**4. Generation of NSPC from various sources**

#### **4.1. Adult tissue derived NSPCs**

NSPCs can be derived from various regions along the neuroaxis during embryonic develop‐ ment and in adult life [66, 67]. These cells retain their mulipotentiality and can generate neural cells in culture. NPSCs have been isolated from the subependymal zone of the adult mammali‐ anbrainandfromependymal andnon-ependymalregions ofthe adultmammalianspinal cord. [68-70] Single adult NPSCs can be isolated *in vitro* in the presence of growth factors (epithelial growth factor, EGF; fibroblast growth factor, FGF) that enable the proliferation and formation of clonally-derived free-floating colonies. The differentiation and survival of cellular subpopu‐ lations canbepromoted*invitro*byexposure tobonemorphogeneticproteins (BMPs)toproduce astrocytes [71, 72], insulin-like growth factor(IGF)-I, interleukin-1 (IL-1), Neuregulin-1 (Nrg-1) to generate oligodendrocyte [73, 74], and neurogenin-2 to produce neurons [75, 76].

For experimental purposes neurospheres can be generated from the germinal zone of the adult mouse brain, according to well-established techniques [77, 78]. Briefly, the subventricular zone (SVZ) of the forebrain of mice can be dissected and transferred to a low calcium aCSF solution. Cells are plated on uncoated tissue culture flasks in serum free medium containing FGF2 and EGF for 7 days. The neurospheres are passaged weekly by mechanical dissociation in the same medium. This method of generating NSPCs has obvious clinical limitations as it requires brain tissue to generate the renewable cell population.

#### **4.2. Embryonic stem cell (ESC) derived-NSPCs**

regenerating receptor neurons in the PNS in the olfactory mucosa to the target neurons in the CNS in the olfactory bulb glomeruli, has made them an excellent candidate for cell treatment strategies in SCI. [58, 59] They may be specifically advantageous when co-transplanted with SCs, since they can overcome SCs' limitations in passing the transplant graft and entering the

OECs create a permissive environment for axonal growth and regeneration by interacting with the astrocytes in the glial scar and promoting angiogenesis. [62] This neuroprotective effect has led to their use in numerous clinical trials outside North America. However, they all report mixed results with none showing a significant benefit. [3, 27] Additionally, there are numerous axonal regeneration claims seen in OEC literature, which have not been confirmed by other studies and there are inconsistent reports on their regenerative capacity *in vivo*. The reasons for such discrepancies are not completely understood but could be attributed to variability in cell sources, cultures, and injury models studied. [63, 64] Thus, there is a need for further studies on the biology of OECs and more refined criteria set for isolation of these cells in order

Neural Stem/Progenitor Cells (NSPCs) are multipotent stem cells that self-renew and differ‐ entiate into lineage-specific neural precursor cells, which can give rise to neurons, astrocytes, and oligodendrocytes through asymmetric cell division. [30] Indeed, neural precursor cells and oligodendroglial precursor cells have been shown to replace damaged cells, secrete trophic factors, regulate gliosis and scar formation, reduce cystic cavity size and axonal destruction, as well as to remyelinate axons.[3, 27, 65] However, the scarcity of adult NSPCs limits the clinical translation of transplanting these cells in injured tissue. Therefore, alternative routes

NSPCs can be derived from various regions along the neuroaxis during embryonic develop‐ ment and in adult life [66, 67]. These cells retain their mulipotentiality and can generate neural cells in culture. NPSCs have been isolated from the subependymal zone of the adult mammali‐ anbrainandfromependymal andnon-ependymalregions ofthe adultmammalianspinal cord. [68-70] Single adult NPSCs can be isolated *in vitro* in the presence of growth factors (epithelial growth factor, EGF; fibroblast growth factor, FGF) that enable the proliferation and formation of clonally-derived free-floating colonies. The differentiation and survival of cellular subpopu‐ lations canbepromoted*invitro*byexposure tobonemorphogeneticproteins (BMPs)toproduce astrocytes [71, 72], insulin-like growth factor(IGF)-I, interleukin-1 (IL-1), Neuregulin-1 (Nrg-1)

to generate oligodendrocyte [73, 74], and neurogenin-2 to produce neurons [75, 76].

For experimental purposes neurospheres can be generated from the germinal zone of the adult mouse brain, according to well-established techniques [77, 78]. Briefly, the subventricular zone

injured CNS environment to produce functional synapses. [3, 60, 61]

for their translation into the clinic to be feasible. [34]

278 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**4. Generation of NSPC from various sources**

*3.1.4. Neural stem/progenitor cells*

to derive NSPCs have been studied.

**4.1. Adult tissue derived NSPCs**

The first isolation of a pluripotent population of cells from the mouse embryo over 30 years ago revolutionized the emerging field of regenerative medicine. [79, 80] Once a similar population of cells were identified and isolated from a human source, [81] the possibilities and potential for clinical application for the derivatives of these cells became limitless. Neurons and glial cells were among the first differentiated cells to be generated from these pluripotent cells. Although there are some differences between human and murine ES cells, such as LIF responsiveness *in vitro* [82], many of the neural differentiation protocols established for mouse ES cells have been adapted for use with human ES cells. There are multiple strategies that are implemented to generate neural cells from ES cells with varying degrees of cell homogeneity, differentiation potential, efficiency and time requirements. In general, two strategies exist to direct ES cells to NSPC: the first uses embryoid body (EB) formation to mimic the physiological neurodevelopment of the embryo and the second involves removal of all cues that would direct the cells to a non-ectoderm lineage by limiting cell-cell interactions with low cell density culture and environmental signals, with serum-free media.

#### *4.2.1. NSPC generation with EB intermediate*

Using EB intermediates to generate NSPCs and their differentiated products mimics the physiological environment that produces neuroectoderm. [83, 84] In brief, the ES cells are transferred from their expansion conditions on feeder cells to a suspension culture and allowed to form aggregates. Within a few days, the EBs formed resemble an anterior pre-streak stage embryo with an epiblast-like core. [85] At this stage the cells are still able to form cells from all three germ layers. Next conditions are such that they drive the neuroectoderm lineage while inhibiting the endoderm and mesoderm fates concentrating the NSPC population and thus yield cells that can differentiate to neurons and glial cells.

The use of retinoic acid (RA) with the EB based neuralization protocols can vastly improve the neural lineage cells produced. [86, 87] Many studies have shown that RA plays a key role during neurogenesis, both *in vitro* conditions as well as physiologically. RA treatment directs cell towards those of the posterior CNS. [88] The general culturing process involves EB formation, as described above, for 4 days, followed by RA treatment (4-/4+) and then 1 week of adherent conditions. A high proportion of the differentiated cells have neural properties, with positive identification for both glutamatergic and motor neurons. [88, 89]

Other neuralization protocols that use an EB intermediate, such as culturing with carcinoma or stromal cell-line conditioned media or via selection in defined media conditions, can be used to generate NSPCs with relatively high efficiency. [90, 91] However, a key limitation with NSPCs generated from EB intermediates exists; despite success with these approaches, concerns related to the restricted potential of NSPCs remain. The involvement of EB formation creates a risk for the persistence of non-neural cells in the final population. This has been associated with teratoma formation and increase tumorigenicity. The persistence of non-neural cell can be traced back to the non-specific differentiation pattern associated with the initial aggregation of EB.

animals were perfused and spinal tissue was fixed with 4% PFA. Cryosections of tissue were immunohistochemically labeled for neural cell markers. The ES-dNSPCs were able to survive and integrate into the host tissue in a similar pattern to that previously described with aNPCs. The transplanted cells preferentially migrated to the white matter tracts and differentiated to oligodendrocytes with multiple processes that expressed MBP. The quantification of the differentiation pattern showed that the transplanted cells become primary mature APC+ oligodendrocytes with limited astroglial and neuronal fates. The variability between *in vivo* and *in vitro* differentiation potential can be attributed to the instructive nature of the spinal cord niche. The *Shiverer* spinal cord has been shown to have greater amounts of NG2-express‐ ing oligodendrocyte progenitors. [96] The environment that leads to the excess of these progenitors is also acting on the transplanted cells. This instructive/permissive niche could be a result of signaling molecules such as GRO-1 [97] and neuregulin (NGR) [98], both of which

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ES cells are the most defined source of readily available pluripotent cells that can be used in cell-based treatment for SCI. Although, they have some safety and ethical concerns, this

**Figure 2.** Default neural differentiation of embryonic stem cells. (A) Schematic representation of neural induction of ESCs through the default pathway: Individual ESCs when cultured at low density in minimal serum-free media con‐ taining LIF acquire a neural identity through a default mechanism. These neural stem cells colonies are termed pNSPCs, are LIF- dependent, and divide to form clonally-produced floating neurosphere colonies. pNSPC-derived neu‐ rospheres can be dissociated into single cells and passaged indefinitely in serum-free media containing LIF or can be passaged into serum-free media containing FGF2 to produce a distinct population of FGF2-dependent cells termed dNSPCs that also divide to form clonally-produced neurospheres. These spheres can also be passaged indefinitely and when differentiated produce all three cell types of the neural lineage. (B) Phase-contrast image of a neurosphere colo‐ ny generated by a pNSPC. (C) Phase-contrast image of a neurosphere colony generated by a dNSPC. (D) Phase-con‐ trast image of a neurosphere generated by adult neural stem cells derived from the subependymal layer of the adult mouse forebrain. For B–D, bar equals 50 mm. ESCs, embryonic stem cells; LIF, leukemia inhibitory factor; dNSPC, defi‐ nitve neural stem cell; pNSPC, primitive neural stem cell; FGF2, basic fibroblast growth factor; Olig2, oligodendrocyte transcription factor 2. \*from *Rowland et al., 2011, Stem cell and Develop. 20(11); 1829-1845, with permission.*

population will remain extremely relevant in the future of spinal cord regeneration.

are released by astrocytes and neurons.

#### *4.2.2. NSPC generation from default pathway*

In order to circumvent EB intermediates, and its limitations, during neuralization of pluripo‐ tent cells, our lab has opted to use the default pathways to direct ES cells to a neural lineage. ([92]; Figure 2) This pathway relies on the fact that, in the absence of extrinsic signaling to form non-ectoderm lineage, the cells will adopt a neuroectoderm fate. [93, 94] The default mecha‐ nism is based on studies that showed the inhibition of BMP signaling by protein inhibitors or by gene expression manipulation leads to neural lineage commitment. A small percentage (0.2%) of single ESCs cultured under serum-free, low cell density condition proliferate in the presence of LIF to form floating neurospheres of cells that express the neuroepithelial markers Nestin and Sox1, but downregulate the ES cell markers Oct4 and SSEA1. [93, 94] Moreover, a small proportion of cells derived from primary neurospheres can generate secondary colonies when subcloned, which are independent of LIF, but are dependent on FGF2. These cells have been termed definitive (dNSPC). [94]

In order to assess the clinical potential of the NSPCs generated by this method, with specific focus on SCI, ourlab has extensively characterized the ES-derived dNSPCs *in vitro*.[92]In brief, EScellsweredirectedtoprimitiveanddefinitiveNSPCfates.TheirmRNAprofilewasevaluated using RT-PCR and *in vitro* differentiation patterns were quantified and compared to aNSPCs populations isolatedfromtheSVZ.TheES-dNSPCpopulationswere similartoaNPCs analyzed atthemRNAlevelwithasignificantdecreaseinpluripotency(*Nanog,Oct4*)andstemness (*Tdgf1, Dnmt3b, Gaf3*) markers with increased transcription of neural-specific markers (*Pax6, Nestin, Olig2, Synaphysin*). The pNSPC retained many of the pluripotency and stem markers associat‐ ed with undifferentiated ES cells reflecting the importance of driving the NSPC to a definitive state. To assess the *in vitro*differentiation pattern ofthe ES-derived NSPCs and aNSPC controls, the cells were cultured on matrigel for one week with 1% FBS media. The cells were immunola‐ belled for NSPCs (Nestin), neurons (BetaIII Tubulin), astrocytes (GFAP), and oligodendro‐ cytes (Olig2,PDGFRa,CNPase).BothES-dNSPCs andaNSPCsyieldedprimarilydifferentiated neural cells with the majority being astrocytes (~65%). The largest proportion of cells from the ES-pNSPCs were undefined or retained the NSPC marker Nestin. Although astrocytic differentiation may not be the desired cell, this differentiation into astrocytic cells demon‐ strates that the *in vitro* environment is very different from the *in vivo* niche.

The potential of these cells to survive, integrate, and differentiate *in vivo* is critical to evaluating their role in regenerative medicine applications. Since remyelination following SCI is a likely potential mechanism of recovery, our lab uses a dysmyelinated *Shiverer* mouse to assess the *in vivo* potential of our ES-dNSPC. *Shiverer* mice lack compact myelin basic protein (MBP) and therefore are an ideal candidate to evaluate the myelination ability of these cells. [95] In general, the Shiverer mouse, under isoflurane anaesthesia, received a T6-T7 laminectomy followed by 4 intraspinal injections of ES-dNSPC (100,000 in 2ul of media). The mice were immune suppressed with continued cyclosporine A treatment. Six weeks following transplantation the animals were perfused and spinal tissue was fixed with 4% PFA. Cryosections of tissue were immunohistochemically labeled for neural cell markers. The ES-dNSPCs were able to survive and integrate into the host tissue in a similar pattern to that previously described with aNPCs. The transplanted cells preferentially migrated to the white matter tracts and differentiated to oligodendrocytes with multiple processes that expressed MBP. The quantification of the differentiation pattern showed that the transplanted cells become primary mature APC+ oligodendrocytes with limited astroglial and neuronal fates. The variability between *in vivo* and *in vitro* differentiation potential can be attributed to the instructive nature of the spinal cord niche. The *Shiverer* spinal cord has been shown to have greater amounts of NG2-express‐ ing oligodendrocyte progenitors. [96] The environment that leads to the excess of these progenitors is also acting on the transplanted cells. This instructive/permissive niche could be a result of signaling molecules such as GRO-1 [97] and neuregulin (NGR) [98], both of which are released by astrocytes and neurons.

creates a risk for the persistence of non-neural cells in the final population. This has been associated with teratoma formation and increase tumorigenicity. The persistence of non-neural cell can be traced back to the non-specific differentiation pattern associated with the initial

In order to circumvent EB intermediates, and its limitations, during neuralization of pluripo‐ tent cells, our lab has opted to use the default pathways to direct ES cells to a neural lineage. ([92]; Figure 2) This pathway relies on the fact that, in the absence of extrinsic signaling to form non-ectoderm lineage, the cells will adopt a neuroectoderm fate. [93, 94] The default mecha‐ nism is based on studies that showed the inhibition of BMP signaling by protein inhibitors or by gene expression manipulation leads to neural lineage commitment. A small percentage (0.2%) of single ESCs cultured under serum-free, low cell density condition proliferate in the presence of LIF to form floating neurospheres of cells that express the neuroepithelial markers Nestin and Sox1, but downregulate the ES cell markers Oct4 and SSEA1. [93, 94] Moreover, a small proportion of cells derived from primary neurospheres can generate secondary colonies when subcloned, which are independent of LIF, but are dependent on FGF2. These cells have

In order to assess the clinical potential of the NSPCs generated by this method, with specific focus on SCI, ourlab has extensively characterized the ES-derived dNSPCs *in vitro*.[92]In brief, EScellsweredirectedtoprimitiveanddefinitiveNSPCfates.TheirmRNAprofilewasevaluated using RT-PCR and *in vitro* differentiation patterns were quantified and compared to aNSPCs populations isolatedfromtheSVZ.TheES-dNSPCpopulationswere similartoaNPCs analyzed atthemRNAlevelwithasignificantdecreaseinpluripotency(*Nanog,Oct4*)andstemness (*Tdgf1, Dnmt3b, Gaf3*) markers with increased transcription of neural-specific markers (*Pax6, Nestin, Olig2, Synaphysin*). The pNSPC retained many of the pluripotency and stem markers associat‐ ed with undifferentiated ES cells reflecting the importance of driving the NSPC to a definitive state. To assess the *in vitro*differentiation pattern ofthe ES-derived NSPCs and aNSPC controls, the cells were cultured on matrigel for one week with 1% FBS media. The cells were immunola‐ belled for NSPCs (Nestin), neurons (BetaIII Tubulin), astrocytes (GFAP), and oligodendro‐ cytes (Olig2,PDGFRa,CNPase).BothES-dNSPCs andaNSPCsyieldedprimarilydifferentiated neural cells with the majority being astrocytes (~65%). The largest proportion of cells from the ES-pNSPCs were undefined or retained the NSPC marker Nestin. Although astrocytic differentiation may not be the desired cell, this differentiation into astrocytic cells demon‐

The potential of these cells to survive, integrate, and differentiate *in vivo* is critical to evaluating their role in regenerative medicine applications. Since remyelination following SCI is a likely potential mechanism of recovery, our lab uses a dysmyelinated *Shiverer* mouse to assess the *in vivo* potential of our ES-dNSPC. *Shiverer* mice lack compact myelin basic protein (MBP) and therefore are an ideal candidate to evaluate the myelination ability of these cells. [95] In general, the Shiverer mouse, under isoflurane anaesthesia, received a T6-T7 laminectomy followed by 4 intraspinal injections of ES-dNSPC (100,000 in 2ul of media). The mice were immune suppressed with continued cyclosporine A treatment. Six weeks following transplantation the

strates that the *in vitro* environment is very different from the *in vivo* niche.

aggregation of EB.

*4.2.2. NSPC generation from default pathway*

280 Trends in Cell Signaling Pathways in Neuronal Fate Decision

been termed definitive (dNSPC). [94]

ES cells are the most defined source of readily available pluripotent cells that can be used in cell-based treatment for SCI. Although, they have some safety and ethical concerns, this population will remain extremely relevant in the future of spinal cord regeneration.

**Figure 2.** Default neural differentiation of embryonic stem cells. (A) Schematic representation of neural induction of ESCs through the default pathway: Individual ESCs when cultured at low density in minimal serum-free media con‐ taining LIF acquire a neural identity through a default mechanism. These neural stem cells colonies are termed pNSPCs, are LIF- dependent, and divide to form clonally-produced floating neurosphere colonies. pNSPC-derived neu‐ rospheres can be dissociated into single cells and passaged indefinitely in serum-free media containing LIF or can be passaged into serum-free media containing FGF2 to produce a distinct population of FGF2-dependent cells termed dNSPCs that also divide to form clonally-produced neurospheres. These spheres can also be passaged indefinitely and when differentiated produce all three cell types of the neural lineage. (B) Phase-contrast image of a neurosphere colo‐ ny generated by a pNSPC. (C) Phase-contrast image of a neurosphere colony generated by a dNSPC. (D) Phase-con‐ trast image of a neurosphere generated by adult neural stem cells derived from the subependymal layer of the adult mouse forebrain. For B–D, bar equals 50 mm. ESCs, embryonic stem cells; LIF, leukemia inhibitory factor; dNSPC, defi‐ nitve neural stem cell; pNSPC, primitive neural stem cell; FGF2, basic fibroblast growth factor; Olig2, oligodendrocyte transcription factor 2. \*from *Rowland et al., 2011, Stem cell and Develop. 20(11); 1829-1845, with permission.*

#### **4.3. Induced pluripotent stem cells (IPSC) derived NSPCs**

GiventheaforementionedconcernswithEScells combinedwiththe immunogenicitythatarises from allograft transplantation, the search for a patient-specific and accessible cell source has been a principle endeavor of regenerative medicine. Historically, many techniques and strategies have been developed to accomplish this aim, most notably somatic cell nuclear transfers (SCNT). SCNTis theprocessbywhichthenucleusofthe somatic cellbeingreprogram‐ med is transferred to an enucleated ooctye [99]. This technique became famous in the late 1990s when Dr. Ian Wilmut cloned the first mammal, a sheep named "Dolly". [100] Although there is no obvious mechanism that would preclude SCNT from reprogramming human cells, this technique has yet to be successfully applied in human cells. Furthermore, the requirement for donor ooctyes, combined with an inefficient and technically difficult processes makes SCNT unlikely to be a viable option for clinical application even in the most ideal circumstances.

specifically histone methylation.[101] Epigenetic memory of iPS cells can influence the differentiation potential and the differentiation of the iPS cells, favouring the identity of the tissue of origin. [114] The impact of epigenetic variability still remains an area of debate and

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The conceptual outline of how iPS cells may be used in the treatment of SCI is described in figure 3. [28] Many of the *in vitro* neuralization protocols outlined above for generating NSPCs and terminally differentiated neural cells, neurons, astroglia and oligodendrocyte, from ES cells can be applied to iPS cells. iPS cell have been used in models of Parkinson's [110, 115], spinal muscular atrophy (SMA) [116], amyotrophic lateral sclerosis (ALS) [117], and SCI [118]. Although there have been positive results using iPS-derived neural cells for cell-based treatment, the variation in iPS cells has resulted in variability in the safety and neuralization of these cells. A comprehensive evaluation of NSPCs from mouse iPS cell lines generated from various tissue sources and reprogramming methods has shown differences in tumorigenesis

The variation in neuralization potential between iPS cell lines and their ES cell counterparts represents anareaofregenerativemedicine that,ifnot addressed, couldbe anobstacle toclinical translation. Our lab specifically examined the difference in neuralization potential of multi‐ ple iPS cells lines. [120] We used iPS cells generated using piggyBac transposon technology to eliminate variation that could be attributed to the resurgence of the silenced transgenes or due toinsertionmutations.[108,109]Furthermore,thedefaultpathwayofneuralizationwasutilized to avoid issues associated with the EB formation as an intermediate step in generating NSPCs from a pluripotent cell source. Even with the additional precautions to help generate safe and clinically-relevant cells, there was retention of plurioptency markers as well as persistence of non-ectoderm lineage markers. In order for the potential for iPS technology to be realized,

methods to effectively and consistently generate definitive cells must be developed.

We examined the role of BMP antagonism during the initial neuralization of the pluripotent IPS cells. We identited this as a possible area where IPS cell neuralization could be improved. Noggin, a BMP antagonist, has previously been shown to increase the number of neurospheres generated using the default pathway and ESCs.[94] BMPs are involved in directing pluripotent cells to an endoderm lineage fate. Although the inclusion of Noggin during the *in vitro* culture of primary primitive NSPCs did increase the number of neurospheres, it did not affect the character of the NSPCs as determined by a battery of gene markers analyzed by RT-PCR. Furthermore, there was no difference on the subsequent mRNA profile between definitive NSPCs with or without Noggin treatment. These data led us to conclusion that the variability in the PB-IPS cells' response to the default pathway is not a result of poor initial neuralization but likely due to incomplete transition from primitive NSPC to definitive NSPCs state.

The NOTCH pathway has been shown to be involved in many aspects of neurodevelopment and its role persists in the adult CNS. [121, 122] Disruption in NOTCH signaling leads to a

methods to optimize iPS cells are underway.

between iPS cell lines compared to NSPCs from ES cell lines. [119]

*4.3.2. Improving the IPS neuralization using NOTCH pathway agonism*

*4.3.1. Neuralization of IPS cells*

Thediscovery ofIPS celltechnology hasmade a significant stride towards realizing thepromise of patient-specific regenerative medicine. IPS cells are somatic cells that have been reprogram‐ med to ectopically express certain transcription factors thatinduce an ES cell-like state, in terms of their differentiation potential and response to *in vitro* culture conditions. [28] This technolo‐ gyallows for a constant andrelativelyeasymethodofgeneratingcells for autologous transplan‐ tation from readily available cell sources such as skin cells. In 2006, Takahashi and Yamanaka usedretrovirus transfected with 24 transcription factors inmouse embryonic fibroblasts (MEF). [101] Through careful elimination they were able to reduce the required genes to four: OCT4, Sox2, KLF4 and c-myc. The expression of these four genes was sufficient to revert the MEFs to an undifferentiated, pluripotent state that was verified using teratoma formation and contribu‐ tion to a chimeric mouse. Since the initial characterization of the iPS cells there has been a tremendous amount of research further expand and refine the technology. The Yamanaka factors have been used to reprogrammed various cells from tissues from a variety of species including mice [101], rats [102], rhesus monkeys [103], and humans [104-107].

The first generation of iPS cell technology is not without its shortcomings and clinical obstacles. The process of reprogramming can be slow and inefficient and includes oncogenic potential of the factors themselves, insertion mutatgenesis, and the risks associated with the use of viral vectors. All of this can contribute to limited clinical translational potential. Fortunately, substantial research has rapidly developed iPS techniques that are viral vector and mutation free. For example, piggyBac transposition is a viral-free system that can be used to deliver the reprogramming factors in both human and mice fibroblasts [108, 109]. A single transposon containing all four iPS transcription factors is introduced to the cells and reprogrammed colonies are selected. IPS cell lines with a single insertion site are identified and transposon is seamlessly excised yielding stable, reprogrammed cell lines that are do not contain any exogenous DNA or have any insertion mutation. Other viral-free and mutation-free methods exist including using lentiviruses [110, 111],an episomal system [112], and the use of recombi‐ nant proteins [113], to generate more clinically relevant iPS cells.

Even with the advancement in iPS cell generation, intrinsic differences between ES and iPS cells exist. IPS and ES are often described as "indistinguishable", however, key differences have been identified. The initial characterization of the iPS cells from Takahashi and Yamanaka noted variation in global gene expression as well as differences in epigenetic characters, specifically histone methylation.[101] Epigenetic memory of iPS cells can influence the differentiation potential and the differentiation of the iPS cells, favouring the identity of the tissue of origin. [114] The impact of epigenetic variability still remains an area of debate and methods to optimize iPS cells are underway.

#### *4.3.1. Neuralization of IPS cells*

**4.3. Induced pluripotent stem cells (IPSC) derived NSPCs**

282 Trends in Cell Signaling Pathways in Neuronal Fate Decision

GiventheaforementionedconcernswithEScells combinedwiththe immunogenicitythatarises from allograft transplantation, the search for a patient-specific and accessible cell source has been a principle endeavor of regenerative medicine. Historically, many techniques and strategies have been developed to accomplish this aim, most notably somatic cell nuclear transfers (SCNT). SCNTis theprocessbywhichthenucleusofthe somatic cellbeingreprogram‐ med is transferred to an enucleated ooctye [99]. This technique became famous in the late 1990s when Dr. Ian Wilmut cloned the first mammal, a sheep named "Dolly". [100] Although there is no obvious mechanism that would preclude SCNT from reprogramming human cells, this technique has yet to be successfully applied in human cells. Furthermore, the requirement for donor ooctyes, combined with an inefficient and technically difficult processes makes SCNT unlikely to be a viable option for clinical application even in the most ideal circumstances.

Thediscovery ofIPS celltechnology hasmade a significant stride towards realizing thepromise of patient-specific regenerative medicine. IPS cells are somatic cells that have been reprogram‐ med to ectopically express certain transcription factors thatinduce an ES cell-like state, in terms of their differentiation potential and response to *in vitro* culture conditions. [28] This technolo‐ gyallows for a constant andrelativelyeasymethodofgeneratingcells for autologous transplan‐ tation from readily available cell sources such as skin cells. In 2006, Takahashi and Yamanaka usedretrovirus transfected with 24 transcription factors inmouse embryonic fibroblasts (MEF). [101] Through careful elimination they were able to reduce the required genes to four: OCT4, Sox2, KLF4 and c-myc. The expression of these four genes was sufficient to revert the MEFs to an undifferentiated, pluripotent state that was verified using teratoma formation and contribu‐ tion to a chimeric mouse. Since the initial characterization of the iPS cells there has been a tremendous amount of research further expand and refine the technology. The Yamanaka factors have been used to reprogrammed various cells from tissues from a variety of species

The first generation of iPS cell technology is not without its shortcomings and clinical obstacles. The process of reprogramming can be slow and inefficient and includes oncogenic potential of the factors themselves, insertion mutatgenesis, and the risks associated with the use of viral vectors. All of this can contribute to limited clinical translational potential. Fortunately, substantial research has rapidly developed iPS techniques that are viral vector and mutation free. For example, piggyBac transposition is a viral-free system that can be used to deliver the reprogramming factors in both human and mice fibroblasts [108, 109]. A single transposon containing all four iPS transcription factors is introduced to the cells and reprogrammed colonies are selected. IPS cell lines with a single insertion site are identified and transposon is seamlessly excised yielding stable, reprogrammed cell lines that are do not contain any exogenous DNA or have any insertion mutation. Other viral-free and mutation-free methods exist including using lentiviruses [110, 111],an episomal system [112], and the use of recombi‐

Even with the advancement in iPS cell generation, intrinsic differences between ES and iPS cells exist. IPS and ES are often described as "indistinguishable", however, key differences have been identified. The initial characterization of the iPS cells from Takahashi and Yamanaka noted variation in global gene expression as well as differences in epigenetic characters,

including mice [101], rats [102], rhesus monkeys [103], and humans [104-107].

nant proteins [113], to generate more clinically relevant iPS cells.

The conceptual outline of how iPS cells may be used in the treatment of SCI is described in figure 3. [28] Many of the *in vitro* neuralization protocols outlined above for generating NSPCs and terminally differentiated neural cells, neurons, astroglia and oligodendrocyte, from ES cells can be applied to iPS cells. iPS cell have been used in models of Parkinson's [110, 115], spinal muscular atrophy (SMA) [116], amyotrophic lateral sclerosis (ALS) [117], and SCI [118]. Although there have been positive results using iPS-derived neural cells for cell-based treatment, the variation in iPS cells has resulted in variability in the safety and neuralization of these cells. A comprehensive evaluation of NSPCs from mouse iPS cell lines generated from various tissue sources and reprogramming methods has shown differences in tumorigenesis between iPS cell lines compared to NSPCs from ES cell lines. [119]

#### *4.3.2. Improving the IPS neuralization using NOTCH pathway agonism*

The variation in neuralization potential between iPS cell lines and their ES cell counterparts represents anareaofregenerativemedicine that,ifnot addressed, couldbe anobstacle toclinical translation. Our lab specifically examined the difference in neuralization potential of multi‐ ple iPS cells lines. [120] We used iPS cells generated using piggyBac transposon technology to eliminate variation that could be attributed to the resurgence of the silenced transgenes or due toinsertionmutations.[108,109]Furthermore,thedefaultpathwayofneuralizationwasutilized to avoid issues associated with the EB formation as an intermediate step in generating NSPCs from a pluripotent cell source. Even with the additional precautions to help generate safe and clinically-relevant cells, there was retention of plurioptency markers as well as persistence of non-ectoderm lineage markers. In order for the potential for iPS technology to be realized, methods to effectively and consistently generate definitive cells must be developed.

We examined the role of BMP antagonism during the initial neuralization of the pluripotent IPS cells. We identited this as a possible area where IPS cell neuralization could be improved. Noggin, a BMP antagonist, has previously been shown to increase the number of neurospheres generated using the default pathway and ESCs.[94] BMPs are involved in directing pluripotent cells to an endoderm lineage fate. Although the inclusion of Noggin during the *in vitro* culture of primary primitive NSPCs did increase the number of neurospheres, it did not affect the character of the NSPCs as determined by a battery of gene markers analyzed by RT-PCR. Furthermore, there was no difference on the subsequent mRNA profile between definitive NSPCs with or without Noggin treatment. These data led us to conclusion that the variability in the PB-IPS cells' response to the default pathway is not a result of poor initial neuralization but likely due to incomplete transition from primitive NSPC to definitive NSPCs state.

The NOTCH pathway has been shown to be involved in many aspects of neurodevelopment and its role persists in the adult CNS. [121, 122] Disruption in NOTCH signaling leads to a reduction in NSPCs while conversely the induction of this pathway promotes NSPCs *in vivo*. [123, 124] The NSPC niche of the SVZ expresses receptors and ligands of the NOTCH pathway. [125] Delta-like ligands (DLL) or Jagged are the principle ligands of the NOTCH pathway, and interact with the membrane-bound NOTCH receptor. The NOTCH intracellular domain (NICD) is cleaved by gamme-sectrase. NICD is translocated to the nucleus to facilitate the transcription of targets such as Hair and enhancer of split, HES genes. [126] Furthermore, NOTCH appears to play a critical role in transition from primitive to definitive neural state. Primitive NSPCs were readily generated in LIF-dependent culture conditions from ES cells as well as NSPCs isolated from E7.5 embyros from NOTCH-deficient sources. However, passage of these cells to a definitive state was disrupted indicating a crucial role for NOTCH. [124, 127] Using these data we hypothesized that agonizing the NOTCH pathway during the neuralization of PB-iPSs would improve the neural character of the definitive NSPCs generated and thus, improve the clinical relevance and translation potential of the cells.

We demonstrated that the addition of recombinant mouse DLL4 to the definitive culture conditions of the default pathway of neuralization improved the generation of definitive NSPCs compare to those cells grown in parallel using standard default conditions. [120] DLL4 was selected to this pathway since DLL4 is most avid ligand for the NOTCH1 receptor.[128] The definitive neurospheres treated with DLL4 produced a greater number of spheres that retained a free-floating phenotype while untreated spheres showed extensive adhesion and signs of differentiation. Also, the mRNA profile of the DLL4 treated dNSPC showed a reduction in pluripotency markers (*Lin28, Nanog, Oct3/4)* as well as reduction or elimination of endodermal markers (*Gata6, Afp*) compared to control cells. This pattern was confirmed at the protein level with immuncytochemistry. The dNSPCs were also cultured in chamberslides matrigel in SFM containing 1% FBS to induce the differentiation of the cells. The differentiation profile of the DLL4-treated definitive NSPCs reflected the ES-derived dNSPCs as well as aNSPCs. Primarily differentiated neural cells were identified following 1 week of differentia‐ tion with mostly GFAP+ astrocytes in the iPS-dNSPC(+DLL4) group compared to non-treated iPS-dNSPCs that were primarily positive for the undifferentiated NSPC marker Nestin or were not labeled by any of the neural markers used. Lastly, the neurons, oligodendrocytes and astrocytes from DLL4-treated dNSPCs were shown to be electrophysiologically functional.

**Figure 3.** A general schematic representation of the generation of iPS cells, the promotion of neural precursor cells, and their use in spinal cord remyelination. A: Following a spinal cord injury, demyelination occurs resulting in vulnera‐ ble axons and impaired CNS function. B: The patient's fibroblasts, or skin cells, are harvested. C: Reprogramming con‐ ditions/factors are introduced to induce self-renewal and pluripotency properties. D: The cells are now iPS cells. E: When full and independent reprogramming is achieved, reprogramming condition/factors are removed. F: Neuraliza‐ tion of the iPS cell to NPCs under minimal conditions. G: Growth factors (i.e., FGF) further differentiate the cells to be‐ come definitive neurospheres. H: iPS cell-derived neural stem/precursor cells can be injected into the injured spinal cord. I: Once transplanted, these cells can differentiate CNS cells such as myelinating oligodendrocytes. J: iPS-NPCmediated remyelination, or by some unidentified neuroprotective effect, can result in functional recovery following

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http://dx.doi.org/10.5772/55054

**5. Transplantation of neural stem/progenitor cells into the injured spinal**

The *in vivo* regenerative and neuroprotective effect of the cell-based treatments must be evaluated in preclinical animal models. The rodent clip compression model of SCI developed in our lab mimics injuries observed in human SCI, in terms of primary and secondary injury processes and, inparticular, with regards to lesion andcavity formation.This creates a situation where we can optimize the cell culture and transplantation paradigm while extensively

spinal cord injury. \* from *Salewski et al., 2010, J. Cell Physiol. 222; 515-521, with permission*

**5.1. Model of SCI and NSPC transplantation**

**cord**

InadditiontotheNOTCHpathway,SonicHedgehog(SHH)andWNTsignalingarebothknown to play roles in neurodevelopment. These pathways have been shown to have independent and interconnected mechanisms of action and there is evidence of considerable crosstalk with NOTCH signaling. SHH expression can be up-regulated in a time dependent profile with the Jagged1 in NSPCs *in vitro*. [125] WNT signaling has been shown to influence the transition of primitive NSPCs to definitive state through the manipulation of *Hes* expression. [129, 130]

iPS cell technology combined with the default pathway of neuralization has tremendous potential to revolutionize the treatment of SCI. Patients could someday use their own skin cells to regenerate and repair their injury (Figure 3).

**Figure 3.** A general schematic representation of the generation of iPS cells, the promotion of neural precursor cells, and their use in spinal cord remyelination. A: Following a spinal cord injury, demyelination occurs resulting in vulnera‐ ble axons and impaired CNS function. B: The patient's fibroblasts, or skin cells, are harvested. C: Reprogramming con‐ ditions/factors are introduced to induce self-renewal and pluripotency properties. D: The cells are now iPS cells. E: When full and independent reprogramming is achieved, reprogramming condition/factors are removed. F: Neuraliza‐ tion of the iPS cell to NPCs under minimal conditions. G: Growth factors (i.e., FGF) further differentiate the cells to be‐ come definitive neurospheres. H: iPS cell-derived neural stem/precursor cells can be injected into the injured spinal cord. I: Once transplanted, these cells can differentiate CNS cells such as myelinating oligodendrocytes. J: iPS-NPCmediated remyelination, or by some unidentified neuroprotective effect, can result in functional recovery following spinal cord injury. \* from *Salewski et al., 2010, J. Cell Physiol. 222; 515-521, with permission*
