Showing papers by "Hossein Baharvand published in 2016"

Application of Mesenchymal Stem Cells in Melanoma: A Potential Therapeutic Strategy for Delivery of Targeted Agents.

Abstract: Melanoma is a leading cause of mortality from skin cancer and has a poor prognosis. Despite rapid advances in the treatment of this tumor type, the efficacy of current chemo-/targeted-therapies is still limited owing to the lack of sufficient drug accumulation in the tumor tissue and development of chemo-resistance. Recently, the application of mesenchymal stem cells (MSCs) in cancer therapy has gained substantial attention, suggesting their potential roles as an intriguing vehicle in improving the delivery of targeted agents. MSCs are genetically modified with suicide tumor suppressor genes to inhibit cell signaling pathways associated with the progression and metastatic features of melanoma. Here we describe the clinical application of MSCs in melanoma with a particular emphasis on recent findings on the role of MSC expressing a distinct set of biologically functional chemokines and tumor suppressing agents. Accumulating data has shown the tumor- oriented homing capacity of MSCs and their applications as a vehicle (e.g., adipose derived mesenchymal stem cells expressing TRAIL, interferon-α/γ, pigment epithelium-derived factor and cytosine deaminase). Several questions regarding possible potential and intrinsic mechanisms that might induce tumorigenesis and drug resistance are yet to be addressed for tailoring MSC-nbased treatment of melanoma.

Effect of Secreted Molecules of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells on Acute Hepatic Failure Model

Abstract: Adult tissue-derived mesenchymal stem cells (MSCs) show tremendous promise for a wide array of therapeutic applications predominantly through paracrine activity Recent reports showed that human embryonic stem cell (ESC)-derived MSCs are an alternative for regenerative cellular therapy due to manufacturing large quantities of MSCs from a single donor However, no study has been reported to uncover the secretome of human ESC-MSCs as treatment of an acute liver failure (ALF) mouse model We demonstrated that human ESC-MSCs showed similar morphology and cell surface markers compared with bone marrow-derived MSCs ESC-MSCs exhibited a higher growth rate during early in vitro expansion, along with adipogenic and osteogenic differentiation potential Treatment with ESC-MSC-conditioned medium (CM) led to statistically significant enhancement of primary hepatocyte viability and increased immunomodulatory interleukin-10 secretion from lipopolysaccharide-induced human blood mononuclear cells Analysis of the MSCs secretome by a protein array screen showed an association between higher frequencies of secretory proteins such as vascular endothelial growth factor (VEGF) and regulation of cell proliferation, cell migration, the development process, immune system process, and apoptosis In this thioacetamide-induced mouse model of acute liver injury, we observed that systemic infusion of VEGF led to significant survival These data have provided the first experimental evidence of the therapeutic potential of human ESC-MSC-derived molecules These molecules show trophic support to hepatocytes, which potentially creates new avenues for the treatment of ALF, as an inflammatory condition

Conversion of Human Fibroblasts to Stably Self-Renewing Neural Stem Cells with a Single Zinc-Finger Transcription Factor

Abstract: Direct conversion of somatic cells into neural stem cells (NSCs) by defined factors holds great promise for mechanistic studies, drug screening, and potential cell therapies for different neurodegenerative diseases. Here, we report that a single zinc-finger transcription factor, Zfp521, is sufficient for direct conversion of human fibroblasts into long-term self-renewable and multipotent NSCs. In vitro, Zfp521-induced NSCs maintained their characteristics in the absence of exogenous factor expression and exhibited morphological, molecular, developmental, and functional properties that were similar to control NSCs. In addition, the single-seeded induced NSCs were able to form NSC colonies with efficiency comparable with control NSCs and expressed NSC markers. The converted cells were capable of surviving, migrating, and attaining neural phenotypes after transplantation into neonatal mouse and adult rat brains, without forming tumors. Moreover, the Zfp521-induced NSCs predominantly expressed rostral genes. Our results suggest a facilitated approach for establishing human NSCs through Zfp521-driven conversion of fibroblasts.

Vitamin C enhances epigenetic modifications induced by 5-azacytidine and cell cycle arrest in the hepatocellular carcinoma cell lines HLE and Huh7

Abstract: Background 5-Azacytidine (5-AZA), a DNA methyl transferase inhibitor, is a clinically used epigenetic drug for cancer therapy. Recently, we have shown that 5-AZA upregulates ten-eleven translocation (TET) protein expression in hepatocellular carcinoma (HCC) cells, which induce active demethylation. Vitamin C facilitates TET activity and enhances active demethylation. The aim of this study is to investigate whether vitamin C is able to enhance the effect of 5-AZA on active demethylation and to evaluate its consequence in HCC cell lines.

Intraportal Infusion of Bone Marrow Mononuclear or CD133+ Cells in Patients With Decompensated Cirrhosis: A Double-Blind Randomized Controlled Trial

Abstract: The present study assessed the effects of intraportal infusions of autologous bone marrow-derived mononuclear cells (MNCs) and/or CD133 + cells on liver function in patients with decompensated cirrhosis. We randomly assigned 27 eligible patients to a placebo, MNCs, and/or CD133 + cells. Cell infusionswereperformedatbaselineandmonth3.WeconsideredtheabsolutechangesintheModelfor End-Stage Liver Disease (MELD) scores at months 3 and 6 after infusion as the primary outcome. The participants and those who assessed the outcomes were unaware of the treatment intervention assignments. After 6 months, 9 patients were excluded because of liver transplantation (n = 3), hepatocellular carcinoma (n = 1), loss to follow-up (n = 3), and death (n = 2). The final analysis included 4 patients from the CD133 + group, 8 from the MNC group, and 6 from the placebo group. No improvement was seen in the MELD score at month 6 using either CD133 + cells or MNC infusions compared with placebo. However, at month 3 after infusion, a trend was seen toward a higher mean absolute changeintheMELDscoreinpatientswhohadreceivedCD133 + cellscomparedwithplacebo(22.0061.87

Derivation of Pluripotent Cells from Mouse SSCs Seems to Be Age Dependent

Abstract: Here, we aimed to answer important and fundamental questions in germ cell biology with special focus on the age of the male donor cells and the possibility to generate embryonic stem cell- (ESC-) like cells. While it is believed that spermatogonial stem cells (SSCs) and truly pluripotent ESC-like cells can be isolated from adult mice, it remained unknown if the spontaneous conversion of SSCs to ESC-like cells fails at some age. Similarly, there have been differences in the literature about the duration of cultures during which ESC-like cells may appear. We demonstrate the possibility to derive ESC-like cells from SSC cultures until they reach adolescence or up to 7 weeks of age, but we point out the impossibility to derive these cells from older, mature adult mice. The inability of real adult SSCs to shift to a pluripotent state coincides with a decline in expression of the core pluripotency genes Oct4, Nanog, and Sox2 in SSCs with age. At the same time genes of the spermatogonial differentiation pathway increase. The generated ESC-like cells were similar to ESCs and express pluripotency markers. In vitro they differentiate into all three germ lineages; they form complex teratomas after transplantation in SCID mice and produce chimeric mice.

Human Hair Reconstruction: Close, But Yet So Far.

Abstract: Billions of dollars are annually invested in pharmaceutical industry and cosmetic sector with intent to develop new drugs and treatment strategies for alopecia. Because the hair looks an important characteristic of humans-an effective appendage in perception, expression of beauty, and preservation of self-esteem-the global market for hair loss treatment products is exponentially increasing. However, current methods to treat hair loss endure yet multiple challenges, such as unfavorable outcomes, nonpermanent and patient-dependent results, as well as unpredictable impacts, which limit their application. Over recent years, remarkable advances in the fields of regenerative medicine and hair tissue engineering have raised new hopes for introducing novel cell-based approaches to treat hair loss. Through cell-based approaches, it is possible to produce hair-like structures in the laboratory setting or manipulate cells in their native niche (in vivo lineage reprogramming) to reconstruct the hair follicle. However, challenging issues still exist with the functionality of cultured human hair cells, the proper selection of nonhair cell sources in cases of shortage of donor hair, and the development of defined culture conditions. Moreover, in the case of in vivo lineage reprogramming, selecting appropriate induction factors and their efficient delivery to guide resident cells into a hair fate-with the aim of reconstructing functional hair-still needs further explorations. In this study, we highlight recent advances and current challenges in hair loss treatment using cell-based approaches and provide novel insights for crucial steps, which must be taken into account to develop reproducible, safe, and efficient cell-based treatment.

Modification of PDMS to fabricate PLGA microparticles by a double emulsion method in a single microfluidic device

Abstract: We present an easy, durable method to generate a partially hydrophilic/hydrophobic poly(dimethylsiloxane) (PDMS) microfluidic device. The functionality of this device was assessed in a double flow focusing design to fabricate core–shell structured poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs).

Safety and Efficacy of Repeated Bone Marrow Mononuclear Cell Therapy in Patients with Critical Limb Ischemia in a Pilot Randomized Controlled Trial.

Abstract: BACKGROUND: Critical limb ischemia is a manifestation of peripheral arterial disease characterized by insufficient arterial blood flow for maintaining tissue viability in the lower extremities. Therapeutic angiogenesis is used for peripheral arterial disease patients who are not candidates for surgical revascularization or radiological intervention. There is accumulating evidence for the beneficial impact of autologous bone marrow mononuclear cell transplantation for treatment of critical limb ischemia in humans. This study aims to investigate the safety and efficacy of repeated bone marrow mononuclear cell injections in comparison with a single bone marrow mononuclear cell injection in critical limb ischemia patients. METHODS: Patients with critical limb ischemia (n = 22) were randomized (http://clinicaltrials.gov/ct2 show/NCT01480414) to receive either a single (n = 11) or four (n = 11) intramuscular injections of bone marrow mononuclear cells as a cell therapy product. RESULTS: There were no reported adverse events during the 24-week follow-up period after cell delivery. Efficacy assessment indicated that after cell injections, there was significant improvement in Ankle-Brachial Index, Visual Analog Scale, pain-free walking distance, and Wagner stage as well as reduction in ulcer size. There was no significant difference between the two groups in terms of clinical parameters. However, by the 24th week the pain-free walking distance improved significantly in the group who received four injections of cells. CONCLUSION: Favorable clinical outcomes strongly indicate the long-term benefit of bone marrow mononuclear cell transplantation, either as one or several injections, for retrieval from critical limb ischemia. Repeated cell injections have shown increased improvement of pain-free walking distance in patients. These findings warrant further exploration in later-phase clinical trials with repeated injections.

Repeated Intraportal Injection of Mesenchymal Stem Cells in Combination with Pioglitazone in Patients with Compensated Cirrhosis: A Clinical Report of Two Cases.

Abstract: Background Transplantation of mesenchymal stem cells (MSCs) in combination with pioglitazone, an agonist of peroxisome proliferator-activated receptor-γ (PPAR-γ), can reduce liver fibrosis in models of liver injury. In this study, we conducted a pilot study of intraportal infusion of autologous MSCs in combination with pioglitazone to assess safety, feasibility, and effectiveness in patients with compensated cirrhosis. Methods Two patients with compensated cirrhosis were enrolled in this study. Intraportal autologous bone marrow-derived MSCs were transplanted twice (6 months interval) to the patients. Meanwhile, 30 mg/day pioglitazone was prescribed for 12 months. Patients were assessed at baseline and months 1, 3, 6, and 12 post-infusion. Results Procedural complications or any major adverse effects did not occur in this pilot study. The patients' clinical conditions remained stable with no evidence of deterioration during the course of the study. A transient improvement in the Model for End-Stage Liver Disease (MELD) score was observed at month 3 post-infusion in one patient, which eventually returned to baseline at month 12. Conclusion The combination of pioglitazone with MSCs is safe and feasible. The data justify further study of the combination therapy in cirrhotic patients.

Nurr1 and PPARγ protect PC12 cells against MPP+ toxicity: involvement of selective genes, anti-inflammatory, ROS generation, and antimitochondrial impairment

Abstract: Parkinson's disease (PD) can degenerate dopaminergic (DA) neurons in midbrain, substantia-nigra pars compacta. Alleviation of its symptoms and protection of normal neurons against degeneration are the main aspects of researches to establish novel therapeutic strategies. PPARγ as a member of PPARs have shown neuroprotection in a number of neurodegenerative disorders such as Alzheimer's disease and PD. Nuclear receptor related 1 protein (Nurr1) is, respectively, member of NR4A family and has received great attentions as potential target for development, maintenance, and survival of DA neurons. Based on neuroprotective effects of PPARγ and dual role of Nurr1 in anti-inflammatory pathways and development of DA neurons, we hypothesize that PPARγ and Nurr1 agonists alone and in combined form can be targets for neuroprotective therapeutic development for PD in vitro model. 1-Methyl-4-phenylpyridinium (MPP(+)) induced neurotoxicity in PC12 cells as an in vitro model for PD studies. Treatment/cotreatment with PPARγ and Nurr1 agonists 24 h prior to MPP(+) induction enhanced the viability of PC12 cell. The viability of PC12 cells was determined by MTS test. Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) were detected by flow cytometry. In addition, the relative expression of four genes including TH (the marker of DA neurons), Ephrin A1, Nurr1, and Ferritin light chain were assessed by RT-qPCR. In the MPP(+)-pretreated PC12 cells, PPARγ and Nurr1 agonists and their combined form resulted in a decrease in the cell death rate. Moreover, production of intracellular ROS and MMP modulated by MPP(+) was decreased by PPARγ and Nurr1 agonists' treatment alone and in the combined form.

Effect of autologous muscle-derived cells in the treatment of urinary incontinence in female patients with intrinsic sphincter deficiency and epispadias: A prospective study

Abstract: Objectives To evaluate the effect of autologous muscle-derived cells injection in the treatment of complicated stress urinary incontinence in female patients. Methods Female patients presenting with severe and complicated stress urinary incontinence secondary to the bladder neck and/or urethral trauma or congenital epispadias (with or without exstrophy) were enrolled in this prospective study. They underwent transurethral injection of autologous muscle-derived cells. In selected cases, another injection was given after 6 months, as per the surgeon's assessment. All patients were monitored for 1 year, and the effect of autologous muscle-derived cells was evaluated by cough stress test, 1-h pad test and Incontinence Impact Questionnaire-short form score. A multichannel urodynamic study and maximum urethral closure pressure were carried out before and 12 months after the last treatment session. Cough stress test, 1-h pad test and uroflowmetry were repeated 36 months after the last injection. Severity and occurrence of complications were recorded at each visit. Results All 10 patients who completed the study were monitored for 36 months. Three patients were cured, four had improved and three did not respond to the treatment. There was no major adverse effect related to the treatment. Conclusions Muscle-derived cell therapy might represent a minimally-invasive and a safe procedure in the treatment of patients with severe and complicated stress urinary incontinence.

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol.

Abstract: Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-β, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing > 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Systematic selection of small molecules to promote differentiation of embryonic stem cells and experimental validation for generating cardiomyocytes

Abstract: Small molecules are being increasingly used for inducing the targeted differentiation of stem cells to different cell types. However, until now no systematic method for selecting suitable small molecules for this purpose has been presented. In this work, we propose an integrated and general bioinformatics- and cheminformatics-based approach for selecting small molecules which direct cellular differentiation in the desired way. The approach was successfully experimentally validated for differentiating stem cells into cardiomyocytes. All predicted compounds enhanced expression of cardiac progenitor (Gata4, Nkx2-5 and Mef2c) and mature cardiac markers (Actc1, myh6) significantly during and post-cardiac progenitor formation. The best-performing compound, Famotidine, increased the percentage of Myh6-positive cells from 33 to 56%, and enhanced the expression of Nkx2.5 and Tnnt2 cardiac progenitor and cardiac markers in protein level. The approach employed in the study is applicable to all other stem cell differentiation settings where gene expression data are available.

Is TGFβ as an anti-inflammatory cytokine required for differentiation of inflammatory TH17 cells?

Abstract: T-Helper 17 (TH17) cells are a CD4+ TH subset that plays a critical role in the pathophysiology of inflammatory disorders, especially chronic forms. It seems that the derivation of TH17 cells from their precursors take place in inflammatory microenvironment. The role of transforming growth factor (TGF)-β as an anti-inflammatory cytokine in TH17 cell differentiation is controversial. To address some of the discrepancies that exist among different studies, this study was undertaken to more clarify the TGFβ role in human TH17 cell differentiation. Here, CD4+ T-cells were isolated from peripheral blood samples and cultured in X-VIVO 15 serum-free medium. Purified cells were then treated with different combinations of polarizing cytokines (interleukin [IL]1-β, -6, and -23, with or without TGFβ), neutralizing anti-interferon (IFN)-γ and anti-IL-4 antibodies and polyclonal stimulators anti-CD3 and -CD28 antibodies, and then analyzed for IL-17, IFNγ, Foxp3, and CD25 expression by flow cytometry and for release of IL-17, -21, -22, and -10 into culture media by ELISA. The effects of selective inhibition of TGFβ signaling pathway on TH17 cell polarization were also determined by using small molecules SB-431542 and A83-01. The current study found that a combination of pro-inflammatory cytokines, including IL-1β, -6, and -23, but not TGFβ, could be used as a cytokine combination to induce development of human TH17 cells. It was also shown that TGFβ acted as a negative regulator in this regard and also led to reduced IL-17 and IL-22 production while inducing Foxp3 expression. Indeed, blocking of TGFβ signaling pathways by selective inhibitors up-regulated TH17 cell differentiation. From the data here, we concluded that TGFβ down-regulates human TH17 cell differentiation and that a presence of pro-inflammatory cytokines (along with IFNγ and IL-4 neutralizing antibodies) is sufficient for optimal differentiation of human TH17 cells.

The Therapeutic Potential of Differentiated Lung Cells from Embryonic Stem Cells in Lung Diseases.

Abstract: Lung diseases cause great morbidity and mortality. The choice of effective medical treatment is limited and the number of lung diseases are difficult to treat with current treatments. The embryonic stem cells (ESCs) have the potential to differentiate into cell types of all three germinal layers, including lung epithelial cells. So they can be a potential source for new cell therapies for hereditary or acquired diseases of the airways and lungs. One method for treatment of lung diseases is cell therapy and the use of ESCs that can replace the damaged epithelial and endothelial cells. Progress using ESCs has developed slowly for lung regeneration because differentiation of lung cells from ESCs is more difficult as compared to differentiation of other cells. The review studies the therapeutic effects of differentiated lung cells from embryonic stem cells in lung diseases. There are few studies of differentiation of ESCs into a lineage of respiratory and then investigation of this cell in experimental model of lung diseases.

Comparative epigenetic evaluation of human embryonic stem and induced pluripotent cells.

Abstract: Histone H3 lysine 9 methylation has been shown to be a critical barrier to efficient cell reprogramming. This discovery allows the assessment of the cell pluripotency state by considering the extent of H3K9 methylation vs. acetylation at the same position. A set of pluripotent and differentiated human cells including embryonic stem cells, their differentiated and reprogrammed counterparts, along with human fibroblasts and their derived reprogrammed cells, were used to evaluate the ratio of total H3K9 methylation over acetylation using a quantitative ELISA-based approach. Also, the occurrence of the H3K4me3 and H3K27me3 bivalent marks was evaluated. Additionally, using ChIP-qPCR the occurrence of these histone marks on the regulatory regions of stemness genes (Nanog, Oct4 and Sox2) as well as on genes indicating fibroblast differentiation (Vim, COL1A1 and THY1) was evaluated. We evidence remarkably high ratios of H3K9ac/K9me2 in ES and iPS cells vs. differentiated cells. In iPSCs, a direct relationship between the ratios of total H3K9ac/H3K9me2 and the ratios of these marks on pluripotency gene regulatory regions and their expression was observed. In differentiated cells, in contrast, the ratios of global H3K9ac/K9me2 is low but the active genes escape this general situation and bear higher amounts of H3K9ac vs. H3K9me. Total H3K4me3/K27me3 ratios presented the same trends, but with reduced amplitudes. We propose that the rapid quantitative measurements of relative amounts of H3K9ac and K9me2 in iPS cells compared to the parental differentiated cells constitute a reliable and convenient criterion to rapidly assess the cell pluripotency potentials and the efficiency of cell reprogramming.

Influence of decellularized pericardium matrix on the behavior of cardiac progenitors

Abstract: Following myocardial infarction, heart muscle has a limited capacity of self-healing. Biological platforms providing the natural biochemical and biophysical cues of the native myocardium might be crucial to address current therapeutic shortcomings. The aim of this study was to assess the effect of decellularized human pericardium (DPc), as a bioactive platform, on viability, attachment, proliferation and differentiation of human cardiac progenitors (CPs), and evaluate the possibility of using DPc as a substitute of tissue culture polystyrene (TCPS) substrate for culturing CPs in vitro. The decellularization process removed more than 99% of the cellular components from Pc, yet well preserved its macro-/micro-structure and extracellular collagen and glycosaminoglycan content. DPc supported the viability, attachment, metabolism and proliferation of CPs, and enhanced their differentiation into mature cardiomyocytes compared to TCPS. Decellularized pericardium appeared thus to have a high potential for cardiac cell culture and could be applied as a superior alternative to common TCPS. DPc could be then utilized for further tissue engineering applications. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43255.

Zinc finger protein 521 overexpression increased transcript levels of Fndc5 in mouse embryonic stem cells.

Abstract: Zinc finger protein 521 is highly expressed in brain, neural stem cells and early progenitors of the human hematopoietic cells. Zfp521 triggers the cascade of neurogenesis in mouse embryonic stem cells through inducing expression of the early neuroectodermal genes Sox1, Sox3 and Pax6. Fndc5, a precursor of Irisin has inducing effects on the expression level of brain derived neurotrophic factor in hippocampus. Therefore, it is most likely that Fndc5 may play an important role in neural differentiation. To exhibit whether the expression of this protein is under regulation with Zfp521, we overexpressed Zfp521 in a stable transformants of mESCs expressing EGFP under control of Fndc5 promoter. Increased expression of Zfp521 enhanced transcription levels of both EGFP and endogenous Fndc5. This result was confirmed by overexpression the aforementioned vectors in HEK cells and indicated that Zfp521 functions upstream of Fndc5 expression. It is most likely that Zfp521 may act through the binding to its response element on Fndc5 core promoter. Therefore it is concluding that an enhanced expression of Fndc5 in neural progenitor cells is stimulated by Zfp521 overexpression in these cells.

Analysis of Promyelocytic Leukemia in Human Embryonic Carcinoma Stem Cells During Retinoic Acid-Induced Neural Differentiation.

Abstract: Background Promyelocytic leukemia protein (PML) is a tumor suppressor protein that is involved in myeloid cell differentiation in response to retinoic acid (RA). In addition, RA acts as a natural morphogen in neural development. Objectives This study aimed to examine PML gene expression in different stages of in vitro neural differentiation of NT2 cells, and to investigate the possible role of PML in pluripotency and/or neural development. Materials and methods RA was used as a neural inducer for in vitro neural differentiation of NT2 cells. During this process PML mRNA and protein levels were assessed by quantitative real time RT-PCR (QRT-PCR) and Immunoblotting, respectively. Furthermore bisulfite sequencing PCR (BSP) was used to assess PML promoter methylation in NT2 cells and NT2 derived neuronal precursor cells (NT2.NPCs). Results QRT-PCR results showed that, PML had maximum expression with significant differences in NT2 derived neuronal precursor cells relative to NT2 cells and NT2 derived neural cells (NT2.NCs). Numerous isoforms of PML with different intensities appeared in immunoblots of pluripotent NT2 cells, NT2.NPCs, and NT2.NCs. Furthermore, the methylation of the PML promoter in NT2.NCs was 2.6 percent lower than NT2 cell. Conclusions The observed differences in PML expression in different cellular stages possibly could be attributed to the fact that PML in each developmental state might be involved in different cell signaling machinery and different functions. The appearance of different PML isoforms with more intensity in neural progenitor cells; may suggest apossible role for this protein in neural development.

Comparison of BAX and Bcl-2 Expression During Human Embryonic Stem Cell Differentiation into Cardiomyocytes and Doxorubicin-induced Apoptosis

Abstract: Back ground: Although the cell differentiation is an inseparable part of development in multicellular organisms, the regulating molecular pathway of it still is not fully defined. In the other hand, apoptosis is a fundamental physiological process which plays an essential role in a variety of biological events during development. Moreover, recent studies have found that apoptosis shows several common features with differentiation but that how cells distinguish apoptosis and differentiation from each other is not clear. In this study two critical members of the Bcl-2 family, BAX (an apoptosis promoter) and Bcl-2 (an apoptosis inhibitor) ratio was investigated in both apoptosis and differentiation processes. BAX/ Bcl-2 ratio is one of the important characteristics of the apoptosis process but its role during differentiation is still unknown. Materials and methods: At the present study hESC (human embryonic stem cell) line RH5 was used for induction of differentiation into the beating cardiomyocytes and also doxorubicin induced apoptosis. Samples were collected at different time points and their total RNA was extracted and RT-PCR was performed to assess the expression of genes of interest. Results: During apoptosis BAX mRNA level increased but Bcl-2 mRNA level decreased and consequently BAX /Bcl-2 ratio increased significantly (p<0.05) about 8 fold, 24 hours after apoptosis induction. But interestingly BAX /Bcl-2 ratio fluctuated during differentiation of beating cardiomyocytes. Conclusion : It seems that BAX /Bcl-2 ratio and the time of its engagement may contribute to the choice between cell death and differentiation in hESC.

Substrate-mediated commitment of human embryonic stem cells for hepatic differentiation.

Abstract: Human embryonic stem cell (hESC)-derived endodermal cells are of interest for the development of cellular therapies to treat disorders such as liver failure. The soluble form of activin A (Act) has been widely used as an in vitro inducer of definitive endoderm (DE). In this study, we have developed a nanofibrous poly (ɛ-caprolactone) substrate, biofunctionalized with Act, for directed differentiation of hESCs into DE. Bioconjugation of Act on nanofibrous meshes was confirmed by enzyme-linked immunosorbent assay (ELISA) and immunostaining. In order to investigate the bioactivity of immobilized Act (iAct), hESCs were cultivated on the Act-conjugated nanofibers for five days. The nanofibers with covalent iAct significantly increased expression levels of the endodermal markers SOX17, FOXA2, and CXCR4, compared with physically adsorbed Act (aAct) or without Act (noAct). In addition, iAct retained its bioactivity after storage for five days in the absence of cell seeding. The capability of cultivated cells to generate the DE-derived lineage was evaluated through further differentiation of seeded cells into hepatocyte-like cells (HLCs). Interestingly, the iAct sample showed a higher level of hepatic markers compared to the aAct sample. We also demonstrated that iAct in the presence of soluble Act (sAct) could improve the conventional protocol to generate HLCs from hESCs. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2861-2872, 2016.