Stem Cell Clinic

Induced pluripotent stem cells and Parkinson’s disease …

Review Authors

Correspondence: T. Wang, Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Road, Wuhan 430022, Hubei, China; E-mail: wangtaowh@hust.edu.cn

Many neurodegenerative disorders, such as Parkinson's disease (PD), are characterized by progressive neuronal loss in different regions of the central nervous system, contributing to brain dysfunction in the relevant patients. Stem cell therapy holds great promise for PD patients, including with foetal ventral mesencephalic cells, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Moreover, stem cells can be used to model neurodegenerative diseases in order to screen potential medication and explore their mechanisms of disease. However, related ethical issues, immunological rejection and lack of canonical grafting protocols limit common clinical use of stem cells. iPSCs, derived from reprogrammed somatic cells, provide new hope for cell replacement therapy. In this review, recent development in stem cell treatment for PD, using hiPSCs, as well as the potential value of hiPSCs in modelling for PD, have been summarized for application of iPSCs technology to clinical translation for PD treatment.

adenoviral iPSCs

cluster of differentiation

cynomolgus macaque

dopamine

dopamine transporter

deep brain stimulation

flow cytometric analysis

glucocerebrosidase

human embryonic stem cells

human induced pluripotent stem cells

Lewy bodies

leucine-rich repeat kinase 2

mouse embryonic fibroblasts

major histocompatibility complex

mitochondrial DNA

neural stem cells

Parkinson's disease

subgranular zone

-synuclein

substantia nigra pars compacta

subventricular zone

valproic acid

zinc finger nuclease

zonisamide

Parkinson's disease (PD) is the second most common neurodegenerative disorder, and concerns progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) of the midbrain [1]. The crucial pathological feature of PD is presence of Lewy bodies (LBs), which are abnormal aggregates of -synuclein (SNCA) protein. Reported standardized incidence rates of PD are 818 per 100 000 person-years worldwide [2]. In China, prevalence of PD for those aged 65 years is 1.7% [3]. PD patients suffer from motor symptoms such as rest tremor, bradykinesia, rigidity and abnormal gait. Non-motor symptoms, such as olfactory dysfunction, psychiatric changes and sleep disorders, further impair PD patients' quality of life. Up to now, multiple factors have been found to involve pathogenesis of PD, including genetic susceptibility, environmental toxins, interruption of autophagy, neuroinflammation and most importantly advancing age. Although the precise mechanisms underlying the pathogenesis of PD are not well understood, interactions within these pathogenic factors give rise to loss of DA neurons within the SNpc. Unfortunately, current pharmacological and surgical treatments provide only insufficient symptomatic relief, but cannot reverse nor slow down the underlying loss of midbrain DA neurons. Stem cell transplantation, however, holds great promise in the treatment of PD.

Neural stem cells (NSCs) provide a potential endogenous source for neuron replacement therapy in neurodegenerative disorders such as PD. One of the most important essential features of NSCs is their proliferation potential. It has already been indicated that NSCs can differentiate directly into DA neurons and Suksuphew and Noisa have shown that they have high possibility for producing two undifferentiated daughter cells at early stages of development (symmetric division), and later cell division for production of differentiated neurons plus glial cells (asymmetric division) [4]. A further feature of NSCs' multipotency is their potential to differentiate into astrocytes and oligodendrocytes, as well as into neurons [5, 6]. NSCs in the subventricular zone (SVZ) can differentiate into olfactory neurons, while those of the subgranular zone (SGZ) can differentiate into granular neurons of the dentate gyrus [7]. Furthermore, when implanted into developing eyes, hippocampal NSCs have exhibited several morphological and immunological properties of retinal cells, including photoreceptors [7]. Differentiation of adult NSCs can be influenced by their local environment as well as by intrinsic programmes [8].

Human ESCs were the first human stem cells to be identified and cultured, by Thomson et al. in 1998 [9], and were proven at that time to be self-renewing and pluripotent. These properties indicated hESCs as having great promise for cell transplantation therapy. However, ethical concerns arose immediately as generation of hESCs requires destruction of the fertilized human embryo. A further significant problem with transplanting stem cells is associated with immunological rejection after transplantation of specific cells derived from allogeneic hESCs. In 2006, Yamanaka et al. reported generation of ES-like pluripotent stem cells from somatic fibroblasts, the so-called iPSCs [10]. Since then, many methods have been explored to generate hiPSCs from a wide variety of easily accessible source tissues, including skin, adipose and blood cells [11-14]. Unlike hESCs, there are no ethical issues preventing use of iPSCs. Refinement of reprogramming methods now allows for iPSC generation without genomic integration of reprogramming factors, using expression plasmids, non-integrating viruses, recombinant proteins, small molecules and synthetically modified mRNAs or miRNAs [15]. Here, we review the existing iPSC-based models and treatments, with particular emphasis on PD, and explore the challenges associated with cell therapies using iPSCs-derived DA neurons, which have thus far hindered expansion of this research.

Cell replacement therapy with foetal ventral midbrain (VM) DA neurons has been shown, in some ways, to be beneficial to PD patients. Dopaminergic neurons lost in PD are primarily of the VM, and VM DA neurons arise from floor plate cells during embryonic development. The earliest study of DA neuron differentiation from mouse ESCs was performed by Lee et al. in 2000 [16]. This group generated CNS progenitor populations from ESCs, expanded the cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogens and specific signalling molecules. Their differentiation involved a number of steps: generation of embryoid bodies (EBs) without retinoic acid (RA) treatment in a serum-containing medium, use of a defined medium to select for CNS stem cells, proliferation of CNS stem cells in the presence of mitogen, basic fibroblast growth factor (bFGF), and differentiation of the stem cells by removal of the mitogen in serum-free medium [16]. Finally, the differentiation medium consisted of N2 medium supplemented with laminin, cAMP and ascorbic acid (AA). Sonic hedgehog (SHH), FGF8 and AA also enhanced differentiation to DA fate, and increased yield of ES-derived TH+ neurons. The cells were incubated under differentiation conditions for 615 days at the last stage, in order to increase numbers of TH+ neurons and DA level [16]. While this protocol succeeded in generating DA neurons at relatively high efficiency, extensive studies in Parkinsonian animals are needed to further assess complete function and safety of ESC-derived DA neurons in vivo [16]. Efficiency and purification of generated cell populations also needs to be improved by genetic methods.

After some months, Kawasaki and colleagues introduced an efficient method for generating neurons from ESCs by using PA6-derived stromal cell-derived inducing activity (SDIA) in a serum-free condition requiring neither EBs nor RA treatment [17]. High proportions of TH+ neurons producing DA were obtained from SDIA-treated ESCs. When transplanted, SDIA-induced dopaminergic neurons integrated into the mouse striatum and remained positive for TH expression [17]. In accordance with Lee's group, Kawasaki et al. also avoided using RA in their experiment, as RA seemed to perturb neural patterning and neuronal identities in EBs, as a strong teratogen. Efficiency of DA neuron induction in the SDIA method is as high as maximum efficiency (~30%) obtained by Lee's method with SHH, FGF8 and ascorbate treatment [17]. Neural induction by SDIA provided a new powerful tool for both basic neuroscience research and therapeutic applications.

Low efficiency of generation of DA neurons from primary cultures of foetal neonatal cells, or adult stem cells, limits their therapeutic potential as donor cells [18]. In effort to improve efficiency of DA neuron generation, survival and maturation in vitro, Kim et al. used a cytomegalovirus plasmid (pCMV) driving expression of rat Nurr1 complementary DNA modified to establish stable Nurr1 ESC lines [18]. Nurr1 ESCs raised the proportion of TH+ neurons up to 78%, combined with their previous five-stage differentiation method [16, 18]. They also demonstrated that these DA neurons from ESCs could functionally integrate into host tissue as well as lead to recovery in a rodent model of PD [18].

These results have subsequently been replicated using hESCs with some modifications, but efficiency was not satisfactory [19-21]. Perrier et al. have reported that co-culture of hESCs on MS5 stroma can yield highly efficient differentiation into midbrain DA neurons [22]. Neural differentiation of hESCs was induced by means of co-culture on MS5, MS5-Wnt or S2 stroma at comparable efficiencies. Growth factors were added in various combinations and at various time points. Rosette structures were harvested mechanically from feeder layers on day 28 of differentiation and gently replanted on 15 g/ml polyornithine/1 g/ml laminin-coated culture dishes in N2 medium supplemented with SHH, FGF8, AA and BDNF. These cells were resuspended in N2 medium, and replated again on to polyornithine/laminin-coated culture dishes in the presence of SHH, FGF8, AA and BDNF. After additional 79 days culture, cells were found to have differentiated in the absence of SHH and FGF8 but in the presence of BDNF, glial cell line-derived neurotrophic factor, transforming growth factor type 3, dibutyryl cAMP and AA [22]. The workers observed that exposure to SHH and FGF8 from day 12 to day 20 differentiation, followed by differentiation in the presence of AA and BDNF, resulted in 3-fold increase in TH+ cells. Up to 79% of all the neurons express TH, the rate-limiting enzyme in the synthesis of DA. In addition to TH expression, cells in these cultures expressed key markers associated with normal midbrain DA neurons. However, the high-yield midbrain DA neuron derivation protocol reported here need to be transplantated into pre-clinical animal models of PD [22]. Beyond this, cell survival and long-term maintenance of phenotype are essential parameters for testing in vivo.

In 2008, a group of Korean scientists reported a method for differentiating hESCs into functional TH+ neurons, with up to near 86% total hESC-derived neurons, the highest purity ever reported [23]. The unique feature of their protocol was generation of pure spherical neural masses (SNMs). These SNMs could be expanded for long periods without losing their differentiation capability and could be coaxed into DA neurons efficiently within a relatively short time (approximately 2 weeks) when needed. SNM culture and DA neuron derivation from the SNMs did not need feeder cells, which reduced risks of contamination by unwanted cells and pathogens. More importantly, their hESC-derived DA neurons induced clear behavioural recovery after transplantation in a Parkinsonian rat model, indicating their functionality in vivo [23].

It has been reported that bone marrow mesenchymal stem cells (BMSCs) can differentiate into not only osteogenic, adipogenic, chondrogenic cells, but also into other lineages including myogenic, hepatic and neurogenic cells [24]. Furthermore, they are inducible to differentiate into cells with the DA neuronal phenotype suggested by expression of TH, DAT markers, as well as synthesis and secretion of DA after appropriate stimuli [25]. Previous studies have shown that human BMSC engraftment can alleviate motor dysfunction in Parkinsonian animal models, but with limited efficacy and but few engrafted cells surviving. Our team transplanted equal amounts of hBMSCs into hemi-lesioned Parkinsonian rats with supplementation of bFGF, to assess whether a combination of bFGF and hBMSC therapy would enhance treatment effectiveness in PD rat models [26]. As a result, bFGF promoted hBMSCs to transdifferentiate towards neural-like lineages in vitro [26]. In addition, hBMSC transplantation alleviated motor functional asymmetry, and prevented DA neurons from loss in the PD model, while bFGF administration enhanced neurodifferentiation capacity and therapeutic effect [26].

Similar strategies have been applied for differentiating hiPSCs into DA neurons. Cooper et al. postulated that a major limitation for experimental studies of current ESC/iPSC differentiation protocols, was lack of VM DA neurons of stable phenotype, as defined by expression marker code FOXA2/TH/-tubulin [27]. They demonstrated a combination of three modifications that were required to produce VM DA neurons. First, early and specific exposure to low-dose RA improved regional identity of neural progenitor cells derived from pluripotent stem cells. Secondly, a high activity form of human SHH established a sizeable FOXA2+ neural progenitor cell population in vitro. Thirdly, early exposure to FGF8a, rather than FGF8b, and WNT1 were required for robust differentiation of the FOXA2+ floor plate-like human neural progenitor cells into FOXA2+ DA neurons [27]. FOXA2+ DA neurons were also generated when this protocol was adapted to feeder-free conditions. In summary, their new human ESC and iPSC differentiation protocol can generate human VM DA neurons as required for relevant new bioassays, drug discovery and cell-based therapies for PD [27].

The majority of PD cases are sporadic with unknown cause. Age, oxidative stress, toxin and environmental factors are risk factors [2], and remaining 10% is familial PD, where several causative genes have been identified [28]. Before stem cell modelling appeared, the most used cell or animal models for PD were generated with toxins such as rotenone, 6-OHDA, MPTP or genetic models. The relationship between MPTP and PD was found in a cluster of young drug addicts, by Davis et al. in 1979 [29]. MPTP easily crosses the bloodbrain barrier (BBB) where it is oxidized in glial cells into MPP+. MPP+ competes with DA for the DA transporter and after entering neurons, it exerts its toxic effect by inactivating complex I of the ETC [30]. MPTP is commonly used to model for PD in primates and rodents in that the drug kills dopaminergic neurons, allowing researchers to study neuronal circuitry with reduced dopaminergic involvement [31]. Many workers have demonstrated that MPTP administration is able to reproduce most, but not all, the clinical and pathological hallmarks of PD in monkeys [32-34] and, at least degeneration of dopaminergic neurons, in mice [35]. Similar to MPTP, the pesticide rotenone disrupts complex I function of mitochondria [36]. Our team has demonstrated that rotenone models for PD appear to mimic most clinical features of idiopathic PD and recapitulate the slow and progressive loss of DA neurons and LB formation in the nigral-striatal system [36]. Both MPTP and rotenone have been important for establishment of PD animal models. However, while they promote dopaminergic neuron death with associated motor impairment, their side effects and lack of specificity are major drawbacks [31]. 6-OHDA, a selective catecholaminergic neurotoxin, is used to generate lesions in the nigrostriatal DA neurons in rats [37]. Unlike MPTP, 6-OHDA cannot cross the BBB. So, 6-OHDA must be injected into the SNc, medial forebrain bundle or striatum, to induce Parkinsonism, rather than systemic administration [38]. Intrastriatal injection of 6-OHDA causes progressive retrograde neuronal degeneration in the SNc and VTA [39, 40]. Genetic models provide us with better understanding of underlying genetic forms of PD, even though their pathological and behavioural phenotypes are often quite different from the human condition [41]. Many genetic variant models, including SNCA, LRRK2, PINK, Parkin, DJ-1 and Glucocerebrosidase (GBA), have been generated to explore inherent mechanisms in PD [42-45]. The well-established genetic models are able to interpret pathogenesis of only 510% familial PD, however, without replicating the entire genetic background of the patients in vitro [46]. Moreover, differences between species in displaying neurodegenerative phenotypes make it difficult to extrapolate results obtained from animal models to humans [47]. The discovery of iPSCs has for the first time enabled us to reproduce DA neurons from individuals who suffered from familial or sporadic PD [47, 48]. Moreover, these iPSC models allow us to explore pathogenic factors and discover interactions between genetic and exogenous factors involved in the pathogenesis of PD. As individuals' responses to drug compounds varies, patient-specific iPSCs may be used to distinguish between those individuals likely to respond to new therapeutics and those who are not, and more accurately predict toxicity and efficacy in screening drugs, from mechanisms, in comparison to animal models.

Differentiation of DA neurons from iPSCs has been demonstrated to be relatively robust and reproducible, allowing for generation of disease models from patients carrying a variety of mutations in key genes implicated in familial PD, including PARK2, PINK1, LRRK2, SNCA and GBA [49, 50]. Among genetic risk factors, Parkin (PARK2) is the most frequently mutated gene that has causally been linked to autosomal recessive early-onset familial PD [51]. In patients with PD onset before the age 45, PARK2 mutations are seen in up to 50% of familial cases and about 15% of sporadic cases [52]. Parkin knockout mouse models display some abnormalities, but do not fully recapitulate the pathophysiology of human PARK2. Jiang et al. generated iPSCs from normal subjects and PD patients with Parkin mutations and demonstrated that loss of Parkin in human midbrain DA neurons greatly increased expression of monoamine oxidases and oxidative stress, significantly reduced DA uptake and increased spontaneous DA release [53]. These results suggest that Parkin controls dopamine utilization in human midbrain DA neurons by enhancing the precision of DA neurotransmission and suppressing DA oxidation [53]. Imaizumi et al. used PARK2 patients' specific iPSCs-derived neurons to recapitulate pathogenic changes in the brain of PARK2 patients [54]. The data indicated that PARK2 iPSC-derived neurons exhibited increased oxidative stress, impaired mitochondrial homoeostasis and SNCA accumulation [54]. Recently, Ren et al. showed that the complexity of neuronal processes and microtubule stability were significantly reduced in iPSC-derived neurons from PD patients with Parkin mutations [55], suggesting that Parkin maintains morphological complexity of human neurons by stabilizing microtubules [55]. Shaltouki et al. observed reduced DA differentiation, accompanied by reduced mitochondrial volume ratio and abnormal mitochondrial ultrastructure, consistent with the current model of PARK2 mutations [48].

PINK1 functions upstream of Parkin, and is involved in recruiting Parkin to damaged mitochondria, for example, following mitochondrial depolarization [56]. Mutations in either PARK2 or PINK1 result in impaired mitophagy. Cooper et al. found that PINK1 mutant iPSC-derived DA neuronal cells were more sensitive to cell death and production of ROS elicited by mitochondrial and oxidative stressors, and further showed increased basal oxygen consumption and proton leakage suggestive of intrinsically damaged mitochondria [57]. Moreover, cell vulnerability associated with mitochondrial function in iPSC-derived neural cells could be rescued with coenzyme Q10, rapamycin or LRRK2 kinase inhibitor GW5074. These data demonstrate that iPSC-derived neural cells are sensitive models for measuring vulnerability and doseresponses of candidate neuroprotective molecules and might help to identify disease causes and better individualize treatment efficacy [57].

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with sporadic and familial forms of PD. Nguyen et al. have reported that DA neurons derived from G2019S mutation-iPSCs have high levels of expressions of key oxidative stress-response genes and SNCA protein [58]. The mutant neurons were more sensitive to caspase-3 activation and cell death caused by exposure to stress agents, such as hydrogen peroxide, MG-132 and 6-hydroxydopamine [58]. Cooper et al. indicated that LRRK2 G2019S and R1441C mutations reduced availability of substrates for oxidative phosphorylation, and were associated with disrupted mitochondrial movement in PD patient-specific iPSCs [57]. Laurie et al. further demonstrated the mechanisms by which LRRK2 mutations lead to loss of mitochondrial function. Their data revealed that mitochondrial DNA (mtDNA) damage was induced in neural cells by PD-associated mutations in LRRK2, and this phenotype could be functionally reversed or prevented by zinc finger nuclease (ZFN)-mediated genome editing in iPSCs [59]. These results indicate that mtDNA damage is likely to be a critical early event in neuronal dysfunction that leads ultimately to LRRK2-related PD [59].

The first genetic cause identified for familial PD was SNCA, as PD can be caused by mutations in SNCA or by overexpression of normal SNCA via gene duplication or triplication, consistent with a gain-of-function mechanism. iPSC-derived midbrain DA cultures from SNCA triplication patients exhibit several disease-related phenotypes in culture, including accumulation of SNCA, inherent overexpression of markers of oxidative stress and sensitivity to peroxide-induced oxidative stress [60]. iPSCs, reprogrammed from patients with the most common A53T-SNCA mutation, had high nitric oxide and 3-NT levels compared to controls [61].

GBA mutations, which cause the lysosomal storage disorder Gaucher disease, have recently been linked to a 5-fold greater risk of developing Parkinsonism than non-carrier individuals [62], and are the strongest genetic risk factor for PD known to date. GBA1 mutated iPSC-derived neurons have low glucocerebrosidase activity and protein levels, and high SNCA levels as well as autophagic and lysosomal defects [63]. Mutant neurons display dysregulation of calcium homoeostasis and increased vulnerability to stress responses involving elevation of cytosolic calcium [63]. These findings using iPSC technology, have provided evidence for a link between GBA1 mutations and complex changes in autophagic/lysosomal system and intracellular calcium homoeostasis, underlying vulnerability to neurodegeneration. As monozygotic twins share identical genetic makeup, twin studies have been valuable for dissecting complex gene-environmental interactions in PD. Woodard et al., using iPSC technology [64], investigated a unique set of monozygotic twins and found that SNCA clearance was impaired in midbrain DA neurons carrying GBA N370S regardless of disease status. Moreover, DA levels of twins discordant for PD were different, suggesting that non-genetic factors further perturbed DA homoeostasis in addition to GBA mutations. These results verified the interactions between genetic and environmental factors in the progress of PD, and offer a theoretical basis for personalized medicine in PD (Table 1).

R42P

EX3DEL R275W

More sensitive to cell death and production of ROS elicited by mitochondrial and oxidative stressors, and increased basal oxygen consumption and proton leakage

Phenotype rescue using coenzyme Q10, rapamycin or the LRRK2 kinase inhibitor GW5074

Increased expression of key oxidative stress-response genes and -synuclein

More sensitive to caspase-3 activation and cell death caused by exposure to stress agents

L444P,

N370S

Reduced glucocerebrosidase activity and protein levels, increased -synuclein levels as well as autophagic and lysosomal defects

Dysregulation of calcium homoeostasis and increased vulnerability to stress responses involving elevation of cytosolic calcium

Although the cause of sporadic PD is not fully understood, various factors including environmental toxins, genetic susceptibility and age, have been implicated. Isogenic hiPSC PD models show that toxin-induced nitrosative/oxidative stress results in S-nitrosylation of transcription factor MEF2C and this redox reaction inhibits MEF2C-PGC1 transcriptional network, contributing to mitochondrial dysfunction and apoptotic cell death [65], suggesting that the MEF2C-PGC1 pathway may be a new drug target for PD. The advance of iPSC technology now enables widespread development of PD models for dissecting molecular mechanisms that contribute to its disease pathogenesis.

As previously mentioned, there is currently no cure for PD except for some extent of relieving the symptoms. Current treatments include the use of oral medication of l-DOPA dopamine receptor agonists, MAO-B, apomorphine in more serious cases, continuous intestinal infusion of l-DOPA, and deep brain stimulation (DBS) in subthalamic nucleus and globus pallidus by using surgically implanted electrodes [66]. l-DOPA is the gold standard for treatment of PD. Up to now, no medical nor surgical therapy has been shown to provide superior anti-parkinsonian benefits than can be achieved with l-DOPA [67]. Unfortunately, its therapeutic effect is reduced after around 35 years use [68]. The problems and limitations associated with long-term use of l-DOPA, including on-off fluctuations and emergence of dyskinesia, facilitating exploration of better ways to restore dopamine neurotransmission. Dopamine receptor agonists are used as the first choice to delay initiation of l-DOPA treatment, with longer plasma elimination half-lives than l-DOPA. Their mechanism of action are by stimulation of presynaptic and postsynaptic DA receptors so that their use has therefore been considered to be opportunity to improve continuous drug delivery [67]. Selegiline was the first selective, irreversible inhibitor of monoamine oxidase type B (MAO-B) used in treatment of PD, which can stabilize DA levels in the synaptic cleft [67]. Because of its capacity for interfering with oxidative stress and for blocking MPTP toxicity, selegiline has been tested in the first major trial as a putative disease-modifying agent [67]. Rasagiline is another MAO-B inhibitor, with different metabolites than selegiline, successfully developed for PD therapy. Good tolerance to rasagiline and its ease of use make it an appealing option at the start of therapy [67]. DBS as surgical treatment has some serious limitations. It is costly and can produce cognitive disorders, which may be permanent [68]. All of these treatments have considerable side effects such as ultimate loss of drug effect (wearing off) during disease progression, occurrence of dyskinesia (notably with l-DOPA) use, and appearance of non-motor symptoms that are largely refractory to dopaminergic medication [69]. The concept of using cell transplantation to substitute for loss of DA neurons in the brains of PD patients has evolved. In addition to conventional clinical treatments, such as pharmaceutical drugs and DBS, cell replacement therapy has offered a novel basis for development of effective therapeutic strategies for PD. In 1987, Brundin et al. first transplanted human VM tissue into the striatum of PD patients in Sweden, and the era of cell therapy for PD patients started [70]. Various source tissues have been assessed for therapeutic replacement of DA neurons, such as hESC, hiPSC or DA grafts directly converted from somatic cells. Our team also has transplanted DiI-labelled human umbilical cord mesenchymal stem cells (HUMSCs) to rotenone-induced hemiparkinsonian rats [71]. We showed that intra-CPu transplantation of DiI-labelled HUMSCs 4 weeks after rotenone administration ameliorated APO-induced rotations gradually over a period of 12 months, indicating long-term therapeutic effect of this approach [71]. By monitoring red fluorescence of DiI, we found that HUMSCs migrated in the lesioned cerebral hemisphere, from CPu to SNc, or even to the opposite hemisphere through the corpus callosum. HUMSCs survived for up to 12 months after transplantation, and differentiated into Nestin-, NSE-, GFAP- and TH-positive cells in the CPu and TH+ cells in the SNc. No tumour-like structures was observed in implanted CPu [71]. As reported, vascular endothelial growth factor (VEGF) is a neurotrophic factor which has been proven to promote growth and survival of DA neurons in VM explants and animal models for PD [72-74]. Our previous work has also indicated that relatively low-level expression of VEGF in the striatum protects DA neurons of Parkinsonian rats [75]. Next, we developed a more effective neurorestorative and neuroregenerative therapy combining VEGF and HUMSC [76]. As a result, intrastriatal infusion VEGF-expressing HUMSCs to rotenone-induced Parkinsonian rats provided a significant behavioural improvement, more significant than HUMSC transplantation alone, and resulted in revival of TH immunoreactivity in the lesioned striatum and SNc [76]. Importantly, VEGF expression enhanced neuroprotective effects by promoting DA neuron-orientated differentiation of the HUMSCs. Thus, our findings have presented the suitability of HUMSC as a vector for gene therapy and suggested that stem cell engineering with VEGF may improve transplantation strategies for PD treatment [76].

iPSCs, induced pluripotent cells, have the potential capacity for self-renewal and are able to differentiate into any somatic cells, including DA neurons [77]. Alternatively, iPSCs have properties similar to ESCs but can be generated from adult human cells such as skin, adipose tissue and fibroblasts [10]. Thus, they are ethically more acceptable than some other stem cells sources. In theory, iPSCs from patients are without risk of immunological rejection for autografting [78].

Before successfully generating hiPSCs, many efforts had been made in animal experiments. In 2006, Yamanaka et al. generated iPSCs from mouse embryonic fibroblasts (MEF) and adult mouse tail-tip fibroblasts, by retrovirus-mediated transfection of four transcription factors, Oct3/4, Sox2, c-Myc and Klf4 [10]. One year later, iPSCs were derived by the same group [79] by viral reprogramming of human skin fibroblasts with the same four factors. These studies opened a new avenue for generating patient- and disease-specific pluripotent stem cells. Wernig et al. used 6-OHDA-lesioned rats to examine whether DA neurons derived from directly reprogrammed fibroblasts had therapeutic potential for PD animals [78]. As a result, in the striatum of rats grafted with differentiated iPSCs, a large number of TH+ cells with complex morphologies have been observed, these grafted stem cells were also positive for En1, VMAT2 and DAT. Four of the five transplanted animals which contained large numbers of TH+ neurons showed marked recovery of rotation behaviour 4 weeks after transplantation [78].

Kikuchi et al. first grafted hiPSC-derived DA neurons into the brains of an MPTP-lesioned Parkinsonian monkey, which survived as DA neurons as long as 6 months [80]. In order to reduce immune rejection, Deleidi et al. generated iPSCs from cynomolgus macaque (CM) skin fibroblasts carrying specific major histocompatibility complex (MHC) haplotypes, observing that neither tumour formation nor inflammatory reactions occurred in the transplanted animals, up to 6 months after transplantation [81]. Concerning the aspect of directed differentiation of iPSC into DA neurons, Snchez-Danes et al. indicated lentiviral vectors driving controlled expression of LMX1A was an efficient way to generate enriched populations of human VM DA neurons [82]. However, Mak et al. revealed that the protocol using dorsomorphin and SB431542 to replace SHH with purmorphamine or smoothened agonist could greatly improve conversion of hiPSCs to the neuronal lineage [83]. These histocompatible iPSCs may allow pre-clinical validation of safety and efficacy of iPSCs for PD.

Two commonly used anti-convulsants drugs, valproic acid (VPA) and zonisamide (ZNS), have been tested to promote differentiation of iPSC-derived DA neurons [84]. As iPSC-derived donor cells inevitably contain tumourigenic or inappropriate cells, finding better protocols to purify and sort iPSCs is urgent. Doi et al. have shown that hiPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN [85]. When transplanted into 6-OHDA-lesioned rats, CORIN+ cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement in motor behaviour, without tumour formation [85]. Recently, Hallett et al. analysed CM iPSC-derived midbrain DA neurons for up to 2 years following autologous transplantation in a PD model. They observed that unilateral engraftment of CM-iPSCs provided gradual onset of functional motor improvement, and increased motor activity, without any need for immunosuppression. Postmortem analyses demonstrated robust survival of midbrain-like DA neurons and extensive outgrowth into the transplanted putamen [86]. These experiments offered strong immunological, functional and biological rationales for using midbrain DA neurons derived from iPSCs for future cell replacement in PD (Table 2).

Although pre-clinical studies concerning iPSCs-derived cell therapies have shown great achievement, yet, some limitations hinder clinical usage of iPSCs for PD treatment. Tumourigenicity of iPSCs is an an important putative problem. Murine iPSCs and ESCs both form teratomas when transplanted into syngeneic mice. Also, hiPSCs and hESCs generate teratomas when injected into immunodeficient mice [87]. A standardized sensitive teratoma assay to detect low numbers of tumour-forming cells within a therapeutic cell preparation would be highly valuable. Gropp et al. presented detailed characterization of an efficient, quantitative, sensitive and easy-to-perform teratoma assay [87]. These tumours may be benign (also, they may be malignant), although even so can become fatal when very large. In some studies, when teratomas have been removed from mice, the animals survived [88]. Importantly, aggressiveness of teratocarcinomas from iPSCs is greater than that of ESCs [88]. Differences in oncogenicity between ESCs and iPSCs might be due to their different approach of being derived [88]. As hiPSCs have been first derived by transduction of human dermal fibroblasts with integrating viruses carrying four transcription factors Oct4, Sox2, c-Myc and Klf4 [79], c-Myc is a well-established oncogene while the other three transcription factors are known to be highly expressed in various types of cancer [89-91]. Yamanaka et al. subsequently reported a further Myc family member, L-Myc, as well as C-Myc mutants (W136E and dN2), all of which indicated little transformational activity, promoting hiPSC generation more efficiently and specifically compared to WT C-Myc [32]. A further cause of tumourigenicity may be attributed to random integration of foreign DNA into the host genome disrupting important genes or activating oncogenes, potentially leading to uncontrollable growth of cells [92].

Stadtfeld et al. generated mouse iPSCs from fibroblasts and liver cells by using non-integrating adenoviruses transiently expressing Oct4, Sox2, Klf4 and c-Myc [93]. These adenoviral iPSCs (adeno-iPSCs) showed DNA demethylation characteristic of reprogrammed cells, expressed endogenous pluripotency genes and formed teratomas [93]. Their work indicated that insertional mutagenesis was not required for in vitro reprogramming [93]. Thus, more than 2 years after establishment of iPSC technology by Yamanaka's group, these newly generated adeno-iPSCs have been the first reported reprogrammed pluripotent stem cells with evidence of complete lack of viral transgene integration [94]. Yamanaka et al. described an alternative method to generate iPSCs from MEFs by continual transfection of plasmid vectors free from plasmid integration [95]; this protocol took around 2 months to complete, from MEF isolation to iPSC establishment. The virus-free technique reduced safety concerns for iPSC generation and application, and have provided a source of cells for investigation of mechanisms underlying reprogramming and pluripotency [95]. Introducing mRNA directly into host cells without altering their genomic makeup or using episomal DNA-based vectors which seldom integrate into the host genome, holds the potential to solve this problem, by providing sufficient reprogramming factor expression for successful transformation of somatic cells [96-98].

Anokye-Danso et al. showed that expression of the miR302/367 cluster rapidly and efficiently reprogramed mouse and human somatic cells to an iPSC state without requirement for exogenous transcription factors [99]. This miRNA-based reprogramming approach was two orders of magnitude more efficient than standard Oct4/Sox2/Klf4/Myc-mediated methods, and the miR302/367 iPSCs displayed characteristics similar to the Oct4/Sox2/Klf4/Myc-iPSCs [99].

A further problem which hinders iPSCs treatment is that the therapeutic effect can be influenced by inherent pathogenic features of PD. Interactions between genetic and exogenous factors result in its pathogenesis. A general concern about use of autologous iPSC transplantation is whether underlying PD-associated genetic mutations presented in transplanted neurons increases vulnerability of iPSC-derived midbrain DA neurons to disease pathology. Environmental factors and age contribute largely to the pathogenesis of PD. Thus, iPSC-derived neurons represent a reasonable strategy for more advantages. It has been shown that LBs, the pathological features of PD, can be found in grafts of foetal VM tissue [100, 101]. The first reason is that LB pathology is a reaction to inflammation from host brain tissues, possibly mediated by cell stress induced by reactive microglia [102]. Second, the spread of SNCA into the graft from the host may contribute to these pathological changes [103, 104]. However, Barker et al. believe that these pathological changes are not likely to limit widespread adoption of cell treatments, as numbers of cells with LB-like pathology in the grafts were small compared to numbers of healthy cells [105-107]. Patients can still be functionally stable, more than a decade after such a graft, at a time when accumulation of SNCA had been observed [108, 109].

Apart from the potential risk of tumourigenicity or inherent pathogenic features deriving from donor cells, pluripotent stem cell-derived cell populations for therapies also confer a risk for the contamination of transplantation cell populations with residual pluripotent cells [110]. In order to resolve this issue, several sorting methods have been developed for enrichment of differentiated neural cell populations and elimination of pluripotent stem cells, using flow cytometric analysis (FACS) or MACS [111]. Different combinations of CD markers have been explored to purify heterogeneous pluripotent stem cell-derived neural cell populations. Pruszak et al. identified a cluster of differentiation (CD) surface antigen code for the neural lineage, based on combinatorial FACS of three distinct populations derived from hESCs: combinatorial CD15/CD24/CD29 marker profiles [112]. They found that CD15(+)/CD29(HI)/CD24(LO) surface antigen expression defined NSCs, and this could eliminate tumour formation in vivo, resulting in pure neuronal grafts [112]. Yuan et al. performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on pluripotent stem cells [113]. From this analysis they isolated a population of NSC that were CD184(+)/CD271(-)/CD44(-)/CD24(+) from neural induction cultures of hESCs and hiPSCs [113]. To improve the sorting method, Sundberg et al. sorted primate iPSC-derived neural cell population with NCAM+/CD29low selection [111]. They demonstrated that teh NCAM+/CD29low selection method enriched the FOXA2/TH and EN1/TH+ DA neurons in vitro compared to unsorted cell populations from >10% prior to sorting to > 35% after sorting. Importantly, sorting with NCAM+/CD29low selection prior to transplantation eliminated non-neural tumourigenic cells from the grafts and significantly increased the number of TH+ cells in the cell grafts compared to unsorted cell populations [111].

In this review, we have summarized a number of scientific and ethical issues in modelling and treatment with iPSCs for PD. iPSCs can be employed as relevant Parkinsonian cell models, for drug screening, studying disease progression and most importantly for treatment of PD by transplantation techniques. Compared with other stem cells, iPSCs stand out for their powerful pluripotency, few ethical issues and less immune rejection, although there are still several issues that need to be solved prior to translation of iPSCs into the clinical setting. First, the exact mechanisms of how transplanted cells restore host brain function and how to connect them with circumjacent brain tissues have not been yet elucidated. Second, tumourigenicity of iPSCs may surpass their therapeutic effects. Ultimately, iPSC, derived from autologous PD patients, may contain pathogenic gene mutations that affect prognosis of transplantation therapy. With improvements in differentiation methodologies and better understanding of pathogenesis of PD through patient-specific iPSCs, iPSC therapy can be a potential alternative for PD treatment combined with traditional drug development platforms and gene therapy.

This work was supported by grants 31171211 and 81471305 from the National Natural Science Foundation of China (to TW), grant 81200983 from the National Natural Science Foundation of China (to NX), grant 81301082 from the National Natural Science Foundation of China (to JSH), grant 2012B09 from China Medical Foundation (to NX) and grant 0203201343 from Hubei Molecular Imaging Key Laboratory (to NX). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

There are no actual or potential conflicts of interest.

2016 John Wiley & Sons Ltd

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Induced Pluripotent Stem Cells: A New Frontier for Stem …

Induced pluripotent stem cells (iPSCs) are the newest member of a growing list of stem cell populations that hold great potential for use in cell-based treatment approaches in the dental field. This review summarizes the dental tissues that have successfully been utilized to generate iPSC lines, as well as the potential uses of iPSCs for tissue regeneration in different dental applications. While iPSCs display great promise in a number of dental applications, there are safety concerns with these cells that need to be addressed before they can be used in clinical settings. This review outlines some of the apprehensions to the use of iPSCs clinically, and it details approaches that are being employed to ensure the safety and efficacy of these cells. One of the major approaches being investigated is the differentiation of iPSCs prior to use in patients. iPSCs have successfully been differentiated into a wide range of cells and tissue types. This review focuses on 2 differentiation approaches-the differentiation of iPSCs into mesenchymal stem cells and the differentiation of iPSCs into osteoprogenitor cells. Both these resulting populations of cells are particularly relevant to the dental field.

International & American Associations for Dental Research 2015.

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Induced Pluripotent Stem Cells (iPS) – UCLA Broad Stem Cell

iPSC are derived from skin or blood cells that have been reprogrammed back into an embryonic-like pluripotent state that enables the development of an unlimited source of any type of human cell needed for therapeutic purposes. For example, iPSC can be prodded into becoming beta islet cells to treat diabetes, blood cells to create new blood free of cancer cells for a leukemia patient, or neurons to treat neurological disorders.

In late 2007, a BSCRC team of faculty, Drs. Kathrin Plath, William Lowry, Amander Clark, and April Pyle were among the first in the world to create human iPSC. At that time, science had long understood that tissue specific cells, such as skin cells or blood cells, could only create other like cells. With this groundbreaking discovery, iPSC research has quickly become the foundation for a new regenerative medicine.

Using iPSC technology our faculty have reprogrammed skin cells into active motor neurons, egg and sperm precursors, liver cells, bone precursors, and blood cells. In addition, patients with untreatable diseases such as, ALS, Rett Syndrome, Lesch-Nyhan Disease, and Duchenne's Muscular Dystrophy donate skin cells to BSCRC scientists for iPSC reprogramming research. The generous participation of patients and their families in this research enables BSCRC scientists to study these diseases in the laboratory in the hope of developing new treatment technologies.

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Induced Pluripotent Stem Cells (iPS) - UCLA Broad Stem Cell

Induced stem cells – Wikipedia

Induced stem cells (iSC) are stem cells derived from somatic, reproductive, pluripotent or other cell types by deliberate epigenetic reprogramming. They are classified as either totipotent (iTC), pluripotent (iPSC) or progenitor (multipotentiMSC, also called an induced multipotent progenitor celliMPC) or unipotent(iUSC) according to their developmental potential and degree of dedifferentiation. Progenitors are obtained by so-called direct reprogramming or directed differentiation and are also called induced somatic stem cells.

Three techniques are widely recognized:[1]

In 1895 Thomas Morgan removed one of a frog's two blastomeres and found that amphibians are able to form whole embryos from the remaining part. This meant that the cells can change their differentiation pathway. In 1924 Spemann and Mangold demonstrated the key importance of cellcell inductions during animal development.[20] The reversible transformation of cells of one differentiated cell type to another is called metaplasia.[21] This transition can be a part of the normal maturation process, or caused by an inducement.

One example is the transformation of iris cells to lens cells in the process of maturation and transformation of retinal pigment epithelium cells into the neural retina during regeneration in adult newt eyes. This process allows the body to replace cells not suitable to new conditions with more suitable new cells. In Drosophila imaginal discs, cells have to choose from a limited number of standard discrete differentiation states. The fact that transdetermination (change of the path of differentiation) often occurs for a group of cells rather than single cells shows that it is induced rather than part of maturation.[22]

The researchers were able to identify the minimal conditions and factors that would be sufficient for starting the cascade of molecular and cellular processes to instruct pluripotent cells to organize the embryo. They showed that opposing gradients of bone morphogenetic protein (BMP) and Nodal, two transforming growth factor family members that act as morphogens, are sufficient to induce molecular and cellular mechanisms required to organize, in vivo or in vitro, uncommitted cells of the zebrafish blastula animal pole into a well-developed embryo.[23]

Some types of mature, specialized adult cells can naturally revert to stem cells. For example, "chief" cells express the stem cell marker Troy. While they normally produce digestive fluids for the stomach, they can revert into stem cells to make temporary repairs to stomach injuries, such as a cut or damage from infection. Moreover, they can make this transition even in the absence of noticeable injuries and are capable of replenishing entire gastric units, in essence serving as quiescent "reserve" stem cells.[24] Differentiated airway epithelial cells can revert into stable and functional stem cells in vivo.[25]

After injury, mature terminally differentiated kidney cells dedifferentiate into more primordial versions of themselves and then differentiate into the cell types needing replacement in the damaged tissue[26] Macrophages can self-renew by local proliferation of mature differentiated cells.[27][28] In newts, muscle tissue is regenerated from specialized muscle cells that dedifferentiate and forget the type of cell they had been. This capacity to regenerate does not decline with age and may be linked to their ability to make new stem cells from muscle cells on demand.[29]

A variety of nontumorigenic stem cells display the ability to generate multiple cell types. For instance, multilineage-differentiating stress-enduring (Muse) cells are stress-tolerant adult human stem cells that can self-renew. They form characteristic cell clusters in suspension culture that express a set of genes associated with pluripotency and can differentiate into endodermal, ectodermal and mesodermal cells both in vitro and in vivo.[30][31][32][33][34]

Other well-documented examples of transdifferentiation and their significance in development and regeneration were described in detail.[35][36]

Induced totipotent cells can be obtained by reprogramming somatic cells with somatic-cell nuclear transfer (SCNT). The process involves sucking out the nucleus of a somatic (body) cell and injecting it into an oocyte that has had its nucleus removed[3][5][37][38]

Using an approach based on the protocol outlined by Tachibana et al.,[3] hESCs can be generated by SCNT using dermal fibroblasts nuclei from both a middle-aged 35-year-old male and an elderly, 75-year-old male, suggesting that age-associated changes are not necessarily an impediment to SCNT-based nuclear reprogramming of human cells.[39] Such reprogramming of somatic cells to a pluripotent state holds huge potentials for regenerative medicine. Unfortunately, the cells generated by this technology, potentially are not completely protected from the immune system of the patient (donor of nuclei), because they have the same mitochondrial DNA, as a donor of oocytes, instead of the patients mitochondrial DNA. This reduces their value as a source for autologous stem cell transplantation therapy, as for the present, it is not clear whether it can induce an immune response of the patient upon treatment.

Induced androgenetic haploid embryonic stem cells can be used instead of sperm for cloning. These cells, synchronized in M phase and injected into the oocyte can produce viable offspring.[40]

These developments, together with data on the possibility of unlimited oocytes from mitotically active reproductive stem cells,[41] offer the possibility of industrial production of transgenic farm animals. Repeated recloning of viable mice through a SCNT method that includes a histone deacetylase inhibitor, trichostatin, added to the cell culture medium,[42] show that it may be possible to reclone animals indefinitely with no visible accumulation of reprogramming or genomic errors[43] However, research into technologies to develop sperm and egg cells from stem cells raises bioethical issues.[44]

Such technologies may also have far-reaching clinical applications for overcoming cytoplasmic defects in human oocytes.[3][45] For example, the technology could prevent inherited mitochondrial disease from passing to future generations. Mitochondrial genetic material is passed from mother to child. Mutations can cause diabetes, deafness, eye disorders, gastrointestinal disorders, heart disease, dementia and other neurological diseases. The nucleus from one human egg has been transferred to another, including its mitochondria, creating a cell that could be regarded as having two mothers. The eggs were then fertilised and the resulting embryonic stem cells carried the swapped mitochondrial DNA.[46] As evidence that the technique is safe author of this method points to the existence of the healthy monkeys that are now more than four years old and are the product of mitochondrial transplants across different genetic backgrounds.[47]

In late-generation telomerase-deficient (Terc/) mice, SCNT-mediated reprogramming mitigates telomere dysfunction and mitochondrial defects to a greater extent than iPSC-based reprogramming.[48]

Other cloning and totipotent transformation achievements have been described.[49]

Recently some researchers succeeded to get the totipotent cells without the aid of SCNT. Totipotent cells were obtained using the epigenetic factors such as oocyte germinal isoform of histone.[50] Reprogramming in vivo, by transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice, confers totipotency features. Intraperitoneal injection of such in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic (trophectodermal) markers.[51]

iPSc were first obtained in the form of transplantable teratocarcinoma induced by grafts taken from mouse embryos.[52] Teratocarcinoma formed from somatic cells.[53]Genetically mosaic mice were obtained from malignant teratocarcinoma cells, confirming the cells' pluripotency.[54][55][56] It turned out that teratocarcinoma cells are able to maintain a culture of pluripotent embryonic stem cell in an undifferentiated state, by supplying the culture medium with various factors.[57] In the 1980s, it became clear that transplanting pluripotent/embryonic stem cells into the body of adult mammals, usually leads to the formation of teratomas, which can then turn into a malignant tumor teratocarcinoma.[58] However, putting teratocarcinoma cells into the embryo at the blastocyst stage, caused them to become incorporated in the inner cell mass and often produced a normal chimeric (i.e. composed of cells from different organisms) animal.[59][60][61] This indicated that the cause of the teratoma is a dissonance - mutual miscommunication between young donor cells and surrounding adult cells (the recipient's so-called "niche").

In August 2006, Japanese researchers circumvented the need for an oocyte, as in SCNT. By reprograming mouse embryonic fibroblasts into pluripotent stem cells via the ectopic expression of four transcription factors, namely Oct4, Sox2, Klf4 and c-Myc, they proved that the overexpression of a small number of factors can push the cell to transition to a new stable state that is associated with changes in the activity of thousands of genes.[7]

Reprogramming mechanisms are thus linked, rather than independent and are centered on a small number of genes.[62] IPSC properties are very similar to ESCs.[63] iPSCs have been shown to support the development of all-iPSC mice using a tetraploid (4n) embryo,[64] the most stringent assay for developmental potential. However, some genetically normal iPSCs failed to produce all-iPSC mice because of aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster.[18]

An important advantage of iPSC over ESC is that they can be derived from adult cells, rather than from embryos. Therefore, it became possible to obtain iPSC from adult and even elderly patients.[9][65][66]

Reprogramming somatic cells to iPSC leads to rejuvenation. It was found that reprogramming leads to telomere lengthening and subsequent shortening after their differentiation back into fibroblast-like derivatives.[67] Thus, reprogramming leads to the restoration of embryonic telomere length,[68] and hence increases the potential number of cell divisions otherwise limited by the Hayflick limit.[69]

However, because of the dissonance between rejuvenated cells and the surrounding niche of the recipient's older cells, the injection of his own iPSC usually leads to an immune response,[70] which can be used for medical purposes,[71] or the formation of tumors such as teratoma.[72] The reason has been hypothesized to be that some cells differentiated from ESC and iPSC in vivo continue to synthesize embryonic protein isoforms.[73] So, the immune system might detect and attack cells that are not cooperating properly.

A small molecule called MitoBloCK-6 can force the pluripotent stem cells to die by triggering apoptosis (via cytochrome c release across the mitochondrial outer membrane) in human pluripotent stem cells, but not in differentiated cells. Shortly after differentiation, daughter cells became resistant to death. When MitoBloCK-6 was introduced to differentiated cell lines, the cells remained healthy. The key to their survival, was hypothesized to be due to the changes undergone by pluripotent stem cell mitochondria in the process of cell differentiation. This ability of MitoBloCK-6 to separate the pluripotent and differentiated cell lines has the potential to reduce the risk of teratomas and other problems in regenerative medicine.[74]

In 2012 other small molecules (selective cytotoxic inhibitors of human pluripotent stem cellshPSCs) were identified that prevented human pluripotent stem cells from forming teratomas in mice. The most potent and selective compound of them (PluriSIn #1) inhibits stearoyl-coA desaturase (the key enzyme in oleic acid biosynthesis), which finally results in apoptosis. With the help of this molecule the undifferentiated cells can be selectively removed from culture.[75][76] An efficient strategy to selectively eliminate pluripotent cells with teratoma potential is targeting pluripotent stem cell-specific antiapoptotic factor(s) (i.e., survivin or Bcl10). A single treatment with chemical survivin inhibitors (e.g., quercetin or YM155) can induce selective and complete cell death of undifferentiated hPSCs and is claimed to be sufficient to prevent teratoma formation after transplantation.[77] However, it is unlikely that any kind of preliminary clearance,[78] is able to secure the replanting iPSC or ESC. After the selective removal of pluripotent cells, they re-emerge quickly by reverting differentiated cells into stem cells, which leads to tumors.[79] This may be due to the disorder of let-7 regulation of its target Nr6a1 (also known as Germ cell nuclear factor - GCNF), an embryonic transcriptional repressor of pluripotency genes that regulates gene expression in adult fibroblasts following micro-RNA miRNA loss.[80]

Teratoma formation by pluripotent stem cells may be caused by low activity of PTEN enzyme, reported to promote the survival of a small population (0,1-5% of total population) of highly tumorigenic, aggressive, teratoma-initiating embryonic-like carcinoma cells during differentiation. The survival of these teratoma-initiating cells is associated with failed repression of Nanog as well as a propensity for increased glucose and cholesterol metabolism.[81] These teratoma-initiating cells also expressed a lower ratio of p53/p21 when compared to non-tumorigenic cells.[82] In connection with the above safety problems, the use iPSC for cell therapy is still limited.[83] However, they can be used for a variety of other purposes - including the modeling of disease,[84] screening (selective selection) of drugs, toxicity testing of various drugs.[85]

It is interesting to note that the tissue grown from iPSCs, placed in the "chimeric" embryos in the early stages of mouse development, practically do not cause an immune response (after the embryos have grown into adult mice) and are suitable for autologous transplantation[86] At the same time, full reprogramming of adult cells in vivo within tissues by transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs.[51] Furthermore, partial reprogramming of cells toward pluripotency in vivo in mice demonstrates that incomplete reprogramming entails epigenetic changes (failed repression of Polycomb targets and altered DNA methylation) in cells that drive cancer development.[87]

Determining the unique set of cellular factors that is needed to be manipulated for each cell conversion is a long and costly process that involved much trial and error. As a result, this first step of identifying the key set of cellular factors for cell conversion is the major obstacle researchers face in the field of cell reprogramming. An international team of researchers have developed an algorithm, called Mogrify(1), that can predict the optimal set of cellular factors required to convert one human cell type to another. When tested, Mogrify was able to accurately predict the set of cellular factors required for previously published cell conversions correctly. To further validate Mogrify's predictive ability, the team conducted two novel cell conversions in the laboratory using human cells and these were successful in both attempts solely using the predictions of Mogrify.[89][90][91] Mogrify has been made available online for other researchers and scientists.

By using solely small molecules, Deng Hongkui and colleagues demonstrated that endogenous "master genes" are enough for cell fate reprogramming. They induced a pluripotent state in adult cells from mice using seven small-molecule compounds.[17] The effectiveness of the method is quite high: it was able to convert 0.02% of the adult tissue cells into iPSCs, which is comparable to the gene insertion conversion rate. The authors note that the mice generated from CiPSCs were "100% viable and apparently healthy for up to 6 months". So, this chemical reprogramming strategy has potential use in generating functional desirable cell types for clinical applications.[92][93]

In 2015th year a robust chemical reprogramming system was established with a yield up to 1,000-fold greater than that of the previously reported protocol. So, chemical reprogramming became a promising approach to manipulate cell fates.[94]

The fact that human iPSCs capable of forming teratomas not only in humans but also in some animal body, in particular in mice or pigs, allowed to develop a method for differentiation of iPSCs in vivo. For this purpose, iPSCs with an agent for inducing differentiation into target cells are injected to genetically modified pig or mouse that has suppressed immune system activation on human cells. The formed teratoma is cut out and used for the isolation of the necessary differentiated human cells[95] by means of monoclonal antibody to tissue-specific markers on the surface of these cells. This method has been successfully used for the production of functional myeloid, erythroid and lymphoid human cells suitable for transplantation (yet only to mice).[96] Mice engrafted with human iPSC teratoma-derived hematopoietic cells produced human B and T cells capable of functional immune responses. These results offer hope that in vivo generation of patient customized cells is feasible, providing materials that could be useful for transplantation, human antibody generation and drug screening applications. Using MitoBloCK-6[74] and/or PluriSIn # 1 the differentiated progenitor cells can be further purified from teratoma forming pluripotent cells. The fact, that the differentiation takes place even in the teratoma niche, offers hope that the resulting cells are sufficiently stable to stimuli able to cause their transition back to the dedifferentiated (pluripotent) state and therefore safe. A similar in vivo differentiation system, yielding engraftable hematopoietic stem cells from mouse and human iPSCs in teratoma-bearing animals in combination with a maneuver to facilitate hematopoiesis, was described by Suzuki et al.[97] They noted that neither leukemia nor tumors were observed in recipients after intravenous injection of iPSC-derived hematopoietic stem cells into irradiated recipients. Moreover, this injection resulted in multilineage and long-term reconstitution of the hematolymphopoietic system in serial transfers. Such system provides a useful tool for practical application of iPSCs in the treatment of hematologic and immunologic diseases.[98]

For further development of this method animal in which is grown the human cell graft, for example mouse, must have so modified genome that all its cells express and have on its surface human SIRP.[99] To prevent rejection after transplantation to the patient of the allogenic organ or tissue, grown from the pluripotent stem cells in vivo in the animal, these cells should express two molecules: CTLA4-Ig, which disrupts T cell costimulatory pathways and PD-L1, which activates T cell inhibitory pathway.[100]

See also: US 20130058900 patent.

In the near-future, clinical trials designed to demonstrate the safety of the use of iPSCs for cell therapy of the people with age-related macular degeneration, a disease causing blindness through retina damaging, will begin. There are several articles describing methods for producing retinal cells from iPSCs[101][102] and how to use them for cell therapy.[103][104] Reports of iPSC-derived retinal pigmented epithelium transplantation showed enhanced visual-guided behaviors of experimental animals for 6 weeks after transplantation.[105] However, clinical trials have been successful: ten patients suffering from retinitis pigmentosa have had their eyesight restoredincluding a woman who had only 17 percent of her vision left.[106]

Chronic lung diseases such as idiopathic pulmonary fibrosis and cystic fibrosis or chronic obstructive pulmonary disease and asthma are leading causes of morbidity and mortality worldwide with a considerable human, societal and financial burden. So there is an urgent need for effective cell therapy and lung tissue engineering.[107][108] Several protocols have been developed for generation of the most cell types of the respiratory system, which may be useful for deriving patient-specific therapeutic cells.[109][110][111][112][113]

Some lines of iPSCs have the potentiality to differentiate into male germ cells and oocyte-like cells in an appropriate niche (by culturing in retinoic acid and porcine follicular fluid differentiation medium or seminiferous tubule transplantation). Moreover, iPSC transplantation make a contribution to repairing the testis of infertile mice, demonstrating the potentiality of gamete derivation from iPSCs in vivo and in vitro.[114]

The risk of cancer and tumors creates the need to develop methods for safer cell lines suitable for clinical use. An alternative approach is so-called "direct reprogramming" - transdifferentiation of cells without passing through the pluripotent state.[115][116][117][118][119][120] The basis for this approach was that 5-azacytidine - a DNA demethylation reagent - can cause the formation of myogenic, chondrogenic and adipogeni clones in the immortal cell line of mouse embryonic fibroblasts[121] and that the activation of a single gene, later named MyoD1, is sufficient for such reprogramming.[122] Compared with iPSC whose reprogramming requires at least two weeks, the formation of induced progenitor cells sometimes occurs within a few days and the efficiency of reprogramming is usually many times higher. This reprogramming does not always require cell division.[123] The cells resulting from such reprogramming are more suitable for cell therapy because they do not form teratomas.[120] For example, Chandrakanthan et al., & Pimanda describe the generation of tissue-regenerative multipotent stem cells (iMS cells) by treating mature bone and fat cells transiently with a growth factor (platelet-derived growth factorAB (PDGF-AB)) and 5-Azacytidine. These authors claim that: "Unlike primary mesenchymal stem cells, which are used with little objective evidence in clinical practice to promote tissue repair, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner without forming tumors" and so "has significant scope for application in tissue regeneration."[124][125][126]

Originally only early embryonic cells could be coaxed into changing their identity. Mature cells are resistant to changing their identity once they've committed to a specific kind. However, brief expression of a single transcription factor, the ELT-7 GATA factor, can convert the identity of fully differentiated, specialized non-endodermal cells of the pharynx into fully differentiated intestinal cells in intact larvae and adult roundworm Caenorhabditis elegans with no requirement for a dedifferentiated intermediate.[127]

The cell fate can be effectively manipulated by directly activating of specific endogenous gene expression with CRISPR-mediated activator. When dCas9 (that has been modified so that it no longer cuts DNA, but still can be guided to specific sequences and to bind to them) is combined with transcription activators, it can precisely manipulate endogenous gene expression. Using this method, Wei et al., enhanced the expression of endogenous Cdx2 and Gata6 genes by CRISPR-mediated activators, thus directly converted mouse embryonic stem cells into two extraembryonic lineages, i.e., typical trophoblast stem cells and extraembryonic endoderm cells.[128] An analogous approach was used to induce activation of the endogenous Brn2, Ascl1, and Myt1l genes to convert mouse embryonic fibroblasts to induced neuronal cells.[129] Thus, transcriptional activation and epigenetic remodeling of endogenous master transcription factors are sufficient for conversion between cell types. The rapid and sustained activation of endogenous genes in their native chromatin context by this approach may facilitate reprogramming with transient methods that avoid genomic integration and provides a new strategy for overcoming epigenetic barriers to cell fate specification.

Another way of reprogramming is the simulation of the processes that occur during amphibian limb regeneration. In urodele amphibians, an early step in limb regeneration is skeletal muscle fiber dedifferentiation into a cellulate that proliferates into limb tissue. However, sequential small molecule treatment of the muscle fiber with myoseverin, reversine (the aurora B kinase inhibitor) and some other chemicals: BIO (glycogen synthase-3 kinase inhibitor), lysophosphatidic acid (pleiotropic activator of G-protein-coupled receptors), SB203580 (p38 MAP kinase inhibitor), or SQ22536 (adenylyl cyclase inhibitor) causes the formation of new muscle cell types as well as other cell types such as precursors to fat, bone and nervous system cells.[130]

The researchers discovered that GCSF-mimicking antibody can activate a growth-stimulating receptor on marrow cells in a way that induces marrow stem cells that normally develop into white blood cells to become neural progenitor cells. The technique[131] enables researchers to search large libraries of antibodies and quickly select the ones with a desired biological effect.[132]

Schlegel and Liu[133] demonstrated that the combination of feeder cells[134][135][136] and a Rho kinase inhibitor (Y-27632) [137][138] induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro. This process occurs without the need for transduction of exogenous viral or cellular genes. These cells have been termed "Conditionally Reprogrammed Cells (CRC)". The induction of CRCs is rapid and results from reprogramming of the entire cell population. CRCs do not express high levels of proteins characteristic of iPSCs or embryonic stem cells (ESCs) (e.g., Sox2, Oct4, Nanog, or Klf4). This induction of CRCs is reversible and removal of Y-27632 and feeders allows the cells to differentiate normally.[133][139][140] CRC technology can generate 2106 cells in 5 to 6 days from needle biopsies and can generate cultures from cryopreserved tissue and from fewer than four viable cells. CRCs retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors.[133][141][142]

The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking.[133][141][142] Using CRC technology, researchers were able to identify an effective therapy for a patient with a rare type of lung tumor.[143] Engleman's group[144] describes a pharmacogenomic platform that facilitates rapid discovery of drug combinations that can overcome resistance using CRC system. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host. While initial studies revealed that co-culturing epithelial cells with Swiss 3T3 cells J2 was essential for CRC induction, with transwell culture plates, physical contact between feeders and epithelial cells is not required for inducing CRCs and more importantly that irradiation of the feeder cells is required for this induction. Consistent with the transwell experiments, conditioned medium induces and maintains CRCs, which is accompanied by a concomitant increase of cellular telomerase activity. The activity of the conditioned medium correlates directly with radiation-induced feeder cell apoptosis. Thus, conditional reprogramming of epithelial cells is mediated by a combination of Y-27632 and a soluble factor(s) released by apoptotic feeder cells.[145]

Riegel et al.[146] demonstrate that mouse ME cells isolated from normal mammary glands or from mouse mammary tumor virus (MMTV)-Neuinduced mammary tumors, can be cultured indefinitely as conditionally reprogrammed cells (CRCs). Cell surface progenitor-associated markers are rapidly induced in normal mouse ME-CRCs relative to ME cells. However, the expression of certain mammary progenitor subpopulations, such as CD49f+ ESA+ CD44+, drops significantly in later passages. Nevertheless, mouse ME-CRCs grown in a three-dimensional extracellular matrix gave rise to mammary acinar structures. ME-CRCs isolated from MMTV-Neu transgenic mouse mammary tumors express high levels of HER2/neu, as well as tumor-initiating cell markers, such as CD44+, CD49f+ and ESA+ (EpCam). These patterns of expression are sustained in later CRC passages. Early and late passage ME-CRCs from MMTV-Neu tumors that were implanted in the mammary fat pads of syngeneic or nude mice developed vascular tumors that metastasized within 6 weeks of transplantation. Importantly, the histopathology of these tumors was indistinguishable from that of the parental tumors that develop in the MMTV-Neu mice. Application of the CRC system to mouse mammary epithelial cells provides an attractive model system to study the genetics and phenotype of normal and transformed mouse epithelium in a defined culture environment and in vivo transplant studies.

A different approach to CRC is to inhibit CD47a membrane protein that is the thrombospondin-1 receptor. Loss of CD47 permits sustained proliferation of primary murine endothelial cells, increases asymmetric division and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters. CD47 knockdown acutely increases mRNA levels of c-Myc and other stem cell transcription factors in cells in vitro and in vivo. Thrombospondin-1 is a key environmental signal that inhibits stem cell self-renewal via CD47. Thus, CD47 antagonists enable cell self-renewal and reprogramming by overcoming negative regulation of c-Myc and other stem cell transcription factors.[147] In vivo blockade of CD47 using an antisense morpholino increases survival of mice exposed to lethal total body irradiation due to increased proliferative capacity of bone marrow-derived cells and radioprotection of radiosensitive gastrointestinal tissues.[148]

Differentiated macrophages can self-renew in tissues and expand long-term in culture.[27] Under certain conditions macrophages can divide without losing features they have acquired while specializing into immune cells - which is usually not possible with differentiated cells. The macrophages achieve this by activating a gene network similar to one found in embryonic stem cells. Single-cell analysis revealed that, in vivo, proliferating macrophages can derepress a macrophage-specific enhancer repertoire associated with a gene network controlling self-renewal. This happened when concentrations of two transcription factors named MafB and c-Maf were naturally low or were inhibited for a short time. Genetic manipulations that turned off MafB and c-Maf in the macrophages caused the cells to start a self-renewal program. The similar network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers.[28]

Hence macrophages isolated from MafB- and c-Maf-double deficient mice divide indefinitely; the self-renewal depends on c-Myc and Klf4.[149]

Indirect lineage conversion is a reprogramming methodology in which somatic cells transition through a plastic intermediate state of partially reprogrammed cells (pre-iPSC), induced by brief exposure to reprogramming factors, followed by differentiation in a specially developed chemical environment (artificial niche).[150]

This method could be both more efficient and safer, since it does not seem to produce tumors or other undesirable genetic changes and results in much greater yield than other methods. However, the safety of these cells remains questionable. Since lineage conversion from pre-iPSC relies on the use of iPSC reprogramming conditions, a fraction of the cells could acquire pluripotent properties if they do not stop the de-differentation process in vitro or due to further de-differentiation in vivo.[151]

A common feature of pluripotent stem cells is the specific nature of protein glycosylation of their outer membrane. That distinguishes them from most nonpluripotent cells, although not white blood cells.[152] The glycans on the stem cell surface respond rapidly to alterations in cellular state and signaling and are therefore ideal for identifying even minor changes in cell populations. Many stem cell markers are based on cell surface glycan epitopes including the widely used markers SSEA-3, SSEA-4, Tra 1-60 and Tra 1-81.[153] Suila Heli et al.[154] speculate that in human stem cells extracellular O-GlcNAc and extracellular O-LacNAc, play a crucial role in the fine tuning of Notch signaling pathway - a highly conserved cell signaling system, that regulates cell fate specification, differentiation, leftright asymmetry, apoptosis, somitogenesis, angiogenesis and plays a key role in stem cell proliferation (reviewed by Perdigoto and Bardin[155] and Jafar-Nejad et al.[156])

Changes in outer membrane protein glycosylation are markers of cell states connected in some way with pluripotency and differentiation.[157] The glycosylation change is apparently not just the result of the initialization of gene expression, but perform as an important gene regulator involved in the acquisition and maintenance of the undifferentiated state.[158]

For example, activation of glycoprotein ACA,[159] linking glycosylphosphatidylinositol on the surface of the progenitor cells in human peripheral blood, induces increased expression of genes Wnt, Notch-1, BMI1 and HOXB4 through a signaling cascade PI3K/Akt/mTor/PTEN and promotes the formation of a self-renewing population of hematopoietic stem cells.[160]

Furthermore, dedifferentiation of progenitor cells induced by ACA-dependent signaling pathway leads to ACA-induced pluripotent stem cells, capable of differentiating in vitro into cells of all three germ layers.[161] The study of lectins' ability to maintain a culture of pluripotent human stem cells has led to the discovery of lectin Erythrina crista-galli (ECA), which can serve as a simple and highly effective matrix for the cultivation of human pluripotent stem cells.[162]

Cell adhesion protein E-cadherin is indispensable for a robust pluripotent phenotype.[163] During reprogramming for iPS cell generation, N-cadherin can replace function of E-cadherin.[164] These functions of cadherins are not directly related to adhesion because sphere morphology helps maintaining the "stemness" of stem cells.[165] Moreover, sphere formation, due to forced growth of cells on a low attachment surface, sometimes induces reprogramming. For example, neural progenitor cells can be generated from fibroblasts directly through a physical approach without introducing exogenous reprogramming factors.

Physical cues, in the form of parallel microgrooves on the surface of cell-adhesive substrates, can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells' epigenetic state. Specifically, "decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)a subunit of H3 methyltranferaseby microgrooved surfaces lead to increased histone H3 acetylation and methylation". Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves, suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state.[166]

Substrate rigidity is an important biophysical cue influencing neural induction and subtype specification. For example, soft substrates promote neuroepithelial conversion while inhibiting neural crest differentiation of hESCs in a BMP4-dependent manner. Mechanistic studies revealed a multi-targeted mechanotransductive process involving mechanosensitive Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosin cytoskeleton integrity and contractility.[167]

Mouse embryonic stem cells (mESCs) undergo self-renewal in the presence of the cytokine leukemia inhibitory factor (LIF). Following LIF withdrawal, mESCs differentiate, accompanied by an increase in cellsubstratum adhesion and cell spreading. Restricted cell spreading in the absence of LIF by either culturing mESCs on chemically defined, weakly adhesive biosubstrates, or by manipulating the cytoskeleton allowed the cells to remain in an undifferentiated and pluripotent state. The effect of restricted cell spreading on mESC self-renewal is not mediated by increased intercellular adhesion, as inhibition of mESC adhesion using a function blocking anti E-cadherin antibody or siRNA does not promote differentiation.[168] Possible mechanisms of stem cell fate predetermination by physical interactions with the extracellular matrix have been described.[169][170]

A new method has been developed that turns cells into stem cells faster and more efficiently by 'squeezing' them using 3D microenvironment stiffness and density of the surrounding gel. The technique can be applied to a large number of cells to produce stem cells for medical purposes on an industrial scale.[171][172]

Cells involved in the reprogramming process change morphologically as the process proceeds. This results in physical difference in adhesive forces among cells. Substantial differences in 'adhesive signature' between pluripotent stem cells, partially reprogrammed cells, differentiated progeny and somatic cells allowed to develop separation process for isolation of pluripotent stem cells in microfluidic devices,[173] which is:

Stem cells possess mechanical memory (they remember past physical signals)with the Hippo signaling pathway factors:[174] Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding domain (TAZ) acting as an intracellular mechanical rheostatthat stores information from past physical environments and influences the cells' fate.[175][176]

Stroke and many neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis need cell replacement therapy. The successful use of converted neural cells (cNs) in transplantations open a new avenue to treat such diseases.[177] Nevertheless, induced neurons (iNs), directly converted from fibroblasts are terminally committed and exhibit very limited proliferative ability that may not provide enough autologous donor cells for transplantation.[178] Self-renewing induced neural stem cells (iNSCs) provide additional advantages over iNs for both basic research and clinical applications.[118][119][120][179][180]

For example, under specific growth conditions, mouse fibroblasts can be reprogrammed with a single factor, Sox2, to form iNSCs that self-renew in culture and after transplantation can survive and integrate without forming tumors in mouse brains.[181] INSCs can be derived from adult human fibroblasts by non-viral techniques, thus offering a safe method for autologous transplantation or for the development of cell-based disease models.[180]

Neural chemically induced progenitor cells (ciNPCs) can be generated from mouse tail-tip fibroblasts and human urinary somatic cells without introducing exogenous factors, but - by a chemical cocktail, namely VCR (V, VPA, an inhibitor of HDACs; C, CHIR99021, an inhibitor of GSK-3 kinases and R, RepSox, an inhibitor of TGF beta signaling pathways), under a physiological hypoxic condition.[182] Alternative cocktails with inhibitors of histone deacetylation, glycogen synthase kinase and TGF- pathways (where: sodium butyrate (NaB) or Trichostatin A (TSA) could replace VPA, Lithium chloride (LiCl) or lithium carbonate (Li2CO3) could substitute CHIR99021, or Repsox may be replaced with SB-431542 or Tranilast) show similar efficacies for ciNPC induction.[182] Zhang, et al.,[183] also report highly efficient reprogramming of mouse fibroblasts into induced neural stem cell-like cells (ciNSLCs) using a cocktail of nine components.

Multiple methods of direct transformation of somatic cells into induced neural stem cells have been described.[184]

Proof of principle experiments demonstrate that it is possible to convert transplanted human fibroblasts and human astrocytes directly in the brain that are engineered to express inducible forms of neural reprogramming genes, into neurons, when reprogramming genes (Ascl1, Brn2a and Myt1l) are activated after transplantation using a drug.[185]

Astrocytesthe most common neuroglial brain cells, which contribute to scar formation in response to injurycan be directly reprogrammed in vivo to become functional neurons that formed networks in mice without the need of cell transplantation.[186] The researchers followed the mice for nearly a year to look for signs of tumor formation and reported finding none. The same researchers have turned scar-forming astrocytes into progenitor cells called neuroblasts that regenerated into neurons in the injured adult spinal cord.[187]

Without myelin to insulate neurons, nerve signals quickly lose power. Diseases that attack myelin, such as multiple sclerosis, result in nerve signals that cannot propagate to nerve endings and as a consequence lead to cognitive, motor and sensory problems. Transplantation of oligodendrocyte precursor cells (OPCs), which can successfully create myelin sheaths around nerve cells, is a promising potential therapeutic response. Direct lineage conversion of mouse and rat fibroblasts into oligodendroglial cells provides a potential source of OPCs. Conversion by forced expression of both eight[188] or of the three[189] transcription factors Sox10, Olig2 and Zfp536, may provide such cells.

Cell-based in vivo therapies may provide a transformative approach to augment vascular and muscle growth and to prevent non-contractile scar formation by delivering transcription factors[115] or microRNAs[14] to the heart.[190] Cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of cardiac core transcription factors ( GATA4, MEF2C, TBX5 and for improved reprogramming plus ESRRG, MESP1, Myocardin and ZFPM2) after coronary ligation.[115][191] These results implicated therapies that can directly remuscularize the heart without cell transplantation. However, the efficiency of such reprogramming turned out to be very low and the phenotype of received cardiomyocyte-like cells does not resemble those of a mature normal cardiomyocyte. Furthermore, transplantation of cardiac transcription factors into injured murine hearts resulted in poor cell survival and minimal expression of cardiac genes.[192]

Meanwhile, advances in the methods of obtaining cardiac myocytes in vitro occurred.[193][194] Efficient cardiac differentiation of human iPS cells gave rise to progenitors that were retained within infarcted rat hearts and reduced remodeling of the heart after ischemic damage.[195]

The team of scientists, who were led by Sheng Ding, used a cocktail of nine chemicals (9C) for transdifferentiation of human skin cells into beating heart cells. With this method, more than 97% of the cells began beating, a characteristic of fully developed, healthy heart cells. The chemically induced cardiomyocyte-like cells (ciCMs) uniformly contracted and resembled human cardiomyocytes in their transcriptome, epigenetic, and electrophysiological properties. When transplanted into infarcted mouse hearts, 9C-treated fibroblasts were efficiently converted to ciCMs and developed into healthy-looking heart muscle cells within the organ.[196] This chemical reprogramming approach, after further optimization, may offer an easy way to provide the cues that induce heart muscle to regenerate locally.[197]

In another study, ischemic cardiomyopathy in the murine infarction model was targeted by iPS cell transplantation. It synchronized failing ventricles, offering a regenerative strategy to achieve resynchronization and protection from decompensation by dint of improved left ventricular conduction and contractility, reduced scarring and reversal of structural remodelling.[198] One protocol generated populations of up to 98% cardiomyocytes from hPSCs simply by modulating the canonical Wnt signaling pathway at defined time points in during differentiation, using readily accessible small molecule compounds.[199]

Discovery of the mechanisms controlling the formation of cardiomyocytes led to the development of the drug ITD-1, which effectively clears the cell surface from TGF- receptor type II and selectively inhibits intracellular TGF- signaling. It thus selectively enhances the differentiation of uncommitted mesoderm to cardiomyocytes, but not to vascular smooth muscle and endothelial cells.[200]

One project seeded decellularized mouse hearts with human iPSC-derived multipotential cardiovascular progenitor cells. The introduced cells migrated, proliferated and differentiated in situ into cardiomyocytes, smooth muscle cells and endothelial cells to reconstruct the hearts. In addition, the heart's extracellular matrix (the substrate of heart scaffold) signalled the human cells into becoming the specialised cells needed for proper heart function. After 20 days of perfusion with growth factors, the engineered heart tissues started to beat again and were responsive to drugs.[201]

See also: review[202]

The elderly often suffer from progressive muscle weakness and regenerative failure owing in part to elevated activity of the p38 and p38 mitogen-activated kinase pathway in senescent skeletal muscle stem cells. Subjecting such stem cells to transient inhibition of p38 and p38 in conjunction with culture on soft hydrogel substrates rapidly expands and rejuvenates them that result in the return of their strength.[203]

In geriatric mice, resting satellite cells lose reversible quiescence by switching to an irreversible pre-senescence state, caused by derepression of p16INK4a (also called Cdkn2a). On injury, these cells fail to activate and expand, even in a youthful environment. p16INK4a silencing in geriatric satellite cells restores quiescence and muscle regenerative functions.[204]

Myogenic progenitors for potential use in disease modeling or cell-based therapies targeting skeletal muscle could also be generated directly from induced pluripotent stem cells using free-floating spherical culture (EZ spheres) in a culture medium supplemented with high concentrations (100ng/ml) of fibroblast growth factor-2 (FGF-2) and epidermal growth factor.[205]

Unlike current protocols for deriving hepatocytes from human fibroblasts, Saiyong Zhu et al., (2014)[206] did not generate iPSCs but, using small molecules, cut short reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells and subsequently hepatocytes (iMPC-Heps) were efficiently differentiated. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to those of human primary adult hepatocytes. iMPC-Heps did not form tumours, most probably because they never entered a pluripotent state.

These results establish the feasibility of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy.

Complications of Diabetes mellitus such as cardiovascular diseases, retinopathy, neuropathy, nephropathy and peripheral circulatory diseases depend on sugar dysregulation due to lack of insulin from pancreatic beta cells and can be lethal if they are not treated. One of the promising approaches to understand and cure diabetes is to use pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PCSs (iPSCs).[207] Unfortunately, human PSC-derived insulin-expressing cells resemble human fetal cells rather than adult cells. In contrast to adult cells, fetal cells seem functionally immature, as indicated by increased basal glucose secretion and lack of glucose stimulation and confirmed by RNA-seq of whose transcripts.[208]

An alternative strategy is the conversion of fibroblasts towards distinct endodermal progenitor cell populations and, using cocktails of signalling factors, successful differentiation of these endodermal progenitor cells into functional beta-like cells both in vitro and in vivo.[209]

Overexpression of the three transcription factors, PDX1 (required for pancreatic bud outgrowth and beta-cell maturation), NGN3 (required for endocrine precursor cell formation) and MAFA (for beta-cell maturation) combination (called PNM) can lead to the transformation of some cell types into a beta cell-like state.[210] An accessible and abundant source of functional insulin-producing cells is intestine. PMN expression in human intestinal "organoids" stimulates the conversion of intestinal epithelial cells into -like cells possibly acceptable for transplantation.[211]

Adult proximal tubule cells were directly transcriptionally reprogrammed to nephron progenitors of the embryonic kidney, using a pool of six genes of instructive transcription factors (SIX1, SIX2, OSR1, Eyes absent homolog 1(EYA1), Homeobox A11 (HOXA11) and Snail homolog 2 (SNAI2)) that activated genes consistent with a cap mesenchyme/nephron progenitor phenotype in the adult proximal tubule cell line.[212] The generation of such cells may lead to cellular therapies for adult renal disease. Embryonic kidney organoids placed into adult rat kidneys can undergo onward development and vascular development.[213]

As blood vessels age, they often become abnormal in structure and function, thereby contributing to numerous age-associated diseases including myocardial infarction, ischemic stroke and atherosclerosis of arteries supplying the heart, brain and lower extremities. So, an important goal is to stimulate vascular growth for the collateral circulation to prevent the exacerbation of these diseases. Induced Vascular Progenitor Cells (iVPCs) are useful for cell-based therapy designed to stimulate coronary collateral growth. They were generated by partially reprogramming endothelial cells.[150] The vascular commitment of iVPCs is related to the epigenetic memory of endothelial cells, which engenders them as cellular components of growing blood vessels. That is why, when iVPCs were implanted into myocardium, they engrafted in blood vessels and increased coronary collateral flow better than iPSCs, mesenchymal stem cells, or native endothelial cells.[214]

Ex vivo genetic modification can be an effective strategy to enhance stem cell function. For example, cellular therapy employing genetic modification with Pim-1 kinase (a downstream effector of Akt, which positively regulates neovasculogenesis) of bone marrowderived cells[215] or human cardiac progenitor cells, isolated from failing myocardium[216] results in durability of repair, together with the improvement of functional parameters of myocardial hemodynamic performance.

Stem cells extracted from fat tissue after liposuction may be coaxed into becoming progenitor smooth muscle cells (iPVSMCs) found in arteries and veins.[217]

The 2D culture system of human iPS cells[218] in conjunction with triple marker selection (CD34 (a surface glycophosphoprotein expressed on developmentally early embryonic fibroblasts), NP1 (receptor - neuropilin 1) and KDR (kinase insert domain-containing receptor)) for the isolation of vasculogenic precursor cells from human iPSC, generated endothelial cells that, after transplantation, formed stable, functional mouse blood vessels in vivo, lasting for 280 days.[219]

To treat infarction, it is important to prevent the formation of fibrotic scar tissue. This can be achieved in vivo by transient application of paracrine factors that redirect native heart progenitor stem cell contributions from scar tissue to cardiovascular tissue. For example, in a mouse myocardial infarction model, a single intramyocardial injection of human vascular endothelial growth factor A mRNA (VEGF-A modRNA), modified to escape the body's normal defense system, results in long-term improvement of heart function due to mobilization and redirection of epicardial progenitor cells toward cardiovascular cell types.[220]

RBC transfusion is necessary for many patients. However, to date the supply of RBCs remains labile. In addition, transfusion risks infectious disease transmission. A large supply of safe RBCs generated in vitro would help to address this issue. Ex vivo erythroid cell generation may provide alternative transfusion products to meet present and future clinical requirements.[221][222] Red blood cells (RBC)s generated in vitro from mobilized CD34 positive cells have normal survival when transfused into an autologous recipient.[223] RBC produced in vitro contained exclusively fetal hemoglobin (HbF), which rescues the functionality of these RBCs. In vivo the switch of fetal to adult hemoglobin was observed after infusion of nucleated erythroid precursors derived from iPSCs.[224] Although RBCs do not have nuclei and therefore can not form a tumor, their immediate erythroblasts precursors have nuclei. The terminal maturation of erythroblasts into functional RBCs requires a complex remodeling process that ends with extrusion of the nucleus and the formation of an enucleated RBC.[225] Cell reprogramming often disrupts enucleation. Transfusion of in vitro-generated RBCs or erythroblasts does not sufficiently protect against tumor formation.

The aryl hydrocarbon receptor (AhR) pathway (which has been shown to be involved in the promotion of cancer cell development) plays an important role in normal blood cell development. AhR activation in human hematopoietic progenitor cells (HPs) drives an unprecedented expansion of HPs, megakaryocyte- and erythroid-lineage cells.[226] See also Concise Review:[227][228] The SH2B3 gene encodes a negative regulator of cytokine signaling and naturally occurring loss-of-function variants in this gene increase RBC counts in vivo. Targeted suppression of SH2B3 in primary human hematopoietic stem and progenitor cells enhanced the maturation and overall yield of in-vitro-derived RBCs. Moreover, inactivation of SH2B3 by CRISPR/Cas9 genome editing in human pluripotent stem cells allowed enhanced erythroid cell expansion with preserved differentiation.[229] (See also overview.[228][230])

Platelets help prevent hemorrhage in thrombocytopenic patients and patients with thrombocythemia. A significant problem for multitransfused patients is refractoriness to platelet transfusions. Thus, the ability to generate platelet products ex vivo and platelet products lacking HLA antigens in serum-free media would have clinical value. An RNA interference-based mechanism used a lentiviral vector to express short-hairpin RNAi targeting 2-microglobulin transcripts in CD34-positive cells. Generated platelets demonstrated an 85% reduction in class I HLA antigens. These platelets appeared to have normal function in vitro[231]

One clinically-applicable strategy for the derivation of functional platelets from human iPSC involves the establishment of stable immortalized megakaryocyte progenitor cell lines (imMKCLs) through doxycycline-dependent overexpression of BMI1 and BCL-XL. The resulting imMKCLs can be expanded in culture over extended periods (45 months), even after cryopreservation. Halting the overexpression (by the removal of doxycycline from the medium) of c-MYC, BMI1 and BCL-XL in growing imMKCLs led to the production of CD42b+ platelets with functionality comparable to that of native platelets on the basis of a range of assays in vitro and in vivo.[232] Thomas et al., describe a forward programming strategy relying on the concurrent exogenous expression of 3 transcription factors: GATA1, FLI1 and TAL1. The forward programmed megakaryocytes proliferate and differentiate in culture for several months with megakaryocyte purity over 90% reaching up to 2x105 mature megakaryocytes per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as one million starting hPSCs.[233] See also overview[234]

A specialised type of white blood cell, known as cytotoxic T lymphocytes (CTLs), are produced by the immune system and are able to recognise specific markers on the surface of various infectious or tumour cells, causing them to launch an attack to kill the harmful cells. Thence, immunotherapy with functional antigen-specific T cells has potential as a therapeutic strategy for combating many cancers and viral infections.[235] However, cell sources are limited, because they are produced in small numbers naturally and have a short lifespan.

A potentially efficient approach for generating antigen-specific CTLs is to revert mature immune T cells into iPSCs, which possess indefinite proliferative capacity in vitro and after their multiplication to coax them to redifferentiate back into T cells.[236][237][238]

Another method combines iPSC and chimeric antigen receptor (CAR)[239] technologies to generate human T cells targeted to CD19, an antigen expressed by malignant B cells, in tissue culture.[240] This approach of generating therapeutic human T cells may be useful for cancer immunotherapy and other medical applications.

Invariant natural killer T (iNKT) cells have great clinical potential as adjuvants for cancer immunotherapy. iNKT cells act as innate T lymphocytes and serve as a bridge between the innate and acquired immune systems. They augment anti-tumor responses by producing interferon-gamma (IFN-).[241] The approach of collection, reprogramming/dedifferentiation, re-differentiation and injection has been proposed for related tumor treatment.[242]

Dendritic cells (DC) are specialized to control T-cell responses. DC with appropriate genetic modifications may survive long enough to stimulate antigen-specific CTL and after that be completely eliminated. DC-like antigen-presenting cells obtained from human induced pluripotent stem cells can serve as a source for vaccination therapy.[243]

CCAAT/enhancer binding protein- (C/EBP) induces transdifferentiation of B cells into macrophages at high efficiencies[244] and enhances reprogramming into iPS cells when co-expressed with transcription factors Oct4, Sox2, Klf4 and Myc.[245] with a 100-fold increase in iPS cell reprogramming efficiency, involving 95% of the population.[246] Furthermore, C/EBPa can convert selected human B cell lymphoma and leukemia cell lines into macrophage-like cells at high efficiencies, impairing the cells' tumor-forming capacity.[247]

The thymus is the first organ to deteriorate as people age. This shrinking is one of the main reasons the immune system becomes less effective with age. Diminished expression of the thymic epithelial cell transcription factor FOXN1 has been implicated as a component of the mechanism regulating age-related involution.[248][249]

Clare Blackburn and colleagues show that established age-related thymic involution can be reversed by forced upregulation of just one transcription factor - FOXN1 in the thymic epithelial cells in order to promote rejuvenation, proliferation and differentiation of these cells into fully functional thymic epithelium.[250] This rejuvenation and increased proliferation was accompanied by upregulation of genes that promote cell cycle progression (cyclin D1, Np63, FgfR2IIIb) and that are required in the thymic epithelial cells to promote specific aspects of T cell development (Dll4, Kitl, Ccl25, Cxcl12, Cd40, Cd80, Ctsl, Pax1).

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