Stem Cell Rev and Rep DOI 10.1007/s12015-013-9443-6
Progress in Stem Cell Therapy for Major Human Neurological Disorders P. L. Martínez-Morales & A. Revilla & I. Ocaña & C. González & P. Sainz & D. McGuire & I. Liste
# Springer Science+Business Media New York 2013
Abstract Human neurological disorders such as Alzheimer’s disease (AD), Parkinson’s disease, stroke or spinal cord injury are caused by the loss of neurons and glial cells in the brain or spinal cord in the Central Nervous System (CNS). Stem cell technology has become an attractive option to investigate and treat these diseases. Several types of neurons and glial cells have successfully been generated from stem cells, which in some cases, have ameliorated some dysfunctions both in animal models of neurological disorders and in patients at clinical level. Stem cell-based therapies can be beneficial by acting through several mechanisms such as cell replacement, modulation of inflammation and trophic actions. Here we review recent and current remarkable clinical studies involving stem cell-based therapy for AD and stroke and provide an overview of the different types of stem cells available nowadays, their main properties and how they are developing as a possible therapy for neurological disorders. Keywords Stem cells . Neurodegeneration . Cell therapy . Neurogenesis . Alzheimer’s disease . Stroke
Introduction Neurological disorders represent a wide range of acute and chronic conditions in which neurons and glial cells in the P. L. Martínez-Morales and A. Revilla contributed equally to this work P. L. Martínez-Morales : A. Revilla : I. Ocaña : C. González : P. Sainz : D. McGuire : I. Liste (*) Unidad de Regeneración Neural, Unidad Funcional de Investigación de Enfermedades Crónicas, Instituto de Salud Carlos III (ISCIII), 28220 Majadahonda, Madrid, Spain e-mail:
[email protected] P. L. Martínez-Morales e-mail:
[email protected]
brain or spinal cord are lost [1]. Acute neurodegeneration may result from a temporally discrete insult, such as stroke or trauma, where different types of neurons and glial cells die within a restricted brain area in a short period of time due to inadequate blood flow [1, 2]. In contrast, chronic neurodegeneration usually develops over a long period of time and results in the loss of a particular neuronal subtype such as dopaminergic neurons in Parkinson’s disease (PD) or the widespread degeneration of many neuronal types, as is the case of Alzheimer’s disease (AD). In general these disorders are age-related, but arise for unknown reasons, and progress in a relentless manner. The burden of the agerelated neurodegenerative diseases such as AD is expected to increase dramatically as the life expectancy and aging population rise worldwide [2]. Furthermore, the adult brain regeneration potential is very limited. Only a small number of stem cells in very restricted areas can be stimulated to proliferate and differentiate under certain insults, but do not contribute significantly to functional recovery. Among the chronic neurodegenerative disorders, the most common is AD [3] that is characterized by the loss of neurons from the cortex and hippocampus accompanied by massive accumulation of beta-amyloid plaques and tautangles [4]. Unfortunately, in the majority of neurological disorders, conventional therapies with drugs offer only a limited benefit, alleviating certain symptoms. Chronic use of drugs can lead to deleterious side-effects, and none of the treatments appear to alter the natural course of the disease. Recently, cellular therapies have earned increased attention as potential treatment options. The application of stem cell research to neurodegenerative disorders is rapidly increasing; not only for transplantation, but also as prospective models of the disorder “in vitro”, contributing to improve the knowledge of the cellular and molecular mechanisms of neurodegeneration as well as novel drug discovery [5, 6]. Stem cell-based therapies could be beneficial by acting
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through several mechanisms: 1) cell-replacement therapy, through transplantation, to directly replace the lost neural types, 2) trophic support, by promoting survival of affected or remaining neurons, 3) modulation of inflammation, which may be involved in the disease process [1, 2]. In this review, we summarize some general issues related to the experimental and clinical application of stem cells, providing an overview of the different types of stem cells currently available, their properties and how stem cells are becoming a new approach for treating neurological disorders. Finally, we discuss current data and progress in stem cell-based therapies for AD and stroke, the most common neurological disorders.
Stem Cell Classification and Properties Stem cells are unspecialized cells with capacity to proliferate and differentiate into more specialized cellular lineages. They can be classified according to their differentiation potential and also how they have been derived. In order to find potential applications of stem cell technology it is essential to know the characteristics and peculiarities of the different existing cell types. Attending to their plasticity to differentiate into different cellular types, stem cells may be classified basically in two major categories: a) pluripotent stem cells which have the capacity to give rise to specialized cells from the three germ layers, and b) multipotent stem cells that give rise to more restricted lineages than pluripotent cells, depending on the germ layer of origin [7].
crucial to eliminate any danger of contamination, especially the risk of xenogenic contamination (from cell culture reagents or feeder layers). This implies that hESCs-derived neurons or their precursors must be generated and cultured under GLP/GMP (Good Laboratory and Manufacture Procedures) conditions in xenogen-free environment from the time of blastocyst isolation. In addition, due to the ethical controversial, hESC research is subjected to extensive policy intervention. Currently, the U.S. NIH maintains detailed policies and procedures for funding hESC research (http://stemcells.nih.gov/policy/ guidelines.asp). Various U.S. States have developed different laws governing the permissively and funding of hESC research. In general, guidelines (ranging from total prohibition to controlled permissiveness) defining what may be permitted in research with pluripotent stem cells have been issued in countries all over the world [18, 19]. In fact, due in all probability to these challenges, almost no clinical trials have been performed with these cells yet. The first FDA-approved clinical trial with hESCs-derived cells, oligodendrocyte progenitors for spinal cord injury, started in 2010 by Geron company (www.Geron.com) [20]. The company stopped the trial, leaving the stem cell business entirely. Recently, Geron has been purchased by Bio Time Acquisition Corporation (BAC). With this agreement, Geron will contribute to BAC the intellectual property of certain cell lines and other assets, including the Phase 1 clinical trial with hES cell-derived oligodendrocytes in patients with acute spinal cord injury. Hopefully, the clinical trials will be continued and the treated patients will be followed up, which can provide valuable information related to biosecurity and functional outcome.
Human Pluripotent Stem Cells
Human Induced Pluripotent Stem Cells
Human Embryonic Stem Cells
Human induced pluripotent stem cells (hiPSCs) have been successfully established from adult human fibroblasts after over-expression of four transcription factors: Oct3/4, Sox2 and Klf4 and c-Myc (or Nanog, Lin28) [21, 22]. These cells are similar to hESCs in morphology, gene expression profile and differentiation potential [23]. The iPS cell technology offers new possibilities for biomedical research and clinical applications, as these cells could be used as “in vitro” cellular models of neurodegenerative diseases and for autologous transplantation for which theoretically, no immunosuppressive therapy would be necessary. In addition, hiPSCs do not raise ethical concerns since they are derived from somatic cells, following routine tissue donation procedures. However, caution should be exercised, since iPSCs are expected to generate not only the same challenges as hESCs, but additional problems related to the fact that they are patient-derived (bearing the same genetic defects as the patient itself) and the way they are generated, in most cases,
Human embryonic stem cells (hESCs) were first established by James Thomson in 1998 from the inner cell mass of the blastocyst [8]. Because of their properties, these cells may constitute an optimal cell source for cell-replacement therapies. Clinical application of hESCs in the treatment of neurological disorders requires an efficient and strict differentiation of hESCs into the desired neural phenotypes. So far, several neuronal phenotypes have successfully been generated from hESCs in vitro, including dopaminergic neurons [9–12] spinal motor neurons [13, 14] and glial cells [13, 15], encouraging the research aimed at using hESCs. However, there are still several important problems associated with their use including control of cell growth and differentiation to avoid tumor formation, phenotype instability and poor survival of transplanted cells [16, 17]. Likewise, to optimize their use for clinical applications, it is
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genetically modified for the over-expression of cell growthrelated transcription factors. Moreover, recent studies have shown significant reprogramming variability, including structural chromosomal abnormalities, somatic memory and aberrant reprogramming of DNA methylation patterns in hiPSCs [24]. Another important issue is the risk of tumor formation by iPSCs, which due to the reprogramming process, can be even higher than that of ESC [24, 25]. In addition to the forced expression of cell growth related factors, most of the human iPSCs lines have been generated using viral methods, which can generate multiple chromosomal integrations and insertional mutagenesis.
Multipotent Stem Cells Human Neural Stem Cells Human Neural Stem Cells (hNSCs) are the precursors of neurons and glia and generate all the differentiated neural cells of the CNS. These cells can be sourced from the fetal, neonatal, adult brain, or from directed differentiation of pluripotent stem cells. They can be propagated in vitro as free-floating aggregates, called neurospheres, containing a mixture of NSCs and progenitors, in the presence of growth factors such as bFGF and EGF [26, 27]. An alternative approach to expand hNSCs is to combine epigenetic and genetic immortalization strategies, where cells are transduced with an immortalizing gene (e.g., v-Myc, c-Myc or TERT), and their proliferation is supported with growth factors [28–30]. In general, hNSCs (non- modified) present lower risk of tumor formation than pluripotent cells. Human fetal NSCs are being applied in clinical trials carried out by the company ReNeuron in UK for the treatment of patients with ischemic stroke [31–33] (Table 2).
Other Multipotent Stem Cells with Neurogenic Potential: Neural Crest Stem Cells (from Human Hair Follicles or from Human Teeth) and Olfactory Ensheathing Glia Neural Crest Stem Cells Neural crest stem cells (NCSCs) are born during vertebrate embryogenesis within the dorsal margins or the closing neural folds. Initially they are integrated within the neuroepithelium where they are morphologically indistinguishable from the other neural epithelial cells. Following induction, NCSCs are delaminated through an epithelial-tomesenchymal transition and they start migrating extensively to several different locations in the embryo where they contribute, according to microenvironment cues, into a
variety of cell types including cartilage, bone, connective tissue, endocrine cells as well as neurons and glia amongst others [34–37]. Since these cells are generated transiently within the embryo, it is considered that it may be more appropriate to describe the majority of NCSCs as progenitor cells, with a more limited potential to self-renew and differentiate than true stem cells [34, 37, 38]. Despite that, NCSCs continue to attract the interest of the scientists because of their importance in vertebrate development and their potential application in regenerative medicine. The advantages of the use of NCSCs as compared to hESCs are both related to the no need to supply immunosuppressive medication since it can be used the patient’s own cells and that their usage is not ethically controverted. In addition, compared to hiPSCs, there is no need for genetic manipulation. NCSCs can be found in different adult tissues including dermis, hair follicles, heart, cornea or gut. However, the most accessible tissues for possible therapeutic applications are, probably, the skin and dental tissues that we discuss below. Human Epidermal Neural Crest Stem Cells (hEPI-NCSCs) NCSCs have been successfully isolated from murine and human epidermis and/or dermis [35, 39–41]. These cells were designed like epidermal NC stem cells (EPI-NCSCs) by Sieber-Blum M et al., 2004 [40]. This discovery has led, probably, one of the most intriguing and therapeutically significant findings of the last few years in the field of adult NCSCs. The skin is the most accessible tissue of the body, for that reason is a good candidate to be used in autologous cell replacement therapies and regenerative medicine. EPINCSCs are multipotent stem cells, which are derived from the embryonic neural crest and are located in the bulge of hair follicles. Therefore they are readily accessible in the hairy skin by minimal invasive procedure. They are able to generate all major neural crest derivatives, including bone/cartilage cells, myofibroblasts, melanocytes and neurons [34–36, 40, 42]. It has been observed that EPI-NCSCs transplanted in spinal cord injury animal models, integrate and are able to differentiate into Gabaergic neurons and myelinating oligodendrocytes, with no tumour or teratoma formation [43]. Moreover, EPi-NCSCs grafts cause a significant improvement in sensory connectivity and touch perception in the contused mouse spinal cord. The recovery mechanism seems to be related to the expression of neurotrophic and angiogenic factors by EPI-NSCs [42, 44]. Also worth mentioning that hEPI-NCSCs can be isolated reproducibly, with high yield; they can be purified and expanded “ex vivo” into millions of stem cells that remain multipotent. All these advantages have made of these cells an attractive candidate for future cell-based therapies [39, 45].
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Human Dental Pulp Stem Cells Human adult dental pulp stem cells (hDPSCs) reside within the perivascular niche of dental pulp and are thought to originate from migrating cranial neural crest cells [46–48]. They are isolated based on their expression of mesenchymal stem cell associated markers [49]. Several studies have demonstrated the self-renewal potential of DPSCs as well as their multipotency with interesting differentiation and regenerative capabilities [46–50]. Under specific conditions, DPSCs exhibit osteogenic, dentinogenic, adipogenic, chondrogenic and neurogenic derivative potentials [47, 49]. hDPSCs have the capacity to differentiate into mature functional neuronal cells, under inductive conditions, both in vitro [47] and in vivo following transplantation into the avian embryo [48]. In addition, the administration of hDPSCs yielded positive therapeutic outcomes in various animal models of disease. For example, following transplantation into hemisected spinal cord in a rat model of spinal cord injury, hDPSCs were able to rescue motoneurons [51]. Most recently, intracerebral transplantation of hDPSCs 24 h following focal cerebral ischemia in a rodent model resulted in significant improvement in forelimb sensorimotor function at 4 weeks post-treatment [46]. The mechanisms of action of these treatments have been attributed to the secretion of several factors resulting in paracrine effects. Some of these secreted factors described are stromal cell-derived factor-1 (SDF-1) [48], neurotrophic factors like NGF, BDNF and GDNF [51] and VEGF [52]. These factors have been proposed to play important roles in enhancing endogenous restorative processes following injury or disease. Olfactory Ensheathing Cells Olfactory ensheathing cells (OECs) are glial cells that envelop bundles of olfactory axons, both peripherally in the olfactory nerve and within the olfactory nerve layer of the olfactory bulb as well as in the nasal olfactory mucosa. The OECs guide growing axons from the neurons of the nasal cavity of the olfactory mucosa to the olfactory bulb to form synapses in the brain [53, 54]. Because these cells are found in a fairly accessible region of the brain and can be removed from a person without causing any significant harm, they have been considered as a prospective and attractive nonembryonic source of cells with potential for transplantationbased therapy in neurological diseases [55, 56]. A number of studies have shown that when transplanted into the demyelinated spinal cord, OECs can repair the defective myelin, restore conductance in remyelinated axons and improve of motor behavior [57–62]. The precise mechanisms underlying functional improvement after OECs transplantation are not fully understood; however OECs
has been shown to secrete some trophic factors including NGF, BDNF, GDNF, VEGF and SDF-1, depending on culture conditions or in vivo microenvironment [63, 64]. Hence it suggests that the benefit noticed may be related to their ability to regenerate damaged axons and probably also to the angiogenesis promoted by the secretion of growth factors. Human trials have been carried out to analyze the therapeutic effect of OECs transplantation on spinal cord injury and other CNS diseases including amyotrophic lateral sclerosis, multiple sclerosis and ataxia [65, 66]. The results showed that the transplant was safe and there were no adverse effects, however sensory improvement was modest. Transplantation of OECs into animal models of stroke promotes neuroplasticity, facilitates neurite outgrowth and activates the stem cell homing process, thus promoting reversal of the neurological deficit [67]. These results suggest a possible clinical benefit of hOECs after autologous transplantation in stroke patients. Currently, autologous hOECs are being grafted into the peri-infarcted area of the brain in a Phase 1 clinical trial in stroke patients (Table 2; NCT01327768).
Human Mesenchymal Stem Cells Human Mesenchymal Stem Cells (hMSCs) are an alternative source of multipotent self-renewing cells that can be derived from adult bone marrow, peripheral blood, adipose tissue and umbilical cord blood. hMSCs are nonhematopoietic stromal cells, characterized by their adherence to plastic in cell culture, specific surface antigen expression and multi-lineage in vitro differentiation potential into multiple tissues of a mesenchymal origin (osteogenic, chondrogenic, adipogenic) [2, 68, 69]. Although some reports show that they can also generate cells of the neural lineage including cholinergic neurons [69] or dopamine neurons in vitro [2, 70–72]. Previous studies have shown that hMSCs have both paracrine and autocrine activities in damaged tissues, including the brain [73, 74]. In fact, MSCs secrete a variety of cytokines and growth factors with anti-inflammatory, antiapoptotic and immunomodulatory properties, such as vascular endothelial growth factor (VEGF) [75], hepatocyte growth factor (HGF) [75–77] and insulin-like growth factor (IGF-1), which are involved in angiogenesis, healing and tissue repair processes [78]. MSCs utilized in experimental models of stroke have improved the functional recovery of neurological deficits caused by cerebral ischemia [79, 80]. Clinical reports of hMSCs in stroke patients reveal that hMSCs may improve the functional recovery of patients without adverse side effects [81, 82]. However, the underlying mechanism remains unclear. It is known that transplantation of MSCs
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via intravenous, intracarotid or intracerebral delivery, leads to low or modest graft survival rate [83]. Thus, the benefit observed is probably not attributable to the very few MSCs that differentiate into brain cells. A more reasonable explanation for the graft-derived beneficial effects is that MSCs secrete neurotrophic factors (VEGF, BDNF, and IGF-1) that may induce the host brain to activate endogenous repair mechanisms, including angiogenesis and neurogenesis [84, 85]. Another interesting property of MSCs is that when transplanted systemically in animals, they are able to migrate to sites of injury, suggesting that these cells possess migratory capacity [86–89]. This migration may be induced by chemokines, cytokines and growth factors released upon injury that would provide migratory cues that induce upregulation of selectins and activation of integrins of the MSC surface, enabling cells to interact with the endothelium. Stem cells subsequently adhere and transmigrate across the endothelial layer into tissues, similarly to leukocytes of the immunological system [86, 87]. One of the main advantages of the use of hMSCs is the potential circumvention of the need for immunosuppressant in cellular therapies, because they can be derived from an autologous source. They are not ethically controversial as the case in hESCs, are relatively easy to expand, and possess the ability to migrate to damaged areas in the brain, secrete neurorestorative factors and regulate inflammation [90].
Human Hematopoietic Stem Cells Another potential source of multipotent stem cells for cell transplantation are hematopoietic stem cells (HSCs), which are functionally defined by their unique capacity to selfrenew and to differentiate to produce all mature blood types [91]. HSCs have the potential to repopulate removed bone marrow, a characteristic feature demonstrating their proliferative capacity [92]. Furthermore, they are able to differentiate into erytroid, lymphoid and myeloid lineages [91, 93]. Isolation or selection of HSCs is done on the basis of cellsurface marker phenotypes representing different stages of differentiation. The first cell-surface marker used to enrich human HSCs was CD34, a ligand for L-selectin that is expressed by a small percentage of blood or bone marrow cells [93]. HSCs can be obtained from bone marrow, peripheral blood, or umbilical cord blood. The biggest difference in obtaining HSCs from different sources is the quantity of cells generated. While human umbilical cord blood (HUCB) may be a viable source of HSCs, very few HSCs can be obtained, thereby limiting use to children and not adults. Bone marrow offers the best viable option for autologous HSCs administration and higher yields; however this procedure is painful and may require hospital stay. Peripheral
blood also contains circulating HSCs, which can be induced to mobilize from the bone marrow into the blood in response to different insults. The CNS can contribute to this HSCs mobilization via cytokine production, and under stress condition (i.e. stroke), can amplify the recruitment of HSCs into the brain [92, 94]. This process is being employed in clinical protocols, such as treatment with granulocyte-colony stimulating factor (GCSF) for the creation of ample supply of HSCs for brain repair [94]. GCSF is a glycoprotein that regulates the generation, proliferation, survival and maturation of neutrophilic granulocytes [95]. Over the last few years evidence has emerged that GCSF can have a therapeutic potential in neurological disorders such as stroke. It has been shown to exhibit neuroprotective and regenerative activity in experimental stroke models [96, 97] and promising results have been obtained in patients with stroke [98, 99]. Recent data support the notion that human HSCs play key regulatory roles in the maintenance of homeostasis and the repair of nervous system. For instance, systemic administration of human CD34+ cells to mice previously exposed to stroke 48 h earlier induces neovascularization in the ischemic zone [100] thereby creating a permissive microenvironment for survival of both exogenous grafts and endogenous stem cells, essential for neural regeneration [92]. In addition, positive effects of transplantation of HSCs have also been observed in animal models of spinal cord injury [101] or Alzheimer’s disease [102]. HSCs have the advantage that are suitable for both autologous as well as allogenic use, and furthermore are not associated with the ethical issues surrounding hESCs or fetally derived stem cells. Potential disadvantages are related with consistency of number and potency of cells obtained from bone marrow, and the need for “ex vivo” expansion of cells, when using umbilical cord blood as the source.
Stem Cell Therapy in Alzheimer’s Disease Alzheimer’s disease (AD) is the most frequent form of dementia, characterized by memory loss and cognitive decline. As the disease progresses, there is a widespread loss of neurons and synaptic contacts affecting the cortex, hippocampus, amygdala and basal forebrain [3, 103]. The causes of AD are still unknown, but age is the major risk factor for this disorder; its incidence doubles every 5 years after the age of 65 [104]. The main pathological hallmarks of AD include the presence of neurofibrillary tangles (intraneuronal aggregations of hyperphosphorylated tau, a microtubule-associated protein involved in microtubule stabilization), amyloid-β plaques (extracellular deposits consisting primarily of amyloid-β peptides (Aβ peptide)), and cerebrovascular
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amyloidosis (amyloid-β protein surrounding the blood vessels) [105, 106]. In addition, severe inflammation and microglial activation occurs around these amyloid deposits, producing a variety of toxic compounds, including chemokines, neurotoxic cytokines and reactive oxygen and nitrogen species that are deleterious to the CNS. Therefore, a possible therapeutic strategy for treating AD could be to decrease or inhibit this inflammatory reaction, either through drugs or stem cell therapy. In this context, several groups have reported that MSCs have regenerative and paracrine effects; the latter related to the release of soluble factors that modulate apoptosis, and inflammation in damaged tissue. In animal models of AD transplantation of MSCs derived from human umbilical cord blood, improved spatial memory, decreased amyloid plaques in the brain and inhibited the release of proinflammatory cytokines from the microglia [107, 108]. Currently, two different clinical trials are evaluating the safety and efficacy of intracerebral (NEUROSTEM-AD) or intravenous infusion of MSCs derived from human umbilical cord blood in patients with Alzheimer’s type dementia. These trials are still ongoing (Table 1). Among the neuronal populations that degenerate in AD, loss of basal forebrain cholinergic neurons and their cortical projections is particularly severe. This correlates with reduced activity of choline acetyltransferase (ChAT) and acetylcholine release in the brain [109–112]. According to the “cholinergic hypothesis” of AD the loss of cholinergic function contributes to the deterioration in cognitive function observed in patients with AD [113]. In fact, cholinesterase inhibitors are commonly used to counteract this cholinergic deficit. They inhibit the activity of acetylcholinesterase which degrades acetylcholine in the synapse, and they are effective in reducing symptoms, at least temporarily, in approximately half of the prescribed patients [113–117].
Therefore, theoretically, transplantation of stem cellderived cholinergic neurons could be a good option to improve symptoms in AD. Transplantation of hNSCs, murine septal precursors, porcine cholinergic precursors or human fetal basal forebrain neurons into cortical areas of rodents resulted in stable engraftment of the introduced cells, some of which differentiated into cholinergic neurons [118–120]. Whilst this strategy was limited by the inability to generate highly purified basal forebrain cholinergic neurons, Bissonnette and colleagues have recently described a method for the successful generation of this type of neurons from hESCs [121] using diffusible ligands or overexpression of Lhx8 and Gbx1. This approach can provide new candidate cells for transplantation in AD. Another theory proposed to explain the development of AD is the “Neurotrophic factor hypothesis of AD”. This theory suggests that a trophic factor required for cholinergic survival would be insufficiently produced by target regions such as hippocampus, resulting in a higher vulnerability to Aβ peptide accumulation effects or directly results in neuronal degeneration [105, 122]. The main neurotrophic factors candidates proposed are Nerve Growth Factor (NGF) and BDNF (Brain Derived Neurotrophic Factor (BDNF)). NGF was the first nervous system growth factor identified, and potentially stimulates the survival and function of basal forebrain cholinergic neurons. These neurons express the tyrosine kinase receptor A (TrkA) which responds to NGF by promoting and maintaining synaptic contact with the neurons of the hippocampus and cortex. This suggests that NGF could be a means for reducing the cholinergic component of cell degeneration in AD [105, 123]. Indeed, when applied locally, NGF has shown protective and regenerative effects on the basal cholinergic forebrain system. Associated cognitive improvement in several animal models and clinical pilot studies has also been demonstrated. Autologous
Table 1 Stem cells in current clinical trials for Alzheimer’s disease Stem cell type
Transplant type
Delivery administration
Status
Name/ sponsor
MSC from umbilical cord blood
Allogenic
i.c.
Phase 1 NCT01297218
Allogenic
i.c.
Recruiting NCT01696591
Allogenic
i.v.
Phase 1/2 NCT01547689
Allogenic
i.c. (implantation into the basal forebrain nuclei)
Phase 1 NCT01163825
NEUROSTEM-AD/Medipost Co Ldt.* http://medi-post.com/ NEUROSTEM-AD/ Duk Lyul Na* Affiliated Hospital to academy of military medical Science* Ns Gene A/S* http://nsgene.dk/
Encapsulated cell biodelivery device: NsG0202 Fibroblasts genetically modified expressing NGF
Autologous i.c. (implantation into the cholinergic basal forebrain)
Phase 1 Tuszynski, The Shiley Family Trust Sponsor* et al., 2005 [124] NCT00017940
Types of stem cells: Mesenchymal Stem Cells (MSC). Administration routes: intravenous (i.v.), intracerebral implantation (i.c.). * From www.clinicaltrials.com
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fibroblasts genetically engineered to produce NGF were implanted into the basal forebrain in AD patients in a phase 1 clinical trial [124]. Although two of the patients were severely affected by the surgical procedure, the remaining six showed no undesirable effects for up to 22 months posttreatment. PET analysis showed a striking increase of fluorodeoxyglucose uptake in areas receiving cholinergic input from the basal forebrain. The autopsy of four patients from the trial, showed an increase in cholinergic cell growth in response to NGF. In the same way the company NsGene is currently developing a new therapy using encapsulated human epithelial cells to deliver NGF to the cholinergic basal forebrain in order to decrease the degeneration of cholinergic neurons and the cognitive decline in patients with AD. The results have been recently published [125, 126] and are quite encouraging. Neither inflammation nor adverse effects have been observed and some of the patients are showing cognitive benefits. The other relevant trophic factor, BDNF, is a secreted protein vital in the organization of neuronal networks and synaptic plasticity, especially in the hippocampus. This factor is widespread expressed in the entorhinal cortex, being anterogradely trafficked into de hippocampus, where it is involved in plasticity and memory. BDNF levels have been shown to decrease with age and in AD [127, 128]. In animal models of AD an increase of BDNF production by direct infusion [64] or through grafting of neural precursor cells demonstrated to be not only neuroprotective but to improve cognitive function as well [129, 130]; setting the key for future clinical trials. A different approach to stem cell therapy in AD could be the increase of endogenous neurogenesis. In the hippocampus, endogenous neurogenesis decreases as we age, and these effects are even more notorious in AD, contributing to exacerbate neuronal vulnerability to AD and to aggravate memory impairment and other symptoms observed in AD patients [127, 131]. A number of studies suggest that stimulation of endogenous neurogenesis by cellular therapies, physical activity, trophic factors (such as BDNF), drugs or other strategies may help to promote the recovery process or at least delay the progression of AD. Nevertheless neurons thus generated in the hippocampus are too few when compared to the number of degenerated neurons and therefore unlike to achieve a global and complete repair by their own.
Stem Cell Therapy in Stroke A stroke is a sudden death of brain cells in a focalized area due to interrupted blood flow. This disorder is the third leading cause of death and disability in developed countries [132]. There are two types of stroke: a) ischemic stroke, caused by an occlusion of a blood vessel, and b) haemorrhagic stroke, initiated by a rupture of a blood vessel
in the brain. Most of the cases of stroke are ischemic (85– 90 %) as compared to haemorrhagic (10–15 %). A thrombosis, an embolism or systemic hypo-perfusion, all of which result in a restriction of blood flow to the brain, can cause an ischemic stroke, which results in insufficient oxygen and glucose delivery to support cellular homeostasis. This elicits multiple processes that lead to cell death: excitotoxicity, acidotoxicity and ionic imbalance, oxidative/nitrative stress, inflammation, apoptosis and peri-infarct depolarization [1, 133, 134]. At the core of the infarcted area the affected cells die rapidly by necrosis. The surrounding area of tissue, the “penumbra”, consists of tissue in which neurons and glia variably survive or die by a mixture of ischaemic degeneration and apoptosis over an extended time course of between several hours and days or even weeks. Currently, the only accepted treatment for stroke is the administration of antithrombotic agents, but their use is often limited by a narrow therapeutic time window [133, 135]. Therefore the necessity of clinically relevant alternative therapies to promote the recovery of stroke symptoms is clear. One promising alternative approach is stem cell-based therapy. This can be categorized into two strategies. The first one based on mobilization of stem cells already present within the individual (in the brain, blood or bone marrow). The second strategy involves the transplantation of exogenous stem cells delivered locally (e.g. direct intracerebral implantation) or systemically (e.g. intravenous or intra-arterial) and may involve the use of cells from the own patient (autologous) or from a different individual (allogenic) (Table 2). With regard to the first approach, several studies have shown that in cerebral infarcts there is increased cellular proliferation in the subventricular zone (SVZ) of the lateral ventricles [136]. This proliferation can further be pharmacologically stimulated by several compounds including antidepressants or hormones such as estrogen [137], helping to recovery. Interestingly, transplants of human NSCs into the brain of a rat model of stroke enhanced both cellular proliferation and neuroblast formation in the SVZ [138]. Similar results have been observed in humans after stroke [139]. However, the survival of these new cells seems to be poor [98, 140] and many of these neurons or neural precursors either die or remain undifferentiated [141]. It is clear that to become therapeutically valuable, neurogenesis has first to be optimized. Another endogenous stem cell approach uses granulocytecolony stimulating factor (G-CSF), a growth factor that acts on hematopoietic stem cells (CD34+) to regulate neutrophil progenitor proliferation and differentiation which is commonly used to mobilise stem cells of transplants in haematological malignancies. In experimental models of stroke, G-CSF exhibited neuroprotective and regenerative activity, resulting in a reduction of cerebral edemas, improved cell survival and increased sensorimotor functional recovery [142, 143].
Stem Cell Rev and Rep Table 2 Stem cells in current clinical trials for ischemic stroke Stem cell type
Stage of Stroke
Transplant type
Delivery Status administration
Name/sponsor
HSC (CD34+ cells) CS from peripheral CS blood CS HSC (CD34+ cells) CS from umbilical cord blood HSC (CD34+ cells) AS from bone marrow HSC (mononuclear AS cells) from bone AS marrow AS/ SAS
Autologous Autologous Autologous Allogenic
i.c. i.c. i.a. i.c.
Phase 2 NCT00950521 NCT01239602 Phase 1 NCT01518231 Phase 1 NCT01438593
China Medical University Hospital* China Medical University Hospital* AHSCTIS/ Zhejiang Hospital* China Medical University Hospital*
Autologous
i.a.
Phase 1/2 NCT00761982
Hospital Universitario Central de Asturias*
Autologous Autologous Autologous
i.v. i.v. i.v.
Phase 2 NCT01501773 Phase 1 NCT00859014 Phase 1 NCT00473057
Manipal Acunova Ltd.* University of Texas Health Science Center* Federal University of Rio de Janeiro*
MSC from bone marrow
AS
Autologous
i.v.
STARTING/ Korea Health 21 R&D Project
AS AS
Autologous Autologous
i.v. i.v.
Phase 1/2 Bang et al., 2005 [81] Phase 3 NCT01716481 Lee et al., 2010 [82]
AS SAS AS CS
Autologous Autologous Autologous Allogenic
i.v. i.v. i.v. i.v.
Phase 2 NCT01461720 Phase 2 NCT00875654 Phase 1/2 NCT01468064 Phase 1/2 NCT01297413
MSC from adipose tissue
S
Autologous
i.v./i.a.
Phase 1/2 NCT01453829
MSC from umbilical cord blood NSC (CTX0E03)
SAS
Allogenic
i.v.
Phase 2 NCT01389453
General Hospital of Chinese Armed Police Forces*
CS
Allogenic
i.c.
Phase 1 NCT01151124
PISCES/ ReNeuron Limited* http:// www.reneuron.com/
hOEC
CS
Autologous
i.c.
Phase 1 NCT01327768
OECs/ China Medical University Hospital*
STARTING-2/ Samsung Medical Center* Korea Research Foundation and the Korea Health 21 R&D Project National University of Malaysia* ISIS / University Hospital, Grenoble* AMETIS/ Southern Medical University, China* Stemedica Cell Technologies, Inc. * http:// www.stemedica.com/ Ageless Regenerative Institute* http:// www.agelessaestheticinstitute.com/
Stages of stroke: stroke (S), acute ischemic stroke (AS), subacute ischemic stroke (SAS), chronic ischemic stroke (CS) Administration routes: intraarterial (i.a.), intravenous (i.v.), intracerebral implantation (i.c.) Types of stem cells: Hematopietic Stem Cells (HSC), Neural Stem Cells (NSC), Mesenchymal Stem Cells (MSC), Olfactory Ensheathing Cells (OEC). * From www.clinicaltrials.com
Furthermore, it has been shown to be safe in phase 1 clinical trials in patients with subacute stroke [98]. Regarding the second strategy based on exogenous stem cell transplantation, it can be divided into two main categories, depending on the cell types used: a) treatment with hNSCs or Neural-like cells and b) treatment with non-NSCs, mainly bone-marrow-derived stem cells. Treatment with hNSCs or Neural-Like Cells Human ES cell-derived NSCs and human fetal NSCs transplanted into an ischemic murine brain are able to give rise to neurons, to migrate to the ischaemic lesion and, in some cases, improve some sensorimotor functions [144, 145]. At present, human fetal NSCs are being used in clinical trials by the company ReNeuron in UK for the
treatment of patients with ischemic stroke. These hNSCs have a conditional form of the oncogene encoding c-Myc under the control of an estradiol receptor that allows the propagation of the cells. Patients are transplanted with these NSCs 6 to 24 months after stroke using neurosurgical implantation into the brain [31, 32]. According to the company results (http://www.reneuron.com) no cell-related adverse events have been reported in any of the patients treated to date. Moreover, sustained reductions in neurological impairment and spasticity have been observed in these patients (see Table 2). In a different clinical trial, neuronal-like cells obtained from an immortalized human teratocarcinoma cell line (NT2N cells, also known as LSB cells), were implanted into ischemic/haemorragic infarcts which were affecting the basal ganglia and/or the cerebral cortex [146, 147]. This
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experimental trial indicated the safety and feasibility of cell transplantation for patients with stroke, however, evidence of a significant benefit in motor function was not found. Much remain to be seen about the utility of NT2N cells, particularly in a clinical setting. Treatment with non-NSCs or Bone-Marrow-Derived Stem Cells Two types of stem cells can be derived from bone marrow: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). HSCs are the precursors of all blood and lymphoid lineages, while MSCs give rise to the stromal cells of the bone marrow. The cells most widely studied in models of stroke are probably MSCs. Transplantation of MSCs after induction of cerebral ischemia can reduce infarct size and improve functional outcome in rodent models of ischemia [148, 149]. There is little evidence that these cells act by neural/neuronal replacement, however, different mechanisms have been suggested for these beneficial effects: 1) neuroprotective effects, because of the capacity of MSCs to release growth and trophic factors, such as BDNF [150] 2) angiogenic stimulation, by secreting angiogenic cytokines such as VEGF (vascular endothelial growth factor) and Ang-1 (angiopoietin-1) [151] and 3) neurogenesis stimulation, as revealed by the increased number of endogenous progenitor neuronal cells after MSCs treatment [152]. The first clinical study to evaluate the feasibility and safety of a cell therapy approach in stroke patients using autologous MSCs, was carried out by Bang et al. [81, 153]. A 5 year follow-up of the patients, confirmed that there were no adverse effects after the infusion of hMSCs, but functional improvement was modest [82]. Currently, several clinical studies using hMSCs from different sources (bone marrow, adipose, umbilical cord blood, placenta) are ongoing in stroke patients. Likewise, a number of studies are testing the possible use of different sources of HSCs, from bone marrow, peripheral blood, umbilical cord blood, for treating patients suffering from stroke. Preclinical studies of CD34+ HSCs have shown significant benefits in rodent models of stroke, with evidence of functional improvement as well as reduced infarct volume. Some of these studies have demonstrated increased angiogenesis in penumbral zone after transplantation of CD34+ cells, both systemically and intracerebrally. This angiogenesis could be mediating neural plasticity and neurogenesis after stroke injury [100, 154]. More likely mechanisms of action include the release of VEGF and also the increase in endogenous levels of other factors such as BDNF and fibroblast growth factor, that play a role in neovascularisation [155].
Several clinical trials are currently underway investigating the safety, feasibility and tolerability of infusion of autologous bone marrow mononuclear cells (BMMNCs) or CD34+ cells from various sources at different stages of ischemic stroke and using various methods of delivery (see Table 2). Results from these trials are pending. Potential disadvantages of HSCs include issues with consistency of numbers and potency of cells obtained from bone marrow, as well as the need for “ex vivo” expansion of cells, when using umbilical cord blood as a source. Timing and Route of Stem Cell Delivery One of the main advantages of stem cell-based therapy in stroke is the potential for a wide window of opportunity to intervention; since stem cells may be applied days, weeks or even months after the injury. However, at present, the most appropriate time for administration is still unclear. The poststroke brain is characterized by several unique conditions that vary with time, including increased excitotoxicity, the presence of radical oxygen species, inflammation and cell death [156, 157]. According to these conditions, the survival and function of the cells transplanted will be different. The timing for delivery depends mainly on the goal of the treatment, for example, if a treatment strategy focuses on neuroprotective mechanisms, early delivery of the cells after the insult, will be critical. Thus, when the transplanted cells act to enhance endogenous repair mechanisms (i.e. plasticity, angiogenesis and neurogenesis) or require these events in order to survive and integrate, then early stem cell delivery would be better since these processes are more prevalent during the first 2–3 weeks after ischemia [158]. Furthermore, early intravascular infusion of stem cells might benefit from the processes tied to poststroke inflammation, since there is some evidence suggesting that different types of stem cells may use similar migratory mechanisms to those of immune cells such as chemoattraction and transendothelial migration [159–161]. On the contrary, the inflammation process may be detrimental to cells directly transplanted intracerebrally. Therefore, this last option might be useful as a second-line or delayed stem cell treatment for stroke once the lesion has stabilized [156, 162]. However, recent findings show that the best survival of grafted neural stem cells into the brain of stroke animals occurs in the early post-stroke phase, before inflammatory response is established [158]. On the other hand, the ideal stem cell delivery route is also unclear. Stem cells can home to sites of injury in the CNS and induce functional recovery after different transplantation techniques such as intracerebral transplantation [144], intravenous infusion [80, 163] or intraarterial administration [164, 165].
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Some delivery routes, such as intraarterial or intracerebral are more invasive than others but may be more effective as the cells can be grafted directly into the ischaemic or boundary lesion. Systemic delivery like intravenous administration has the advantage of being non-invasive but raises the problem of cell homing to organs other than the target site, such as the lung, liver and spleen and only a very limited number of injected cells arrive to the injury site [162, 166]. The other route of intravascular delivery, intraarterial administration, would circumvent body circulation, since cells injected into the carotid artery would pass directly to the brain. In fact, some studies have explored this route of delivery and have demonstrated functional recovery in animals with stroke [164, 167]. However, intraarterial delivery causes high level of mortality that poses a serious concern for using this route in cell therapy [168]. The above mentioned reasoning is reflected in the clinical trials presented in Table 2. Most of the trials in patients with acute (AS) or semiacute stroke (SAS) (11 trials in total), are using systemic administration of stem cells, especially intravenous (10 trials) and only one intraarterial trial. On the contrary, most of the clinical trials carried out in patients with chronic stroke (7 trials in total) are performed with stem cells administered intracerebrally (6 trials) and only one trial is using intravenous infusion.
Concluding Remarks and Future Perspectives Cell therapy and in particular, stem cell therapy, is an emerging and exciting area of research. Stem cells hold enormous potential and are expanding our current understanding of the molecular mechanisms of neurodegeneration. Furthermore the clinical application of cell therapy is offering promising solutions for the treatment of a vast number of neurological disorders, most of them currently incurable, such as the case of AD or stroke. Results from studies in experimental models for these disorders are encouraging and provide supportive evidence that stem cell-based approaches can be developed into clinically useful strategies to promote recovery. The beneficial effects may be mediated by several mechanisms. Apart from the replacement of cells, stem cells have been shown to lead to improvements of potentially clinical value through immunomodulation, trophic actions, neuroprotection or stimulation of angiogenesis; the possibilities are considerable. However, still, many factors remain uncontrolled and greater insight is imperative for stem cell therapy to be effective, feasible and safe in the future. First, each neurological disorder is distinct and presents its own challenges for stem cell-based therapy. As such, a different approach should be considered for the treatment of each disease. For instance, in Parkinson’s disease only a
specific neuronal type is affected, while in AD and stroke, multiple neuronal systems and neurotransmitters phenotypes are affected, making cell-replacement strategies an extremely challenging approach. Human stem cells would first need to migrate, differentiate and mature into multiple neuronal subtypes. These neurons would also need to reinnervate appropriate targets and establish physiologically relevant afferent and efferent connectivity. Thus, the cell replacement strategy, “per se”, seems unlikely to succeed for diffuse disorders such as AD or stroke. Other important question still to be solved, before boarding a clinical treatment based in stem cell therapies, is to discover which stem cell source (hNSCs, hMSCs, HSCs, etc.) fits the best to treat each neurological disorder. Thereby, it is essential a better knowledge about the mechanisms of action of each source of stem cells such as the control of proliferation, differentiation, survival, function and integration as well as their dosage and route of administration, among others, in order to choose the correct stem cell source. Furthermore, biosafety is another key issue to take into consideration when working with stem cell-based treatments. Therefore, it is very important to bear in mind how the cells must be extracted, prepared and administered in order to reduce risks in the patient. Long-term studies are essential in this respect, particularly with regard to the potential of tumorigenicity, so as to standardize the preparations of stem cells. Finally, more clinical studies are needed to provide safer and efficient stem cell therapies to patients with neurodegenerative disorders. Acknowledgments The authors wish to thank members of their laboratory for their research work and fruitful discussions. Research at the authors’ laboratory was funded by the MICINN-ISCIII (PI-10/ 00291 and MPY1412/09) and Comunidad de Madrid (NEUROSTEMCM consortium; S2010/BMD-2336). PMM was supported by a Posdoctoral Fellowship from the Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico. DM is supported by the program INOV CONTACTO, AICEP, Portugal. Conflict of Interest The authors confirm that there are no conflicts of interest.
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