5.3.2. Autologous olfactory ensheathing glia and olfactory fibroblasts

The olfactory ensheathing glial cells are belong to a class of macroglia, which are involved in ensheathing demyelinated axons of olfactory neurons. In olfactory bulb, typical and transected olfactory axonal structures are able to move in, regenerate and restore damaged synaptic communications with their respective targets [92]. Moreover, transplantation of olfactory ensheathing glial cells has been reported to improve axonal remyelination within a damaged nervous system [92]. A preclinical study has reported a transplantation of adult rat's olfactory bulb-derived ensheathing glia cells in a SCI's site, where the cells filled the lesion gap through regeneration [93]. In addition to the preclinical success of olfactory ensheathing cell transplantation, a study has reported the feasibility of transplantation of autologous olfactory ensheathing cells into three spinal cord injured patients where the cells were found safe without any serious complication for up to 12 months after transplantation [94]. A subsequent study has reported that transplantation of olfactory ensheathing cells is viable and safe for promoting motor and sensory activities [95]. Therapeutically, the olfactory ensheathing cells are most likely to maintain their effects via secretion of specific growth promoting factors that develop new olfactory axons and promote axonal regeneration following SCI [96]. At present, the olfactory ensheathing glial and fibroblastic cells are being evaluated in phase I clinical trial (NCT01231893) for treatment of complete SCIs [44].

iPSCs, the augmented factors binding at promoter regions are linked with elevated level of transcription, signifying the fact that OCT3/4, SOX2 and KLF4 are involved in genes regulation

Cellular Transplantation-Based Therapeutic Strategies for Spinal Cord Injuries: Preclinical and Clinical…

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For induction of cellular reprogramming in adult cells, four strategies can be employed that include nuclear transfer, fusion of cells, cellular explantation and infection through retroviral vectors [114]. In the mechanism of nuclear transfer, direct administration of a viable donor somatic nucleus into an enucleated egg cell, followed by implantation in a surrogate mother, can develop a reproductive clone. If the enucleated egg cell with implanted somatic nucleus is grown in vitro, it can generate inherently matched ESCs population. In mechanism of cell fusion, culture of adult body cells with ESCs can give rise to hybrid progeny of cells that can entirely exhibit pluripotency. In another strategy of cellular explantation, adult body cell cultures can be directly induced to gain pluripotency via reprogramming factors. In fourth strategy of cellular reprogramming, infection through retroviral vectors carrying distinct fac-

A preclinical study has reported that following transplantation of human iPSC-derived neurospheres into spinal cord-injured mice model, a locomotional recovery from SCI was achieved, signifying the importance of human iPSC-derived neurospheres in regenerative medicines [115]. Another study has reported the transplantation of human iPSC-derived neural stem cells into monkeys' specie that promoted locomotory function after SCI, without inducing teratomas [116]. One of a report has stated that iPSCs have the ability to generate three important functional cell types of the CNS, i.e. astrocytes, oligodendrocytes and neurons [117]. An optimization of efficient protocols that can be utilized to generate constant and longterm population of neural stem cells from ESCs and iPSCs has been demonstrated [118, 119]. These types of cells have displayed stable features, for example constant expanding properties, consistent differentiation into neurons and glia and the ability of producing efficient established neurons in vitro. Another study has reported that following transplantation of such long-term stemness-rich cells (iPSC-derived neural stem cells) into SCI mice model, improved remyelination and axonal regrowth were observed with additional support for subsistence of

In one of an important study, cells from a healthy man of 86 years of age were induced to generate iPSCs, from which neural stem cells were generated and transplanted into rats exhibiting immunodeficiency following SCI. After 12 weeks of C5 lateral hemi-sections, iPSCs endured and generated neuronal and glial cells, extending large number of axons from injured area to almost the complete distance of rat CNS that subsequently developed synaptic communications with host neurons [121]. Another study on human iPSC-derived neural progenitors known as IMR90 has reported functional recovery after IMR90 transplantation in SCI rat models. It was shown that iPSCs have the ability to generate functional neurons, which resulted in long-term functional recovery from SCI [122]. Altogether, several studies have reported that following transplantation of iPSC-derived neural stem/precursor cells, locomotory function was improved/recovered in spinal cord-injured animal models [115, 116, 120, 123, 124]. In contrast, other studies have reported that transplantation of clones containing

predominantly as activators of transcription [113].

tors can induce reprogramming to generate iPSCs [114].

5.4.3. Trials involving iPSCs for SCIs

endogenic neurons [120].

#### 5.4. Induced pluripotent stem cells (iPSCs)

Pluripotency, a special ability of a pluripotent cell to differentiate into any type of body cell, was shown to be induced in adult differentiated cells via the activity of only four embryonic transcription factors—Oct3/4, Sox2, Klf4 and c-Myc. These induced adult cells gaining the ability of pluripotency were termed as "induced pluripotent stem cells" (iPSCs) [97]. This discovery offered a very forthright procedure of how to induce pluripotency in mature cells in a basic laboratory setup, yet the scope of this cell-rewinding discovery was extraordinarily vast in biomedical sciences and regenerative medicine, which earned the principle investigator, Yamanaka, a Nobel Prize in year 2012 [98].

### 5.4.1. Reprogramming factors

The reprogramming factors, also known as pluripotency markers, are the main regulators of inducing pluripotency in mature cells via a process of activating pluripotent genes expression. The induction of pluripotency can be achieved via expression of only four reprogramming factors, i.e. OCT3/4, SOX2, KLF4 and c-Myc [99]. POU class 5 homeobox 1 (POU5F1 or OCT4) is being reported to play a significant role in developing embryo and maintaining pluripotent status. It has been observed to bind an octamer motif (ATGCAAAT) of DNA, where it is involved in regulation of several genes that play important part in pluripotency. Oct4 has been observed to frequently regulate in association with Sox2 [100–102]. SRY (sex determining region Y)-box 2 (SOX2) is a transcription factor encoded by SOX2 intronless gene. It plays an important role to regulate embryonic development and determine cell fate and is usually expressed in developing embryo and neuronal cells [100]. It has been reported that expression of SOX2 is fundamental for maintaining pluripotent status of evolving embryos, whereas its downregulation is associated with mesodermal and endodermal differentiation. Embryos with no expression of SOX2 were found unable to grow and proliferate after implantation [102, 103]. Kruppel-like factor 4 (KLF4 or EZF), a zinc finger protein is a transcription factor that is involved in regular growth of the barrier properties of body skin [100]. KLF4 has been reported to have a higher expression in non-dividing cells and is associated with induction of cell cycle arrest [104, 105]. KLF4 has been shown to specifically regulate genetic stability [106, 107] and promote cellular growth and survival [108]; however, in some cases, KLF4 has been reported to induce cell death [109, 110]. c-Myc, a nuclear phosphoprotein, has been recognized to play multiple functions, including cell cycle multiplication, programmed cell death and cellular propagation, via transcriptional regulation of particular genes [100]. The efficacy of OCT3/4, SOX2 and KLF4 is shown to be enhanced by the expression of an enhancer factor c-Myc [111].

#### 5.4.2. Mechanisms of cellular reprogramming

In mechanism of cellular reprogramming, three pluripotency markers (OCT3/4, SOX2 and KLF4) have been observed to greatly influence multiple genes expression in iPSCs [112]. In iPSCs, the augmented factors binding at promoter regions are linked with elevated level of transcription, signifying the fact that OCT3/4, SOX2 and KLF4 are involved in genes regulation predominantly as activators of transcription [113].

For induction of cellular reprogramming in adult cells, four strategies can be employed that include nuclear transfer, fusion of cells, cellular explantation and infection through retroviral vectors [114]. In the mechanism of nuclear transfer, direct administration of a viable donor somatic nucleus into an enucleated egg cell, followed by implantation in a surrogate mother, can develop a reproductive clone. If the enucleated egg cell with implanted somatic nucleus is grown in vitro, it can generate inherently matched ESCs population. In mechanism of cell fusion, culture of adult body cells with ESCs can give rise to hybrid progeny of cells that can entirely exhibit pluripotency. In another strategy of cellular explantation, adult body cell cultures can be directly induced to gain pluripotency via reprogramming factors. In fourth strategy of cellular reprogramming, infection through retroviral vectors carrying distinct factors can induce reprogramming to generate iPSCs [114].
