epicenter cavities following chronic human spinal cord injury. Exp. Neurol. 192:384–393; 2005. 126. Gutierrez-Aranda, I.; Ramos-Mejia, V.; Bueno, C.; MunozLopez, M.; Real, P. J.; Macia, A.; Sanchez, L.; Ligero, G.; Garcia-Parez, J. L.; Menendez, P. Human induced pluripotent stem cells develop teratoma more efficiently and faster than human embryonic stem cells regardless the site of injection. Stem Cells 28:1568–1570; 2010. 127. Han, J.; Yuan, P.; Yang, H.; Zhang, J.; Soh, B. S.; Li, P.; Lim, S. L.; Cao, S.; Tay, J.; Orlov, Y. L.; Lufkin, T.; Ng, H.-H.; Tam, W.-L.; Lim, B. Tbx3 improves the germ-line competency of induced pluripotent stem cells. Nature 463:1096–1101; 2010. 128. Han, J. W.; Yoon, Y.-S. Induced pluripotent stem cells: Emerging techniques for nuclear reprogramming. Antioxid. Redox Signal. 15:1799–1820; 2011.
iPSC TECHNOLOGY FOR SCI
129. Han, S. S.; Williams, L. A.; Eggan, K. C. Constructing and deconstructing stem cell models of neurological disease. Neuron 70:626–644; 2011. 130. Hanna, J.; Saha, K.; Pando, B.; van Zon, J.; Lengner, C. J.; Creyghton, M. P.; van Oudenaarden, A.; Jaenisch, R. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462:595–603; 2009. 131. Hanna, J.; Wernig, M.; Markoulaki, S.; Sun, C.; Meissner, A.; Cassady, J. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318:1920–1923; 2007. 132. Hargus, G.; Cooper, O.; Deleidi, M.; Levy, A.; Lee, K.; Marlow, E.; Yow, A.; Soldner, F.; Hockemeyer, D.; Hallett, P. J.; Osborn, T.; Jaenisch, R.; Isacson, O. Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats. Proc. Natl. Acad. Sci. USA 107:15921–15926; 2010. 133. Harvey, A. R. Labeling and Identifying Grafted Cells. In: Dunnett, S. B.; Boulton, A. A.; Baker, G. B., eds. Neural Transplantation Methods: Totowa, NJ: Humana Press Inc; 2000:319–361. 134. Harvey, A. R.; Symons, N. A.; Pollett, M. A.; Brooker, G. J.; Bartlett, P. F. Fate of adult neural precursors grafted to adult cortex monitored with a Y-chromosome marker. Neuroreport 8:3939–3943; 1997. 135. Hawkins, R. D.; Hon, G. C.; Lee, L. K.; Ngo, Q.; Lister, R.; Pelizzola, M.; Edsall, L. E.; Kuan, S.; Luu, Y.; Klugman, S.; Antosiewicz-Bourget, J.; Ye, Z.; Espinoza, C.; Agarwahl, S.; Shen, L.; Ruotti, V.; Wang, W.; Stewart, R.; Thomson, J. A.; Ecker, J. R.; Ren, B. Distinct epigenomic landscapes of pluripotent and lineage-committed human cells. Cell Stem Cell 6:479–491; 2010. 136. Hayashi, K.; Hashimoto, M.; Yamazaki, M.; Okawa, A.; Koda, M.; Sakuma, T.; Takahashi, H. Transplantation of astrocytes derived from induced pluripotent stem cells on an experimental spinal cord injury in rats. J. Neurotrauma 26:P83; 2009. 137. Henon, P. R. Human embryonic or adult stem cells: An overview on ethics and perspectives for tissue engineering.Adv. Exp. Med. Biol. 534:27–45; 2003. 138. Hester, M. E.; Murtha, M. J.; Song, S.; Rao, M.; Miranda, C. J.; Meyer, K.; Tian, J.; Boulting, G.; Schaffer, D. V.; Zhu, M. X.; Pfaff, S. L.; Gage, F. H.; Kaspar, B. K. Rapid and efficient generation of functional motor neurons from human pluripotent stem cells using gene delivered transcription factor codes. Mol. Ther. 19:1905–1912; 2011. 139. Hill, C. Contusive spinal cord injury: Endogenous responses of descending systems and effects of acute transplantation of glial restricted precursor cells. Graduate School of the Ohio State University: Ohio State University; 2002:176. 140. Himes, B. T.; Neuhuber, B.; Coleman, C.; Kushner, R.; Swanger, S. A.; Kopen, G. C.; Wagner, J.; Shumsky, J. S.; Fischer, I. Recovery of function following grafting of human bone marrow-derived stromal cells into the injured spinal cord. Neurorehabil. Neural Repair 20:278–296; 2006. 141. Hirami, Y.; Osakada, F.; Takahashi, K.; Okita, K.; Yamanaka, S.; Ikeda, H.; Yoshimura, N.; Takahashi, M. Generation of retinal cells from mouse and human induced pluripotent stem cells. Neurosci. Lett. 458:126– 131; 2009. 142. Hiratsuka, M.; Uno, N.; Ueda, K.; Kurosaki, H.; Imaoka, N.; Kazuki, K.; Ueno, E.; Akakura, Y.; Katoh, M.; Osaki,
607
M.; Kazuki, Y.; Nakagawa, M.; Yamanaka, S.; Oshimura, M. Integration-free iPS cells engineered using human artificial chromosome vectors. PLoS One 6:e25961; 2011. 143. Hjelm, B. E.; Rosenberg, J. B.; Szelinger, S.; Sue, L. I.; Beach, T. G.; Huentelman, M. J.; Craig, D. W. Induction of pluripotent stem cells from autopsy donor-derived somatic cells. Neurosci. Lett. 502:219–224; 2011. 144. Hochedlinger, K.; Jaenisch, R. Nuclear reprogramming and pluripotency. Nature 441:1061–1067; 2006. 145. Hochedlinger, K.; Yamada, Y.; Beard, C.; Jaenisch, R. Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell 121:465–477; 2005. 146. Hodgetts, S. I.; Simmons, P. J.; Plant, G. W. Human mesenchymal precursor cells (stro-1+) from spinal cord injury patients improve functional recovery and tissue sparing in an acute spinal cord injury rat model. Cell Transplant. In Press; 2012. 147. Hong, H.; Takahashi, K.; Ichisaka, T.; Aoi, T.; Kanagawa, O.; Nakagawa, M.; Okita, K.; Yamanaka, S. Suppression of induced pluripotent stem cell generation by the p53– p21 pathway. Nature 460:1132–1136; 2009. 148. Houle, J.; Tessler, A. Repair of chronic spinal cord injury. Exp. Neurol. 182:247–260; 2003. 149. Hu, B.; Weick, J.; Yu, J.; Ma, L.; Zhang, X.; Thomson, J. Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc. Natl. Acad. Sci. USA 107:4335– 4340; 2010. 150. Huang, J.; Wang, F.; Okuka, M.; Liu, N.; Ji, G.; Ye, X.; Zuo, B.; Li, M.; Liang, P.; Ge, W. W.; Tsibris, J. C. M.; Keefe, D. L.; Liu, L. Association of telomere length with authentic pluripotency of ES/iPS cells. Cell Res. 21:779– 792; 2011. 151. Huangfu, D.; Osafune, K.; Maehr, R.; Guo, W.; Eijkelenboom, A.; Chen, S.; Muhlestein, W.; Melton, D. A. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat. Biotechnol. 26:1269–1275; 2008. 152. Hussein, S. M.; Batada, N. N.; Vuoristo, S.; Ching, R. W.; Autio, R.; Narva, E.; Ng, S.; Sourour, M.; Hamalainen, R.; Olsson, C.; Lundin, K.; Mikkola, M.; Trokovic, R.; Peitz, M.; Brustle, O.; Bazett-Jones, D. P.; Alitalo, K.; Lahesmaa, R.; Nagy, A.; Otonkoski, T. Copy number variation and selection during reprogramming to pluripotency. Nature 471:58–62; 2011. 153. Ichida, J. K.; Blanchard, J.; Lam, K.; Son, E. Y.; Chung, J. E.; Egli, D.; Loh, K. M.; Carter, A. C.; Di Giorgio, F. P.; Koszka, K.; Huangfu, D.; Akutsu, H.; Liu, D. R.; Rubin, L. L.; Eggan, K. A small-molecule inhibitor of TGF-b signaling replaces sox2 in reprogramming by inducing nanog. Cell Stem Cell 5:491–503; 2009. 154. Imaizumi, M.; Nomoto, Y.; Sugino, T.; Miyake, M.; Wada, I.; Nakamura, T.; Omori, K. Potential of induced pluripotent stem cells for the regeneration of the tracheal wall. Ann. Otol. Rhinol. Laryngol. 119:697–703; 2010. 155. Inman, D.; Steward, O. Ascending sensory, but not other long-tract axons, regenerate into the connective tissue matrix that forms at the site of a spinal cord injury in mice. J. Comp. Neurol. 462:431–449; 2003. 156. Ishikawa, K.; Toyama-Sorimachi, N.; Nakada, K.; Morimoto, M.; Imanishi, H.; Yoshizaki, M.; Sasawatari, S.; Niikura, M.; Takenaga, K.; Yonekawa, H.; Hayashi, J.
608 kramer ET AL.
The innate immune system in host mice targets cells with allogenic mitochondrial DNA. J. Exp. Med. 207:2297– 2305; 2010. 157. Iwanami, A.; Kaneko, S.; Nakamura, M.; Kanemura, Y.; Mori, H.; Kobayashi, S. Transplantation of human neural stem cells for spinal cord injury in primates. J. Neurosci. Res. 80:182–190; 2005. 158. Jaenisch, R.; Young, R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132:567–582; 2008. 159. Jain, K. K. Ethical and regulatory aspects of embryonic stem cell research. Expert Opin. Biol. Ther. 5:153–162; 2005. 160. James, D.; Noggle, S. A.; Swigut, T.; Brivanlou, A. H. Contribution of human embryonic stem cells to mouse blastocysts. Dev. Biol. 295:90–102; 2006. 161. Jang, J.; Kang, H. C.; Kim, H. S.; Kim, J. Y.; Huh, Y. J.; Kim, D. S.; Yoo, J. E.; Lee, J. A.; Lim, B.; Lee, J.; Yoon, T. M.; Park, I. H.; Hwang, D. Y.; Daley, G. Q.; Kim, D. W. Induced pluripotent stem cell models from X-linked adrenoleukodystrophy patients. Ann. Neurol. 70:402–409; 2011. 162. Jia, F.; Wilson, K.; Sun, N.; Gupta, D.; Huang, M.; Li, Z. A nonviral minicircle vector for deriving human iPS cells. Nat. Methods 7:197–199; 2010. 163. Jiang, J.; Chan, Y.-S.; Loh, Y.-H.; Cai, J.; Tong, G.-Q.; Lim, C.-A.; Robson, P.; Zhong, S.; Ng, H.-H. A core Klf circuitry regulates self-renewal of embryonic stem cells. Nat. Cell Biol. 10:353–360; 2008. 164. Jiang, M.; Lv, L.; Ji, H.; Yang, X.; Zhu, W.; Cai, L.; Gu, X.; Chai, C.; Huang, S.; Sun, J.; Dong, Q. Induction of pluripotent stem cells transplantation therapy for ischemic stroke. Mol. Cell. Biochem. 354:67–75; 2011. 165. Jones, L.; Sajed, D.; Tuszynski, M. Axonal regeneration through regions of chondroitin sulfate proteoglycan deposition after spinal cord injury: A balance of permissiveness and inhibition. J. Neurosci. 23:9276–9288; 2003. 166. Judson, R. L.; Babiarz, J. E.; Venere, M.; Blelloch, R. Embryonic stem cell-specific microRNAs promote induced pluripotency. Nat. Biotechnol. 27:459–461; 2009. 167. Kaji, K.; Norrby, K.; Paca, A.; Mileikovsky, M.; Mohseni, P.; Woltjen, K. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 458:771–776; 2009. 168. Kakulas, B. A. Neuropathology: The foundation for new treatments in spinal cord injury. Spinal Cord 42:549–563; 2004. 169. Kang, L.; Wang, J.; Zhang, Y.; Kou, Z.; Gao, S. iPS cells can support full-term development of tetraploid blastocystcomplemented embryos. Cell Stem Cell 5:135–138; 2009. 170. Kapadia, M.; Sakic, B. Autoimmune and inflammatory mechanisms of CNS damage. Prog. Neurobiol. 95:301– 333; 2011. 171. Karow, M.; Chavez, C. L.; Farruggio, A. P.; Geisinger, J. M.; Keravala, A.; Jung, W. E.; Lan, F.; Wu, J. C.; Chen-Tsai, Y.; Calos, M. P. Site-specific recombinase strategy to create induced pluripotent stem cells efficiently with plasmid DNA. Stem Cells 29:1696–1704; 2011. 172. Karumbayaram, S.; Novitch, B.; Patterson, M.; Umbach, J.; Richter, L.; Lindgren, A. Directed differentiation of human-induced pluripotent stem cells generates active motor neurons. Stem Cells 27:806–811; 2009. 173. Kawai, H.; Yamashita, T.; Ohta, Y.; Deguchi, K.; Nagotani, S.; Zhang, X.; Ikeda, Y.; Matsuura, T.; Abe, K. Tridermal
tumorigenesis of induced pluripotent stem cells transplanted in ischemic brain. J. Cereb. Blood Flow Metab. 30:1487–1493; 2010. 174. Kawamura, T.; Suzuki, J.; Wang, Y. V.; Menendez, S.; Batlle Morera, L.; Raya, A.; Wahl, G. M.; Izpisua Belmonte, J. C. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460:1140–1145; 2009. 175. Keane, R. W.; Davis, A. R.; Dietrich, W. D. Inflammatory and apoptotic signaling after spinal cord injury. J. Neuro trauma 23:335–344; 2006. 176. Keirstead, H. S.; Nistor, G.; Bernal, G.; Totoiu, M.; Cloutier, F.; Sharp, K.; Steward, O. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J. Neurosci. 25:4694–4705; 2005. 177. Kigerl, K. A.; McGaughy, V. M.; Popovich, P. G. Com parative analysis of lesion development and intraspinal inflammation in four strains of mice following spinal contusion injury. J. Comp. Neurol. 494:578–594; 2006. 178. Kim, D.-S.; Lee, J. S.; Leem, J. W.; Huh, Y. J.; Kim, J. Y.; Kim, H.-S.; Park, I.-H.; Daley, G. Q.; Hwang, D.-Y.; Kim, D.-W. Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity. Stem Cell Rev. 6:270– 281; 2010. 179. Kim, D.; Kim, C.; Moon, J.; Chung, Y.; Chang, M.; Han, B. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4:472–476; 2009. 180. Kim, E. Y.; Jeon, K.; Park, H. Y.; Han, Y. J.; Yang, B. C.; Park, S. B.; Chung, H. M.; Park, S. P. Differences between cellular and molecular profiles of induced pluripotent stem cells generated from mouse embryonic fibroblasts. Cell. Reprogram. 12:627–639; 2010. 181. Kim, J.-E.; O’Sullivan, M. L.; Sanchez, C. A.; Hwang, M.; Israel, M. A.; Brennand, K.; Deerinck, T. J.; Goldstein, L. S. B.; Gage, F. H.; Ellisman, M. H.; Ghosh, A. Investigating synapse formation and function using human pluripotent stem cell-derived neurons. Proc. Natl. Acad. Sci. USA 108: 3005–3010; 2011. 182. Kim, J.; Chu, J.; Shen, X.; Wang, J.; Orkin, S. H. An extended transcriptional network for pluripotency of embryonic stem cells. Cell 132:1049–1061; 2008. 183. Kim, J. B.; Sebastiano, V.; Wu, G.; Arauzo-Bravo, M. J.; Sasse, P.; Gentile, L.; Ko, K.; Ruau, D.; Ehrich, M.; van den Boom, D.; Meyer, J.; Huebner, K.; Bernemann, C.; Ortmeier, C.; Zenke, M.; Fleischmann, B. K.; Zaehres, H.; Schoeler, H. R. Oct4-induced pluripotency in adult neural stem cells. Cell 136:411–419; 2009. 184. Kim, K.; Doi, A.; Wen, B.; Ng, K.; Zhao, R.; Cahan, P.; Kim, J.; Aryee, M. J.; Ji, H.; Ehrlich, L. I. R.; Yabuuchi, A.; Takeuchi, A.; Cunniff, K. C.; Hongguang, H.; McKinneyFreeman, S.; Naveiras, O.; Yoon, T. J.; Irizarry, R. A.; Jung, N.; Seita, J.; Hanna, J.; Murakami, P.; Jaenisch, R.; Weissleder, R.; Orkin, S. H.; Weissman, I. L.; Feinberg, A. P.; Daley, G. Q. Epigenetic memory in induced pluripotent stem cells. Nature 467:285–290; 2010. 185. Kitazawa, A.; Shimizu, N. Differentiation of mouse induced pluripotent stem cells into neurons using conditioned medium of dorsal root ganglia. Nat. Biotechnol. 28:326–333; 2011. 186. Klapka, N.; Muller, H. Collagen matrix in spinal cord injury. J. Neurotrauma 23:422–435; 2006.
iPSC TECHNOLOGY FOR SCI
187. Knoepfler, P. S. Deconstructing stem cell tumorigenicity: A roadmap to safe regenerative medicine. Stem Cells 27:1050–1056; 2009. 188. Koch, C. A.; Geraldes, P.; Platt, J. L. Immunosuppression by embryonic stem cells. Stem Cells 26:89–98; 2008. 189. Koehler, K. R.; Tropel, P.; Theile, J. W.; Kondo, T.; Cummins, T. R.; Viville, S.; Hashino, E. Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells. BMC Neurosci. 12:1–14; 2011. 190. Kofidis, T.; deBruin, J. L.; Tanaka, M.; Zwierzchoniewska, M.; Weissman, I.; Fedoseyeva, E.; Haverich, A.; Robbins, R. C. They are not stealthy in the heart: Embryonic stem cells trigger cell infiltration, humoral and T-lymphocytebased host immune response. Eur. J. Cardiothorac. Surg. 28:461–466; 2005. 191. Kooreman, N. G.; Wu, J. C. Tumorigenicity of pluripotent stem cells: Biological insights from molecular imaging. J. R. Soc. Interface 7:S753–S763; 2010. 192. Krencik, R.; Weick, J. P.; Liu, Y.; Zhang, Z.-J.; Zhang, S.-C. Specification of transplantable astroglial subtypes from human pluripotent stem cells. Nat. Biotech. 29:528– 534; 2011. 193. Kwon, B.; Hillyer, J.; Tetzlaff, W. Translational research in spinal cord injury: A survey of opinion from the SCI community. J. Neurotrauma 27:21–33; 2010. 194. Lacroix, S.; Chang, L.; Rose-John, S.; Tuszynski, M. H. Delivery of hyper-interleukin 6 to the injured spinal cord increases neutrophil and macrophage infiltration and inhibits axonal growth. J. Comp. Neurol. 454:213–228; 2002. 195. Lake, B. B.; Fink, J.; Klemetsaune, L.; Fu, X.; Jeffers, J. R.; Zambetti, G. P.; Xu, Y. Context-dependent enhancement of induced pluripotent stem cell reprogramming by silencing puma. Stem Cells 30:888–897; 2012. 196. Lamba, D. A.; Karl, M. O.; Ware, C. B.; Reh, T. A. Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 103:12769– 12774; 2006. 197. Laurent, L. C.; Ulitsky, I.; Slavin, I.; Tran, H.; Schork, A.; Morey, R.; Lynch, C.; Harness, J. V.; Lee, S.; Barrero, M. J.; Ku, S.; Martynova, M.; Semechkin, R.; Galat, V.; Gottesfeld, J.; Belmonte, J. C. I.; Murry, C.; Keirstead, H. S.; Park, H.-S.; Schmidt, U.; Laslett, A. L.; Muller, F.-J.; Nievergelt, C. M.; Shamir, R.; Loring, J. F. Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell Stem Cell 8:106–118; 2011. 198. Lee, A. S.; Xu, D.; Plews, J. R.; Nguyen, P. K.; Nag, D.; Lyons, J. K.; Han, L.; Hu, S.; Lan, F.; Liu, J.; Huang, M.; Narsinh, K. H.; Long, C. T.; de Almeida, P. E.; Levi, B.; Kooreman, N.; Bangs, C.; Pacharinsak, C.; Ikeno, F.; Yeung, A. C.; Gambhir, S. S.; Robbins, R. C.; Longaker, M. T.; Wu, J. C. Preclinical derivation and imaging of autologously transplanted canine induced pluripotent stem cells. J. Biol. Chem. 286:32697–32704; 2011. 199. Lee, C. H.; Kim, J.-H.; Lee, H. J.; Jeon, K.; Lim, H.; Choi, H. Y.; Lee, E.-R.; Park, S. H.; Park, J.-Y.; Hong, S.; Kim, S.; Cho, S.-G. The generation of iPS cells using nonviral magnetic nanoparticle based transfection. Biomaterials 32:6683–6691; 2011. 200. Lee, E. X.; Lam, D. H.; Wu, C.; Yang, J.; Tham, C. K.; Ng, W. H.; Wang, S. Glioma gene therapy using induced pluripotent stem cell derived neural stem cells. Mol. Pharm. 8:1515–1524; 2011.
609
201. Lee, G.; Chambers, S. M.; Tomishima, M. J.; Studer, L. Derivation of neural crest cells from human pluripotent stem cells. Nat. Protoc. 5:688–701; 2010. 202. Lee, G.; Papapetrou, E. P.; Kim, H.; Chambers, S. M.; Tomishima, M. J.; Fasano, C. A.; Ganat, Y. M.; Menon, J.; Shimizu, F.; Viale, A.; Tabar, V.; Sadelain, M.; Studer, L. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461:402– 406; 2009. 203. Lee, J. P.; Tsai, D. J.; In Park, K.; Harvey, A. R.; Snyder, E. Y. The dynamics of long-term transgene expression in engrafted neural stem cells. J. Comp. Neurol. 515:83– 92; 2009. 204. Lee, S. B.; Seo, D.; Choi, D.; Park, K.-Y.; Holczbauer, A.; Marquardt, J. U.; Conner, E. A.; Factor, V. M.; Thorgeirsson, S. S. Contribution of hepatic lineage stagespecific donor memory to the differential potential of induced mouse pluripotent stem cells. Stem Cells 30:997– 1007; 2012. 205. Leskovar, A.; Moriarty, L. J.; Turek, J. J.; Schoenlein, I. A.; Borgens, R. B. The macrophage in acute neural injury: Changes in cell numbers over time and levels of cytokine production in mammalian central and peripheral nervous systems. J. Exp. Biol. 203:1783–1795; 2000. 206. Li, H.; Collado, M.; Villasante, A.; Strati, K.; Ortega, S.; Canamero, M.; Blasco, M. A.; Serrano, M. The Ink4/ Arf locus is a barrier for iPS cell reprogramming. Nature 460:1136–1139; 2009. 207. Li, L.; Baroja, M. L.; Majumdar, A.; Chadwick, K.; Rouleau, A.; Gallacher, L.; Ferber, I.; Lebkowski, J.; Martin, T.; Madrenas, J.; Bhatia, M. Human embryonic stem cells possess immune-privileged properties. Stem Cells 22:448–456; 2004. 208. Li, W.; Wei, W.; Zhu, S.; Zhu, J.; Shi, Y.; Lin, T.; Hao, E.; Hayek, A.; Deng, H.; Ding, S. Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 4:16–19; 2009. 209. Li, Y.; Yokohama-Tamaki, T.; Tanaka, T. S. Short-term serum-free culture reveals that inhibition of Gsk3b induces the tumor-like growth of mouse embryonic stem cells. PLoS One 6:e21355; 2011. 210. Lian, Q.; Zhang, Y.; Zhang, J.; Zhang, H.; Wu, X. Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice. Circulation 121:1113–1123; 2010. 211. Liang, G.; Taranova, O.; Xia, K.; Zhang, Y. Butyrate promotes induced pluripotent stem cell generation. J. Biol. Chem. 285:25516–25521; 2010. 212. Liao, B.; Bao, X.; Liu, L.; Feng, S.; Zovoilis, A.; Liu, W.; Xue, Y.; Cai, J.; Guo, X.; Qin, B.; Zhang, R.; Wu, J.; Lai, L.; Teng, M.; Niu, L.; Zhang, B.; Esteban, M. A.; Pei, D. MicroRNA cluster 302–enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition. J. Biol. Chem. 286:17359–17364; 2011. 213. Liao, J.; Cui, C.; Chen, S.; Ren, J.; Chen, J.; Gao, Y. Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 4:11–15; 2009. 214. Lin, S.-L.; Chang, D. C.; Lin, C.-H.; Ying, S.-Y.; Leu, D.; Wu, D. T. S. Regulation of somatic cell reprogramming through inducible mir-expression. Nucleic Acids Res. 39:1054–1065; 2011. 215. Lin, T.; Ambasudhan, R.; Yuan, X.; Li, W.; Hilcove, S.; Abujarour, R.; Lin, X.; Hahm, H. S.; Hao, E.; Hayek, A.;
610 kramer ET AL.
Ding, S. A chemical platform for improved induction of human iPSCs. Nat. Methods 6:805–808; 2009. 216. Lister, R.; Pelizzola, M.; Kida, Y. S.; Hawkins, R. D.; Nery, J. R.; Hon, G.; Antosiewicz-Bourget, J.; O’Malley, R.; Castanon, R.; Klugman, S.; Downes, M.; Yu, R.; Stewart, R.; Ren, B.; Thomson, J. A.; Evans, R. M.; Ecker, J. R. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471:68–73; 2011. 217. Liu, H.; Kim, Y.; Sharkis, S.; Marchionni, L.; Jang, Y.-Y. In vivo liver regeneration potential of human induced pluripotent stem cells from diverse origins. Sci. Transl. Med. 3:82ra39; 2011. 218. Liu, J.; Balehosur, D.; Murray, B.; Kelly, J. M.; Sumer, H.; Verma, P. J. Generation and characterization of reprogrammed sheep induced pluripotent stem cells. Theriogenology 77:338–346; 2012. 219. Liu, X. Z.; Xu, X. M.; Hu, R.; Du, C.; Zhang, S. X.; McDonald, J. W.; Dong, H. X.; Wu, Y. J.; Fan, G. S.; Jacquin, M. F.; Hsu, C. Y.; Choi, D. W. Neuronal and glial apoptosis after traumatic spinal cord injury. J. Neurosci. 17:5395–5406; 1997. 220. Liu, Y.; Wang, L.; Kikuiri, T.; Akiyama, K.; Chen, C.; Xu, X.; Yang, R.; Chen, W.; Wang, S.; Shi, S. Mesenchymal stem cell-based tissue regeneration is governed by recipient T lymphocytes via IFN-g and TNF-a. Nat. Med. 17:1594–1601; 2011. 221. Loehle, M.; Hermann, A.; Glass, H.; Kempe, A.; Schwarz, S. C.; Kim, J. B.; Poulet, C.; Ravens, U.; Schwarz, J.; Schoeler, H. R.; Storch, A. Differentiation efficiency of induced pluripotent stem cells depends on the number of reprogramming factors. Stem Cells 30:570–579; 2012. 222. Loewer, S.; Cabili, M. N.; Guttman, M.; Loh, Y.-H.; Thomas, K.; Park, I. H.; Garber, M.; Curran, M.; Onder, T.; Agarwal, S.; Manos, P. D.; Datta, S.; Lander, E. S.; Schlaeger, T. M.; Daley, G. Q.; Rinn, J. L. Large intergenic noncoding RNA-RoR modulates reprogramming of human induced pluripotent stem cells. Nat. Genet. 42:1113–1137; 2010. 223. Loh, Y. H.; Wu, Q.; Chew, J. L.; Vega, V. B.; Zhang, W. W.; Chen, X.; Bourque, G.; George, J.; Leong, B.; Liu, J.; Wong, K. Y.; Sung, K. W.; Lee, C. W. H.; Zhao, X. D.; Chiu, K. P.; Lipovich, L.; Kuznetsov, V. A.; Robson, P.; Stanton, L. W.; Wei, C. L.; Ruan, Y. J.; Lim, B.; Ng, H. H. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat. Genet. 38:431–440; 2006. 224. Lopez, M.; Valenzuela, J.; Alvarez, F.; Lopez-Alvarez, M.; Cecilia, G.; Paricio, P. Long-term problems related to immunosuppression. Transpl. Immunol. 17:31–35; 2006. 225. Lowry, W. E.; Richter, L.; Yachechko, R.; Pyle, A. D.; Tchieu, J.; Sridharan, R.; Clark, A. T.; Plath, K. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc. Natl. Acad. Sci. USA 105:2883–2888; 2008. 226. Lui, K. O.; Boyd, A. S.; Cobbold, S. P.; Waldmann, H.; Fairchild, P. J. A role for regulatory T cells in acceptance of ESC-derived tissues transplanted across an major histocompatibility complex barrier. Stem Cells 28:1905–1914; 2010. 227. Lyssiotis, C. A.; Foreman, R. K.; Staerk, J.; Garcia, M.; Mathur, D.; Markoulaki, S.; Hanna, J.; Lairson, L. L.; Charette, B. D.; Bouchez, L. C.; Bollong, M.; Kunick, C.; Brinker, A.; Cho, C. Y.; Schultz, P. G.; Jaenisch, R. Reprogramming of murine fibroblasts to induced pluri-
potent stem cells with chemical complementation of Klf4. Proc. Natl. Acad. Sci. USA 106:8912–8917; 2009. 228. MacArthur, B. D.; Please, C. P.; Oreffo, R. O. C. Stochasticity and the Molecular Mechanisms of Induced Pluripotency. PLoS One 3:e3086; 2008. 229. Madhavan, L.; Collier, T. A synergistic approach for neural repair: Cell transplantation and induction of endogenous precursor cell activity. Neuropharmacology 58:835–844; 2010. 230. Maekawa, M.; Yamaguchi, K.; Nakamura, T.; Shibukawa, R.; Kodanaka, I.; Ichisaka, T.; Kawamura, Y.; Mochizuki, H.; Goshima, N.; Yamanaka, S. Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. Nature 474:225–230; 2011. 231. Magill, G.; Neaves, W. B. Ontological and ethical implications of direct nuclear reprogramming. Kennedy Inst. Ethics J. 19:23–32; 2009. 232. Maherali, N.; Sridharan, R.; Xie, W.; Utikal, J.; Eminli, S.; Arnold, K.; Stadtfeld, M.; Yachechko, R.; Tchieu, J.; Jaenisch, R.; Plath, K.; Hochedlinger, K. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1:55–70; 2007. 233. Maier, L.; Schwab, J. M. Sprouting, regeneration and circuit formation in the injured spinal cord: Factors and activity. Philos. Trans. R. Soc. Lond. Biol. Sci. 381:1611– 1684; 2006. 234. Major, T.; Menon, J.; Auyeung, G.; Soldner, F.; Hocke meyer, D.; Jaenisch, R.; Tabar, V. Transgene excision has no impact on in vivo integration of human iPS derived neural precursors. PLoS One 6:e24687; 2011. 235. Mali, P.; Chou, B.-K.; Yen, J.; Ye, Z.; Zou, J.; Dowey, S.; Brodsky, R. A.; Ohm, J. E.; Yu, W.; Baylin, S. B.; Yusa, K.; Bradley, A.; Meyers, D. J.; Mukherjee, C.; Cole, P. A.; Cheng, L. Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes. Stem Cells 28:713–720; 2010. 236. Marchetto, M.; Yeo, G.; Kainohana, O.; Marsala, M.; Gage, F.; Muotri, A. Transcriptional signature and memory retention of human induced pluripotent stem cells. PLoS One 4:e7076; 2009. 237. Marchetto, M. C.; Brennand, K. J.; Boyer, L. F.; Gage, F. H. Induced pluripotent stem cells (iPSCs) and neurological disease modeling: Progress and promises. Hum. Mol. Genet. 20:R109–R115; 2011. 238. Marchetto, M. C. N.; Carromeu, C.; Acab, A.; Yu, D.; Yeo, G. W.; Mu, Y.; Chen, G.; Gage, F. H.; Muotri, A. R. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143:527–539; 2010. 239. Marion, R. M.; Blasco, M. A. Telomere rejuvenation during nuclear reprogramming. Curr. Opin. Biotechnol. 20:190–196; 2010. 240. Marion, R. M.; Strati, K.; Li, H.; Murga, M.; Blanco, R.; Ortega, S.; Fernandez-Capetillo, O.; Serrano, M.; Blasco, M. A. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature 460:1149–1155; 2009. 241. Markman, J. L.; Silvers, R. M.; Ndoye, A. M. N.; Geary, K. R.; Alvarado, D.; Smith, J. A.; Daniel, R. HIV-1 integration site preferences in pluripotent cells. Front. Biosci. (Elite Ed.) 3:453–462; 2011.
iPSC TECHNOLOGY FOR SCI
242. Marson, A.; Foreman, R.; Chevalier, B.; Bilodeau, S.; Kahn, M.; Young, R. A.; Jaenisch, R. Wnt signaling promotes reprogramming of somatic cells to pluripotency. Cell Stem Cell 3:132–135; 2008. 243. Martins-Taylor, K.; Nisler, B. S.; Taapken, S. M.; Compton, T.; Crandall, L.; Montgomery, K. D.; Lalande, M.; Xu, R.-H. Recurrent copy number variations in human induced pluripotent stem cells. Nat. Biotechnol. 29:488– 491; 2011. 244. Martins-Taylor, K.; Xu, R.-H. Concise review: Genomic stability of human induced pluripotent stem cells. Stem Cells 30:22–27; 2012. 245. Mathew, R.; Jia, W.; Sharma, A.; Zhao, Y.; Clarke, L. E.; Cheng, X.; Wang, H.; Salli, U.; Vrana, K. E.; Robertson, G. P.; Zhu, J.; Wang, S. Robust activation of the human but not mouse telomerase gene during the induction of pluripotency. FASEB J. 24:2702–2715; 2010. 246. Mayshar, Y.; Ben-David, U.; Lavon, N.; Biancotti, J.-C.; Yakir, B.; Clark, A. T.; Plath, K.; Lowry, W. E.; Benvenisty, N. Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell 7:521–531; 2010. 247. McDonald, J. W.; Althomsons, S. P.; Hyrc, K. L.; Choi, D. W.; Goldberg, M. P. Oligodendrocytes from forebrain are highly vulnerable to AMPA/kainate receptor-mediated excitotoxicity. Nat. Med. 4:291–297; 1998. 248. McDonald, J. W.; Belegu, V. Demyelination and remyelination after spinal cord injury. J. Neurotrauma 23:345– 359; 2006. 249. McDonald, J. W.; Howard, M. J. Repairing the damaged spinal cord: A summary of our early success with embryonic stem cell transplantation and remyelination. Prog. Brain Res. 137:299–309; 2002. 250. McKeon, R. J.; Höke, A.; Silver, J. Injury-induced proteoglycans inhibit the potential for laminin-mediated axon growth on astrocytic scars. Exp. Neurol. 136:32–43; 1995. 251. Meissner, A.; Jaenisch, R. Mammalian nuclear transfer. Dev. Dyn. 235:2460–2469; 2006. 252. Meissner, A.; Wernig, M.; Jaenisch, R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat. Biotechnol. 25:1177–1181; 2007. 253. Melton, C.; Blelloch, R. MicroRNA regulation of embryonic stem cell self-renewal and differentiation. Adv. Exp. Med. Biol. 695:105–117; 2010. 254. Mitsui, T.; Shumsky, J.; Lepore, A.; Murray, M.; Fischer, I. Transplantation of neuronal and glial restricted precursors into contused spinal cord improves bladder and motor functions, decreases thermal hypersensitivity, and modifies intraspinal circuitry. J. Neurosci. 25:9624–9636; 2005. 255. Miura, K.; Okada, Y.; Aoi, T.; Okada, A.; Takahashi, K.; Okita, K.; Nakagawa, M.; Koyanagi, M.; Tanabe, K.; Ohnuki, M.; Ogawa, D.; Ikeda, E.; Okano, H.; Yamanaka, S. Variation in the safety of induced pluripotent stem cell lines. Nat. Biotechnol. 27:743–745; 2009. 256. Moloney, T. C.; Dockery, P.; Windebank, A. J.; Barry, F. P.; Howard, L.; Dowd, E. Survival and immunogenicity of mesenchymal stem cells from the green fluorescent protein transgenic rat in the adult rat brain. Neurorehabil. Neural Repair 24:645–656; 2010. 257. Moon, J.-H.; Heo, J. S.; Kim, J. S.; Jun, E. K.; Lee, J. H.; Kim, A.; Kim, J.; Whang, K. Y.; Kang, Y.-K.; Yeo, S.; Lim, H.-J.; Han, D. W.; Kim, D.-W.; Oh, S.; Yoon, B. S.; Schoeler, H. R.;
611
You, S. Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1. Cell Res. 21:1305–1315; 2011. 258. Moriguchi, H.; Chung, R. T.; Sato, C. Tumorigenicity of human induced pluripotent stem cells depends on the balance of gene expression between p21 and p53. Hepatology 51:1088–1089; 2010. 259. Nagy, K.; Sung, H.-K.; Zhang, P.; Laflamme, S.; Vincent, P.; Agha-Mohammadi, S.; Woltjen, K.; Monetti, C.; Michael, I. P.; Smith, L. C.; Nagy, A. Induced pluripotent stem cell lines derived from equine fibroblasts. Stem Cell Rev. 7:693–702; 2011. 260. Nakagawa, M.; Koyanagi, M.; Tanabe, K.; Takahashi, K.; Ichisaka, T.; Aoi, T. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol. 26:101–106; 2008. 261. Nakagawa, M.; Takizawa, N.; Narita, M.; Ichisaka, T.; Yamanaka, S. Promotion of direct reprogramming by transformation-deficient Myc. Proc. Natl. Acad. Sci. USA 107:14152–14157; 2010. 262. Nakayama, T.; Momoki-Soga, T.; Inoue, N. Astrocytederived factors instruct differentiation of embryonic stem cells into neurons. Neurosci. Res. 46:241–249; 2003. 263. Narsinh, K. H.; Plews, J.; Wu, J. C. Comparison of human induced pluripotent and embryonic stem cells: Fraternal or identical twins? Mol. Ther. 19:635–638; 2011. 264. Nashmi, R.; Fehlings, M. G. Mechanisms of axonal dysfunction after spinal cord injury: With an emphasis on the role of voltage-gated potassium channels. Brain Res. Rev. 38:165–191; 2001. 265. Nelson, T. J.; Martinez-Fernandez, A.; Yamada, S.; PerezTerzic, C.; Ikeda, Y.; Terzic, A. Repair of acute myocardial infarction by human stemness factors induced pluripotent stem cells. Circulation 120:408–416; 2009. 266. Neveu, P.; Kye, M. J.; Qi, S.; Buchholz, D. E.; Clegg, D. O.; Sahin, M.; Park, I.-H.; Kim, K.-S.; Daley, G. Q.; Kornblum, H. I.; Shraiman, B. I.; Kosik, K. S. MicroRNA profiling reveals two distinct p53-related human pluripotent stem cell states. Cell Stem Cell 7:671–681; 2010. 267. Newman, A. M.; Cooper, J. B. Lab-specific gene expression signatures in pluripotent stem cells. Cell Stem Cell 7:258–262; 2010. 268. Nguyen, H. N.; Byers, B.; Cord, B.; Shcheglovitov, A.; Byrne, J.; Gujar, P.; Kee, K.; Schule, B.; Dolmetsch, R. E.; Langston, W.; Palmer, T. D.; Pera, R. R. LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress. Cell Stem Cell 8:267–280; 2011. 269. Nishimura, K.; Sano, M.; Ohtaka, M.; Furuta, B.; Umemura, Y.; Nakajima, Y.; Ikehara, Y.; Kobayashi, T.; Segawa, H.; Takayasu, S.; Sato, H.; Motomura, K.; Uchida, E.; Kanayasu-Toyoda, T.; Asashima, M.; Nakauchi, H.; Yamaguchi, T.; Nakanishia, M. Development of defective and persistent sendai virus vector: A unique gene delivery/ expression system ideal for cell reprogramming. J. Biol. Chem. 286:4760–4771; 2011. 270. Norenberg, M. D.; Smith, J.; Marcillo, A. The pathology of human spinal cord injury: Defining the problems. J. Neurotrauma 21:429–440; 2004. 271. Nori, S.; Okada, Y.; Yasuda, A.; Tsuji, O.; Takahashi, Y.; Kobayashi, Y.; Fujiyoshi, K.; Koike, M.; Uchiyama, Y.; Ikeda, E.; Toyama, Y.; Yamanaka, S.; Nakamura, M.; Okano, H. Grafted human-induced pluripotent stem-cellderived neurospheres promote motor functional recovery
612 kramer ET AL.
after spinal cord injury in mice. Proc. Natl. Acad. Sci. USA 108:16825–16830; 2011. 272. Nussbaum, J.; Minami, E.; Laflamme, M. A.; Virag, J. A. I.; Ware, C. B.; Masino, A.; Muskheli, V.; Pabon, L.; Reinecke, H.; Murry, C. E. Transplantation of undifferentiated murine embryonic stem cells in the heart: Teratoma formation and immune response. FASEB J. 21:1345–1357; 2007. 273. Oda, Y.; Yoshimura, Y.; Ohnishi, H.; Tadokoro, M.; Katsube, Y.; Sasao, M.; Kubo, Y.; Hattori, K.; Saito, S.; Horimoto, K.; Yuba, S.; Ohgushi, H. Induction of pluripotent stem cells from human third molar mesenchymal stromal cells. J. Biol. Chem. 285:29270–29278; 2010. 274. Ogawa, S.-I.; Tokumoto, Y.; Miyake, J.; Nagamune, T. Immunopanning selection of A2B5-positive cells increased the differentiation efficiency of induced pluripotent stem cells into oligodendrocytes. Neurosci. Lett. 489:79–83; 2011. 275. Ogawa, S.-I.; Tokumoto, Y.; Miyake, J.; Nagamune, T. Induction of oligodendrocyte differentiation from adult human fibroblast-derived induced pluripotent stem cells. In Vitro Cell. Dev. Biol. Anim. 47:464–469; 2011. 276. Ohm, J. E.; Mali, P.; Van Neste, L.; Berman, D. M.; Liang, L.; Pandiyan, K.; Briggs, K. J.; Zhang, W.; Argani, P.; Simons, B.; Yu, W.; Matsui, W.; Van Criekinge, W.; Rassool, F. V.; Zambidis, E.; Schuebel, K. E.; Cope, L.; Yen, J.; Mohammad, H. P.; Cheng, L.; Baylin, S. B. Cancer-related epigenome changes associated with reprogramming to induced pluripotent stem cells. Cancer Res. 70:7662–7673; 2010. 277. Oki, K.; Tatarishvili, J.; Woods, J.; Koch, P.; Wattananit, S.; Mine, Y.; Monni, E.; Prietro, D. T.; Ahlenius, H.; Ladewig, J.; Brustle, O.; Lindvall, O.; Kokaia, Z. Human induced pluripotent stem cells form functional neurons and improve recovery after grafting in stroke-damaged brain. Stem Cells 30:1120–1133; 2012. 278. Okita, K.; Hong, H.; Takahashi, K.; Yamanaka, S. Gene ration of mouse-induced pluripotent stem cells with plasmid vectors. Nat. Protoc. 5:418–428; 2010. 279. Okita, K.; Ichisaka, T.; Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448:313–317; 2007. 280. Okita, K.; Matsumura, Y.; Sato, Y.; Okada, A.; Morizane, A.; Okamoto, S.; Hong, H.; Nakagawa, M.; Tanabe, K.; Tezuka, K.; Shibata, T.; Kunisada, T.; Takahashi, M.; Takahashi, J.; Saji, H.; Yamanaka, S. A more efficient method to generate integration-free human iPS cells. Nat. Methods 8:409–412; 2011. 281. Okita, K.; Nakagawa, M.; Hong, H. J.; Ichisaka, T.; Yamanaka, S. Generation of mouse induced pluripotent stem cells without viral vectors. Science 322:949–953; 2008. 282. Onorati, M.; Camnasio, S.; Binetti, M.; Jung, C. B.; Moretti, A.; Cattaneo, E. Neuropotent self-renewing neural stem (NS) cells derived from mouse induced pluripotent stem (iPS) cells. Mol. Cell. Neurosci. 43:287–295; 2010. 283. Osafune, K.; Caron, L.; Borowiak, M.; Martinez, R. J.; Fitz-Gerald, C. S.; Sato, Y.; Cowan, C. A.; Chien, K. R.; Melton, D. A. Marked differences in differentiation propensity among human embryonic stem cell lines. Nat. Biotechnol. 26:313–315; 2008. 284. Panopoulos, A. D.; Yanes, O.; Ruiz, S.; Kida, Y. S.; Diep, D.; Tautenhahn, R.; Herrerias, A.; Batchelder, E. M.; Plongthongkum, N.; Lutz, M.; Berggren, W. T.; Zhang,
K.; Evans, R. M.; Siuzdak, G.; Izpisua Belmonte, J. C. The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming. Cell Res. 22:168–177; 2012. 285. Papapetrou, E. P.; Sadelain, M. Generation of transgenefree human induced pluripotent stem cells with an excisable single polycistronic vector. Nat. Protocols 6:1251–1273; 2011. 286. Papapetrou, E. P.; Tomishima, M. J.; Chambers, S. M.; Mica, Y.; Reed, E.; Menon, J.; Tabar, V.; Mo, Q.; Studer, L.; Sadelain, M. Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation. Proc. Natl. Acad. Sci. USA 106:12759–12764; 2009. 287. Parameswaran, S.; Balasubramanian, S.; Babai, N.; Qiu, F.; Eudy, J. D.; Thoreson, W. B.; Ahmad, I. Induced pluripotent stem cells generate both retinal ganglion cells and photoreceptors: Therapeutic implications in degenerative changes in glaucoma and age-related macular degeneration. Stem Cells 28:695–703; 2010. 288. Park, E.; Velumian, A. A.; Fehlings, M. G. The role of excitotoxicity in secondary mechanisms of spinal cord injury: A review with an emphasis on the implications for white matter degeneration. J. Neurotrauma 21:754–774; 2004. 289. Park, I.-H.; Lerou, P. H.; Zhao, R.; Huo, H.; Daley, G. Q. Generation of human-induced pluripotent stem cells. Nat. Protoc. 3:1180–1186; 2008. 290. Pasterkamp, R. J.; Anderson, P. N.; Verhaagen, J. Peripheral nerve injury fails to induce growth of lesioned ascending dorsal column axons into spinal cord scar tissue expressing the axon repellent Semaphorin3A. Eur. J. Neurosci. 13:457–471; 2001. 291. Pearl, J. I.; Lee, A. S.; Leveson-Gower, D. B.; Sun, N.; Ghosh, Z.; Lan, F.; Ransohoff, J.; Negrin, R. S.; Davis, M. M.; Wu, J. C. Short-term immunosuppression promotes engraftment of embryonic and induced pluripotent stem cells. Cell Stem Cell 8:309–317; 2011. 292. Pedrosa, E.; Sandler, V.; Shah, A.; Carroll, R.; Chang, C.; Rockowitz, S.; Guo, X.; Zheng, D.; Lachman, H. M. Development of patient-specific neurons in schizophrenia using induced pluripotent stem cells. J. Neurogenet. 25: 88–103; 2011. 293. Pfaff, N.; Fiedler, J.; Holzmann, A.; Schambach, A.; Moritz, T.; Cantz, T.; Thum, T. miRNA screening reveals a new miRNA family stimulating iPS cell generation via regulation of Meox2. EMBO Rep. 12:1153–1159; 2011. 294. Pick, M.; Stelzer, Y.; Bar-Nur, O.; Mayshar, Y.; Eden, A.; Benvenisty, N. Clone- and gene-specific aberrations of parental imprinting in human induced pluripotent stem cells. Stem Cells 27:2686–2690; 2009. 295. Plath, K.; Lowry, W. E. Progress in understanding reprogramming to the induced pluripotent state. Nat. Rev. Gen. 12:253–265; 2011. 296. Polo, J. M.; Liu, S.; Figueroa, M. E.; Kulalert, W.; Eminli, S.; Tan, K. Y.; Apostolou, E.; Stadtfeld, M.; Li, Y.; Shioda, T.; Natesan, S.; Wagers, A. J.; Melnick, A.; Evans, T.; Hochedlinger, K. Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nat. Biotechnol. 28:848–855; 2010. 297. Popovich, P. G.; Jones, T. B. Manipulating neuroinflammatory reactions in the injured spinal cord: Back to basics. Trends Pharmacol. Sci. 24:13–17; 2003.
iPSC TECHNOLOGY FOR SCI
298. Popovich, P. G.; Wei, P.; Stokes, B. T. Cellular inflammatory response after spinal cord injury in Sprague–Dawley and Lewis rats. J. Comp. Neurol. 377:443–464; 1997. 299. Pouya, A.; Satarian, L.; Kiani, S.; Javan, M.; Baharvand, H. Human induced pluripotent stem cells differentiation into oligodendrocyte progenitors and transplantation in a rat model of optic chiasm demyelination. PLoS One 6:e27925; 2011. 300. Pralong, D.; Trounson, A. O.; Verma, P. J. Cell fusion for reprogramming pluripotency—Toward elimination of the pluripotent genome. Stem Cell Rev. 2:331–340; 2006. 301. Prigione, A.; Adjaye, J. Modulation of mitochondrial biogenesis and bioenergetic metabolism upon in vitro and in vivo differentiation of human ES and iPS cells. Int. J. Dev. Biol. 54:1729–1741; 2010. 302. Prigione, A.; Lichtner, B.; Kuhl, H.; Struys, E. A.; Wamelink, M.; Lehrach, H.; Ralser, M.; Timmermann, B.; Adjaye, J. Human induced pluripotent stem cells harbor homoplasmic and heteroplasmic mitochondrial DNA mutations while maintaining human embryonic stem cell-like metabolic reprogramming. Stem Cells 29:1338–1348; 2011. 303. Profyris, C.; Cheema, S. S.; Zang, D.; Azari, M. F.; Boyle, K.; Petratos, S. Degenerative and regenerative mechanisms governing spinal cord injury. Neurobiol. Dis. 15:415–436; 2004. 304. Puckett, W. R.; Hiester, E. D.; Norenberg, M. D.; Marcillo, A. E.; Bunge, R. P. The astroglial response to Wallerian degeneration after spinal cord injury in humans. Exp. Neurol. 148:424–432; 1997. 305. Ramos-Mejia, V.; Munoz-Lopez, M.; Garcia-Perez, J. L.; Menendez, P. iPSC lines that do not silence the expression of the ectopic reprogramming factors may display enhanced propensity to genomic instability. Cell Res. 20:1092–1095; 2010. 306. Reier, P. J.; Houle, J. D. The Glial Scar: It’s bearing on axonal elongation and transplantion approaches to CNS repair. Adv. Neurol. 47:87–138; 1988. 307. Rhee, Y.-H.; Ko, J.-Y.; Chang, M.-Y.; Yi, S.-H.; Kim, D.; Kim, C.-H.; Shim, J.-W.; Jo, A. Y.; Kim, B.-W.; Lee, H.; Lee, S.-H.; Suh, W.; Park, C.-H.; Koh, H.-C.; Lee, Y.-S.; Lanza, R.; Kim, K.-S.; Lee, S.-H. Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease. J. Clin. Invest. 121:2326–2335; 2011. 308. Rideout, W. M.; Eggan, K.; Jaenisch, R. Nuclear cloning and epigenetic reprogramming of the genome. Science 293:1093–1098; 2001. 309. Risling, M.; Fried, K.; Linda, H.; Carlstedt, T.; Cullheim, S. Regrowth of motor axons following spinal-cord lesions. Distribution of laminin and collagen in the CNS scar tissue. Brain Res. Bull. 30:405–414; 1993. 310. Robertson, J. A. Ethics and policy in embryonic stem cell research. Kennedy Inst. Ethics J. 9:109–136; 1999. 311. Robertson, N. J.; Brook, F. A.; Gardner, R. L.; Cobbold, S. P.; Waldmann, H.; Fairchild, P. J. Embryonic stem cellderived tissues are immunogenic but their inherent immune privilege promotes the induction of tolerance. Proc. Natl. Acad. Sci. USA 104:20920–20925; 2007. 312. Rodriguez-Piza, I.; Richaud-Patin, Y.; Vassena, R.; Gonzalez, F.; Jose Barrero, M.; Veiga, A.; Raya, A.; Izpisua Belmonte, J. C. Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions. Stem Cells 28:36–44; 2010.
613
313. Romano, G. Stem cell transplantation therapy: Controversy over ethical issues and clinical relevance. Drug News Perspect. 17:637–645; 2004. 314. Ronaghi, M.; Erceg, S.; Moreno-Manzano, V.; Stojkovic, M. Challenges of stem cell therapy for spinal cord injury: Human embryonic stem cells, endogenous neural stem cells, or induced pluripotent stem cells? Stem Cells 28:93–99; 2010. 315. Ron-Bigger, S.; Bar-Nur, O.; Isaac, S.; Bocker, M.; Lyko, F.; Eden, A. Aberrant epigenetic silencing of tumor suppressor genes is reversed by direct reprogramming. Stem Cells 28:1349–1354; 2010. 316. Ross, P. J.; Suhr, S. T.; Maria Rodriguez, R.; Chang, E.-A.; Wang, K.; Siripattarapravat, K.; Ko, T.; Bernardo Cibelli, J. Human-induced pluripotent stem cells produced under xeno-free conditions. Stem Cells Dev. 19:1221–1229; 2010. 317. Rossi, S. L.; Keirstead, H. S. Stem cells and spinal cord regeneration. Curr. Opin. Biotechnol. 20:552–562; 2009. 318. Rowland, J. W.; Hawryluk, G. W. J.; Kwon, B.; Fehlings, M. G. Current status of acute spinal cord injury pathophysiology and emerging therapies: Promise on the horizon. Neurosurg. Focus 25:E2; 2008. 319. Roy, N. S.; Cleren, C.; Singh, S. K.; Yang, L.; Beal, M. F.; Goldman, S. A. Functional engraftment of human ES cellderived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat. Med. 13:385–385; 2007. 320. Rudge, J. S.; Silver, J. Inhibition of neurite outgrowth on astroglial scars in vitro. J. Neurosci. 10:3594–3603; 1990. 321. Sanchez-Danes, A.; Consiglio, A.; Richaud, Y.; RodriguezPiza, I.; Dehay, B.; Edel, M.; Bove, J.; Memo, M.; Vila, M.; Raya, A.; Izpisua Belmonte, J. C. Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of Lmx1A in human embryonic stem cells and iPS cells. Hum. Gene Ther. 23:56–69; 2012. 322. Sartore, R. C.; Campos, P. B.; Trujillo, C. A.; Ramalho, B. L.; Negraes, P. D.; Paulsen, B. S.; Meletti, T.; Costa, E. S.; Chicaybam, L.; Bonamino, M. H.; Ulrich, H.; Rehen, S. K. Retinoic acid-treated pluripotent stem cells undergoing neurogenesis present increased aneuploidy and micronuclei formation. PLoS One 6:e20667; 2011. 323. Sasaki, N.; Hirano, T.; Kobayashi, K.; Toyoda, M.; Miyakawa, Y.; Okita, H.; Kiyokawa, N.; Akutsu, H.; Umezawa, A.; Nishihara, S. Chemical inhibition of sulfation accelerates neural differentiation of mouse embryonic stem cells and human induced pluripotent stem cells. Biochem. Biophys. Res. Commun. 401:480–486; 2010. 324. Schwab, M. E.; Bartholdi, D. Degeneration and regeneration of axons in the lesioned spinal cord. Physiol. Rev. 76:319–370; 1996. 325. Seibler, P.; Graziotto, J.; Jeong, H.; Simunovic, F.; Klein, C.; Krainc, D. Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 induced pluripotent stem cells. J. Neurosci. 31:5970–5976; 2011. 326. Sekhon, L. H. S.; Fehlings, M. G. Epidemiology, demographics, and pathophysiology of acute spinal cord injury. Spine 26:S2–S12; 2001. 327. Seki, T.; Yuasa, S.; Oda, M.; Egashira, T.; Yae, K.; Kusumoto, D.; Nakata, H.; Tohyama, S.; Hashimoto, H.; Kodaira, M.; Okada, Y.; Seimiya, H.; Fusaki, N.; Hasegawa, M.; Fukuda, K. Generation of induced
614 kramer ET AL.
pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell 7:11–14; 2010. 328. Sharp, J.; Frame, J.; Siegenthaler, M.; Nistor, G.; Keirstead, H. S. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants improve recovery after cervical spinal cord injury. Stem Cells 28:152–163; 2010. 329. Shi, Y.; Desponts, C.; Do, J. T.; Hahm, H. S.; Schoeler, H. R.; Ding, S. Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell 3:568–574; 2008. 330. Shi, Y.; Do, J. T.; Desponts, C.; Hahm, H. S.; Schoeler, H. R.; Ding, S. A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell 2:525–528; 2008. 331. Shields, C. B.; Zhang, Y. P.; Shields, L. B. E.; Han, Y.; Burke, D. A.; Mayer, N. W. The therapeutic window for spinal cord decompression in a rat spinal cord injury model. J. Neurosurg. Spine 3:302–307; 2005. 332. Shimada, H.; Hashimoto, Y.; Nakada, A.; Shigeno, K.; Nakamura, T. Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia. Biochem. Biophys. Res. Commun. 417:659–664; 2012. 333. Silver, J.; Miller, J. H. Regeneration beyond the glial scar. Nat. Rev. Neurosci. 5:146–156; 2004. 334. Singh, A. M.; Dalton, S. The cell cycle and Myc intersect with mechanisms that regulate pluripotency and reprogramming. Cell Stem Cell 5:141–149; 2009. 335. Singla, D. K.; Long, X.; Glass, C.; Singla, R. D.; Yan, B. Induced pluripotent stem (iPS) cells repair and regenerate infarcted myocardium. Mol. Pharm. 8:1573–1581; 2011. 336. Skene, L. Recent developments in stem cell research: Social, ethical, and legal issues for the future. Ind. J. Global Legal Stud. 17:211–244; 2010. 337. Smith, K. P.; Luong, M. X.; Stein, G. S. Pluripotency: Toward a gold standard for human ES and iPS cells. J. Cell. Physiol. 220:21–29; 2009. 338. Soldner, F.; Hockemeyer, D.; Beard, C.; Gao, Q.; Bell, G. W.; Cook, E. G.; Hargus, G.; Blak, A.; Cooper, O.; Mitalipova, M.; Isacson, O.; Jaenisch, R. Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136:964–977; 2009. 339. Sommer, C. A.; Sommer, A. G.; Longmire, T. A.; Christo doulou, C.; Thomas, D. D.; Gostissa, M.; Alt, F. W.; Murphy, G. J.; Kotton, D. N.; Mostoslavsky, G. Excision of reprogramming transgenes improves the differentiation potential of iPS cells generated with a single excisable vector. Stem Cells 28:64–74; 2010. 340. Sommer, C. A.; Stadtfeld, M.; Murphy, G. J.; Hochedlinger, K.; Kotton, D. N.; Mostoslavsky, G. Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells 27:543–549; 2009. 341. Sperger, J. M.; Chen, X.; Draper, J. S.; Antosiewicz, J. E.; Chon, C. H.; Jones, S. B.; Brooks, J. D.; Andrews, P. W.; Brown, P. O.; Thomson, J. A. Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc. Natl. Acad. Sci. USA 100:13350– 13355; 2003. 342. Sroga, J. M.; Jones, T. B.; Kigerl, K. A.; McGaughy, V. M.; Popovich, P. G. Rats and mice exhibit distinct inflammatory reactions after spinal cord injury. J. Comp. Neurol. 462:223–240; 2003. 343. Stadtfeld, M.; Apostolou, E.; Akutsu, H.; Fukuda, A.; Follett, P.; Natesan, S.; Kono, T.; Shioda, T.; Hochedlinger,
K. Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 465:175–181; 2010. 344. Stadtfeld, M.; Maherali, N.; Breault, D. T.; Hochedlinger, K. Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2:230– 240; 2008. 345. Stadtfeld, M.; Nagaya, M.; Utikal, J.; Weir, G.; Hochedlinger, K. Induced pluripotent stem cells generated without viral integration. Science 322:945–949; 2008. 346. Stelzer, Y.; Yanuka, O.; Benvenisty, N. Global analysis of parental imprinting in human parthenogenetic induced pluripotent stem cells. Nat. Struct. Mol. Biol. 18:735–741; 2011. 347. Stripecke, R.; Carmen Villacres, M.; Skelton, D.; Satake, N.; Halene, S.; Kohn, D. Immune response to green fluorescent protein: Implications for gene therapy. Gene Ther. 6:1305–1312; 1999. 348. Studer, L. The Nervous System. In: Lanza, R., ed. Essentials of stem cell biology. San Diego, CA: Elsevier; 2009:169–178. 349. Subramanyam, D.; Lamouille, S.; Judson, R. L.; Liu, J. Y.; Bucay, N.; Derynck, R.; Blelloch, R. Multiple targets of miR-and miR-promote reprogramming of human fibroblasts to induced pluripotent stem cells. Nat. Biotechnol. 29:443–448; 2011. 350. Sugii, S.; Kida, Y.; Kawamura, T.; Suzuki, J.; Vassena, R.; Yin, Y.-Q.; Lutz, M. K.; Berggren, T.; Izpisua Belmonte, J. C.; Evans, R. M. Human and mouse adipose-derived cells support feeder-independent induction of pluripotent stem cells. Proc. Natl. Acad. Sci. USA 107:3558–3563; 2010. 351. Suhr, S. T.; Chang, E. A.; Rodriguez, R. M.; Wang, K.; Ross, P. J.; Beyhan, Z.; Murthy, S.; Cibelli, J. B. Telomere dynamics in human cells reprogrammed to pluripotency. PLoS One 4:e8124; 2009. 352. Sullivan, G. J.; Bai, Y.; Fletcher, J.; Wilmut, I. Induced pluripotent stem cells: Epigenetic memories and practical implications. Mol. Hum. Reprod. 16:880–885; 2010. 353. Sun, Y.; Kong, W.; Falk, A.; Hu, J.; Zhou, L.; Pollard, S.; Smith, A. CD(prominin) negative human neural stem cells are clonogenic and tripotent. PLoS One 4:e5498; 2009. 354. Suzuki, H.; Shibata, R.; Kito, T.; Ishii, M.; Li, P.; Yoshikai, T.; Nishio, N.; Ito, S.; Numaguchi, Y.; Yamashita, J. K.; Murohara, T.; Isobe, K. Therapeutic angiogenesis by transplantation of induced pluripotent stem cell-derived Flk-1 positive cells. BMC Cell Biol. 11:72; 2010. 355. Svendsen, C. N.; ter Borg, M. G.; Armstrong, R. J. E.; Rosser, A. E.; Chandran, S.; Ostenfeld, T.; Caldwell, M. A. A new method for the rapid and long term growth of human neural precursor cells. J. Neurosci. Methods 85:141– 152; 1998. 356. Swijnenburg, R.-J.; Schrepfer, S.; Govaert, J. A.; Cao, F.; Ransohoff, K.; Sheikh, A. Y.; Haddad, M.; Connolly, A. J.; Davis, M. M.; Robbins, R. C.; Wu, J. C. Immu nosuppressive therapy mitigates immunological rejection of human embryonic stem cell xenografts. Proc. Natl. Acad. Sci. USA 105:12991–12996; 2008. 357. Swijnenburg, R.-J.; Tanaka, M.; Vogel, H.; Baker, J.; Kofidis, T.; Gunawan, F.; Lebl, D. R.; Caffarelli, A. D.; de Bruin, J. L.; Fedoseyeva, E. V.; Robbins, R. C. Embry onic stem cell immunogenicity increases upon differentiation after transplantation into ischemic myocardium. Circulation 112:I166–I172; 2005. 358. Swistowski, A.; Peng, J.; Liu, Q.; Mali, P.; Rao, M. S.; Cheng, L.; Zeng, X. Efficient generation of functional
iPSC TECHNOLOGY FOR SCI
dopaminergic neurons from human induced pluripotent stem cells under defined conditions. Stem Cells 28:1893– 1904; 2010. 359. Taapken, S. M.; Nisler, B. S.; Newton, M. A.; SampsellBarron, T. L.; Leonhard, K. A.; McIntire, E. M.; Mont gomery, K. D. Karyotypic abnormalities in human induced pluripotent stem cells and embryonic stem cells. Nat. Biotechnol. 29:313–314; 2011. 360. Takahashi, K. Direct reprogramming 101. Dev. Growth Differ. 52:319–333; 2010. 361. Takahashi, K.; Tanabe, K.; Ohnuki, M.; Narita, M.; Ichisaka, T.; Tomoda, K.; Yamanaka, S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872; 2007. 362. Takahashi, K.; Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676; 2006. 363. Tang, C.; Lee, A. S.; Volkmer, J. P.; Sahoo, D.; Nag, D.; Mosley, A. R.; Inlay, M. A.; Ardehali, R.; Chavez, S. L.; Pera, R. R.; Behr, B.; Wu, J. C.; Weissman, I. L.; Drukker, M. An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells. Nat. Biotechnol. 29:829–834; 2011. 364. Tang, X. F.; Davies, J. E.; Davies, S. J. A. Changes in distribution, cell associations, and protein expression levels of NG2, neurocan, phosphacan, brevican, versican V2, and tenascin-C during acute to chronic maturation of spinal cord scar tissue. J. Neurosci. Res. 71:427–444; 2003. 365. Taoka, Y.; Okajima, K.; Uchiba, M.; Murakami, K.; Kushi moto, S.; Johno, M.; Naruo, M.; Okabe, H.; Takatsuki, K. Role of neutrophils in spinal cord injury in the rat. Neuroscience 79:1177–1182; 1997. 366. Teichroeb, J. H.; Betts, D. H.; Vaziri, H. Suppression of the imprinted gene NNAT and X-chromosome gene activation in isogenic human iPS cells. PLoS One 6:e23436; 2011. 367. Tetzlaff, W.; Okon, E. B.; Karimi-Abdolrezaee, S.; Hill, C. E.; Sparling, J. S.; Plemel, J. R.; Plunet, W. T.; Tsai, E. C.; Baptiste, D.; Smithson, L. J.; Kawaja, M. D.; Fehlings, M. G.; Kwon, B. K. A systematic review of cellular transplantation therapies for spinal cord injury. J. Neurotrauma 28:1611–1682; 2011. 368. Tokumoto, Y.; Ogawa, S.; Nagamune, T.; Miyake, J. Comparison of efficiency of terminal differentiation of oligodendrocytes from induced pluripotent stem cells versus embryonic stem cells in vitro. J. Biosci. Bioeng. 109:622–628; 2010. 369. Totoiu, M. O.; Keirstead, H. S. Spinal cord injury is accompanied by chronic progressive demyelination. J. Comp. Neurol. 486:373–383; 2005. 370. Touraine, J. L.; Sanhadji, K. Transplantation tolerance induced in humans at the fetal or the neonatal stage. J. Transplant. 2011:760319; 2011. 371. Tsuji, O.; Miura, K.; Okada, Y.; Fujiyoshi, K.; Mukaino, M.; Nagoshi, N.; Kitamura, K.; Kumagai, G.; Nishino, M.; Tomisato, S.; Higashi, H.; Nagai, T.; Katoh, H.; Kohda, K.; Matsuzaki, Y.; Yuzaki, M.; Ikeda, E.; Toyama, Y.; Nakamura, M.; Yamanaka, S.; Okano, H. Therapeutic potential of appropriately evaluated safe-induced pluripotent stem cells for spinal cord injury. Proc. Natl. Acad. Sci. USA 107:12704–12709; 2010. 372. Tucker, B. A.; Park, I. H.; Qi, S. D.; Klassen, H. J.; Jiang, C. H.; Yao, J.; Redenti, S.; Daley, G. Q.; Young, M. J. Transplantation of adult mouse iPS cell-derived
615
photoreceptor precursors restores retinal structure and function in degenerative mice. PLoS One 6:e18992; 2011. 373. Uemura, T.; Takamatsu, K.; Ikeda, M.; Okada, M.; Kazuki, K.; Ikada, Y.; Nakamura, H. Transplantation of induced pluripotent stem cell-derived neurospheres for peripheral nerve repair. Biochem. Biophys. Res. Commun. 419:130– 135; 2012. 374. Utikal, J.; Polo, J. M.; Stadtfeld, M.; Maherali, N.; Kulalert, W.; Walsh, R. M.; Khalil, A.; Rheinwald, J. G.; Hochedlinger, K. Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature 460:1145–1148; 2009. 375. Vaziri, H.; Chapman, K. B.; Guigova, A.; Teichroeb, J.; Lacher, M. D.; Sternberg, H.; Singec, I.; Briggs, L.; Wheeler, J.; Sampathkumar, J.; Gonzalez, R.; Larocca, D.; Murai, J.; Snyder, E.; Andrews, W. H.; Funk, W. D.; West, M. D. Spontaneous reversal of the developmental aging of normal human cells following transcriptional reprogramming. Regen. Med. 5:345–363; 2010. 376. Wang, A.; Tang, Z.; Park, I.-H.; Zhu, Y.; Patel, S.; Daley, G. Q.; Li, S. Induced pluripotent stem cells for neural tissue engineering. Biomaterials 32:5023–5032; 2011. 377. Wang, J. T.; Medress, Z. A.; Barres, B. A. Axon degeneration: Molecular mechanisms of a self-destruction pathway. J. Cell Biol. 196:7–18; 2012. 378. Wang, Q.; Xu, X.; Li, J.; Liu, J.; Gu, H.; Zhang, R.; Chen, J.; Kuang, Y.; Fei, J.; Jiang, C.; Wang, P.; Pei, D.; Ding, S.; Xie, X. Lithium, an anti-psychotic drug, greatly enhances the generation of induced pluripotent stem cells. Cell Res. 21:1424–1435; 2011. 379. Wang, W.; Lin, C.; Lu, D.; Ning, Z.; Cox, T.; Melvin, D.; Wang, X.; Bradley, A.; Liu, P. Chromosomal transposition of PiggyBac in mouse embryonic stem cells. Proc. Natl. Acad. Sci. USA 105:9290–9295; 2008. 380. Wang, Y.; Baskerville, S.; Shenoy, A.; Babiarz, J. E.; Baehner, L.; Blelloch, R. Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat. Genet. 40:1478–1483; 2008. 381. Warren, L.; Manos, P. D.; Ahfeldt, T.; Loh, Y.-H.; Li, H.; Lau, F.; Ebina, W.; Mandal, P. K.; Smith, Z. D.; Meissner, A.; Daley, G. Q.; Brack, A. S.; Collins, J. J.; Cowan, C.; Schlaeger, T. M.; Rossi, D. J. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7:618–630; 2010. 382. Waxman, S. Demyelination in spinal cord injury. J. Neurol. Sci. 91:1–14; 1989. 383. Wernig, M.; Benninger, F.; Schmandt, T.; Rade, M.; Tucker, K. L.; Bussow, H.; Beck, H.; Brustle, O. Functional integration of embryonic stem cell-derived neurons in vivo. J. Neurosci. 24:5258–5268; 2004. 384. Wernig, M.; Lengner, C. J.; Hanna, J.; Lodato, M. A.; Steine, E.; Foreman, R.; Staerk, J.; Markoulaki, S.; Jaenisch, R. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat. Biotechnol. 26:916–924; 2008. 385. Wernig, M.; Meissner, A.; Cassady, J. P.; Jaenisch, R. c-Myc is dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell 2:10–12; 2008. 386. Wernig, M.; Meissner, A.; Foreman, R.; Brambrink, T.; Ku, M.; Hochedlinger, K.; Bernstein, B. E.; Jaenisch, R. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448:318–324; 2007.
616 kramer ET AL.
387. Wernig, M.; Zhao, J.; Pruszak, J.; Hedlund, E.; Fu, D.; Soldner, F. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease. Proc. Natl. Acad. Sci. USA 105:5856–5861; 2008. 388. White, R.; and Jakeman, L. Don’t fence me in: Harnessing the beneficial roles of astrocytes for spinal cord repair. Restor. Neurol. Neurosci. 26:197–214; 2008. 389. Wilson, K. D.; Venkatasubrahmanyam, S.; Jia, F.; Sun, N.; Butte, A. J.; Wu, J. C. MicroRNA profiling of humaninduced pluripotent stem cells. Stem Cells Dev. 18:749–757; 2009. 390. Winkler, T.; Cantilena, A.; Metais, J.-Y.; Xu, X.; Nguyen, A.-D.; Borate, B.; Antosiewicz-Bourget, J. E.; Wolfsberg, T. G.; Thomson, J. A.; Dunbar, C. E. No evidence for clonal selection due to lentiviral integration sites in human induced pluripotent stem cells. Stem Cells 28:687–694; 2010. 391. Wobus, A. M. The Janus face of pluripotent stem cells— Connection between pluripotency and tumourigenicity. Bioessays 32:993–1002; 2010. 392. Woltjen, K.; Michael, I.; Mohseni, P.; Desai, R.; Mileikovsky, M.; Hamalainen, R. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458:766–771; 2009. 393. Wong, D. J.; Liu, H.; Ridky, T. W.; Cassarino, D.; Segal, E.; Chang, H. Y. Module map of stem cell genes guides creation of epithelial cancer stem cells. Cell Stem Cell 2:333–344; 2008. 394. Wong, R. C.-B.; Donovan, P. J.; Pebay, A. Molecular mechanism involved in the maintenance of pluripotent stem cells. In: Rosales, D. W.; Mullen. Q., eds. Pluripotent Stem Cells. New York, NY: Nova Science Publisher; 2010:47–79. 395. Wu, Z.; Chen, J.; Ren, J.; Bao, L.; Liao, J.; Cui, C.; Rao, L.; Li, H.; Gu, Y.; Dai, H.; Zhu, H.; Teng, X.; Cheng, L.; Xiao, L. Generation of pig induced pluripotent stem cells with a druginducible system. J. Mol. Cell Biol. 1:46–54; 2009. 396. Xu, D.; Alipio, Z.; Fink, L. M.; Adcock, D. M.; Yang, J.; Ward, D. C.; Ma, Y. Phenotypic correction of murine hemophilia A using an iPS cell-based therapy. Proc. Natl. Acad. Sci. USA 106:808–813; 2009. 397. Xu, J.; Wang, H.; Liang, T.; Cai, X.; Rao, X.; Huang, Z.; Sheng, G. In vitro induction of mouse meningeal-derived ips cells into neural-like cells. Chin. Sci. Bull. 56:1556– 1561; 2011. 398. Yakubov, E.; Rechavi, G.; Rozenblatt, S.; Givol, D. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors. Biochem. Biophys. Res. Commun. 394:189–193; 2010. 399. Yamanaka, S. Elite and stochastic models for induced pluripotent stem cell generation. Nature 460:49–52; 2009. 400. Yamanaka, S. Pluripotency and nuclear reprogramming. Philos. Trans. R. Soc. Lond. Biol. Sci. 363:2079–2087; 2008. 401. Yamanaka, S.; Blau, H. M. Nuclear reprogramming to a pluripotent state by three approaches. Nature 465:704–712; 2010. 402. Yamashita, T.; Kawai, H.; Tian, F.; Ohta, Y.; Abe, K. Tumorigenic development of induced pluripotent stem cells in ischemic mouse brain. Cell Transplant. 20:883– 891; 2011. 403. Yan, B.; Abdelli, L. S.; Singla, D. K. Transplanted induced pluripotent stem cells improve cardiac function and induce
neovascularization in the infarcted hearts of db/db mice. Mol. Pharm. 8:1602–1610; 2011. 404. Yang, J.; Cai, J.; Zhang, Y.; Wang, X.; Li, W.; Xu, J.; Li, F.; Guo, X.; Deng, K.; Zhong, M.; Chen, Y.; Lai, L.; Pei, D.; Esteban, M. A. Induced pluripotent stem cells can be used to model the genomic imprinting disorder PraderWilli syndrome. J. Biol. Chem. 285:40303–40311; 2010. 405. Yao, X.-L.; Liu, Q.; Ye, C.-H.; Li, Z.-P.; Lu, X.-L.; Li, P.-L.; Li, X.-B.; Li, W.-Q. Neuronal differentiation potential of mouse induced pluripotent stem cells. Neuroreport 22:689–695; 2011. 406. Ye, L.; Chang, J. C.; Lin, C.; Qi, Z.; Yu, J.; Kan, Y. W. Generation of induced pluripotent stem cells using sitespecific integration with phage integrase. Proc. Natl. Acad. Sci. USA 107:19467–19472; 2010. 407. Yehezkel, S.; Rebibo-Sabbah, A.; Segev, Y.; Tzukerman, M.; Shaked, R.; Huber, I.; Gepstein, L.; Skorecki, K.; Selig, S. Reprogramming of telomeric regions during the generation of human induced pluripotent stem cells and subsequent differentiation into fibroblast-like derivatives. Epigenetics 6:63–75; 2011. 408. Yoshida, Y.; Takahashi, K.; Okita, K.; Ichisaka, T.; Yamanaka, S. Hypoxia enhances the generation of induced pluripotent stem cells. Cell Stem Cell 5:237–241; 2009. 409. Yu, J.; Hu, K.; Smuga-Otto, K.; Tian, S.; Stewart, R.; Slukvin, I. Human induced pluripotent stem cells free of vector and transfene sequences. Science 324:797–801; 2009. 410. Yu, J.; Vodyanik, M.; Smuga-Otto, K.; AntosiewiczBourget, J.; Frane, J.; Tian, S.; Nie, J.; Jonsdottir. G. A.; Ruotti, V.; Stewart, R.; Slukvin, I. I.; Thomson, J. A. Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920; 2007. 411. Yusa, K.; Rad, R.; Takeda, J.; Bradley, A. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat. Methods 6:363–369; 2009. 412. Yusa, K.; Zhou, L.; Li, M. A.; Bradley, A.; Craig, N. L. A hyperactive piggyBac transposase for mammalian applications. Proc. Natl. Acad. Sci. USA 108:1531–1536; 2011. 413. Zeng, H.; Guo, M.; Martins-Taylor, K.; Wang, X.; Zhang, Z.; Park, J. W.; Zhan, S.; Kronenberg, M. S.; Lichtler, A.; Liu, H.-X.; Chen, F.-P.; Yue, L.; Li, X.-J.; Xu, R.-H. Specification of region-specific neurons including forebrain glutamatergic neurons from human induced pluri potent stem cells. PLoS One 5:e11853; 2010. 414. Zeuschner, D.; Mildner, K.; Zaehres, H.; Schoeler, H. R. Induced pluripotent stem cells at nanoscale. Stem Cells Dev. 19:615–620; 2010. 415. Zhang, G.; Shang, B.; Yang, P.; Cao, Z.; Pan, Y.; Zhou, Q. Induced pluripotent stem cell consensus genes: Implication for the risk of tumorigenesis and cancers in induced pluripotent stem cell therapy. Stem Cells Dev. 21:955–964; 2012. 416. Zhang, N.; An, M. C.; Montoro, D.; Ellerby, L. M. Charac terization of human huntington’s disease cell model from induced pluripotent stem cells. PLoS Curr. 2:RRN1193; 2010. 417. Zhang, Y. Z.; Wang, D.; Chen, M. L.; Yang, B.; Zhang, F. X.; Cao, K. J. Intramyocardial transplantation of undifferentiated rat induced pluripotent stem cells causes tumorigenesis in the heart. PLoS One 6:e19012; 2011. 418. Zhang, Z.; Guth, L. Experimental spinal cord injury: Walle rian degeneration in the dorsal column is followed by revascularization, glial proliferation, and nerve regeneration. Exp. Neurol. 147:159–171; 1997.
iPSC TECHNOLOGY FOR SCI
419. Zhao, T.; Zhang, Z.-N.; Rong, Z.; Xu, Y. Immunogenicity of induced pluripotent stem cells. Nature 474:212–215; 2011. 420. Zhao, X.-Y.; Li, W.; Lv, Z.; Liu, L.; Tong, M.; Hai, T.; Hao, J.; Guo, C.-L.; Ma, Q.-W.; Wang, L.; Zeng, F.; Zhou, Q. iPS cells produce viable mice through tetraploid complementation. Nature 461:86–90; 2009. 421. Zhao, Y.; Yin, X.; Qin, H.; Zhu, F.; Liu, H.; Yang, W.; Zhang, Q.; Xiang, C.; Hou, P.; Song, Z.; Liu, Y.; Yong, J.; Zhang, P.; Cai, J.; Liu, M.; Li, H.; Li, Y.; Qu, X.; Cui, K.; Zhang, W.; Xiang, T.; Wu, Y.; Zhao, Y.; Liu, C.; Yu, C.; Yuan, K.; Lou, J.; Ding, M.; Deng, H. Two supporting factors greatly improve the efficiency of human iPSC generation. Cell Stem Cell 3:475–479; 2008. 422. Zhong, B.; Trobridge, G. D.; Zhang, X.; Watts, K. L.; Ramakrishnan, A.; Wohlfahrt, M.; Adair, J. E.; Kiem, H.-P. Efficient generation of nonhuman primate induced pluripotent stem cells. Stem Cells Dev. 20:795–807; 2011. 423. Zhong, B.; Watts, K. L.; Gori, J. L.; Wohlfahrt, M. E.; Enssle, J.; Adair, J. E.; Kiem, H.-P. Safeguarding nonhuman primate iPS cells with suicide genes. Mol. Ther. 19:1667–1675; 2011.
617
424. Zhou, H.; Wu, S.; Joo, J.; Zhu, S.; Han, D.; Lin, T. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4:381–384; 2009. 425. Zhou, J.; Su, P.; Li, D.; Tsang, S.; Duan, E.; Wang, F. Highefficiency induction of neural conversion in human ESCs and human induced pluripotent stem cells with a single chemical inhibitor of transforming growth factor-b superfamily receptors. Stem Cells 28:1741–1750; 2010. 426. Zhou, L.; Wang, W.; Liu, Y.; de Castro, J. F.; Ezashi, T.; Telugu, B. P. V. L.; Roberts, R. M.; Kaplan, H. J.; Dean, D. C. Differentiation of induced pluripotent stem cells of swine into rod photoreceptors and their integration into the retina. Stem Cells 29:972–980; 2011. 427. Zhou, W.; Freed, C. R. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells 27:2667–2674; 2009. 428. Zhu, S.; Li, W.; Zhou, H.; Wei, W.; Ambasudhan, R.; Lin, T.; Kim, J.; Zhang, K.; Ding, S. Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell Stem Cell 7:651–655; 2010.