**3. Cell populations for SCI treatment**

Even though modern surgical and medical treatment approaches have dramatically decreased mortality rate due to SCI, current clinical techniques provide only modest efficacy in improv‐ ing outcomes for patients, especially during the chronic stages of the disease. Thus, there is a need for new techniques that could improve recovery for SCI patients. Cell replacement strategies use stem cells because of their long-term proliferative abilities and the trophic support they provide to the regenerating tissue. In the following sections we will briefly review different types of stem cells that are being investigated as cell sources in therapeutic ap‐ proaches for SCI.

#### **3.1 Cell types used in SCI therapy**

#### *3.1.1. Mesenchymal stem cells*

Multipotent mesenchymal stem cells (MSCs) are non-hematopoietic multipotential progenitor cells that were initially characterized by Caplan as cells with self-renewing capacity that can give rise to skeleton and other lineages of the mesenchyme through the mesengenic process [30, 31]. However, with the exception of a few stem cells, which are progenitors with "clonal self-renewal and multilineage potential", MSCs represent a non-homogenous population of cells and not all of the cells have the same potential for differentiation. Therefore, the Interna‐ tional Society for Cellular Therapy (ISCT), suggests usage of the term, "Multipotent mesen‐ chymal stromal cells", for the plastic adherent population of cells isolated from stromal tissue of bone. [32] Additionally, in 2006 Dominici et al., from the International Society for Cellular Therapy, defined a minimum set of criteria for MSCs some of which include lacking hemato‐ poietic cell surface markers such as CD34 (endothelial marker) and CD45 (pan-leukocyte marker), and containing cell surface markers such as CD105 (endoglin), CD90 (Thy-1), and CD73 (ecto 5' nucleotidase) and to be able to generate at least three lineages of the mesenchyme [33]. MSCs are primarily isolated from bone in adults but they also reside in adipose tissue, cartilage, synovium, periosteum, muscles and fetal tissue such as placenta and umbilical cord.

There are many studies on the application of MSCs to SCI models with variable reports on the ability of the cells to survive, integrate, and differentiate into neuronal lineages. [34] MSCs from both rats and humans have been shown to be capable of generating neural morphology and expressing neural and astrocytic markers *in vitro*. However, they have similar differen‐ tiation profiles only when transplanted into an embryonic or developing CNS. When trans‐ planted into adult rat CNS, neither rat nor human MSCs were able to differentiate into neuronal cells. Mostly, they generated perivascular macrophages in the rat brain. [35-37]

There are other studies in which transplanted MSCs were tracked in rat or mouse injury models through GFP or electromagnetic tagging coupled with immunochemistry and MR imaging. Even though both types of studies report functional improvements, only a fraction of cells are reported to show expression of neuronal or astrocytic markers and only a very small number of cells are reported as having differentiated into neurons. [37-39] There are differing reports on the extent of MSC transplantation benefits in functional recovery. MSCs have shown to be effective at 4 weeks up to 1 year after the injury [41], depending on the timing of transplantation from the time of injury [40] to 1 week [37, 38, 41], or 3 months post injury [42]. Other studies show lack of any functional benefit observable through the Basso Beattie Bresnahan (BBB) open field locomotor scoring system. [43] However, all of the mentioned studies report on the ability of MSC transplants to preserve the injured tissue, both gray and white matter, and to provide micro environmental cues supportive of axonal growth, rather than replacing lost cells. [42, 43] Given the inconsistency seen in the results of transplanting MSCs into SCI models in rats, mice and primates, it is necessary to have a better understanding of the type of cells in the MSC population that are being transplanted[44] and investigate all the possible mechanisms behind the functional recovery observed in these models.

#### *3.1.2. Schwann cells*

Two main aims of cell-based treatment for SCI are replacing lost or injured cells such as oligodendrocytes and neurons, or providing the cells with a microenvironment that supports or enhances the neuroprotective and regenerative ability of cells within the lesion. Stem and progenitor cells, and Schwann cells are examples of cells that are being used in studies to replace lost cells. Additionally, many studies have shown that these cells have the ability to remyelinate axons in the injury site while also providing them with supportive growth and

Even though modern surgical and medical treatment approaches have dramatically decreased mortality rate due to SCI, current clinical techniques provide only modest efficacy in improv‐ ing outcomes for patients, especially during the chronic stages of the disease. Thus, there is a need for new techniques that could improve recovery for SCI patients. Cell replacement strategies use stem cells because of their long-term proliferative abilities and the trophic support they provide to the regenerating tissue. In the following sections we will briefly review different types of stem cells that are being investigated as cell sources in therapeutic ap‐

Multipotent mesenchymal stem cells (MSCs) are non-hematopoietic multipotential progenitor cells that were initially characterized by Caplan as cells with self-renewing capacity that can give rise to skeleton and other lineages of the mesenchyme through the mesengenic process [30, 31]. However, with the exception of a few stem cells, which are progenitors with "clonal self-renewal and multilineage potential", MSCs represent a non-homogenous population of cells and not all of the cells have the same potential for differentiation. Therefore, the Interna‐ tional Society for Cellular Therapy (ISCT), suggests usage of the term, "Multipotent mesen‐ chymal stromal cells", for the plastic adherent population of cells isolated from stromal tissue of bone. [32] Additionally, in 2006 Dominici et al., from the International Society for Cellular Therapy, defined a minimum set of criteria for MSCs some of which include lacking hemato‐ poietic cell surface markers such as CD34 (endothelial marker) and CD45 (pan-leukocyte marker), and containing cell surface markers such as CD105 (endoglin), CD90 (Thy-1), and CD73 (ecto 5' nucleotidase) and to be able to generate at least three lineages of the mesenchyme [33]. MSCs are primarily isolated from bone in adults but they also reside in adipose tissue, cartilage, synovium, periosteum, muscles and fetal tissue such as placenta and umbilical cord. There are many studies on the application of MSCs to SCI models with variable reports on the ability of the cells to survive, integrate, and differentiate into neuronal lineages. [34] MSCs from both rats and humans have been shown to be capable of generating neural morphology and expressing neural and astrocytic markers *in vitro*. However, they have similar differen‐ tiation profiles only when transplanted into an embryonic or developing CNS. When trans‐

neuroprotective factors. [1, 19, 27-29]

276 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**3.1 Cell types used in SCI therapy**

*3.1.1. Mesenchymal stem cells*

proaches for SCI.

**3. Cell populations for SCI treatment**

Schwann cells are the myelinating cells in the peripheral nervous system. Since the first transplantation of SCs into SCI by Duncan et al. in 1981 [34], these cells have been used in many different SCI models and have been shown to be able to remyelinate axons and provide a permissive environment for them to regenerate and grow. Binge et al. [45] showed that SCs also have a small but significant effect on increasing functional recovery. However, their remyelinating and regenerating ability does not go beyond the growth-permissive surface, which explains why there is only a slight improvement in functional recovery. [3, 44-46] Additionally, it has been shown that axons in the corticospinal tract (CST) remain unaffected by SC transplants. [34] Also, they have been shown to generate a more active astrocytic response compared to other cell types such as NPCs which results in less efficient integration of these cells into the injured spinal cord. [34] As a result, the clinical application of SCs alone for SCI treatment appears extremely limited.

To overcome this limitation, many combinatorial strategies have evolved which associate SCs with growth factors or bioengineering scaffolds and other cells types to enhance recovery. [4, 47-50] Additionally, new sources for SCs are being investigated such as skin derived precursor cells from the dermis of the skin [51-54] and MSCs. [55-58] However, it is yet to be proven whether these cells hold any advantage in treating SCI compared with SCs that are derived from the PNS.

#### *3.1.3. Olfactory ensheathing cells*

OECs are glial cells derived from the olfactory bulb or lamina propria of the olfactory mucosa and are termed olfactory nerve Schwann cells due to morphological similarities to SCs. Having a lifelong capacity to proliferate and being able to facilitate passage of new axons from 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 injured CNS environment to produce functional synapses. [3, 60, 61]

(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

Regenerative Medicine

279

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

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

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

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,

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

with positive identification for both glutamatergic and motor neurons. [88, 89]

tissue to generate the renewable cell population.

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

and environmental signals, with serum-free media.

yield cells that can differentiate to neurons and glial cells.

*4.2.1. NSPC generation with EB intermediate*

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

#### *3.1.4. Neural stem/progenitor cells*

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 to derive NSPCs have been studied.
