Stem cell research is considered one of the most captivating areas of cell biology mainly due to the unique properties of stem cells and their potential use in cell-based therapies to treat a variety of diseases, including Parkinson's disease (PD), Alzheimer's diseases (AD), Diabetes Mellitus (DM), and many others (Correia et al., 2005; Pagliuca et al., 2014; Sproul, 2015). Given their unique regenerative abilities, these cells provide new potentials in the area of regenerative or reparative medicine.

Embryonic stem cells (ESCs), which are derived from the inner cell mass (ICM) of blastocysts, are pluripotent cells that have the ability to proliferate indefinitely. But still they maintain their pluripotency with the capability to differentiate into cells of all three germ layers: ectoderm, mesoderm and endoderm (Evans and Kaufman, 1981; Martin, 1981). In addition, these cells serve as an internal repair system, limitlessly regenerating into either differentiated cell progeny or additional stem cells (Keller, 1995; Thomson et al., 1998). Since first isolated in 1998, human ESCs have featured high importance as a potential treatment of a variety of diseases like PD, spinal cord injury (SCI) and DM (Thomson et al., 1998). However, the extraction of ESCs raises sharp ethical controversies as they are derived from human embryos and their transplantation in patients may present serious risks with a possibility of rejection (Lo and Parham, 2009).

Alternative approaches to the derivation of ESCs from the ICM of pre-implanted embryos are now available and these tend to avoid ethical issues. Such methodologies directly generate pluripotent stem cell lines from differentiated adult somatic tissue and include nuclear transfer, cell fusion or direct reprogramming (Hochedlinger and Jaenisch, 2006). In 2006, a landmark discovery was published by the Yamanaka group at Kyoto University as they induced the expression of only four pluripotency-associated transcription factors, Oct3/4, Sox2, c-Myc, and Klf4 (OKSM), in mouse fibroblast cells resulting in the generation of ESC-like cells, called induced pluripotent stem cells (iPSCs). These cells are similar to the ESCs in their morphology, gene expression, proliferation and teratoma formation (Hochedlinger and Jaenisch, 2006; Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Wernig et al., 2007; Hadadeh et al., 2012). These iPSCs are now widely used for various applications, such as autologous cell therapy, monogenic and multigenic diseases modeling, and as substrates for drug, toxicity, differentiation and therapeutic screens.

Reprogramming highly depends on the efficient delivery and the suitable expression of certain factors into specific cell types, under particular culture conditions and within a period of time. Although direct reprogramming is a simple technique, it differs depending on the cell type, species and delivery method. It is rather a slow and vulnerable process that may be affected by several factors that hinder the efficiency, reproducibility and the quality of the resulting iPSCs. To date, the most popular donor somatic cells are fibroblasts, being used in more than 80% of all reprogramming experiments published (González et al., 2011). Yet, other cell types have been used in reprogramming such as human primary keratinocytes, cord blood CD133+ cells, and peripheral blood mononuclear cells (Aasen et al., 2008; Giorgetti et al., 2009; Su et al., 2016).

A successful management of the outcome of somatic cell reprograming to iPSCs is related to both the reprograming technique and the type of cells that are used. Here, it is important to note that although fibroblasts are the most widely used cells for iPSCs generation, this does not necessarily mean that they represent the highest efficacy. In fact, since the first approach toward creating iPSCs was obtained from fibroblasts (Takahashi and Yamanaka, 2006), subsequent protocols tried to reproduce that process before other alternatives for fibroblasts were investigated. Fibroblasts are easy to cultivate in culture and much is known about these cells in research. An important characteristic of these cells that makes them great candidates is the low methylation levels of the promotor regions of OCT4 and NANOG that can be associated with the favorable reprogramming of the cells. Furthermore, fibroblasts can be easily obtained from patients through biopsy and are relatively inexpensive and widely commercially available by many companies. However, the fact that fibroblasts are highly proliferative poses few disadvantages as the non-programmed fibroblasts can have the opportunity to overgrow the existing reprogrammed cells and consume the growth factors in the media. This can usually be overcome by using a low passage not exceeding passage 5 in order to avoid accumulated genomic changes (Raab et al., 2014).

Reprogramming can be induced by the co-introduction of some genes that are expressed early during development, such as OCT4, SOX2, NANOG, UTF1, and SALL4, and which are implicated in the maintenance of the pluripotent potential of the ICM (Niwa, 2007; Zhao et al., 2008; Tsubooka et al., 2009). Supplementation with other genes such as c-MYC, KLF4, TERT, and SV40LT can enhance cell proliferation in a direct or indirect manner (Park et al., 2008b). Additionally, microRNAs (miRNAs) have been implicated in pluripotency and reprogramming, such as the miR-290 cluster and miR-302 cluster miRNAs (Wang et al., 2008; Mallanna and Rizzino, 2010). On the other hand, there are several chemical compounds that have proven to enhance reprogramming in different cell types. Those compounds are known to alter DNA methylation or cause chromatin modifications and they include DNA methyltransferase inhibitor 5′-azacytidine or histone deacetylase (HDAC) inhibitors (such as hydroxamic acid (SAHA), trichostatin A (TSA), and valproic acid (VPA)) (Huangfu et al., 2008). The delivery of the OKSM transcription factors into mouse or human fibroblasts is achieved using different viral and non-viral constructs, as well as integrative and non-integrative systems approaches, the latter of which have presented major problems for iPSCs generation. Four main groups of different non-integrative approaches are available: integration-defective viral delivery, episomal delivery, RNA delivery and protein delivery (González et al., 2011). There is no best reprogramming strategy that can be used to fit all purposes. The choice of the strategy highly depends on the purpose of the research; whether it focuses on understanding the mechanisms of reprogramming or on generating clinically relevant iPSCs. Integrative methods with lentiviruses can be sufficient for the former use while non-integrative approaches should be used for the latter to limit genomic modifications.

Understanding and treating many diseases have been constrained by the absence of in vitro models, especially because culturing primary cells affected by the diseases is very challenging. Limitations primarily lie in the access to patient's tissues as the priority goes for diagnosis, in addition to the complications in obtaining some cell types, such as neural or cardiac tissues, and to maintaining these cells in vitro. However, the development of stem cell studies and the novel discovery of iPSCs provided an important source of cells to conduct in vitro studies (Unternaehrer and Daley, 2011). Such establishment of human iPSCs (hiPSCs) has led to new clinical strategies for using them as universal sources in regeneration therapy of damaged organs and tissues (Pei et al., 2010). Moreover, iPSCs generated from a patient affected by a certain disease possibly reproduces the disease phenotype (Egashira et al., 2011). In view of this, different kinds of patient-specific iPSCs have been generated to model human neurodegenerative diseases, such as Parkinson's disease (PD) (Byers et al., 2012), Huntington's disease (HD) (Nekrasov et al., 2016), Amyotrophic lateral sclerosis (ALS) (Chestkov et al., 2014), and Alzheimer's disease (AD) (Mungenast et al., 2016).

The ectoderm is the first germ layer to emerge during gastrulation, which is initiated by the formation of the primitive streak within the epiblast. Cell lineages derived from the ectoderm differentiate to form mainly the epidermis (including skin, hair, nails, and sweat and sebaceous cutaneous glands) and the nervous system (central and peripheral). The development of the vertebrate nervous system is shown to be regulated temporally and spatially by gradients of signaling molecules that may have either inhibitory or activating roles. These molecules are important for neuronal migration (Khodosevich and Monyer, 2011), axonal guidance and outgrowth (Chilton, 2006), interneuronal synapses (Scheiffele, 2003) and neuron-glia interaction (Fields and Stevens-Graham, 2002). Subsequently, experiments have demonstrated that this process is under the control of a combination of small-molecule endogenous inhibitors of bone morphogenic protein (BMP) and TGFβ/activin/nodal signaling (Morizane et al., 2011), which promote highly efficient neural induction from both human ESCs and iPSCs. Additionally, it was shown that DLK1 has a role in stimulating neurogenesis of human and mouse iPSC-derived neural progenitors via modulating Notch and BMP signaling (Surmacz et al., 2012). Using such small molecules to induce differentiation of iPSCs into a specified lineage shows a potent approach to generate specific cell types in order to better understand the biological function and disease processes, as well as to use these cells in drug screening and cell therapy.

Recently, efforts have been dedicated to generate defined lineages of neural cells from ESCs and iPSCs. These cells serve to better understand the molecular mechanisms underlying the pathophysiology of many intractable neurodegenerative diseases such as PD, HD, ALS, and AD aiming for the development of effective therapies. The present lack of precise models of these diseases conveys the significant discrepancies in understanding the mechanisms underlying their pathophysiology. In the last decade, the ability to reprogram somatic cells into iPSCs have enhanced the effectiveness of human in vitro models of neurological diseases (Mungenast et al., 2016). Moreover, the differentiation of these iPSCs into disease-relevant cell types have allowed comprehensive molecular analyses of multiple disease states. Indeed, neurons differentiated from patient-specific iPSCs provide a valuable tool to model specific molecular phenotypes of neurodegenerative diseases in vitro (Heman-Ackah et al., 2016). In this aspect, the introduction of human iPSCs with disease-specific genetic backgrounds requires precise and flexible genome engineering tools.

Among different groundbreaking experiments, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) endonuclease is considered the less cumbersome and the most flexible system to execute precise genome editing in human pluripotent stem cells (Kime et al., 2016). The genomic revolution made by this system and others, including the Zinc Finger Nucleases (ZFN) and Site specific nucleases (SPN), offered the simplest and most powerful approach toward manipulating the produced iPSCs for mimicking any disease and adding more advancement and efficiency to the resulting cells (Mandegar et al., 2016). Recently, Rubio et al. described a new platform where neurons can be generated in vitro and manipulated using CRISPR/Cas9 to inactivate specific genes associated with different neuropathologies in humans (Rubio et al., 2016). Moreover, in a recent study, it was shown that CRISPR can be used to exert precise alterations in the expression of the critical PD-related gene, SNCA, in human iPSC-derived neurons (Heman-Ackah et al., 2016). Remarkably, Paquet et al. (2016) established a procedure that allows the introduction of specific point mutations into iPSCs using CRISPR, thus generating human iPSCs with specific combinations of homozygous and heterozygous early-onset Alzheimer's-associated mutations. Certainly, the rapid development of iPSCs and genome-editing technologies are important tools for disease modeling that hold promise for applications in gene therapy.

In this review we will focus on the reprogramming of somatic cells into patient-specific hiPSCs to model four neurodegenerative diseases, namely Parkinson's disease (PD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD), and other applications in neurotrauma, through summarizing the different protocols that are used in each for the reprogramming and differentiation processes (Figure 1).

In case of injury, the spinal cord does not spontaneously regenerate itself and till now there is not one available treatment that can provide the least functional recovery from SCI. The level at which the injury is present determines the symptoms and consequent motor manifestations. To date, cervical SCIs account for the majority of presented SCI cases (Doulames and Plant, 2016).

Many previous cell transplantation therapies have been approached such as peripheral nerve bridges, Schwann cells, olfactory glia, mesenchymal stem cells and NSCs (Plant et al., 2001; Barry and Murphy, 2004; Ramer et al., 2004; Cummings et al., 2005; Parr et al., 2008). Such therapies function to ameliorate the existing damage and prevent its further exacerbation by providing a scaffold to bridge the lesion, replace the host dead or lost neurons or glia, promote axonal regeneration and overcome glial scar formation (Bregman et al., 1997; Jones et al., 2001; Raisman, 2001; Ikegami et al., 2005; Donnelly and Popovich, 2008). In SCI, the main drawback to regeneration is the inability of the CNS neurons to regenerate axons that can cross through the inhibitory milieu of the glial scar and the injured lesion (Horner and Gage, 2000). Certain studies on rats showed promise that this can be overcome by using iPSCs as these reprogrammed iPSC neurons can extend many axons over very long distances and form synapses with host rat neurons (Parr et al., 2008; Romanyuk et al., 2015).

The implementation of iPSCs offers a great mean of cell-based therapy capable of bypassing the usual ethical dilemma associated with ESCs. The iPSCs have the ability to differentiate into tissue-specific neurons which leads to long-term restoration of the lesioned tissue (Romanyuk et al., 2015). Moreover, these cells can be obtained in a non-invasive patient-specific manner that makes iPSC an even more approachable attractive candidate for SCI therapy. This cell replacement therapeutic approach has opened a new era in the field of regenerative medicine.

Jin Young Hong and colleagues generated self-renewable induced NSCs from somatic fibroblasts and engrafted them in a rat model of SCI. The engrafted cells were able to restore axonal regeneration resulting in recovery of motor, sensory and autonomic functions (Hong et al., 2014). In another study, Pajer et al. performed avulsion of the lumbar 4 (L4) ventral root in rats, an injury that is known to induce the death of majority of affected motor neurons. Afterwards, they transplanted murine iPSCs into the injured spinal cord segment. Their observation included improved re-innervation by the host motor neurons as compared to controls with no iPSC transplantation procedure. It also seemed that the observed morphological re-innervation resulted in functional recovery as the grafted rats exhibited more motor movement units in their re-innervated limb than controls. This study also established that the grafting of iPSCs downregulated astroglial activation in the injured site and was able to conclude that the observed motor neuron survival and regeneration came as a result of neurotrophic and cytokine modulatory mechanisms (Pajer et al., 2015). Furthermore, a study performed on mice suggested that the neurons derived from the transplanted cells functioned as interneurons in the mouse spinal cord which in turn contributed to the reconstruction of neural circuits (Nakamura and Okano, 2013). Moreover, neural precursors derived from a clone of hiPSCs (IMR90) were used to treat a rat spinal cord lesion 1 week after induction. These hiPSC-neural precursors robustly survived in the lesion, migrated, and partially filled the lesion cavity during the entire period of observation. Transplanted animals displayed significant motor improvement already from the second week after the transplantation (Romanyuk et al., 2015).

The application of iPSCs seems so good so far and all these mentioned results raise great clinical expectations. However, it should be noted that safety-related concerns for such iPSCs cell therapy should be resolved prior to clinical application. A main concern after iPSC therapy is tumor formation as a result of residual or remaining undifferentiated iPSCs that were not successfully induced into differentiation. Moreover, there is the chance that the reprogramming was not necessarily complete (Nakamura and Okano, 2013; Kim et al., 2014). Long term safety issues also include deteriorated motor function accompanied by a tumor formation (Nori et al., 2015). However, Tsuji et al. used the neurospheres 3D culturing of iPSCs obtained from mice fibroblasts, and injected them into the injured spinal cord. Those neurospheres were able to differentiate into three neuronal lineages: astrocytes, oligodendrocytes, and neurons, promoting recovery and improving locomotor functional loss with no tumor formation for the observation period of more than 120 days (Tsuji et al., 2010). Besides, an in vivo study has shown that 3 rats have died out of 12 rats that received transplantation with DA neurons derived from protein based iPSCs (Rhee et al., 2011). The rats that died showed tumor formation after 8 weeks from grafting. Moreover, in a comparison between transplanted secondary neurospheres derived from iPSCs generated in 11 different ways and neurospheres from ESCs, the former showed a significant teratoma formation propensity most likely correlated with the persistence of undifferentiated cells (Miura et al., 2009). All this further halts the use of such a therapeutic tool in humans as much still needs to be optimized.

Researchers at the University of California, San Diego School of Medicine launched a 5-year clinical trial program starting 2014 in order to investigate the safety of NSCs transplantation in patients with chronic SCIs (University of California 2014). A recent clinical trial on humans, the SciStar study, has been using oligodendrocyte progenitors to treat people with recent SCIs. This study not only has proven to be effective so far but has lately passed major safety issues and therefore was approved to expand by increasing the number of both enrolled patients and the transplanted cells per patient (California Institute for Regenerative Medicine (CIRM) 2016). Although this study aims at evaluating the safety and effectiveness of AST-OPC1 agent, which consists of oligodendrocyte progenitor cells produced from human ESCs, in patients with recent SCI, results can be promising as regards potential use of iPSCs instead of human ESCs in this context.

Oki et al. (2012) provided the first evidence that transplantation of hiPSC-derived cells is a safe and efficient approach to promote recovery after stroke and can be used to supply the injured brain with new neurons for replacement. They transplanted neuroepithelial-like stem cells, generated from adult human fibroblast-derived iPSCs, into stroke-damaged mouse and rat striatum or cortex. The transplanted cells stopped proliferating after a while but have at least shown that they can survive without forming tumors for at least a period of 4 months. This 4-months observation period warrants lack of rejection of the transplanted cells and creates an optimal setting to evaluate tumorigenicity. The iPSCs successfully differentiated neurons in intrastriatal grafts sent axonal projections to the globus pallidus. Moreover, the grafted cells exhibited electrophysiological properties of mature neurons and most importantly received synaptic input from host neurons (Oki et al., 2012). There is not much data and research on the iPSCs regenerative ability in brain injury. Therefore, in order to establish its safety and effectiveness, much more studies and effort have to be done in that domain. After all, transforming such data to clinical trials in humans would be a great achievement toward finding a treatment and getting more insight on the brain circuitry itself.

While the iPSCs technology holds the promise to becoming an efficient therapy for many neurodegenerative diseases that currently have no cure, there remains the risk of encountering multiple unanticipated outcomes when applying them on humans. The risks range from unwanted biological effects and immune response, toxicity, neoplasm formation, disease transmission, reactivation of latent viruses, to rejection of the cells by the body. It is difficult to determine and pinpoint the risks as they depend on several factors, including the cells that are used to achieve pluripotency, the status of the differentiated cells, their proliferation capacity, the technique of administration of the pluripotency genes, the level of manipulation, the growth factors used, the dilemma of retaining epigenetic memory, the intended site of injection, the reversibility or even irreversibility of the applied treatment, the susceptibility of the administered cells for disease, the incomplete suppression of the four transgenes after differentiation, the persistence of undifferentiated cells, and the survival of the transplanted cells in vivo (Okita et al., 2007). The known risks so far that were obtained mostly from animal models include tumor formation, unwanted immune responses and the transmission of certain adventitious agents (Herberts et al., 2011; Okano et al., 2013). Unfortunately, an estimated 20% of mice that received iPSCs were found to develop tumors (Abdullah et al., 2012). Furthermore, it has been hypothesized that the sustained expression of the transgenes might have the ability to change the expression of certain oncogenes or tumor suppressor genes thus altering the tumorigenic potential of the cells. The c-Myc is a risk factor by itself as it is upregulated in naturally occurring tumors.

An important risk of iPSC therapy is associated with using lentiviruses or retroviruses. These viruses, although are genetically tailored to hold the genes required for an iPSC state transformation, will integrate into the host cell genome and consequently add multiple viral integration sites and be the cause for several safety issues (Howe et al., 2008). However, it should be noted that much control was gained over this process as the viral integration site can be determined in iPSCs using Cre-mediated strategies for instance or by using adenoviruses, plasmids, transposons, recombinant proteins, Sendai virus vectors and modified RNA (Herberts et al., 2011). It should be further noted that there has been worries of a state of dedifferentiation or dedifferentiation into an unwanted cell type once transplanted to humans, though this remains clinically unclear. Therefore, there has to be a thorough consideration of all the suspected risk factors before setting into iPSCs human clinical trials.

Trans-differentiated neurons offer a way to bypass the tumorigenicity risk associated with passing through a pluripotency state. This method allows lineage reprogramming as in directly converting a somatic cell type into another through transgenic expression of transcription factors or miRNAs. On the other hand, the procedure of re-differentiating iPSCs into specific cell types is considered lengthy, costly and arduous. In addition to that, having multiple stages before reaching the intended outcome may lower the efficiency of the generated cell type. So far, in this context, trans-differentiation seems to be also an interesting path in research that also offers the same concept of therapeutic approaches and disease modeling as iPSCs. Trans-differentiated cells show no tumorigenicity when transplanted in vivo, plus these cells show similar functionality to cells derived from iPSCs (Lopez-Leon et al., 2014; Hou and Lu, 2016). In the presence of an insult or a lesion, or even in AD animal models, glial cells can be successfully transformed to functional neurons by single transcription factor intervention (Guo et al., 2014). For example, in the presence of SOX2 in the injured adult spinal cord, astrocytes convert to double cortin (DCX) positive neuroblasts (Lau et al., 2014). However, the major limitation for trans-differentiation to be a therapeutic approach is that the obtained population of induced neurons has little to no proliferation rate, therefore directly restricting the efficiency and expansion of this technique (Hou and Lu, 2016). Hence, a better investment for a drug or therapeutic or modeling purposes would still be iPSCs, but this does not overthrow the importance of trans-differentiation. The research in this field is still young but so vivid and fertile that we can witness iPSCs applied in neurodegenerative diseases treatments in not the so far future. Yet, until this day, there is no recorded iPSCs treatment application on humans (Trounson and DeWitt, 2016).

Although, iPSCs research has been a revolution in the scientific field as it provides new hope for the treatment of many diseases, protocols describing the differentiation of iPSCs into neural cells in neurodegenerative diseases and in the context of neurotrauma are still being modified and studied to assess the effect of this process on gene mutations in these cells and vice versa. Advances have been made recently to uncover the underlying molecular pathways of several neurodegenerative diseases, yet more work has to be done before one can say complete cure from such disorders is possible.

A recent approach of generating 3D brain tissues, namely “cerebral organoids”, closely reproduces the endogenous developmental program. This approach can give rise to retinal identities, ventral telencephalon, developing cerebral cortex, and choroid plexus, within 1–2 months (Lancaster and Knoblich, 2014). Recent development of these 3D brain organoids derived from human iPSCs is a promising technology for understanding the development of in vitro disease models and investigating in particular the human polygenic disorders where animal models are not sufficient (Lindborg et al., 2016; Quadrato et al., 2016).

Yet, many hitches like mutations, incomplete epigenetic reprogramming and tumors formation, which accompany the use of iPSCs, should be solved as well. Therefore, further understanding of iPSCs, including a genome-wide epigenetic characterization of those cells and further studying of their genomic stability, is needed before beginning their clinical applications in the area of regenerative medicine for treating human diseases, mainly the intractable ones (Table 5). Indeed, these patient-specific hiPSCs will serve in the future as precursors for transplantation and tissue regeneration therapy, as well as a copious resource for in vitro and in vivo disease modeling and drug and genetic screening (Figure 2).

HB, OH, FC, and WA worked on study conception and design. HB, OH, FC, KC, BD, and AM screened titles for relevance and abstracted the data from the eligible full text articles. HB, OH, and WA analyzed and interpreted the data. HB, OH, FC, and WA drafted the manuscript. HB, OH, and WA critically revised the manuscript with input from the entire team. All authors have read and approved the final draft.