Background: The use of stem cells is considered as an appropriate source in cell therapy and tissue engineering. Differentiation of human induced Pluripotent Stem Cells (HIPSCs) to Hepatocyte-like Cells (HLCs) on mouse embryonic fibroblasts (MEFs) feeders is confronted with several problems that hinder the clinical applications of these differentiated cells for the treatment of liver injuries. Safe appropriate cells for stem cell-based therapies could create new hopes for liver diseases. This work focused on the determination of a capacity/efficiency for the differentiation of the HIPSCs into Hepatocyte-like Cells on a novel human adult bone marrow mesenchymal stem cells (hMSCs) feeder.Materials and Methods: Undifferentiated human iPSCs were cultured on mitotically inactivated human adult bone marrow mesenchymal stem cells. A three-step differentiation process has been performed in presence of activin A which added for 3 days to induce a definitive endoderm formation. In the second step, medium was exchanged for six days. Subsequently, cells were treated with oncostatin M plus dexamethasone for 9 days to generate hepatic cells. Endodermic and liver-specific genes were assessed via quantitative reverse transcription-polymerase chain reaction and RT-PCR, moreover, immunocytochemical staining for liver proteins including albumin and alpha-fetoprotein. In addition, functional tests for glycogen storage, oil red examination, urea production and alpha-fetoprotein synthesis, as well as, cells differentiated with a hepatocyte-like morphology was also performed.Results: Our results show that inactivated human adult bone marrow mesenchymal stem cell feeders could support the efficient differentiation of HIPSCs into HLCs. This process induced differentiation of iPSCs into definitive endocrine cells that expressed sox17, foxa2 and expression of the specific genes profiles in hepatic-like cells. In addition, immunocytochemical analysis confirmed albumin and alpha-fetoprotein protein expression, as well as, the HIPSCs-derived Hepatocyte-like Cells on human feeder exhibited a typical morphology.Conclusions: we suggested a successful and efficient culture for differentiation and maturation of hepatocytes on alternative human feeders, this is an important step to generate safe and functional hepatocytes that is vital for regenerative medicine and transplantation on the cell-based therapies.

Objective: Induced pluripotent stem cells (iPSCs) with the possibility of patient-specific cell lineage transplantation represent an attractive source for cell therapy in regenerative medicine. This study aimed to assess therapeutic potential of HIPSC-oligodendrocyte progenitors (HIPSC-OPs) in a severe injured spinal cord model in rats.Materials and Methods: The HIPSCs-OPs derived cells expressing PDGFRa, NG2, A2B5, O4 specific markers were transplanted into severe injured spinal cord rats seven days following contusion injury. The severity of model was confirmed by histological assessments. The behavioral tests (plantar and BBB score) were examined for sensory response and locomotor function for 5 weeks after SCI.Results: Behavioral tests (plantar and BBB) during 5 weeks following transplantation showed non-significant improvement in functional recovery and sensory responses.Conclusion: Taken together, it seems that oligodendrocyte progenitors alone are not to be sufficient completely overcoming the biological complications after severe SCI.

Objective: The neuronal repressor REST (RE1-silencing transcription factor) is expressed at high levels in embryonic stem cells (ESC) with a critical role in self-renewal and pluripotency signaling network of these cells. REST is an essential element for brain development and also neuronal differentiation of ESC in vitro. Although REST is a well-known regulatory element in embryonic stem cells, but according to our knowledge, it has not been evaluated in induced pluripotent stem cell (iPSC) till now.Materials and Methods: The quantitative expression of REST in each step was evaluated by real-time PCR and the presence of REST was showed by immunofluorescent assay.Results: Q-PCR analysis showed that the expression level of REST decreased significantly during differentiation of HIPSCs to neural precursor cells, and remained stable until neuronal differentiation. immunofluorescent data revealed the both nuclear as cytoplasmic presence of REST in HIPSCs as well as NSCs, while in the maturated neurons it was only detectable in the cytoplasm.Conclusion: Our findings show a distinct expression pattern for REST during hESCs and HIPSCs neural differentiation. This study opens a new window for further experiments in this field to receive efficient differentiation of neuronal cells.

Objective: Biomaterial technology, when combined with emerging human induced pluripotent stem cell (HIPSC) technology, provides a promising strategy for patient-specific tissue engineering. In this study, we have evaluated the physical effects of collagen scaffolds fabricated at various freezing temperatures on the behavior of HIPSC-derived neural progenitors (HIPSC-NPs). In addition, the coating of scaffolds using different concentrations of laminin was examined on the cells.Materials and Methods: Initially, in this experimental study, the collagen scaffolds fabricated from different collagen concentrations and freezing temperatures were characterized by determining the pore size, porosity, swelling ratio, and mechanical properties. Effects of cross-linking on free amine groups, volume shrinkage and mass retention was also assessed. Then, HIPSC-NPs were seeded onto the most stable three-dimensional collagen scaffolds and we evaluated the effect of pore structure. Additionally, the different concentrations of laminin coating of the scaffolds on HIPSC-NPs behavior were assessed.Results: Scanning electron micrographs of the scaffolds showed a pore diameter in the range of 23-232 μm for the scaffolds prepared with different fabrication parameters. Also porosity of all scaffolds was>98% with more than 94% swelling ratio. HIPSC-NPs were subsequently seeded onto the scaffolds that were made by different freezing temperatures in order to assess for physical effects of the scaffolds. We observed similar proliferation, but more cell infiltration in scaffolds prepared at lower freezing temperatures. The laminin coating of the scaffolds improved NPs proliferation and infiltration in a dose-dependent manner. Immunofluorescence staining and scanning electron microscopy confirmed the compatibility of undifferentiated and differentiated HIPSC-NPs on these scaffolds.Conclusion: The results have suggested that the pore structure and laminin coating of collagen scaffolds significantly impact cell behavior. These biocompatible three-dimensional laminin-coated collagen scaffolds are good candidates for future HIPSC-NPs biomedical nerve tissue engineering applications.

Background: The sensory and mobility failure associated with spinal cord injury (SCI) is desperately complicated due to the pathological events that occur sequentially in consequent to the injury. Objectives: Herein, we applied neural stem cells, derived from human iPSCs (HIPSC-NSCs), to ameliorate the behavioral complications of contusive SCI in Rhesus monkeys, in sub-acute phase. Methods: HIPSC-NSCs were maintained and characterized in vitro for general NSCs as well as hind-limb specific gene and protein expression prior to transplantation. Moreover, Masson's trichrome staining (MTS) in addition to luxol fast blue (LFB) were performed to determine the fibrotic scar reduction and myelination respectively. Tarlov’s scale were utilized to score the motor improvement, plus, sensory perception evaluation throughout six months following the injury. Results: HIPSC-NSCs were identified to own NSCs’ exclusive properties in vitro by SOX2, DCX and NESTIN in addition to NESTIN , PAX6 , SOX1 , HOXA2 and HOXB2 protein and gene expression. Additionally, HIPSC-NSCs caused remarkable depletion in fibrotic scar and enhance myelination; spinal shock, sensory responses, reflexes and motor function were improved over six months. Conclusions: Our findings suggest that HIPSC-NSCs lead to promising recovery after SCI, therefore, this source of NSCs provide a therapeutic potential in clinical studies.

Background. Periodontal regeneration aims to recapitulate the crucial stages of wound healing associated with periodontal development in order to restore lost tissues to their original form and function and for regeneration to occur. The aim of this study was to investigate the effects of human-induced pluripotent stem cell-derived mesenchymal stem cells (HIPSC-MSCs) co-cultured with endothelial cells for on calcium phosphate cement (CPC) scaffold on periodontal regeneration in vivo for the first time. Methods. Twenty number of immunocompromised rats were generated periodontal defects caused by periodontitis by binding wire around teeth and subsequently inoculating them with Porphyromonas gingivalis and tested in a periodontal split mouth defect model. The rat’ s jaws divided in four part and treated with (1) CPC scaffold alone (CPC control); (2) endothelial cells with CPC; (3) HIPSC-MSC with CPC (CPC-HIPSC-MSC); and (4) endothelilal co-cultured with HIPSC-MSCs on CPC scaffolds (co-culture group). Results. After 12 weeks, the results showed that TNFα , IFNγ , and IL1β deccreased co-culture group in comparision to other groups. Histopathological results showed that the new bone and periodontal ligament in the periodontal defects in the co-culutre group were regenerated to the highest levels compared with other groups. Soft x-ray results showed more bone formation in the co-culture group compared with that other groups. Conclusions. In conclusion, endothelial co-cultured with HIPSC-MSCs substantially promoted periodontal regeneration. The novel construct of endothelial co-cultured with HIPSC-MSCs delivered via CPC scaffolds is promising to enhance bone and vascular regeneration in orthopedic applications.

Background: Autophagy is a vital cell survival mechanism that authorizes cells to assort to metabolic stress and is essential for the development and maintenance of cellular and tissue homeostasis, as well as the prevention of human disease. It has also been shown that autophagy plays a significant role in the development and differentiation of stem cells, as well as induced pluripotent stem cells (iPSCs). Objectives: The present study aimed to examine the mRNA expression of the ATG5 gene, one of the key markers of autophagy in human iPSCs (HIPSCs) during endoderm induction. Methods: In this study, wecultured thehumaniPSC line (R1-HIPSC1)onmitomycin-C, inactivatedmouseembryonic fibroblasts (MEF) layer, and used hanging drop protocol to generate embryoid body (EB) and expose differentiation. The Real-time PCR method was used to examine the mRNA expression level of ATG5 in HIPSC during endoderm induction. Results: Our results demonstrated the high mRNA expression of ATG5 in the mesendoderm induction (MEI) stage, which shows the high rate of autophagy in MEI days rather than the other stages of differentiation. Conclusions: The modification of ATG5 gene expression within HIPSC during endoderm induction shows the importance of autophagy assessments in HIPSC differentiation. Therefore, subsequent studies are needed to clarify the details of autophagy effects on HIPSC differentiation.

Human pluripotent stem cells (hPSCs) represent a powerful and potentially unlimited source for disease modeling and for applications in regenerative medicine. While multiple protocols have been developed to derive specific cell types in vitro, there is considerable variability in the efficiency of generating differentiated lineages among independent hESC and HIPSC lines. Currently there is no method available that predicts a priori the differentiation properties of a given cell line. Therefore extensive optimization of individual lines is required to obtain comparable results across lines. Such variability is particularly problematic for the use of patient-specific iPSC lines in disease modeling as it can mask disease-related phenotypes.Here, to identify markers that predict neural differentiation potential, we characterized 33 human embryonic stem cell (hESC) and human induced pluripotent stem cell (HIPSC) lines that were maintained simultaneously. Quantitative analysis of miR-371-3 expression prospectively identified hESC and HIPSC lines with low versus high neurogenic propensity and in vivo dopamine neuron engraftment potential. Predictive markers corresponded to genes previously shown to distinguish mouse ESCs from epiblast-stem cells (EPI-SCs) and, surprisingly, showed more variability in expression among hESC versus HIPSC lines. Neural induction in lines with low neurogenic propensity was more dependent on exposure to BMP inhibitors.Transient over-expression of KLF4 was sufficient to alter neurogenic behavior and markers of pluripotent state. Suppression of miR-371-3 in KLF4-transduced cells rescued neural differentiation propensity demonstrating both predictive and functional roles for miR-371-3 in human pluripotent stem cell behavior.Many of the differentially expressed markers represent genes previously known to discriminate mouse ESCs versus EPI-SCs, and we further demonstrate that such markers can be used to predict in vitro differentiation behavior of a given cell line, which suggest the existence of different level of pluripotent status among hESC and HIPSC lines. Furthermore, we demonstrate that transient over-expression of KLF4 is capable of altering differentiation behavior and pluripotent marker expression, which possible imply the conversion of pluripotent status by simple genetic manipulation. Our findings indicate that the variable differentiation propensity among hESC and HIPSC lines is in part due to differences in pluripotent state and offer a simple strategy to predict and manipulate cell line properties.

Multiple sclerosis (MS) is an autoimmune disease which affects myelin in the central nervous system (CNS) and leads to serious disability. Currently available treatments for MS mainly suppress the immune system. Regenerative medicine-based approaches attempt to increase myelin repair by targeting endogenous progenitors or transplanting stem cells or their derivatives. Fingolimod exerts anti-inflammatory effects and directly affects neural cells. In this study we assessed the effect of fingolimod on transplanted human induced pluripotent stem cell derived neural progenitors (HIPSC-NPs). HIPSC-NPs were labeled by green fluorescence protein (GFP) and transplanted into the corpus callosum of mice which were chronically demyelinated after cuprizone (CPZ) feedings for 10 weeks. The animals received fingolimod from 1 day prior to NPs transplantation via gavage as well as daily intraperitoneal cyclosporine A from 2 days before cell transplantation until the time of sampling. At either 7 or 21 days after NPs transplantation, the animals were sacrificed and their brains were histologically evaluated for the number of transplanted cells and their fate. In the animals treated with fingolimod, we observed higher numbers of NPs within the injection site compared to the animals who did not receive fingolimod showing that HIPSC- NPs were more efficiently differentiated to the oligodendrocyte lineage. These data have suggested that repetitive treatment with fingolimod, beside its anti-inflammatory effect, may enhance the survival and differentiation of transplanted NPs to oligodendrocyte lineage cells to participate in myelin repair.