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Allogeneic mesenchymal stromal cells (MSCs) have been used extensively in various clinical trials. Nevertheless, there are concerns about their efficacy, attributed mainly to the heterogeneity of the applied populations. Therefore, producing a consistent population of MSCs is crucial to improve their therapeutic efficacy. This study presents a good manufacturing practice (GMP)-compatible and cost-effective protocol for manufacturing, banking, and lot-release of a homogeneous population of human bone marrow-derived clonal MSCs (cMSCs).Here, cMSCs were isolated based on the subfractionation culturing method. Afterward, isolated clones that could reproduce up to passage three were stored as the seed stock. To select proliferative clones, we used an innovative, cost-effective screening strategy based on lengthy serial passaging. Finally, the selected clones re-cultured from the seed stock to establish the following four-tired cell banking system: initial, master, working, and end of product cell banks (ICB, MCB, WCB, and EoPCB).Through a rigorous screening strategy, three clones were selected from a total of 21 clones that were stored during the clonal isolation process. The selected clones met the identity, quality, and safety assessments criteria. The validated clones were stored in the four-tiered cell bank system under GMP conditions, and certificates of analysis were provided for the three-individual ready-to-release batches. Finally, a stability study validated the EoPCB, release, and transport process of the frozen final products.Collectively, this study presents a technical and translational overview of a GMP-compatible cMSCs manufacturing technology that could lead to the development of similar products for potential therapeutic applications.

Introduction Mesenchymal stromal cells (MSCs) have opened a new window to treat inflammatory and non-inflammatory diseases. Nonetheless, their clinical applications require rigorous control and monitoring procedures to ensure full compliance with the principles of good manufacturing practice (GMP). Various evaluations should be passed in conjunction with the development of these newly emerging therapeutic products from bench-to-bedside. These evaluations include in vitro characterization, preclinical studies, and clinical trials to ensure product safety and efficacy. Therefore, a robust and well-designed preclinical study is critical to confirm product safety. This study aims to determine the probable toxicity effects of local and systemic injections of cryopreserved human bone marrow-derived clonal MSCs (BM-cMSCs) during subacute and subchronic periods of time. Methods BM-cMSCs were characterized according to the International Society for Cell and Gene Therapy (ISCT) criteria for MSCs. Both safety and toxicity of the BM-cMSCs population produced under GMP-compatible conditions were assessed in both sexes of Sprague Dawley (SD) rats via systemic intravenous (IV) administration and local injection in intervertebral disc (IVD). Behavioral changes, clinical signs of toxicity, and changes in body weight, water and food consumption were the important variables for product toxicity testing over 14 consecutive days during the subacute period and 90 consecutive days during the subchronic period. At the end of the assessment periods, the rats were killed for histopathology analysis of the target tissues. The BM-cMSCs potential for tumorigenicity was checked in nude mice. Results Single IV and IVD injections of BM-cMSCs did not cause significant signs of clinical toxicity, or changes in laboratory and histopathology data during the subacute (14 day) and subchronic (90 day) periods. Ex vivo-expanded and cryopreserved BM-cMSCs did not induce tumor formation in nude mice. Conclusion The results suggest that local and systemic administrations of xenogeneic BM-cMSCs in both sexes of SD rats do not cause toxicity during the subacute and subchronic periods of time. Also, BM-cMSCs were non-tumorigenic in nude mice.

In the use of bovine fetal serum (FBS) there is concern about the possibility of disease transmission from animal to human. Therefore, it seems necessary to create culture conditions free of animal serum, especially in cell therapy. The aim of this study was to evaluate the feasibility of replacing human umbilical cord serum (hUCS) with FBS for in vitro expansion of umbilical cord mesenchymal stromal/stem cells (UC-MSCs). Here, UC-MSCs were cultured for five days in media supplemented either by hUCS or commercial FBS (Gibco and HyClone) to compare their viability, proliferation, morphology, Immunophenotype and differentiation potential. Our data shows that use of 5% and/or 10% hUCS, resulted in a tenfold increase in the number of MSCs; While in the presence of commercial FBS, this figure reached a maximum of five times. Notably, the rate of cell proliferation in the group containing 2% hUCS was the same as the groups containing 10% commercial FBS. Furthermore, there was no significant difference between groups in terms of viability, surface markers, and multilineage differentiation potential. These results demonstrated that hUCS can efficiently replace FBS for the routine culture of MSCs and can be used ideally in manufacturing process of UC-MSCs in cell therapy industry.

Mesenchymal cells of diverse origins differ in gene and protein expression besides producing varying effects on their organ-matched epithelial cells’ maintenance and differentiation capacity. Co-culture with rodent’s tissue-specific pancreatic mesenchyme accelerates proliferation, self-renewal, and differentiation of pancreatic epithelial progenitors. Therefore, in our study, the impact of three-dimensional (3D) co-culture of human fetal pancreatic-derived mesenchymal cells (hFP-MCs) with human embryonic stem cell-derived pancreatic progenitors (hESC-PPs) development towards endocrine and beta cells was assessed. Besides, the ability to maintain scalable cultures combining hFP-MCs and hESC-PPs was investigated. hFP-MCs expressed many markers in common with bone marrow-derived mesenchymal stem cells (BM-MSCs). However, they showed higher expression of DESMIN compared to BM-MSCs. After co-culture of hESC-PPs with hFP-MCs, the pancreatic progenitor (PP) spheroids generated in Matrigel had higher expression of NGN3 and INSULIN than BM-MSCs co-culture group, which shows an inductive impact of pancreatic mesenchyme on hESC-PPs beta-cells maturation. Pancreatic aggregates generated by forced aggregation through scalable AggreWell system showed similar features compared to the spheroids. These aggregates, a combination of hFP-MCs and hESC-PPs can be applied as an appropriate tool for assessing endocrine-niche interactions and developmental processes by mimicking the pancreatic tissue.

Fibroblast plays a key role in wound healing, and the advantages of mesenchymal stem cells (MSC) secretome in wound healing have previously been reported. In the present study, we investigated the impact of human bone marrow MSC-conditioned media (CM) on skin wound healing in diabetic rats and found that some improvements occurred mainly through fibroblast functions. Then, we scrutinized the impact of MSC-CM treatment on fibroblast cellular behavior by culturing human dermal fibroblasts (HDFs) in a high-glucose (HG) medium, as an in vitro diabetic model. In vivo findings revealed significant improvements in some healing kinetics of diabetic wound which received MSC-CM. Particularly, MSC-CM-treated diabetic wounds reached considerably higher percentages of wound closure. Also, the granulation tissue of these wound had less pronounced inflammatory response, better tissue remodeling, and more vascularization compared with non-treated diabetic ones. Gene expression analyses indicated that MSC-CM treatment leads to upregulation of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) genes. In addition, a significantly higher cell viability/proliferation, migration, and bFGF gene expression were observed when MSC-CM was used to treat HDFs in HG culture media. Based on these findings, it is suggested that MSC-CM could promote wound repair and skin regeneration, in some major processes, via improvement of cellular behaviors of fibroblasts in the diabetic microenvironment. The beneficial advantages of mesenchymal stem cells-conditioned media on fibroblast cellular behaviors and wound healing may lead to establish a novel approach as an alternative therapeutic procedure to cure chronic wounds in diabetic conditions.

OCT4 is a major regulator of pluripotency which has several spliced variants and expressed pseudogenes. Here, we are reporting the existence of two additional novel spliced variants of OCT4 , OCT4C and OCT4C1, which lack Exon1 (E1) but start at a novel exon (E0) located ∼14 kb upstream of E2. OCT4C/C1 is highly expressed in ES and iPS cells, and their expression was sharply turned off, upon the induction of neural differentiation. The long non-coding RNA (lncRNA) PSORS1C3 , is located ∼9 kb downstream of the E0 of OCT4C/C1 . PSORS1C3 is vigorously spliced to generate nine novel variants, however, none of its exons incorporated in alternatively spliced variants of OCT4. Interestingly, the exons of OCT4 and PSORS1C3 are intertwined, with a novel exon (E0) of PSORS1C3 located ∼4 kb upstream of OCT4 E0. This exon participates in generating some more variants of PSORS1C3 (variants 10–24). OCT4C/C1 knock-down in ES and iPS cell lines caused a slight downregulation of PSORS1C3 and OCT4A , a slight upregulation of OCT4B1 , and a dramatic upregulation of OCT4B . Altogether, our data revisited the current view of OCT4 gene structure and regulation, and revealed its complex genomic features and expression regulation in stem and tumor cells.

Purpose The aim of this study was to investigate the cardiac repair effect of human bone marrow mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) after intramyocardial injection in free form or encapsulated within a self-assembling peptide hydrogel modified with SDKP motif, in a rat model of myocardial infarction (MI). Methods MSC-EVs were isolated by ultracentrifuge and characterized for physical parameters and surface proteins. Furthermore, cellular uptake and cardioprotective effects of MSC-EVs were evaluated in vitro using neonatal mouse cardiomyocytes (NMCMs). In vivo effects of MSC-EVs on cardiac repair were studied in rat MI model by comparing the vehicle group (injected with PBS), EV group (injected with MSC-EVs) and Gel + EV group (injected with MSC-EVs encapsulated in (RADA)4-SDKP hydrogel) with respect to cardiac function and fibrotic area using echocardiography and Masson’s trichrome staining, respectively. Histological sections were assessed by α-SMA and CD68 immunostaining to investigate the angiogenic and anti-inflammatory effects of the MSC-EVs. Results We observed the uptake of MSC-EVs into NMCMs which led to NMCMs protection against H2O2-induced oxidative stress by substantial reduction of apoptosis. In myocardial infarcted rats, cardiac function was improved after myocardial injection of MSC-EVs alone or in conjunction with (RADA)4-SDKP hydrogel. This functional restoration coincided with promotion of angiogenesis and decrement of fibrosis and inflammation. Conclusion These data demonstrated that MSC-EVs can be used alone as a potent therapeutic agent for improvement of myocardial infarction.

Background & Aim: Allogeneic mesenchymal stromal cells (MSCs) are valuable therapeutic candidates used extensively in various clinical trials. However, the findings of Phase III clinical trials raise concerns about the efficacy of MSCs, which are rooted mainly in the applied populations’ heterogeneity. Although several factors, one of the most crucial causes of this heterogeneity is the lack of an optimized cell culture technique to manufacture a homogenous cultured MSCs. Moreover, it is necessary to provide a good manufacturing practice (GMP) platform to ensure the safety of final cell therapy products (CTPs) and satisfy the legal requirements for clinical applications. Establishing a tired cell bank system is the final step to support the proper translation of producing MSCs into medicine by ensuring standardization of the entire cell manufacturing process, accredited identity, function and safety assessments, and transparent sharing of standard operating procedures (SOPs) data. Methods, Results & Conclusion: Here, clonal MSCs (cMSCs) were isolated based on the subfractionation culturing method (SCM) protocol under the GMP-compatible condition and through an innovative, cost-effective screening approach. Then, cMSCs were compared with their heterogeneous counterparts in terms of identity and function. The validated clones were stored in a four-tiered cell bank system consisting of an initial, master, working, and end of product cell banks (ICB, MCB, WCB, and EoPCB). Additionally, to further develop towards GMP- compatible cMSCs production, several identities, quality, and safety assessments were performed during all banking phases. Finally, the cells stored in the EoPCB were released as a drug product (DP) for the Phase I/II clinical trial of coronavirus disease-2019 (COVID-19). Regarding senescence assessment and capability to serial passaging, three similar passagable clones were manufactured. Remarkably, the clones’ genomic stability was fully retained after 15 passages and were neither tumorigenic nor immunogenic. Altogether, this study presents a technical and translational overview of GMP-compatible cMSCs manufacturing technology, which could be used as a guide for the development of similar production processes with the therapeutic goals.

16 Pagines, 7 Figures, Pluripotent cells emanate from the inner cell mass (ICM) of the blastocyst and when cultivated under optimal conditions immortalize as embryonic stem cells (ESCs). The fundamental mechanism underlying ESC derivation has, however, remained elusive. Recently, we have devised a highly efficient approach for establishing ESCs, through inhibition of the MEK and TGF-β pathways. This regimen provides a platform for dissecting the molecular mechanism of ESC derivation. Via temporal gene expression analysis, we reveal key genes involved in the ICM to ESC transition. We found that DNA methyltransferases play a pivotal role in efficient ESC generation. We further observed a tight correlation between ESCs and preimplantation epiblast cell-related genes and noticed that fundamental events such as epithelial-to-mesenchymal transition blockage play a key role in launching the ESC self-renewal program. Our study provides a time course transcriptional resource highlighting the dynamics of the gene regulatory network during the ICM to ESC transition., This work was supported by grants from the Ministry of Economy and Competitivity, Spain, MINECO grant BFU 2016-7798-P, and DFG10/15, DFG15/15, and DFG141/16 from Diputación Foral de Gipuzkoa, Spain. This work was also supported by the Royan Institute, the Iranian Council of Stem Cell Research and Technology, the Iran National Science Foundation (INSF), and the Iran Science Elites Federation (to H.B.).

Currently, promising outcomes from clinical trials of allogeneic cells, especially allogeneic mesenchymal stromal cells, fibroblasts, keratinocytes, and human cardiac stem cells, have encouraged research institutions, small and medium enterprises (SMEs), and big pharmaceutical companies to invest and focus on developing allogeneic cell therapy products. Commercial and large-scale production of allogeneic cell therapy products requires unique capabilities to develop technologies that generate safe and effective allogeneic cells/cell lines and their fully characterized master/working banks. In addition, it is necessary to design robust upstream and downstream manufacturing processes, and establish integrated, well-designed manufacturing facilities to produce high quality affordable products in accordance with current GMP regulations for the production of cell therapy products. Areas covered: The authors highlight: the recent advances in the development of allogeneic products, the available options to develop robust manufacturing processes, and facility design considerations. Expert opinion: Currently, there are multiple challenges in development of allogeneic cell therapy products. Indeed, the field is still in its infancy; with technologies and regulations still under development, as is our understanding of the mechanisms of action in the body and their interaction with the host immune system. Their characterization and testing is also an emerging and very complex area.

Human embryonic stem cells (hESCs) provide a new source for hepatocyte production in translational medicine and cell replacement therapy. The reported hESC-derived hepatocyte-like cells (HLCs) were commonly generated on Matrigel, a mouse cell line-derived extracellular matrix (ECM). Here, we performed the hepatic lineage differentiation of hESCs following a stepwise application of growth factors on a newly developed serum- and xeno-free, simple and cost-benefit ECM, designated "RoGel," which generated from a modified conditioned medium of human fibroblasts. In comparison with Matrigel, the differentiated HLCs on both ECMs expressed similar levels of hepatocyte-specific genes, secreted α-fetoprotein, and metabolized ammonia, showed glycogen storage activity as well as low-density lipoprotein and indocyanine green uptake. The transplantation of hESC-HLCs into the carbon tetrachloride-injured liver demonstrated incorporation of the cells into the host mouse liver and the expression of albumin. The results suggest that the xeno-free and cost-benefit matrix may be applicable in bioartificial livers and also may facilitating a clinical application of human pluripotent stem cell-derived hepatocytes in the future.

Embryonic stem (ES) cells are considered to exist in a ground state if shielded from differentiation triggers. Here we show that FGF4 and TGFβ signaling pathway inhibitors, designated R2i, not only provide the ground state pluripotency in production and maintenance of naïve ES cells from blastocysts of different mouse strains, but also maintain ES cells with higher genomic integrity following long-term cultivation compared with the chemical inhibition of the FGF4 and GSK3 pathways, known as 2i. Global transcriptome analysis of the ES cells highlights augmented BMP4 signaling pathway. The crucial role of the BMP4 pathway in maintaining the R2i ground state pluripotency is demonstrated by BMP4 receptor suppression, resulting in differentiation and cell death. In conclusion, by inhibiting TGFβ and FGF signaling pathways, we introduce a novel defined approach to efficiently establish the ground state pluripotency.

Optimization and development of better defined culture methods for human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) will provide an invaluable contribution to the field of regenerative medicine. However, one problem is the vulnerability of hESCs and hiPSCs to apoptosis that causes a low plating efficiency upon passaging. Herein, we have developed a novel hESCs and hiPSCs culture technique that uses ROCK inhibitor (ROCKi) Y-27632 (10 microM) in Matrigel-coated dishes in both serum- and feeder-free culture conditions. This increases plating efficiency during enzymatic and mechanical passaging as compared to its presence solely in culture medium. Under these conditions, hESCs (three lines) and hiPSCs (two lines) retain their typical morphology, a stable karyotype, express pluripotency markers and have the potential to differentiate into derivatives of all three germ layers after long-term culture. Real-time RT-PCR analysis of stemness-related integrins (alphaV, alpha6, and beta1) has demonstrated that their expression increases in the presence of ROCKi. Similar plating efficiencies have been obtained in both hESCs and hiPSCs with a lower concentration of Y-27632 (800 nM) and another ROCKi (HA-1077/Fasudil), thus ruling out the non-specific effects of Y-27632. These results show that addition of ROCKi in the extracellular matrix can increase the plating efficiency of hESCs and hiPSCs during passaging of clusters. This is due not only to an anti-apoptotic effect, but also to an increase in the ECM-cells interaction. Therefore, we believe this method will be useful for both current and future applications of these pluripotent stem cells.

Gene expression analyses of embryonic stem cells (ESCs) will help to uncover or further define signaling pathways and molecular mechanisms involved in the maintenance of self-renewal and pluripotency. We employed a 2-DE-based proteomics approach to analyze human ESC line, Royan H5, in undifferentiated cells and different stages of spontaneous differentiation (days 3, 6, 12, and 20) by embryoid body formation. Out of 945 proteins reproducibly detected on gels, the expression of 96 spots changed during differentiation. Using MS, 87 ESC-associated proteins were identified including several proteins involved in cell proliferation, cell apoptosis, transcription, translation, mRNA processing, and protein folding. Transcriptional changes accompanying differentiation of Royan H5 were also analyzed using microarrays. We developed a comprehensive data set that shows the use of human ESC lines in vitro to mimic gastrulation and organogenesis. Our results showed that proteomics and transcriptomics data are complementary rather than duplicative. Although regulation of many genes during differentiation were observed only at transcript level, modulation of several proteins was revealed only by proteome analysis.

BACKGROUND: An essential prerequisite for the future widespread application of human induced pluripotent (hiPSCs) and embryonic stem cells (hESCs) is the development of efficient cryopreservation methods to facilitate their storage and transportation. METHODS: We developed a simple and effective freezing/thawing method of single dissociated hESCs and hiPSCs in a feeder-free culture in the presence of Rho-associated kinase (ROCK) inhibitor Y-27632. RESULTS: Exposure to ROCK inhibitor Y-27632 in freezing solution alone does not significantly enhance the post-thaw survival rate of single dissociated hESCs and hiPSCs. However, when ROCK inhibitor was added to both pre- and post-thaw culture media, there was an enhancement in the survival rate, which further increased when ROCK inhibitor was added to Matrigel as well. Under these treatments, hESCs and hiPSCs retained typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture. CONCLUSIONS: This method is an effective cryopreservation procedure for single dissociated hESCs in feeder-free culture, which is also applicable for single dissociated hiPSCs using a ROCK inhibitor. The cloning efficiency of hiPSCs and hESCs improves when ROCK inhibitor is added both in Matrigel and in medium in comparison with conventional addition to medium. Therefore, we believe this method would be useful for current and future applications of the pluripotent stem cells.

Stem cell banks with a number of qualified stem cell lines deposited are essential to facilitate biomedical applications of stem cells. Iran, which is active in the field of stem cell research and therapy and to meet the needs, has established stem cell banks. In this chapter, Iranian stem cell banks, which are divided into research and therapeutic banks, will be presented. We introduce here the Royan Stem Cell Bank (RSCB), Iran’s only research stem cell bank, and describe the process used for quality control and documentation. Finally, we discuss the future landscape of stem cell banking in Iran and the required steps for establishing good manufacturing practice (GMP) banks in order to improve the current state in Iranian stem cell therapy centers.

Study question Could selected pluripotency-enhancing small molecules (SMs) lead to efficient derivation of human embryonic stem cells (hESCs) from cleavage embryos-derived single blastomeres (SBs)? Summary answer Inhibition of glycogen synthase kinase β (GSK3β) and Rho-associated kinase (ROCK) signaling can enhance the derivation of hESCs from cleavage embryo-derived SBs. What is known already Parameters involved in sustaining the pluripotency of biopsied blastomeres for generating hESCs without causing injury to a viable embryo have remained obscure. This research seeks to improve the culture conditions for increasing the efficiency of deriving hESCs from SBs from cleavage-stage embryos by using SMs. Study design, size, duration In order to identify SMs which may enhance hESC generation from SBs, 11 pluripotency-enhancing SMs were screened and CHIR99021 (CH), a GSK3β inhibitor, was selected. To optimize culture condition in hESC generation from SMs, we used ROCK inhibitor Y27632 (Y) and basic fibroblast growth factor in combination with CH or its alternative, Kenpaullone, in different time courses over 12 days. We also assessed a critical time point for CH + Y treatment of cleavage embryos from 4- to 8-cell embryo. In total, 224 embryos and 1607 SBs were used in the study. Participants/materials, setting, methods Blastomeres of fair and poor-quality from 6- to 8-cell stage human embryos were mechanically dispersed and individually seeded into a 96-well plate that was precoated with mitotically inactivated feeder cells. Derivation of hESC line from each SB was carried out in hESC defined medium supplemented with SMs. Randomly selected hESC lines were evaluated by immunostaining for pluripotency markers, karyotype analysis and differentiation potential into the three embryonic germ layer derivatives. Main results and the role of chance We found that 3 μM CH was the only SM that was capable of directing SBs from fair and poor-quality 6-8-cell embryos into hESC lines. The application of hESC-conditioned medium had no additive effect on hESC establishment from SBs. Also, we indicated that CH combined with Y improved hESC generation efficiency by up to 31%. By using of Kenpaullone as an alternative to CH, we confirmed the involvement of GSK3 inhibition in hESC derivation from SBs. Interestingly, by treatment of 4-cell embryos, these SMs could enhance the derivation efficiency of SB-derived hESC lines up to 73% and the maximum number of hESC lines from SBs of one embryo was achieved in this state. Limitations, reasons for caution The low quality of the embryos used in this study most likely had an effect on hESC generation. Furthermore, although we attempted to minimize any differences in inter-embryo quality, we cannot exclude the possibility that small differences in starting quality between embryos may have contributed to the differences observed, other than the addition of SMs. Wider implications of the findings This approach would allow the establishment of autogeneic or allogeneic matched cells from embryos fertilized in vitro without destroying them. Study funding/competing interest(s) This study was financially supported by the National Elite Foundation and the Royan Institute for Stem Cell Biology and Technology. The authors have no conflict of interest to declare.

Induced pluripotent stem cells (iPSCs) provide an invaluable resource for drug or toxicology screening, medical research and patient-specific cell therapy. However, the potential applications of iPSCs are largely dependent on the degree of similarity between iPSCs and embryonic stem cells (ESCs). In the present study, we analyzed the proteome of human ESCs and hiPSCs with different genetic background. We carried out an orthogonal contrast analysis of the proteome pattern of two human ESC lines (Royan H5 and Royan H6) and two hiPSC lines from a normal individual, three hiPSC lines from a normal individual with Bombay blood group phenotype, and two hiPSC lines from a patient with tyrosinemia. Forty-nine protein spots showed statistically significant differences between two human ESC lines and seven human iPSCs. Mass spectrometry analysis resulted in the identification of 48 proteins belonging to different biological processes, including cytoskeleton organization, energy and metabolic processes, protein synthesis and processing, signal transduction, cell growth and proliferation, cellular trafficking, transcription, calcium binding and immune response. Our results showed that hESCs and hiPSCs had subtle differences at the proteome level thus warranting more detailed and systematic examinations of these cells.

Proteomics showed to be a powerful approach to comprehensively unravel the regulatory networks of differentiation. We applied a two dimensional gel electrophoresis based proteomic approach followed by mass spectrometry to analyze the proteome of two Human ESC lines, Royan H5 and Royan H6, at 0, 3, 6, 12 and 20 days after differentiation initiation. Out of 127 differentiation associated proteins detected in two lines, 35 were common. Mass spectrometry analysis of these protein spots led to identification of 92 proteins. Our results showed that proteins involved in signal transduction, metabolism, cell motility and transport are the major ESC-associated proteins. The expression of several proteins were further confirmed by western blotting and immunocytochemistry. Transcriptome analysis revealed down-regulation of OCT4, NANOG, UTF1, ZNF206 key transcription factors in hESCs and up-regulation of three lineage specific markers VIM, MAP2, OLIG2 (Ectodermic), GATA4, INS, FOXA2 (Endodermic).,HAND1, COL2A1 (Mesodermic). Integration of transcripteome and proteome data could be useful for finding of new mechanisms that controls differentiation. Several novel ESC-associated genes have been identified in this study which warrants further investigation with respect to the etiology of temness.

Objective: Human basic fibroblast growth factor (bFGF) plays an important role in cellular proliferation, embryonic development, and angiogenesis as well as in several signaling pathways of various cell types. bFGF is an essential growth factor for the maintenance of undifferentiated human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Materials and Methods: In this experimental study, we present a straightforward method to produce biologically active recombinant human bFGF protein in E. coli that has long-term storage ability. Results: This procedure provides a rapid, cost effective purification of a soluble human bFGF protein that is biologically active and functional as measured in hESCs and hiPSCs in vitro and in vivo. Conclusion: The results show no significant difference in function between our in-house produced and commercialized bFGF.