Pluripotent stem cells - A review of the current status in neural regeneration
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.84338
Source of Support: None, Conflict of Interest: None
Pharmacological or neurosurgical therapies currently in practice to treat the damage in various neurodegenerative disorders are not efficient in preventing progression or cure of these progressive neurodegenerative processes. Recently, a new approach, cell therapy using stem cell, is being evaluated. However, the use of this therapy in the treatment of these neurological diseases is highly restricted, mainly owing to several technical difficulties and limitations. The strategy of isolation and characterization of neural stem cells from various sources will probably provide a major impetus and open up an interesting, novel therapeutic modality for several neurodegenerative disorders. The high regenerative potential of damaged neural tissues suggests that various embryonic/adult sources serve as a proxy for neural stem cells for cell-based therapy.
Keywords: Neural stem cells, neurological disorders, pluripotent stem cells, regeneration
The discovery of stem cells in central and peripheral nervous system is relatively a recent event. Neural stem cells' (NSCs) generation is a lifelong event and is by the process of neurogenesis. New neurons are generated from subventricular zone (SVZ) of lateral ventricles and dentate gyrus of the hippocampus. SVZ is a region with the highest number of NSCs and these cells have the capacity to generate both neural and glial cells. Cell therapy using these cells has imbibed a ray of hope in the effective management of several neurological disorders.
According to their developmental status, stem cells generally are divided into embryonic and adult stem cells.
Embryonic stem cells (ESCs) are capable of giving rise to all the three derivatives of primary germ layers: ectoderm, endoderm, and mesoderm [Figure 1]. Human ESCs are derived from the inner cell mass of blastocyst of discarded and non-transferred human embryos. NSCs derived from embryo can differentiate into neurons, astrocytes and oligodendrocytes and are capable of forming mature progeny  and also dopamine neurons  both in vitro and in vivo. In comparison to the adult stem cells, ESCs are clinically very effective. However, the limitations of ESCs include: somatic cell nuclear transfer (SCNT), not all of the donor cell's genetic information is transferred, and the resulting hybrid cells retain those mitochondrial structures which originally belonged to the egg. More than 120 ESC lines have been reported worldwide. As the lineage passes, ESC lines tend to degrade and may not be useful for research.  Some of the cell lines are genetically identical to each other, thus creating ethical issues and restricting the number of available cell lines for research to 11. Moreover, these cell lines are grown on mouse feeder layers and are not suitable for clinical applications as there is always an associated risk of viral transmission. In clinical therapy, large number of eggs required for somatic cell nuclear transfer and the human embryos required as a source of ESCs are extremely sensitive ethical issues. Currently, in India, the spare human embryos from in vitro fertilization (IVF) programmers are permitted for research activity. 
Adult stem cells (ASCs) are found in all the body tissues and function in tissue homeostasis and repair. They can be isolated and propagated from bone marrow (BM), liver, brain, dental pulp, hair follicles, skin, skeletal muscle, adipose tissue, and blood ,,, [Figure 2]. In vitro ASCs have been shown to differentiate into a wide variety of cell types such as osteoblasts, chondrocytes, endothelial cells, skeletal myocytes, glia, neurons, and cardiac myocytes. Unlike ESCs, the use of ASCs in research and therapeutics is less controversial. ASCs were mainly studied in humans and experimental models such as mice and rats.
NSCs were isolated and characterized from various regions of the adult central nervous system (CNS) from biopsies and post-mortem tissues. In several studies, adult tissue-derived NSCs were transplanted in animal models and studied as functional engraftment, supporting their potential use for a possible cell therapy. 
Origin of neural stem cells
The SVZ, a remnant of the embryonic germinal neuroepithelium, is the most active compartment of NSCs. This region exists throughout the life as an active mitotic layer in the wall of the telencephalic lateral ventricles and along its rostral extension toward the olfactory bulb. A complete turnover of resident proliferating cell population occurs every 12-28 days in the SVZ and about 30,000 new neuronal precursors (neuroblasts) are produced every day and migrate to olfactory bulb. 
Astrocytes and ependymal cells in the SVZ may produce certain molecules that affect the neuronal or glial fate of the stem/progenitor cells. The proximity of the SVZ with cerebrospinal fluid (CSF), presence of enlarged intercellular spaces, reduced cell-cell contacts, and presence of molecules linked to water co-transport contribute to the cytoarchitectural/biochemical niche in the SVZ. This niche is very different from the environment of mature CNS parenchyma.  In recent years, neurogenesis has been reported to occur in other regions of adult brain. However, other research groups were not able to replicate some of these reports.  The organization of the adult SVZ in humans is different from that of other mammalian species. The lateral ventricular wall consists of four layers: (1) a monolayer of ependymal cells (layer I); (2) a hypocellular gap containing astrocytic processes (layer II); (3) a ribbon of cells composed of astrocytes (layer III); and (4) a transitional zone into the brain parenchyma (layer IV). Individual cells might migrate separately to olfactory bulb. When compared to rodents, precursor cells in human olfactory bulb are rare but not totally absent. However, these endogenous NSCs are very difficult to isolate and use.
Isolation and culturing of neural stem cells
NSCs were first isolated and expanded from the embryonic and adult mouse striatum in early 1990s.  Later, it was found that not only embryonic CNS but also adult CNS possesses the ability to generate neurosphere forming cells in vitro, including neural epithelial progenitor cells, radial glial cells, SVZ cells, and ependymal cells.  However, NSC cultures from adult murine hippocampus form as monolayer in the presence of basic fibroblast growth factor (bFGF). 
Neurospheres on repeated passages produce self-renewing, proliferating and differentiating cells, typically presenting prominin-1 cell surface antigen (CD133) and these cells are uniquely separated directly by magnetic beads conjugated with antibodies (MACS) or fluorescence assorted cell sorting (FACS) by negative selection of CD34− and CD45− antigen markers cells (CD133 + CD34− CD45− ). These cells, upon transplantation into brains of immunodeficient neonatal mice (the sorted/expanded CD133 + ), showed potent engraftment, proliferation, migration, and neural differentiation.  However, previously, stem-like cells have been purified from various organs as side population (SP) cells, based on their property to exclude Hoechst 33342. 
Characteristics of neural stem cells
Proliferating cell population in adult CNS shares the expression of a number of stem cell markers such as Nestin, Notch1, Sox-2. Notch pathway appears to play an essential role in the maintenance of stem/progenitor cell pool. Expression of Notch1 or one of its downstream regulators such as HES-1 inhibits neural differentiation and results in the maintenance of a progenitor state.  Numerous specific genes/pathways have been identified as important regulators of NSC proliferation; some of these are: Bmi-I, P21, nucleostemin, maternal embryonic leucine zipper kinase, P53, Rb, and Akt, among others. 
Olfactory ensheathed cells/olfactory mucosa cells
Nose contains neurons that send signals to brain when triggered by odor molecules. Because olfactory tissue is exposed to the external environment, it contains cells with considerable regeneration potential, including renewable neurons, progenitor/stem cells.
Problems of rejection, overgrowth, disease transmission and ethical issues can be avoided because a person's own olfactory mucosa can be used. Olfactory ensheathed cells (OECs) theoretically promote axonal regeneration by producing insulating myelin sheaths around growing and damaged axons, secreting growth factors, and generating structural and matrix macromolecules that lay the tracks for axonal elongation. These properties have led to an increasing use of OECs in preclinical models of transplantation for spinal cord repair, including complete transection, hemisection, tract lesion, and contusion. ,
With the promising results obtained from animal experiments, several clinical trials were started. A large series recruited more than 400 patients with spinal cord injury for transplantation of fetal olfactory bulb-derived cells and results of 171 operations have been published.  A single-blinded controlled study also demonstrated the safety and feasibility of intraspinal transplantation of autologous olfactory ensheathing cells in human spinal cord injury. 
In Huang's procedure, the undifferentiated nature of the fetal tissue has minimized the immunological rejection.  Feron et al. tested the feasibility and safety of transplantation of autologous OECs into the injured spinal cord in traumatic paraplegic patients.  There was no evidence of spinal cord damage, or cyst, syrinx or tumor formation. This indicates that OEC transplantation may be a safe method for in vitro propagation before transplantation.  However, OEC transplantation was associated with some adverse effects. , Of the 327 patients recruited, 16 (4.9%) experienced various complications: headache, fever, seizure, CNS infection, pneumonia, respiratory failure, urinary tract infection, heart failure, and possible pulmonary embolism and there were 4 (1.2%) deaths. One of the transplant recipients had improvement in light touch and pin prick sensitivity bilaterally, anteriorly and posteriorly over three segments. However, this conclusion should be taken with reservations as the number of patients included in the trial was small. 
BM stroma contains mesenchymal stem cells (MSCs) and these cells have the potential to differentiate into myelin-forming cells. This potential of MSCs has been studied in demyelinated rat spinal cord in vivo using cell transplantation techniques.  Transplanted BM cells showed a characteristic Schwann cell pattern of myelination. Transplantation of CD34 + hematopoietic progenitor cells failed to form myelin. Transplantation of MSCs 1 week after injury demonstrated better results when compared to cells transplanted immediately after injury. MSCs are an easily accessible and expandable cell source for the repair of injured spinal cord.  MSCs, co-cultured with fetal spinal cord-derived neurosphere cells, stimulate the development of extensive processes. Although the number of grafted MSCs may decrease over time, some treated animals showed remarkable functional recovery. 
Using combination approaches (cAMP/NT-3) capable of inducing both neuronal stroma and axon, regeneration of axons across the injured spinal cord segment has been documented.  The tissue matrix formed by MSC grafts supported greater axonal growth than that has been found in studies without grafts. Uniform random sampling of axon profiles revealed that the majority of neurites in MSC grafts were oriented with their long axis parallel to that of the spinal cord, suggesting longitudinally directed growth.  It has been shown that brain-derived neurotrophic factor (BDNF)-expressing marrow stromal cells support extensive axonal growth at spinal cord injury sites. 
Grafting of human MSCs derived from four different donors into a subtotal cervical hemisection in adult female rats showed cell integration at the injured site with little migration away from the graft.  Using enzyme-linked immunosorbent assay (ELISA), variations were found in the secretion patterns of selected growth factors and cytokines between different MSC lots. These results suggest that human MSCs produce factors important for mediating axon outgrowth and recovery after spinal cord injury; however, MSC lots from different donors vary considerably. To qualify MSC lots for future clinical application, such notable differences in donor or lot-lot efficacy highlight the need for establishing adequate characterization, including the development of relevant efficacy assays.  Results demonstrated that transplantation of Schwann cells derived from bone marrow stromal cells (BMSC-SCs) promotes axonal regeneration of injured spinal cord, resulting in recovery of hind limb function in rats. 
However, it is unclear whether BM cells can similarly improve the neurologic functions of complete spinal cord injury in humans. To address this issue, clinical outcome of autologous cell transplantation in conjunction with the administration of granulocyte macrophage-colony stimulating factor (GM-CSF) was analyzed in patients with complete spinal cord injury.  Sensory improvement was noted immediately along with significant motor improvement, 3-7 months after the procedure. Four patients showed neurologic improvements in the Abbreviated Injury Scale (AIS) grades from A to C, and one patient showed improvement in AIS grades from A to B. No immediate worsening of neurological status was found. Adverse effects of GM-CSF treatment, such as fever and myalgia, were observed. Bone marrow transplantation (BMT) and GM-CSF administration has been demonstrated as a safe procedure to manage patients with spinal cord injury, especially in patients with acute complete spinal injury.  However, combined with macroporous polymer hydrogels based on derivatives of poly(2-Hydroxyethyl Methacrylate) are suxitable materials for bridging cavities after spinal cord injuries.  Subarachnoid injection has been recently reported as a minimally invasive method for the transplantation of BM stromal cells in spinal cord injury. It may be, however, less effective than direct injection into the spinal cord in terms of cell delivery. , The safety, therapeutic time window, implantation strategy, method of administration, and functional improvement were investigated in transversal spinal cord injury patients receiving un-manipulated autologous BM. In this study, longer follow-up may be required to determine the safety and confirm the observed beneficial effects due to the cell therapy.  It appears that stem cell transplantation within a therapeutic window of 3-4 weeks following injury will play an important role in the recovery from any type of spinal cord injuries. In addition, clinical trials involving a larger population of patients and different cell types are needed before further conclusions could be drawn.
However, spinal cord injury does not create impenetrable boundaries, and diffusible signals appear to generate robust axonal growth even without resecting chronic scar tissue.  In a rabbit model of spinal cord ischemia, transplantation of MSCs was found to enhance angiogenesis and improve functional recovery. This study also supported the perspective that the therapeutic time window is critical for the therapeutic effects of MSCs.  Koda et al. reported in their study that the number of double positive cells for green fluorescent protein (GFP) and glial markers are larger in the G-CSF treated mice than in the control mice after BM cells transplantation into lethally irradiated C57BL/6 mice.
Umbilical cord blood, which is a rich source for stem cells (CD34+ /45− ), is being increasingly used on an experimental basis as an alternative to BM. Cord blood contains multiple populations of pluripotent stem cells and can be considered as the best alternative to ESCs. However, because of some ethical considerations, their application in humans is prohibited in some countries.
The use of human umbilical cord blood, a rich source of nonembryonic or adult stem cells
Saporta et al. studied umbilical cord blood cells in spinal cord injuries in in vitro model and found cord blood cells only at the site of injury, thus supporting the hypothesis that the cord blood stem cells have the capacity to migrate to the site of injury. In rat spinal cord injury model, intraspinal transplanted CD34 + CB cells achieved a better improvement in functional score when compared to the scores with BMS cells on day 7 and day 14. The conclusion from this study can be that cord blood CD34 + stem cells may be used for routine allogenic and autologous transplantations as a treatment modality for human spinal cord injuries.  In the study by Li et al., intraspinal transplantation of human cord blood CD34 + cells resulted in improvement of neurological function when compared to the control group (P < 0.05). Nishio et al. suggested that transplanted CD34 + fraction cells from hUCB may have therapeutic effects in spinal cord injury. Recently, Dasari et al. showed that hUCB cells can differentiate into several neural phenotypes including neurons, oligodendrocytes and astrocytes. Sequential studies have demonstrated that in the early phase, many differentiated human umbilical cord blood (hUCB) cells expressing characteristic neuronal proteins can be detected, and at 1 month post-grafting, hUCB cells may no longer be detected. 
Rat models with spinal cord injury treated with neurally induced progenitor cells of hUCB showed recovery of somatosensory potentials. The grafted cells especially exhibited oligodendrocytic phenotype around the necrotic cavity. These findings may suggest that neurally induced progenitor cells of hUCB may be a therapeutic option to repair damaged spinal cords.  Data from the published work suggest that umbilical CB stem cells are unique for use in regenerative medicine to treat patients with neurological disease. 
Skin contains precursors that are capable of generating neural cell types.  Skin-derived stem cells (SKPs) can generate both neural and mesodermal cell types. Subpopulations of nestin - vimentin + phenotype of fibroblasts cells have been shown to have neural cell differentiation characters.  Recently, induced pluripotent stem cells (iPS) have been produced from adult human cells. ,, Human fibroblasts have been successfully transformed into pluripotent stem cells using four pivotal genes: Oct-3/4, Sox-2, Klf4, and c-Myc, with a retroviral system. These results demonstrate that defined factors can reprogram human cells to pluripotency. 
Hair follicles are known to contain a well-characterized niche for adult stem cells, the bulge, which contains epithelial and melanocytic stem cells. Nestin + cells were identified in the bulge area in mouse and were found to give rise to neurons, smooth muscle cells, and melanocytes.  Neural-crest-like stem cells have been identified in mouse whisker hair follicles. 
Adipose tissue is a highly complex tissue and consists of mature adipocytes, preadipocytes, fibroblasts, vascular smooth muscle cells, endothelial cells, resident monocytes/macrophages,  and lymphocytes  and is also a rich source of pluripotent adipose tissue-derived stromal cells.  It has been demonstrated that adipose tissue contains stem cells similar to BM-MSCs and lipoaspirate cells.  These processed lipoaspirated cells and clones can be further differentiated into putative neurogenic cells exhibiting a neuronal-like morphology. These putative neurogenic cells express several proteins consistent with the neuronal phenotype. Before clinical application, we need additional in vitro experiments, and preclinical trials are necessary.
Wharton's jelly as a source of primitive cell types was established based on the low levels of collagen expressed in gelatinous connective tissue and the fact that during embryogenesis, totipotent cells, such as primordial germ cells and hematopoetic stem cells, migrate from the yolk sac through this region to populate target tissues in the embryo and fetus. Wharton's jelly cells have telomerase activity, which is usually characteristic of human embryonic stem cells. Colonies of Wharton's jelly cells were also found to express NSE and c-kit. However, the expression of markers for non-neuronal cell lineages by these cells remains to be determined. 
Amniotic placental fluid
Amniotic fluid contains a heterogeneous population of cells mainly contributed by fetal skin, fetal digestive, respiratory, and urinary tract, and placental membranes. ,,, Recent discoveries of stem cell populations in amniotic fluid have postulated that the amniotic fluid is a promising alternative source of fetal stem cells for cellular therapy. ,,,
Recruitment of macrophages is limited in CNS and the resident microglia cells are the main immune cells that are activated after spinal cord injury.  It has been shown that by controlled boosting of local immune response by delivering autologous macrophages, one can promote recovery in spinal cord injury.  The possible mechanisms are activation of infiltrating T cells and increased production of trophic factors and brain-derived neurotrophic factor, , thus leading to removal of inhibitory myelin debris.  Also, it has been shown that this cell therapy was well tolerated in patients with acute spinal cord injury. 
Transplantation of dendritic cells into the injured spinal cord of mice led to better functional recovery when compared to controls.  The implanted dendritic cells induced proliferation of endogenous neural stem/progenitor cells (NSPCs) and led to de novo neurogenesis. This observation was attributed to the action of secreted neurotrophic factors such as neurotrophin-3, cell-attached plasma membrane molecules, and possible activation of microglia/macrophages by implanted dendritic cell. 
Schwann cells originating from dorsal and ventral roots are one of the cellular components that migrate to the site of tissue damage after spinal cord injury. ,, The remyelinating capability of Schwann cells has been demonstrated in a number of studies , and the functioning status of this myelin in conduction of neural impulses has also been confirmed. ,
Fetal-derived multipotent fetal stem cells (FSCs) are more tissue specific than ESCs and are able to generate a more limited number of progenitor types. One of the particular therapeutic advantages of FSCs when compared with ESCs is the fact that FSCs do not form teratomas in vivo. Moreover, the FSCs obtained up to week 12 offer the possibility of transplantation without frequent rejection reactions. Recent work has revealed the possibility of using FSCs or their progenitors, isolated from particular tissues, for multiple therapeutic applications involving tissue regeneration. ,,, FSCs can cross both the placental and blood-brain barrier. These cells can be administered intravenously. SVZ and hippocampus of fetal CNS contains more number of neural progenitor cells, which can be directly isolated and transplanted.
NSCs were extensively found in two areas of brain, the SVZ of the lateral ventricle and the dentate gyrus. This source of progenitor population can be widely employed in the treatment of neurological disorders.  This cell therapy may also support quiescent neurogenesis in brain, leading to stimulation and proliferation of progenitors from these two regions.
Stocum et al. demonstrated that immature neurons originating from the SVZ migrate to the damaged striatal area. Further experiments have given some hope for cell-based therapies in which allogenic NSCs' transplantation in spinal cord injuries showed partial recovery. There is evidence demonstrating that NSCs transplanted in spinal cord injuries differentiate into glial cells and also oligodendrocytes. Of the 32 patients with complete spinal cord injury of 2-12 years duration who received BM cells transplantation, 15 showed modest improvement in the lower extremity function. Mikami et al. reported that transplanted splenic dendritic cells supported proliferation and differentiation of host NSCs and induced axon sprouting, associated with partial recovery from hind limb paralysis in lesioned mouse spinal cords. Stocum et al. reported that adipose tissue generated stromal cells derived oligodendrocyte precursors promoted functional recovery by both remyelination and induction of proliferation and differentiation of host NSCs when transplanted into rat spinal cord lesions. Based on the current knowledge of NSCs from different sources, there is a need to standardize methodology to enable the researchers to harvest a high number of viable and well-characterized cells which can be employed to treat patients with neurological disorders. 
The increasing availability of induced pluripotent stem cells (iPSCs) derived from adult human somatic cells provides new prospects for cell-replacement strategies and disease-related basic research in neurological diseases. iPSC, artificially derived from a non-pluripotent cell, is typically an adult somatic cell through reprogramming. iPSCs were initially derived from mouse embryonic and adult fibroblasts by over-expression of perticular transcription factors (Yamanaka factors) which include 24 candidate genes known to be pluripotency associated. After elimination of irrelevant factors, a minimum of four factors remained: Octamer 3/4 (Oct-3/4), SRY-box containing gene 2 (Sox-2), cytoplasmic Myc protein (c-Myc), and Krueppel-like factor 4 (Klf4). However, currently only fibroblasts have been used to generate iPSCs from patients suffering from neurological diseases. Retroviral and lentiviral vectors have been widely used for the delivery of reprogramming factors. New strategies have been suggested to generate safe and less tumorigenic iPSCs by using non-viral methods or by omitting the oncogenic factors c-Myc and klf4. Therefore, attempts have been made to derive iPSCs by using plasmids rather than viruses. Yamanaka's group has also shown that human iPSCs can differentiate into βIII-tubulin-positive neurons as well as GFAP-positive astrocytes. Park et al.,  for the first time, created patient- as well as disease-specific iPSCs from skin fibroblasts of patients suffering from a variety of genetic diseases, including adenosine deaminase deficiency-related severe combined immunodeficiency, Gaucher disease type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1 diabetes mellitus, Down syndrome (DS)/trisomy 21, and the carrier state of Lesch-Nyhan syndrome.
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