Reprogramming of Somatic Cells to Induced Neural ...

Accepted Manuscript Reprogramming of Somatic Cells to Induced Neural Stem Cells Ebrahim Shahbazi, Fahimeh Mirakhori, Vahid Ezzatizadeh, Hossein Baharvand PII: DOI: Reference:

S1046-2023(17)30119-6 http://dx.doi.org/10.1016/j.ymeth.2017.09.007 YMETH 4320

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Methods

Received Date: Revised Date: Accepted Date:

8 April 2017 2 September 2017 12 September 2017

Please cite this article as: E. Shahbazi, F. Mirakhori, V. Ezzatizadeh, H. Baharvand, Reprogramming of Somatic Cells to Induced Neural Stem Cells, Methods (2017), doi: http://dx.doi.org/10.1016/j.ymeth.2017.09.007

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Reprogramming of Somatic Cells to Induced Neural Stem Cells

Ebrahim Shahbazi1, 2, Fahimeh Mirakhori3, Vahid Ezzatizadeh1, Hossein Baharvand1, 2*

1.

Department of Stem Cells and Developmental Biology, Cell Science Research Center,

Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran 2.

Department of Developmental Biology, University of Science and Culture, Tehran,

Iran 3.

Institute for Cell Engineering, Department of Neurology, Johns Hopkins University

School of Medicine, Baltimore, MD 21205, USA

*Correspondence Hossein Baharvand, Ph.D., Royan Institute, Banihashem Sq., Banihashem St., Resalat Highway, Tehran, Iran. Postal Code: 1665659911, P.O. Box: 16635-148 Tel: +98 21 22306485, Fax: +98 21 23562507. Email: [email protected]

Short title: Somatic cell conversion into iNSC Conflict of interest: The authors declare that they have no conflicts of interests.

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Highlights

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Methods used for generation of induced neural stem cells (iNSCs) from somatic cells are introduced.

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The possible mechanisms of reprogramming of somatic cells to iNSCs are summarized.

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The advantages of iNSCs over induced neurons or iPSC-derived NSCs are discussed.

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The current challenges in iNSC application for treatment of neurological disorders are explained.

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In vivo reprogramming to iNSCs are also described.

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Abstract Recent investigations have demonstrated that defined sets of exogenous factors (chemical and/or biochemical) can convert human and mouse somatic cells into induced neural stem cells (iNSCs). Considering the self-renewal and multi-potential differentiation capabilities of iNSCs, generation of these cells has considerably enhanced cell therapy for treatment of neurodegenerative disorders. These cells can also serve as models for investigation of the mechanism(s) underlying neurodegenerative diseases and as an asset in drug discovery. Meanwhile, using the process of direct conversion/transdifferentiation, by bypassing pluripotent state and consequently reducing tumorigenesis and genetic instability risks, establishment of several desired cells are feasible. In this review, we describe the pros and cons of different methods employed to directly reprogram somatic cells to iNSCs along with the progress of iNSCs applications and the future challenges.

Keywords: Somatic Cells; Neural Stem Cells; Reprogramming; Transdifferentiation

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1.

Introduction

Until very recently, there was not much knowledge about effective application of patientspecific neural stem cell sources in research and practice. Rapid progress in stem cell methodologies, has brought hope for accomplishment of autologous cell replacement therapy. Innovative reprogramming concepts [1, 2] have developed induced pluripotent stem cell (iPSC) technology based on which, more reliable models that directly convert somatic cells into lineage-restricted stem cells, such as induced neural stem cells (iNSCs) (Figure 1) and induced neurons (iNs), have been emerged. iNSCs are similar to other lineage-restricted stem cells in terms of intrinsic self-renewal and multipotent capabilities. However, they retain differentiation potential for both neurons and glial cells, in vitro and in vivo, which makes them a promising holy grail in development of biomedical research and practice. It has been recently demonstrated that introduction of a single gene or small bioactive molecule cocktails can directly induce fibroblast conversion into specific neural-like cell lineages [3-5]. Returning cells back to tissue-specific progenitor cells may be safer, easier and potentially a complement for iPSC technology with less alterations, lower cost, and less time required. Potentially, each of these methods have their own advantages and disadvantages. Of note, the newly generated cells may potentially have clinical benefits for disease modeling, drug development, cell replacement therapy and more importantly, verification of the therapeutic window of drugs in a patient-specific manner. Though humanized animal models could be useful for some clinical assessments, provision of a complete human neurological modeling, is still not feasible. Therefore, a direct reprogramming technology that avoids cross-species issues with a potentially unlimited cell source of a human genome, provides an ethical in vitro tool to be used in clinical trials. Although the current methodologies are too far from being employed in clinical trials, they are tools to model diseases and develop drugs, in vitro. Here, we describe published studies

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where mature somatic cells are directly converted into iNSCs, as well as their related mechanisms and limitations. We also discuss the progress of iNSCs applications and the future challenges.

2.

Methods for obtaining neural stem cells from somatic cells

In comparison with mature neural cells, progenitors or tissue-specific/lineage-restricted stem cells are more favorable in terms of proliferation and differentiation capacities, as well as safety issues. All investigations on the conversion of somatic cells of different developmental origins such as fibroblasts, blood cells, astrocytes, hepatocytes, and Sertoli cells (Figure 1), into iNSCs are based on two main methods namely, direct and indirect reprogramming. Although these strategies generally share similar concepts, they differ in methodology. Indirect reprogramming relies on the employment of classic Yamanaka reprogramming factors (RFs), OCT4, SOX2, KLF4 and c-MYC. In direct approach, less time is required for RF expression to obtain desired cells, as compared to the indirect reprogramming/iPSCs generation. In this approach, by replacing reprogramming medium with the neural medium that contains particular growth factors, the transient unstable intermediate cell population is directed towards a neural stem cell fate. In contrast, direct reprogramming intends to immediately convert mature somatic cells into iNSCs by circumventing the unstable intermediate pluripotent state. In the latter approach, lineage-specific transcription factors are introduced to induce the transformation of somatic cells into NSCs.

2.1 First steps Numerous investigations have been carried out to directly reprogram a mature somatic cell into another one. Davis et al., in 1987, found that forced expression of myogenic differentiation (MyoD) converts mouse fibroblasts into myoblasts [6]. However, the

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conversion of one mature cell into another type with distinctive progenitor properties was not feasible until recently. In 2011, Kim et al., generated induced neural stem/progenitor cells from somatic cells using a modified method adopted from reprogramming to iPSCs [7]. In this experiment, conditional overexpression of RFs, under specific culture conditions adequately generated induced neural progenitor cells [7]. The initial concept of this method was shaped based on the loss of the somatic cells’ epigenetic properties, which generate a non-pluripotent unstable intermediate cell state. The new cell state would be highly responsive to environmental cues before establishment of pluripotency without generating iPSCs. However, the resultant induced neural progenitor cells exhibited limited proliferation (only 3-5 passages) and differentiation capacities as reported by Kim et al. (2011). Thus, these cells were unable to differentiate into oligodendrocytes, indicating their restricted potency to establish different types of relevant mature cell [7]. Thier et al., for the first time, used a similar approach and reported that iNSCs were expanded and differentiated into all three lineages [8]. In parallel, many other groups also demonstrated the same tripotent neural precursor features which were attained from both mouse and human fibroblasts following the overexpression of Yamanaka factors [9-13]. Interestingly, several groups reported that OCT4 expression was alone sufficient to inhibit human fibroblast genes and push somatic cells back into early developmental gene activation [14-18]. Nonetheless, in this approach, the final stage pluripotency marker, Nanog was minimally activated in the generated cells. Thus, several scientists have considered this method as a direct way of fibroblast conversion without going through iPSC reprogramming [7-9, 13, 16, 17, 19]. Consistently, it has been shown that additional lineage-specific signals present in the culture, in a continuous procedure, can remodel the epigenetic fate of transient cells in favor of another specific lineage, instead of iPSCs, which helped boosting the efficiency and quality of cell conversion [20]. Nevertheless, there is still controversy about the mechanisms that underlie this method.

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Molecular lineage tracing to track cell trajectories in the Nanog-GFP or Oct4-GFP reporter systems has indicated that even short-term expression of RFs can heterogeneously produce iPSCs beside the desired tissue-specific stem cells, while JAK1 inhibitor is present without LIF in this method [21, 22]. Advanced molecular analysis has revealed that the majority of these cells undergo rapid, transient iPSC reprogramming followed by differentiation into the desired cell types based on the culture conditions [21, 22]. Further analyses are needed to fully characterize the cells that undergo this type of reprogramming. In addition to the aforementioned method, numerous publications are rooted in the overexpression of some specific TFs that confer NSCs fate to fibroblasts (Figure 1) [3, 4, 2333]. The logic behind this approach is conceptually similar to that of iPSC reprogramming. Lineage-specific transcription factors activate endogenous regulators, not the factors that induce pluripotency. Therefore, overexpression of NSC-specific TFs can direct the transcriptional program towards a totally new level by activation of downstream sequential key neurogenic regulatory loops [34]. In the first report that was published in this regard, the combination of Brn2, FoxG1, and Sox2 exogenes led to generation of tripotent, stably expandable iNSCs [24]. In another study, a self-renewal neural precursor cell population was produced from mesoderm-derived Sertoli cells as soon as nine transcription factors (Ascl1, Ngn2, Hes1, Pax6, Brn2, Sox2, c-Myc and Klf4) were induced. Findings showed that these cells could differentiate into three principal neural cell lineages which were engrafted into mouse brain tissue [35]. More studies were conducted in order to reduce the number of reprogramming factors by testing small molecules [15, 16]. Zhu et al. revealed that chemical associations between A83-01 (TGFβ inhibitor), sodium butyrate (histone deacetylation inhibitor), lysophosphatidic acid (LPA, a phospholipid derivative), SP600125 (a Jun Nterminal kinase (JNK) inhibitor), rolipram (a phosphodiesterase type 4 (PDE4) inhibitor), and CHIR99021 (a GSK3β inhibitor) and ectopically-expressed OCT4 could differentiate adult

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human dermal fibroblast into iNSC colonies [16]. iNSCs which were comparable to native brain NSCs were also produced from mouse and human fibroblasts by using only a single NSC master regulator, SOX2 [3]. Upon transplantation into the mouse brain, these expandable, tripotent cells exhibited the ability to differentiate into different types of neurons (GABAergic and dopaminergic) and glial cells (astrocytes and oligodendrocytes) [3]. Recently, our group demonstrated that ZFP521 alone could be an NSC-like program inducer in mouse and human fibroblasts [4]. We successfully established iNSCs that were directly converted from human and mouse fibroblasts by using a Dox-inducible lentiviral vector which expressed the neurogenic transcription factor ZFP521. The obtained cells exhibited typical NSC morphological features. They were clonogenic and showed rostral regional specificity. Additionally, we demonstrated self-renewal capability as well as tripotency over prolonged passaging and stable karyotype retention. These transdifferentiated cells could survive, migrate, and imitate neural phenotypes subsequent to transplantation into neonatal mouse and adult rat brains with no tumor formation. Although we showed the crucial role of ZFP521 in direct conversion of fetal and neonatal fibroblast into stable iNSC lines, this transcription factor alone could not induce the conversion of adult human fibroblasts to NSClike cells. Ultimately, we could overcome this limitation using a combination of this transcription factor and a small molecule cocktail. Concerning the crucial regulatory role of ZFP521 [4], it would be worthwhile to explore the mechanism(s) that underlie this reprogramming procedure.

2.2

New ways out

Viral integration into the target cell genome has resulted in emergence of mutagenesis risk induced by these two different pioneering techniques. Since therapeutic application of iNSCs

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was not feasible due to the risk of mutagenesis, alternative and non-integrating methods have been developed to exclude the chance of exogenous random integration and improve safety and efficiency. These strategies include generation of progenitor cells using transposon and Cre-recombinase excisable lentviruses, repetitive transient plasmid administration [36], episomal and adenovirus transfections [12, 13], as well as DNA-free methods such as recombinant protein delivery using a cell penetrating peptide [37, 38] or RNA-based cycle Sendai-virus [11]. These new approaches enabled the scientists to generate integration-free iNSCs from fibroblasts [12, 13, 36], albeit with much lower efficiency than viral systems. More recently, rapid direct sustained activation of endogenous genes in their native context by CRISPRCas9 has been introduced as a novel, safe strategy for conversion from one cell type to another. Utilizing this method avoids genomic integration and overcomes epigenetic barriers [39].

Development

of

micro-scale

reprogramming

using microfluidics,

increases

reprogramming efficiency up to 50-fold by traditional modified mRNAs [40]. Along with improving the efficiency, the products of this type of system are more pure and show higher quality and increased reliability to be used in patient-specific high-throughput studies. This process is more feasible, and time and cost effective. MicroRNAs are naturally occurring small noncoding RNAs that play prominent roles in cell developmental processes and can be considered as another safe option. They regulate gene expression by binding to specific sites of their target mRNAs and fine-tune various aspects of cellular behaviors [41]. miR-9 and miR-124 are two highly expressed well-known neural specific microRNAs that have the ability to convert fibroblasts into neurons [42]. Numerous studies have elucidated the central role of microRNAs in NSCs specification, self-renewal, differentiation, and other properties [43, 44]. Among these, miR-145 can fine-tune the Sox2Lin28/Let7 signaling pathway and regulate the fate of NSCs [45]. Despite the extensive

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exploration of microRNA functions in NSCs development and differentiation, they have not been frequently used for direct generation of iNSCs (for more details see [46]). Evidence obtained from successful conversion of somatic cells into neurons by miR-mediated strategy, suggest that numerous microRNA families, particularly neural cell-specific miRs, could induce this reprogramming event similar to defined transcription factors. However, utilizing microRNAs that specifically control certain NSCs fates, along with or without other factors, may help provide more specific target cells in a safer manner. The chemical method is the next safe option. This approach is simple and direct, and allows fine-tuning of a cell’s fate. It can also outperform the aforementioned approaches and represent a more suitable clinically relevant alternative method to generate iNSCs as well as other cell types. To date, several groups have utilized different small molecule combinations to achieve chemically-induced NSCs (ciNSCs) without any genetic manipulation, which avoids ethical issues and the risk of tumor formation (Table 1) [5, 47-51]. Zhang et al. successfully introduced a cocktail of 9 small chemical molecules which make direct conversion of fibroblasts into ciNSCs, possible [49]. These cells presented the patterns of self-renewal, long-term expandability in vitro, multipotency in vitro and in vivo, as well as a gene expression profile comparable to their wild type counterparts. Investigations have demonstrated that this protocol is based on the use of chemical modulators to suppress the fibroblast program and activate endogenous Sox2 as a master NSC gene in the absence of the activity of pluripotency genes (e.g., Nanog, Oct4 and Rex1) [5, 49]. Although the methodology of chemical approach is different from that of the conventionally used exogenous transcription factors, the mechanisms involved in these approaches appear to be consistent with gene alteration methods. In this regard, transcriptome analysis has revealed that such chemical cocktails induce a gradual, specific conversion of fibroblasts toward a neural cell fate by activating the respective downstream signaling pathways and the master

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endogenous neural genes (for review see [52, 53]). Alternatively, small molecules can be used to expand or selectively differentiate desired cell types in vitro or in vivo, thereby facilitating clinical applications. By using chemical inducers, Hu et al. have converted fibroblasts obtained from healthy patients and those with Alzheimer's disease, into neurons (~5% Tuj1-positive) within only 7 days by using a combination of 7 neurogenic small molecules [50]. Li et al. converted fibroblasts into neurons with an efficiency of up to 90% [20]. The latter method offers several important benefits including ease of application, optimization and manufacturing, over the other aforementioned systems in terms of producing desirable cells. This chemical approach can also be readily developed into conventional pharmaceuticals. Advantages of the novel approaches have brought new hope for improvement of iNSC generation in terms of efficiency and safety for research, drug discovery and therapy prospects, as well as tissue-specific stem cell production in vivo.

3.

Mechanisms of iNSC generation and beyond

The exact mechanisms of direct reprogramming are not well understood. Nevertheless, it is known that transcription factors have a pivotal role in this process. Besides, epigenetic modifications (such as DNA methylation, histone modification, and microRNA-based regulation) can affect the reprogramming process. There are many binding sites in the genome for any given transcription factor. Nevertheless, the local chromatin structure restricts transcription factor access to a certain number of binding sites depending on the cell type. For example, the hematopoietic transcription factor, Scl/Tal binds to different regions in different hematopoietic cellular subtype [54]. It has been found that a number of genomic regions, deemed unnecessary, is excluded from transcription factors in the form of heterochromatin [55]. In order to initiate a particular reprogramming event, first, the

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transcription factors should find the appropriate binding site. They must also remodel the chromatin to facilitate binding of additional factors. This requirement elucidates the need for cooperative action of multiple transcription factors to achieve chromatin remodeling in vast regions of the genome. Below, several hypotheses explaining transcription factor binding during the initial stages of the reprogramming process, are summerized. Permissive enhancer model. Binding of transcription factor to permissive enhancer elements (i.e. H3K4me1-enriched) can overcome the silencing effect of polycomb-mediated epigenetic signals (i.e. H3K27me3) at the gene’s promoter, resulting in initiation of subsequent chromatin remodeling events at the promoter [56]. Pioneer factor model. Pioneer reprogramming factors can bind to chromatin in regions where most transcription factors are precluded; therefore, cis-regulatory elements become more readily accessible. Pioneering reprogramming factors can activate non-permissive genes by disassembling the nucleosomal proteins and subsequently recruiting chromatin-modifying enzymes or additional transcription factors [57]. Spontaneous accessibility. Some cis-regulatory elements can be accessed by transcription factors during the short stochastic period of nucleosomal disentanglement resulted from the dynamic balance between the open and closed chromatin conformations in the cell nucleus. Considering the fact that these transiently accessible cis-regulatory elements are pertinent to the reprogramming process, increasing the cellular concentration of appropriate transcription factors will elevate the probability of bona fide binding events. Consequently, the likelihood of undergoing reprogramming process for a cell increases by augmented numbers of exogenous transcription factors. Accessibility during cell division. Since during cell division, chromatin is in an open state, reprogramming factors can bind to the previously inaccessible regulatory elements of the target genes. Furthermore, replacement of old nucleosomes with newly generated and

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unmodified histone proteins, can disrupt previous epigenetic silencing at the respective loci, thus facilitating chromatin remodeling by binding of reprogramming factors and stabilizing the newly introduced reprogramming events. The stimulatory effects of transcription factors as well as the inhibitory effects of epigenetic modifications of chromatin structure, is currently being investigated [58, 59].

4.

The advantages of expandable iNSCs over iNs or iPSC-derived NSCs

A non-invasive approach to obtain an autologous source for cell therapy is highly wanted in biomedical sciences. For this purpose, appropriate numbers of the respective cell source should be generated before transplantation. These cells should immunologically match the host organism, while not causing any malignancy. Considering these demands, generation of autologous iNs, iPSC-derived NSCs and iNSCs from the patients’ own somatic cells, could be considered as an alternative method to be used as a therapeutic approach (Table 2) [60]. iNs can potentially be used as an engaging implement in modeling of brain diseases affected by disruptions in neuronal function. Nonetheless, they cannot be used for glial baseddiseases. iNs are post-mitotic cells like neurons, and they do not have the capability to undergo further expansion; thus, a high quantity of the respective cells is required for transplantation. Detachment of axo-dendritic arborization structure of a mature neuron is extraordinarily harmful for the cells, in vitro. Ultimately, the time-consuming nature of the procedure of cell conversion and accumulation for transplantation purposes as well as heterogeneity of the generated iNs, are other crucial obstacles. These limitations have motivated scientists to convert original cells into iNSC cells which do not have such limitations. The induction efficiency of iNs is comparatively higher (with a yield of up to 90% in some cases) compared to iNSCs [20], however, the small population of the generated cells precludes their use. Also, an immature cell type with a definably intermediating neural

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fate could be used as a feasible alternative to transplantation. iPSCs generation from adult human dermal fibroblasts and their conversion into NSCs have opened a new window to autologous transplantation of the generated neural cells and reduction of the histocompatibility hurdles. Nevertheless, subsequent progression and complete differentiation of these cells into defined lineages, remain a major obstacle for application of iPSCs as they have the potential of tumorigenesis and genomic instability. In comparison with iPSCs-derived NSCs, iNSCs are more therapeutically effective and appear to be safer for transplantation, with less potential for tumorigenesis. iNSCs have selfrenewal capacity in vitro, similar to iPSCs-derived NSCs. Also, iNSCs can differentiate into multiple types of neuronal cells that have a higher survival potential after transplantation [3, 4], enabling them to supersede iNs in cell therapy. Of note, the crucial point for successful iNSCs transplantation for treatment of neurodegenerative disorders is the stepwise stimulation of these cells, which would convert them into desired neuronal cell type (Table 2).

5.

The challenges in iNSC application for treatment of neurological disorders

Although the abovementioned advances are promising, drawbacks exist concerning iNSCs differentiation. In addition, many neurodegenerative disorders are caused by only one certain cell type. Hence, generation of a pure population of the ultimate product is another considerable challenge. The use of current methods has enabled scientists to successfully generate homogenous iNSCs for pan-neural markers (e.g. SOX1 and SOX2), while they show heterogeneous properties for regional markers. This heterogeneity could be due to various combinations of transcription factors that have been used by different researchers. Thus, these combinations could induce NSC-like cells with different differentiation potentials. In this regard,

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development of a novel combination of TFs for generation of regional or more specific iNSCs, could be the key to successful transplantation. Further investigations are required to propose novel methods for therapeutically recruiting iNSCs and neural derivatives in regionally involved neurodegenerative diseases such as Parkinson's disease and multiple sclerosis. Having defined protocols and conditions that allow predictable fate choices to generate desired neuronal cell types from patient fibroblasts, may accelerate the use of iNSCs in practice. Tian et al. have produced lineage-restricted induced dopaminergic precursors by ectopic expressions of Brn2, Sox2, and Foxa2 [61]. They demonstrated that these cells gave rise to dopaminergic neurons in vivo, alleviated motor deficits, and reduced the loss of striatal dopaminergic neuronal axonal termini in an MPTP-induced mouse model of Parkinson's disease [61]. Ai et al. demonstrated that application of a transcription factor cocktail (Oct4, Sox2, and Klf4) in combination with a particular culture medium, could induce fibroblasts from a rhesus monkey into telencephalic neuroepithelial stem cells [62]. They injected the monkey's single C8 cell (as an iNSCs-derived cell) into the left striatum. After two months, their analysis showed that most of the engrafted cells were differentiated into DCX neurons. Pre-treatment of the cells in the striatum with Purmorphamine (Hedgehog pathway chemical agonist), also boosted the cell survival rate, neurons maturation and axons outgrowth, in vivo. In the next step, they assessed the graft site influences on cell survival and effectiveness of integration by injecting the cells into the adult monkey’s prefrontal cortex and white matter. In comparison to the striatum, they observed that fewer cells survived with short axonal outgrowth in the prefrontal cortex region. Nonetheless, these indices in the right prefrontal cortex were improved after treatment of the respective cells with a medium that contained BMP4, a crucial factor involved in cortical cell differentiation and axonal outgrowth. This demonstrates that the in vivo iNSCs fate at least depends on two parameters - the iNSCs

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properties before transplantation as well as the transplantation site [62]. Apart from the underlying mechanism(s), these findings support the perception that different combinations of transcription factors and small molecules not only guarantee NSCs expansion and differentiation, but also assure region-specific NPC production. In addition, it should be taken into account that TFs that are used for iNSCs generation should be cellspecific in a way that proper target neural cells (either specific neuronal sub-types and/or glial cells) could be derived based on the disease phenotype. This requires a deep understanding of gene regulatory networks as well as the time windows among developmental stages in vivo; however, this adds another layer of complexity to this scenario.

6.

In vivo reprogramming

The concept of in vivo transdifferentiation is not a novel idea in the field of stem cell biology. Nevertheless, this concept has been remarkably revolutionized during recent years due to the discovery of further transcription factors that promote cell reprogramming [63, 64]. Considering the impact of the native niche, more efficient transdifferentiation could be achieved in vivo compared to in vitro. This would likely lead to generation of desired functional cell types thanks to essential factors provided by the natural niche. It has been suggested that the similarity between developmental origin of the initial and target cell types could also have a significant impact on the improvement of the transdifferentiation efficiency, in vivo. For example, β-cells have been efficiently transdifferentiated from pancreatic exocrine [65]. As they originated from the same precursor cell, this conversion could requires only a few epigenetic modifications from the initial to the target lineages. In the CNS, it has been determined that resident glial cells which comprise approximately half of the mammalian CNS cells are broadly distributed in the brain and spinal cord and be directly or indirectly converted into functional neurons [66-70]. In response to neural injuries

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and degenerations, these cells are activated, then they proliferate, and generate glial scars which limit the spread of the initial damage. Although this activity could be beneficial for recovery of a neural injury, it could eventually be harmful due to the generation of a physical/chemical barrier which inhibits neuronal regeneration. Hence, it appears that some of these glial cells could be ideal to be reprogrammed into neurons as soon as the neural injury occurs in vivo. Using this approach, it was indicated that induction of Sox2 could also convert brain astrocytes into proliferative neuroblasts in adult mice. Interestingly, treatment of the latter cells with neurotrophic factors or a histone deacetylase inhibitor led to their conversion into mature neurons, while they were functionally integrated into the local neural network [68]. Further investigations showed that the respective functionally mature cells were GABAergic neurons, due to predominant expression of the calcium-binding protein calretinin [71]. Recently, we demonstrated that co-administration of VPA and miR-302/367 could convert astrocytes into neuroblasts both in vitro and in vivo [72]. Both direct and indirect in vivo conversion of resident glial cells into functional neurons have in fact changed scientists’ view points and presented a new approach in cell therapy to treat neurodegenerative diseases. As previously indicated, researchers are currently attempting to replace exogenous genes with small molecules to improve simplicity and safety of cell reprogramming. An ultimate goal in the field of regenerative medicine is the extension of this idea to cell conversion in vivo, where a resident cell may be able to differentiate into a distinctive cell type to recover the damaged tissue.

7.

Conclusion

Thanks to stem cell therapy, a promising way of treatment of several aggressive neurodegenerative disease has been proposed recently. Considering all cell sources, directly induced NSCs seem to be ideal substitutes for defective neurons if their differentiation could

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be controlled toward desired type of neurons. Employing chemical and biochemical agents shed light on determination of the cell fate without genetic manipulation. Small molecules have accurate and adjustable control over protein’s function through correctable activation and inhibition, while microRNAs have the advantages of precise targeting of large amount of genes. In fact, reprogramming could be more efficient if entirely defined chemical conditions are utilized based on a deep understanding of reprogramming mechanism(s). However, we are still far from having a high efficacy protocol for production of cells with determined fate.

Acknowledgements The authors thank Sharif Moradi for proof reading of the manuscript. This work was supported by a grant from Royan Institute, the Iranian Council of Stem Cell Research and Technology, the Iran National Science Foundation (INSF), and Iran Science Elites Federation to H.B.

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24

Figure legends Figure 1. Summary of reported induced neural stem cells (iNSCs).

Different

combinations of neurogenic transcription factors can directly induce various starting cells into iNSCs. Each column represents the details and results of a single report in terms of starting cell types, in vitro or in vivo tests and gene combinations used for reprogramming, in a heatmap-like manner.

25

Table 1. Chemical reprogramming of somatic cells into induced neural stem cells (iNSCs) and their characteristics. Cheng et al. (2014)

Zheng et al. (2016)

Zhang et al. (2016)

Han et al. (2016)

Starting cell

MEF Human urine cells

MEF

MEF

MEF

Small molecules

VPA, CHIR99021, Repsox

A83-01, Purmorphamine, VPA, thiazovivin

LDN, A8301, CHIR99021, RA, HhAg,RG108, Parnate, SMER28 and bFGF

PD032590, Bix01294, RG108, CHIR99021, A83-01, VPA, Vitamin C

Duration

Mouse - 10 days Human - 20 days

12 days

10 days

21 days

Efficiency

40 compact cell colonies emerged from 200,000 cells

149 compact cell colonies emerged from 200,000 cells

24%-30%

Up to 2%

Expansion capacity

>25 passages

>20 passages

>10 passages

Expanded more than 2 years

Electrophysiology

Recorded

Recorded

Recorded

Recorded

Multipotency in vitro

Tripotent (mouse iNSCs) Bipotent, astrocytes and neurons (human induced neural progenitor cells)

Tripotent

Tripotent

Tripotent

Neuronal subtype specification

Not assessed

Dopaminergic, GABAergic and cholinergic neurons

Glutamatergic and GABAergic neurons

Not assessed

Multipotency in vivo

Tripotent

Not assessed

Tripotent

Tripotent

26

Table 2. The advantages of obtaining expandable iNSCs over iNs or iPSC-derived NSCs. advantages iNs

iPS-NSCs

•

Autologous

•

Useful for neuronal

disadvantages •

Cannot be utilized in glial-based diseases

defective disease

•

No self-renewal capacity

modeling

•

Not transplantable

•

Heterogeneity of generated iNs

•

Risk of tumorigenesis due to

•

Autologous

•

Self-renewal capacity

potential iPSC contamination •

Difficulty in controlling differentiation direction after transplantation

iNSCs

•

Autologous

•

Self-renewal capacity

differentiation direction after

•

Lower risk of

transplantation

•

tumorigenesis and genetic instability •

Can be achieved under chemically defined conditions

27

Difficulty in controlling

28