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

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

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

284 Trends in Cell Signaling Pathways in Neuronal Fate Decision

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.

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).

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 characterizing and evaluating the safety and efficiency of our NSPCs in a clinically relevant small animal model prior to their implementation into larger animal models or a clinical trial.

In general, our SCI model involves injured rats that receive a clip compression injury of spinal cord either at the cervical or thoracic level. [19, 65] The model characteristics, histopathology, assessment of axonal integrity, molecular examination of axonal structure, and behavioural assessment have been extensively characterized. [131-134] This model of moderately severe SCI, results in a central cavitation and loss of 80% of axons in the spinal cord white matter, demyelination of the surviving axons in the residual subpial rim and spastic paraparesis. Following acute (2 week) or chronic (6 weeks) SCI, we have administered four intraspinal injections of aNSPCs in growth media to the rats at locations 2mm caudal and 2mm rostral to the injury site. To enhance the survival of the transplanted cells, growth factors [PDGF-AA bFGF and EGF] in a solution containing aCSF, BSA (100 µg/ml) and gentamycine (50 µg/ml) has been infused intrathecally to the area of transplantation for 7 days using a microcatheter connected to an osmotic minipump. A battery of behavioural tests, histological outcomes and electrophysiological measures are used to determine the therapeutic effect aNPC transplan‐ tation.

#### **5.2. Effect of transplant on subacute and chronic injury**

In our study of subacute intervention following SCI, transplantation was delayed to two weeks following injury. [19] The animals were examined using a number of measures of locomotor function, including the BBB scale, ladder walk analysis and gait/coordination assessment, for eight weeks post-SCI. At the eight week endpoint of the experiment, the animals were euthanized and their tissue collected for histological and electron microscopy.

> **Figure 4.** YFP-NPCs in the spinal cord of a subacutely injured rat 8 weeks after transplantation. A, A confocal image from a longitudinal section of an injured spinal cord taken from the dorsal spinal cord of a transplanted rat above the central cavity. A low-magnified image shows the extent of YFP-NPC survival within the injured spinal cord 8 weeks after transplantation. Grafted YFP-NPCs (green) were dispersed along the rostrocaudal axis of the spinal cord 5 mm away from the implantation sites (\*). YFP-NPCs also migrated to the contralateral site of the spinal cord to a lesser extent. Double labeling with the neuronal marker betaIII tubulin (Tuj1) showed that YFP-NPCs reside predominantly in the white-matter area (A–D). Our histological data showed no signs of tumor formation in the spinal cord. E, Confocal image of a transverse section of the spinal cord from a transplanted rat (8 weeks after transplantation) showing the distribution of YFP-NPCs in the lateral columns. F, G, YFP cells mainly showed multipolar morphology and extended numerous branches in the white-matter tissue along the length of axons. WM, White matter; GM, gray matter. \* from

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The successful implementation of the NSPC therapy in the subacute model is a key step forward in spinal cord regenerative medicine. It has a relevant therapeutic window, although narrow, and requires intervention shortly after the initial injury. The chronic injured popula‐ tion represents a large section of the SCI community and currently many of the proposed treatments cannot be used to repair their injuries. The chronic injured environment is charac‐ terized by many additional challenges including cell loss, a cystic cavity and the inhibitory

*Karimi-Abdolrezaee et al, 2006, J Neurosci 26:3377-3389, with permission*

influences of the glial scar. [65]

The transplanted NSPCs had substantial survival (~40%) in the spinal cord when the growth factor mini osmotic pump was used and these cells demonstrated multipotency. In contrast to their *in vitro* differentiation profile, the transplanted cells mostly became oligodendrocytes in the injured spinal cord. In fact, the cells preferentially migrated to the white matter tract and showed extensive myelination. ([19]; Figure 4) The role of myelination will be fully addressed when we discuss potential mechanisms of neuroprotection/neurorepair. Behavioral and functional benefits from aNSPC engraftment were observed. There was significant locomotor recovery compared to the injured control groups. The BBB is a 21 point scale that is used to evaluate hindlimb locomotion in an open field. [43] Immediately following the SCI, all rats were fully paraplegic and exhibited no hindlimb function. The control rats were able to recover to a BBB score of 8, denoting plantar placement of the paw without weight support. The experimental group that received the aNSPC treatment was able to reach a score of 10 on the BBB. Although a 2-point increase may seem negligible, the BBB is a non-linear scale and these animals show occasional weight supported plantar stepping which is a substantial functional improvement. Furthermore, the animals that received the transplanted cells show improve‐ ment on other independent behavioral tests including grid walk and footprint tests. Our results show that the improved recovery of locomotor function in the transplanted groups was a result of the effects of the aNPCs since there was no significant difference in the walking ability of the plain injured or sham controls in all behavioral tests used.

characterizing and evaluating the safety and efficiency of our NSPCs in a clinically relevant small animal model prior to their implementation into larger animal models or a clinical trial.

In general, our SCI model involves injured rats that receive a clip compression injury of spinal cord either at the cervical or thoracic level. [19, 65] The model characteristics, histopathology, assessment of axonal integrity, molecular examination of axonal structure, and behavioural assessment have been extensively characterized. [131-134] This model of moderately severe SCI, results in a central cavitation and loss of 80% of axons in the spinal cord white matter, demyelination of the surviving axons in the residual subpial rim and spastic paraparesis. Following acute (2 week) or chronic (6 weeks) SCI, we have administered four intraspinal injections of aNSPCs in growth media to the rats at locations 2mm caudal and 2mm rostral to the injury site. To enhance the survival of the transplanted cells, growth factors [PDGF-AA bFGF and EGF] in a solution containing aCSF, BSA (100 µg/ml) and gentamycine (50 µg/ml) has been infused intrathecally to the area of transplantation for 7 days using a microcatheter connected to an osmotic minipump. A battery of behavioural tests, histological outcomes and electrophysiological measures are used to determine the therapeutic effect aNPC transplan‐

In our study of subacute intervention following SCI, transplantation was delayed to two weeks following injury. [19] The animals were examined using a number of measures of locomotor function, including the BBB scale, ladder walk analysis and gait/coordination assessment, for eight weeks post-SCI. At the eight week endpoint of the experiment, the animals were

The transplanted NSPCs had substantial survival (~40%) in the spinal cord when the growth factor mini osmotic pump was used and these cells demonstrated multipotency. In contrast to their *in vitro* differentiation profile, the transplanted cells mostly became oligodendrocytes in the injured spinal cord. In fact, the cells preferentially migrated to the white matter tract and showed extensive myelination. ([19]; Figure 4) The role of myelination will be fully addressed when we discuss potential mechanisms of neuroprotection/neurorepair. Behavioral and functional benefits from aNSPC engraftment were observed. There was significant locomotor recovery compared to the injured control groups. The BBB is a 21 point scale that is used to evaluate hindlimb locomotion in an open field. [43] Immediately following the SCI, all rats were fully paraplegic and exhibited no hindlimb function. The control rats were able to recover to a BBB score of 8, denoting plantar placement of the paw without weight support. The experimental group that received the aNSPC treatment was able to reach a score of 10 on the BBB. Although a 2-point increase may seem negligible, the BBB is a non-linear scale and these animals show occasional weight supported plantar stepping which is a substantial functional improvement. Furthermore, the animals that received the transplanted cells show improve‐ ment on other independent behavioral tests including grid walk and footprint tests. Our results show that the improved recovery of locomotor function in the transplanted groups was a result of the effects of the aNPCs since there was no significant difference in the walking ability of

euthanized and their tissue collected for histological and electron microscopy.

tation.

**5.2. Effect of transplant on subacute and chronic injury**

286 Trends in Cell Signaling Pathways in Neuronal Fate Decision

the plain injured or sham controls in all behavioral tests used.

**Figure 4.** YFP-NPCs in the spinal cord of a subacutely injured rat 8 weeks after transplantation. A, A confocal image from a longitudinal section of an injured spinal cord taken from the dorsal spinal cord of a transplanted rat above the central cavity. A low-magnified image shows the extent of YFP-NPC survival within the injured spinal cord 8 weeks after transplantation. Grafted YFP-NPCs (green) were dispersed along the rostrocaudal axis of the spinal cord 5 mm away from the implantation sites (\*). YFP-NPCs also migrated to the contralateral site of the spinal cord to a lesser extent. Double labeling with the neuronal marker betaIII tubulin (Tuj1) showed that YFP-NPCs reside predominantly in the white-matter area (A–D). Our histological data showed no signs of tumor formation in the spinal cord. E, Confocal image of a transverse section of the spinal cord from a transplanted rat (8 weeks after transplantation) showing the distribution of YFP-NPCs in the lateral columns. F, G, YFP cells mainly showed multipolar morphology and extended numerous branches in the white-matter tissue along the length of axons. WM, White matter; GM, gray matter. \* from *Karimi-Abdolrezaee et al, 2006, J Neurosci 26:3377-3389, with permission*

The successful implementation of the NSPC therapy in the subacute model is a key step forward in spinal cord regenerative medicine. It has a relevant therapeutic window, although narrow, and requires intervention shortly after the initial injury. The chronic injured popula‐ tion represents a large section of the SCI community and currently many of the proposed treatments cannot be used to repair their injuries. The chronic injured environment is charac‐ terized by many additional challenges including cell loss, a cystic cavity and the inhibitory influences of the glial scar. [65]

We noted that when aNSPCs were transplanted at 6 weeks post-SCI, the chronically injured spinal cord was not an hospitable environment. To address this, we examined methods to modify the SCI environment to create a permissive environment. Our data suggested that expression of chondroitin sulfate proteoglycans (CSPG) reduces the survival and thus, the therapeutic potential of our aNPC treatment. [65] Chondroitinase ABC (ChABC) was admin‐ istered to the chronic SCI environment to breakdown CSPGs that were upregulated as a result of the injury. We then analyzed the synergistic effect of the transplanted aNSPCs with ChABC and growth factor pump on the repair and plasticity of the chronically injured spinal cord. Survival, integration and migration of the transplanted cells in the chronic SCI with ChABC was extensive compare to treatment without ChABC and growth factors where survival was low and the transplanted cells remained at the injection site. ([65]; Figure 5) The differentiation potential of the aNSPCs was similar to that observed in the subacute injury/transplanted paradigm with the majority of cells displaying oligodendrocyte markers. There was also improvement observed at the functional locomotor level. The animals that received the combinatorial treatment of ChABC followed by aNSPCs and GF pump had a significant improvement on the BBB scale as well as with ladder-walk analysis. These behavioral im‐ provements were also linked to neuroanatomical changes associated with the aNPC therapy. This is the first successful application of an aNSPC-based treatment for chronic SCI and represents a critical step forward improving the lives of patient currently living with SCI.

compared to vehicle treatment (1.40.4mm)(p0.001, Student's t test). We also found a 4.6-fold increase in the total length of rostrocaudal distribution of NPCs in ChABC-treated rats (13.02mmin the best case, average 8.321.5 mm) compared to vehicle-treated rats (2.24mminthe best case, average 1.820.5mm)(p0.005, Student's t test). *\* from* Kari‐

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Although our lab and others have clearly shown that cell-based therapies for SCI can be beneficial, the mechanisms by which the benefits are achieved still remains an area of debate. Trophic factor support [40, 135-137] and remyelination [19, 27, 65] as well as other mechanisms including plasticity and axon integrity have been suggested as possible mechanisms of

Trophin delivery as a potential mechanism of benefit in cell-based treatments, such as the use of bone marrow stromal cells (BMSC) following trauma, was first proposed in response to evidence that there was a degree of neuroprotection despite a lack of survival of transplanted cells. [40, 135-137] Trophic factors have been shown to have apoptotic effects[138], enhance axonal regrowth[139], promote endogenous remyelination[140, 141], and neuronal plastici‐ ty[142, 143]. BMSCs as well as aNSPCs have been shown by our lab and others to produces these trophic factors *in vitro*, however little evidence to show their involvement *in vivo* has been

To study trophin production and involvement *in vivo*, we examine both brain- and spinalderived NPCs, compared them to BMSCs, and transplanted them into injured rat spinal cords. [145] Candidate trophic factor (NGF, BDNF, NT-3, NT-4/5, GDNF, CNTF, PDGF-A, EGF, bFGF, LIF, IGF-1, GGF2, TGF-b1, VEGF-A) expression was examined in spinal tissue homo‐ genates as well as specifically in the transplanted cell population by FAC sorting for the eGFP labeled cells. The neurotrophin levels were increased following SCI and the transplantation of aNSPC with GF pump further elevated specific trophic factors, in particular, GDNF, LIF and bFGF. Furthermore, the analysis of the FAC sorted transplanted population of aNSPC showed increased neurotrophin production, specifically CNTF, EGF and bFGF. The study was the first to shown that both changes in the host tissue as well as the transplanted aNSPCs can influence trophic factor levels *in vivo*. Although further research is required to determine if trophic factor support is the sole mechanism that conveys neuro-protection or neuro-repair, our research does shows that it is a likely contributing factor to the functional improvements observed.

Myelin is a critical component of the CNS and is required for its proper functioning. Myelin is needed for the precise molecular organization of the axon, particularly with regards to nodal architecture. The arrangement of Na+ channels, K+ channels and contactin-associated protein at and around the nodes of Ranvier is closely linked to myelin, and a disruption in myelination results in loss of nodal organization. Furthermore, following SCI there is extensive loss of white matter and often otherwise functional axons are bare and rendered useless. In this situation,

*mi-Abdolrezaee et al, 2010, J Neurosci 30:1657-1676., with permission*

recovery with cell-based treatments for SCI.

*5.3.1. Trophic factor support*

shown. [144]

*5.3.2. Remyelination*

**5.3. Possible mechanism of neurorepair/neuroprotection**

**Figure 5.** ChABC treatment greatly optimizes NPC transplantation in the chronically injured spinal cord. A–D, Confocal images of longitudinal and cross sections of chronically injured spinal cord transplanted with NPCs at 9 weeks after transplantation show a significant increase in the number of surviving NPCs (green) in ChABC-treated spinal cord (C,D) compared to the vehicle-treated spinal cords (A, B). D, The majority of NPCs integrated within white matter areas in‐ cluding dorsal, lateral, and ventral columns. Quantification of transplant volume revealed a 6.2-fold increase in ChABC-treated rats (n6, 3.70.79mm3) compared to vehicle-treated rats (n6, 0.60.24mm3) (p0.01, Student's t test). NPC cell survival analysis also showed a 5.7-fold increase in number of surviving NPCs (YFP/DAPI-positive cells) in ChABC-treated rats (28.256.77%) relative to the vehicle-treated ones (4.91.96%) (p0.01, Student's t test). ChABC treat‐ ment resulted in a significant increase in rostral and caudal migration of NPCs from the injection sites (4.20.50mm) compared to vehicle treatment (1.40.4mm)(p0.001, Student's t test). We also found a 4.6-fold increase in the total length of rostrocaudal distribution of NPCs in ChABC-treated rats (13.02mmin the best case, average 8.321.5 mm) compared to vehicle-treated rats (2.24mminthe best case, average 1.820.5mm)(p0.005, Student's t test). *\* from* Kari‐ *mi-Abdolrezaee et al, 2010, J Neurosci 30:1657-1676., with permission*

#### **5.3. Possible mechanism of neurorepair/neuroprotection**

Although our lab and others have clearly shown that cell-based therapies for SCI can be beneficial, the mechanisms by which the benefits are achieved still remains an area of debate. Trophic factor support [40, 135-137] and remyelination [19, 27, 65] as well as other mechanisms including plasticity and axon integrity have been suggested as possible mechanisms of recovery with cell-based treatments for SCI.

#### *5.3.1. Trophic factor support*

We noted that when aNSPCs were transplanted at 6 weeks post-SCI, the chronically injured spinal cord was not an hospitable environment. To address this, we examined methods to modify the SCI environment to create a permissive environment. Our data suggested that expression of chondroitin sulfate proteoglycans (CSPG) reduces the survival and thus, the therapeutic potential of our aNPC treatment. [65] Chondroitinase ABC (ChABC) was admin‐ istered to the chronic SCI environment to breakdown CSPGs that were upregulated as a result of the injury. We then analyzed the synergistic effect of the transplanted aNSPCs with ChABC and growth factor pump on the repair and plasticity of the chronically injured spinal cord. Survival, integration and migration of the transplanted cells in the chronic SCI with ChABC was extensive compare to treatment without ChABC and growth factors where survival was low and the transplanted cells remained at the injection site. ([65]; Figure 5) The differentiation potential of the aNSPCs was similar to that observed in the subacute injury/transplanted paradigm with the majority of cells displaying oligodendrocyte markers. There was also improvement observed at the functional locomotor level. The animals that received the combinatorial treatment of ChABC followed by aNSPCs and GF pump had a significant improvement on the BBB scale as well as with ladder-walk analysis. These behavioral im‐ provements were also linked to neuroanatomical changes associated with the aNPC therapy. This is the first successful application of an aNSPC-based treatment for chronic SCI and represents a critical step forward improving the lives of patient currently living with SCI.

288 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**Figure 5.** ChABC treatment greatly optimizes NPC transplantation in the chronically injured spinal cord. A–D, Confocal images of longitudinal and cross sections of chronically injured spinal cord transplanted with NPCs at 9 weeks after transplantation show a significant increase in the number of surviving NPCs (green) in ChABC-treated spinal cord (C,D) compared to the vehicle-treated spinal cords (A, B). D, The majority of NPCs integrated within white matter areas in‐ cluding dorsal, lateral, and ventral columns. Quantification of transplant volume revealed a 6.2-fold increase in ChABC-treated rats (n6, 3.70.79mm3) compared to vehicle-treated rats (n6, 0.60.24mm3) (p0.01, Student's t test). NPC cell survival analysis also showed a 5.7-fold increase in number of surviving NPCs (YFP/DAPI-positive cells) in ChABC-treated rats (28.256.77%) relative to the vehicle-treated ones (4.91.96%) (p0.01, Student's t test). ChABC treat‐ ment resulted in a significant increase in rostral and caudal migration of NPCs from the injection sites (4.20.50mm)

Trophin delivery as a potential mechanism of benefit in cell-based treatments, such as the use of bone marrow stromal cells (BMSC) following trauma, was first proposed in response to evidence that there was a degree of neuroprotection despite a lack of survival of transplanted cells. [40, 135-137] Trophic factors have been shown to have apoptotic effects[138], enhance axonal regrowth[139], promote endogenous remyelination[140, 141], and neuronal plastici‐ ty[142, 143]. BMSCs as well as aNSPCs have been shown by our lab and others to produces these trophic factors *in vitro*, however little evidence to show their involvement *in vivo* has been shown. [144]

To study trophin production and involvement *in vivo*, we examine both brain- and spinalderived NPCs, compared them to BMSCs, and transplanted them into injured rat spinal cords. [145] Candidate trophic factor (NGF, BDNF, NT-3, NT-4/5, GDNF, CNTF, PDGF-A, EGF, bFGF, LIF, IGF-1, GGF2, TGF-b1, VEGF-A) expression was examined in spinal tissue homo‐ genates as well as specifically in the transplanted cell population by FAC sorting for the eGFP labeled cells. The neurotrophin levels were increased following SCI and the transplantation of aNSPC with GF pump further elevated specific trophic factors, in particular, GDNF, LIF and bFGF. Furthermore, the analysis of the FAC sorted transplanted population of aNSPC showed increased neurotrophin production, specifically CNTF, EGF and bFGF. The study was the first to shown that both changes in the host tissue as well as the transplanted aNSPCs can influence trophic factor levels *in vivo*. Although further research is required to determine if trophic factor support is the sole mechanism that conveys neuro-protection or neuro-repair, our research does shows that it is a likely contributing factor to the functional improvements observed.

#### *5.3.2. Remyelination*

Myelin is a critical component of the CNS and is required for its proper functioning. Myelin is needed for the precise molecular organization of the axon, particularly with regards to nodal architecture. The arrangement of Na+ channels, K+ channels and contactin-associated protein at and around the nodes of Ranvier is closely linked to myelin, and a disruption in myelination results in loss of nodal organization. Furthermore, following SCI there is extensive loss of white matter and often otherwise functional axons are bare and rendered useless. In this situation, restoring the myelination of these axons can have large effects on the overall outcome and locomotor function since relatively few spared axons can have a tremendous effect on improving neural outcomes.

robust expression of MBP (red) in the white matter of an injured spinal cord. Cell bodies of donor cells are surrounded with MBP. Triple-labeling experiments on longitudinal (D–G) and cross (H–K ) sections of spinal cord white matter showed that MBP-expressing YFP-NPCs ensheathed the injured axons (identified by NF200; blue). These images (D–G) clearly show the oligodendrocyte morphology of one grafted YFP cell (arrowheads) that extends its processes and ex‐ presses MBP around an injured axon and the close proximity of these cells with newly myelinated axons. L, M, Images taken by deconvolution confocal microscopy show a higher-magnification image confirming axonal ensheathment of MBP-expressing YFP-NPCs around the injured axons. \*from *Karimi-Abdolrezaee et al, 2006, J Neurosci 26:3377-3389,*

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Although exogenous myelination has been proposed as a mechanism for aNSPC-mediated recovery, potential endogenous myelination cannot be ruled out. There is still extensive research required to delineate the precise role of remyelination and specifically the role of

In addition to the roles of trophic support and myelination, other mechanisms have been proposed. It is possible that effects on axon integrity and neuronal plasticity play key roles in SCI recovery. We noted positive effects in these areas following our combinatorial strategy of ChABC and aNSPC in the chronically injured spinal cord.[65] Cortiospinal tract (CST) labeling using PKC-g and anterograde tracing showed evidence of enhanced axonal integrity and collateral spouting. This phenomena was seen only at the local level with long-distance CST regeneration beyond the lesion not observed. Promotion of plasticity of serontonergic (5HT) fibers was observed with ChABC and aNSPC therapy as noted by a significant increase in its immunointensity rostral to the injury epicenter. The 5HT positive fibers were observed in areas of aNSPC engraftment. Many mechanisms are likely working in concert to elicit the behavioral and functional outcomes observed by our lab and others after application of aNSPC therapy.

Spinal cord injury pathology creates a situation where cell-based interventions are attractive as potential treatments to promote repair and regeneration. There are a variety of cell sources available for this aim, however, in our research experience, NSPCs are the most likely to provide lasting and appropriate neurological recovery. Both adult brain and spinal tissue are sources of NSPCs for transplantation along with the pluripotent cells (ESC and iPSC) when differentiated *in vitro* to a restricted NSPC identity. Once transplanted into the injured spinal cord, the spinal niche directs the NSPCs to terminally differentiated neural cell types, both neuronal and glial. Our work suggests remyelination via oligodendrocytes is the principal mechanism of recovery although trophic support as well as neuronal lineages that are created are other possible mechanisms by which NSPC treatment is beneficial in SCI. Our results with chronic SCI, which is characterized by cavitation and glial scarring, has shown that stem cell therapy alone may be of limited benefit. Thus, combinatorial stem cell approaches with bioengineered strategies, such as use of chondroitinase, will be a key area of future research

*with permission*

*5.3.3. Other mechanisms*

**6. Conclusions**

remyelination induced by transplanted cells.

in the field of neuro-regenerative medicine.

These data suggest that the remyelination could be the mechanism by which aNSPC trans‐ plantation is able to repair the injured spinal cord. Following our subacute intervention as well as chronic injury with combinatorial therapy, we observed migration of engrafted cells to white matter tracts, mature oligodendrocytic differentiation and expression of myelin basic protein (MBP) by these cells associated with axons. ([19]; Figure 6) Furthermore, the exogenous myelination was confirmed by electron microscopy to form multilayer compact myelin around axons and to restore the nodal architecture as shown by immune labeling for Kv1.2 and panKa.

**Figure 6. Exogenous remyelination following subscute SCI with aNSPC transplantation:** YFP-NPC-derived oligo‐ dendrocytes generate MBP and ensheath the injured axons of the spinal cord. A–C, Confocal images of longitudinal sections of an injured spinal cord 8 weeks after transplantation. The area grafted with YFP-NPCs (green) displays a

robust expression of MBP (red) in the white matter of an injured spinal cord. Cell bodies of donor cells are surrounded with MBP. Triple-labeling experiments on longitudinal (D–G) and cross (H–K ) sections of spinal cord white matter showed that MBP-expressing YFP-NPCs ensheathed the injured axons (identified by NF200; blue). These images (D–G) clearly show the oligodendrocyte morphology of one grafted YFP cell (arrowheads) that extends its processes and ex‐ presses MBP around an injured axon and the close proximity of these cells with newly myelinated axons. L, M, Images taken by deconvolution confocal microscopy show a higher-magnification image confirming axonal ensheathment of MBP-expressing YFP-NPCs around the injured axons. \*from *Karimi-Abdolrezaee et al, 2006, J Neurosci 26:3377-3389, with permission*

Although exogenous myelination has been proposed as a mechanism for aNSPC-mediated recovery, potential endogenous myelination cannot be ruled out. There is still extensive research required to delineate the precise role of remyelination and specifically the role of remyelination induced by transplanted cells.

#### *5.3.3. Other mechanisms*

restoring the myelination of these axons can have large effects on the overall outcome and locomotor function since relatively few spared axons can have a tremendous effect on

These data suggest that the remyelination could be the mechanism by which aNSPC trans‐ plantation is able to repair the injured spinal cord. Following our subacute intervention as well as chronic injury with combinatorial therapy, we observed migration of engrafted cells to white matter tracts, mature oligodendrocytic differentiation and expression of myelin basic protein (MBP) by these cells associated with axons. ([19]; Figure 6) Furthermore, the exogenous myelination was confirmed by electron microscopy to form multilayer compact myelin around axons and to restore the nodal architecture as shown by immune labeling for Kv1.2 and panKa.

**Figure 6. Exogenous remyelination following subscute SCI with aNSPC transplantation:** YFP-NPC-derived oligo‐ dendrocytes generate MBP and ensheath the injured axons of the spinal cord. A–C, Confocal images of longitudinal sections of an injured spinal cord 8 weeks after transplantation. The area grafted with YFP-NPCs (green) displays a

improving neural outcomes.

290 Trends in Cell Signaling Pathways in Neuronal Fate Decision

In addition to the roles of trophic support and myelination, other mechanisms have been proposed. It is possible that effects on axon integrity and neuronal plasticity play key roles in SCI recovery. We noted positive effects in these areas following our combinatorial strategy of ChABC and aNSPC in the chronically injured spinal cord.[65] Cortiospinal tract (CST) labeling using PKC-g and anterograde tracing showed evidence of enhanced axonal integrity and collateral spouting. This phenomena was seen only at the local level with long-distance CST regeneration beyond the lesion not observed. Promotion of plasticity of serontonergic (5HT) fibers was observed with ChABC and aNSPC therapy as noted by a significant increase in its immunointensity rostral to the injury epicenter. The 5HT positive fibers were observed in areas of aNSPC engraftment. Many mechanisms are likely working in concert to elicit the behavioral and functional outcomes observed by our lab and others after application of aNSPC therapy.

#### **6. Conclusions**

Spinal cord injury pathology creates a situation where cell-based interventions are attractive as potential treatments to promote repair and regeneration. There are a variety of cell sources available for this aim, however, in our research experience, NSPCs are the most likely to provide lasting and appropriate neurological recovery. Both adult brain and spinal tissue are sources of NSPCs for transplantation along with the pluripotent cells (ESC and iPSC) when differentiated *in vitro* to a restricted NSPC identity. Once transplanted into the injured spinal cord, the spinal niche directs the NSPCs to terminally differentiated neural cell types, both neuronal and glial. Our work suggests remyelination via oligodendrocytes is the principal mechanism of recovery although trophic support as well as neuronal lineages that are created are other possible mechanisms by which NSPC treatment is beneficial in SCI. Our results with chronic SCI, which is characterized by cavitation and glial scarring, has shown that stem cell therapy alone may be of limited benefit. Thus, combinatorial stem cell approaches with bioengineered strategies, such as use of chondroitinase, will be a key area of future research in the field of neuro-regenerative medicine.
