Derivation Of Human Embryonic Stem Cells Research Articles

Therapeutic Efficacy of Human Embryonic Stem Cell-Derived Multipotent Stem/Stromal Cells in Diabetic Detrusor Underactivity: A Preclinical Study.

Mesenchymal stem/stromal cell (MSC) therapy is a promising approach for treatment of as yet incurable detrusor underactivity (DUA), which is characterized by decreased detrusor contraction strength and/or duration, leading to prolonged bladder emptying. In the present study, we demonstrated the therapeutic potential of human embryonic stem cell (ESC)-derived multipotent MSCs (M-MSCs) in a diabetic rat model of DUA. Diabetes mellitus (DM) was induced by intraperitoneal injection of streptozotocin (STZ) (50 mg/kg) into 8-week-old female Sprague-Dawley rats. Three weeks later, various doses of M-MSCs (0.25, 0.5, and 1 × 106 cells) or an equivalent volume of PBS were injected into the outer layer of the bladder. Awake cystometry, organ bath, histological, and gene expression analyses were evaluated 1 week (short-term) or 2 and 4 weeks (long-term) after M-MSC transplantation. STZ-induced diabetic rats developed DUA, including phenotypes with significantly longer micturition intervals, increased residual urine amounts and bladder capacity, decreased micturition pressure on awake cystometry, and contractile responses to various stimuli in organ bath studies. Muscle degeneration, mast cell infiltration, fibrosis, and apoptosis were present in the bladders of DM animals. A single local transplantation of M-MSCs ameliorated DUA bladder pathology, including functional changes and histological evaluation, and caused few adverse outcomes. Immunostaining and gene expression analysis revealed that the transplanted M-MSCs supported myogenic restoration primarily by engrafting into bladder tissue via pericytes, and subsequently exerting paracrine effects to prevent apoptotic cell death in bladder tissue. The therapeutic efficacy of M-MSCs was superior to that of human umbilical cord-derived MSCs at the early time point (1 week). However, the difference in efficacy between M-MSCs and human umbilical cord-derived MSCs was statistically insignificant at the later time points (2 and 4 weeks). Collectively, the present study provides the first evidence for improved therapeutic efficacy of a human ESC derivative in a preclinical model of DM-associated DUA.

Genetic Manipulation to Improve Athletes Performance - A Critical Review

Genetic manipulation as the name suggests is the modification of genes. It is the process of changing the structure of the genes of a living thing in order to make it healthier, stronger, or more useful. It is the direct manipulation of an organism’s genes using biotechnology used to change the genetic makeup of cells including transfer of genes. The need of genetic modification is to enhance the performance of particular genes, this method is implemented effectively to enhance the quality of food, fight various chronic diseases like cancer, cloning etc. However, this paper is limited to the use of genetic manipulation to enhance Athletics performance of the athletes and healing process of the cells. There are various contradictory thoughts over the use of genetic manipulation in Athletics over its use being ethical or unethical. There have been various normative arguments both in favor and against its use in Athletics. However, no competent authority has prohibited its use in Athletics. There are various methods involved in gene modification, few prominent are discussed in this paper. Genetic manipulation, the process of inducing changes in gene expression and the expression of novel genes, has proven to be an indispensable tool in recent genetic research. The implementation of increasingly powerful genetic tools to mouse embryonic stem (ES) cells has led to an explosion of data concerning the specific properties of an extremely large array of genes. Current available techniques allow for the specific elimination of target gene expression, tissue specific induction of reporter gene expression, the over expression of cellular genes, and more. It is not surprising, therefore, that a short time after the derivation of human ES cells, several studies describing genetic manipulation of these cells have been reported. This focuses on the recent advances in the genetic manipulation of human ES cells, the methods currently available, and their possible utilization. A brief discussion of advanced genetic manipulation techniques not yet demonstrated in these cells is also presented. Through this paper the author has tried to summarize various methods, aspects, approaches and arguments involved with genetic modification in Athletic persons.

Coreceptor blockade targeting CD4 and CD8 allows acceptance of allogeneic human pluripotent stem cell grafts in humanized mice

We have previously demonstrated that short-term coreceptor blockade with non-lytic monoclonal antibodies enables the long-term survival of fully allogeneic embryonic stem cell (ESC) transplants in mice. Here, we describe the use of Hu-PBL humanized mice to determine whether short-term coreceptor blockade with humanized anti-human CD4 and CD8 antibodies can achieve the same outcome towards human ESC derivatives. While control Hu-PBL mice rejected allogeneic hESC-derived transplants within weeks, mice treated with coreceptor blocking antibodies held their grafts for 7 weeks, the duration of the study. Rejection in the control mice was associated with demonstrable infiltrates of human CD45 white blood cells, predominantly of CD8 T-cells, whereas anti-CD4, but not anti-CD8 antibody treated mice showed remarkably reduced lymphocyte infiltration and prolonged allograft survival, indicating that the CD4+ T-cells were crucial to the rejection process. Our results give support to the principle that short-term blockade of T-cell co-receptors can achieve long-term acceptance of regenerative cell transplants in humans.

Human Pluripotent Stem Cells in Neurodegenerative Diseases: Potentials, Advances and Limitations.

Neurodegenerative diseases are progressive and uncontrolled gradual loss of motor neurons function or death of neuron cells in the central nervous system (CNS) and the mechanisms underlying their progressive nature remain elusive. There is urgent need to investigate therapeutic strategies and novel treatments for neural regeneration in disorders like Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Currently, the development and identification of pluripotent stem cells enabling the acquisition of a large number of neural cells in order to improve cell recovery after neurodegenerative disorders. Pluripotent stem cells which consist of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are characterized by their ability to indefinitely self-renew and the capacity to differentiate into different types of cells. The first human ESC lines were established from donated human embryos; while, because of a limited supply of donor embryos, human ESCs derivation remains ethically and politically controversial. Hence, hiPSCs-based therapies have been shown as an effective replacement for human ESCs without embryo destruction. Compared to the invasive methods for derivation of human ESCs, human iPSCs has opened possible to reprogram patient-specific cells by defined factors and with minimally invasive procedures. Human pluripotent stem cells are a good source for cell-based research, cell replacement therapies and disease modeling. To date, hundreds of human ESC and human iPSC lines have been generated with the aim of treating various neurodegenerative diseases. In this review, we have highlighted the recent potentials, advances, and limitations of human pluripotent stem cells for the treatment of neurodegenerative disorders.

Generation of functional human pancreatic organoids by transplants of embryonic stem cell derivatives in a 3D-printed tissue trapper.

Organoids can be regarded as a beneficial tool for discovery of new therapeutics for diabetes and/or maturation of pancreatic progenitors (PP) towards β cells. Here, we devised a strategy to enhance maturation of PP by assembly of three-dimensional (3D) pancreatic organoids (PO) containing human embryonic stem (ES) cell derivatives including ES-derived pancreatic duodenal homeobox 1 (PDX1) + early PP, mesenchymal stem cells, and endothelial cells at an optimized cell ratio, on Matrigel. The PO was placed in a 3D-printed tissue trapper and heterotopically implanted into the peritoneal cavity of immunodeficient mice where it remained for 90 days. Our results indicated that, in contrast to corresponding early PP transplants, 3D PO developed more vascularization as indicated by greater area and number of vessels, a higher number of insulin-positive cells and improvement of human C-peptide secretions. Based on our findings, PO-derived β cells could be considered a novel strategy to study human β-cell development, novel therapeutics, and regenerative medicine for diabetes.

Transcriptional landscape changes during human embryonic stem cell derivation.

What are the transcriptional changes occurring during the human embryonic stem cell (hESC) derivation process, from the inner cell mass (ICM) to post-ICM intermediate stage (PICMI) to hESC stage, that have downstream effects on pluripotency states and differentiation? We reveal that although the PICMI is transcriptionally similar to the hESC profile and distinct from ICM, it exhibits upregulation of primordial germ cell (PGC) markers, dependence on leukemia inhibitory factor (LIF) signaling, upregulation of naïve pluripotency-specific signaling networks and appears to be an intermediate switching point from naïve to primed pluripotency. It is currently known that the PICMI exhibits markers of early and late-epiblast stage. It is suggested that hESCs acquire primed pluripotency features due to the upregulation of post-implantation genes in the PICMI which renders them predisposed towards differentiation cues. Despite this current knowledge, the transcriptional landscape changes during hESC derivation from ICM to hESC and the effect of PICMI on pluripotent state is still not well defined. To gain insight into the signaling mechanisms that may govern the ICM to PICMI to hESC transition, comparative RNA sequencing (RNA-seq) analysis was performed on preimplantation ICMs, PICMIs and hESCs in biological and technical triplicates (n = 3). Primed hESCs (XX) were maintained in feeder-free culture conditions on Matrigel for two passages and approximately 50 cells were collected in biological and technical triplicates (n = 3). For ICM sample collection, Day 3, frozen-thawed human embryos were cultured up to day five blastocyst stage and only good quality blastocysts were subjected to laser-assisted micromanipulation for ICM collection (n = 3). Next, day six expanded blastocysts were cultured on mouse embryonic fibroblasts and manual dissection was performed on the PICMI outgrowths between post-plating Day 6 and Day 10 (n = 3). Sequencing of these samples was performed on NextSeq500 and statistical analysis was performed using edgeR (false discovery rate (FDR) < 0.05). Comparative RNA-seq data analysis revealed that 634 and 560 protein-coding genes were significantly up and downregulated in hESCs compared to ICM (FDR < 0.05), respectively. Upon ICM to PICMI transition, 471 genes were expressed significantly higher in the PICMI compared to ICM, while 296 genes were elevated in the ICM alone (FDR < 0.05). Principle component analysis showed that the ICM was completely distinct from the PICMI and hESCs while the latter two clustered in close proximity to each other. Increased expression of E-CADHERIN1 (CDH1) in ICM and intermediate levels in the PICMI was observed, while CDH2 was higher in hESCs, suggesting a role of extracellular matrix components in facilitating pluripotency transition during hESC derivation. The PICMI also showed regulation of naïve-specific LIF and bone morphogenetic protein signaling, differential regulation of primed pluripotency-specific fibroblast growth factor and NODAL signaling pathway components, upregulation of phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway (PI3K/AKT/mTORC), as well as predisposition towards the germ cell lineage, further confirmed by gene ontology analysis. Hence, the data suggest that the PICMI may serve as an intermediate pluripotency stage which, when subjected to an appropriate culture niche, could aid in enhancing naïve hESC derivation and germ cell differentiation efficiency. Gene Expression Omnibus (GEO) Accession number GSE119378. Owing to the limitation in sample availability, the sex of ICM and PICMI have not been taken into consideration. Obtaining cells from the ICM and maintaining them in culture is not feasible as it will hamper the formation of PICMI and hESC derivation. Single-cell quantitative real-time PCR on low ICM and PICMI cell numbers, although challenging due to limited availability of human embryos, will be advantageous to further corroborate the RNA-seq data on transcriptional changes during hESC derivation process. We elucidate the dynamics of transcriptional network changes from the naïve ICM to the intermediate PICMI stage and finally the primed hESC lines. We provide an in-depth understanding of the PICMI and its role in conferring the type of pluripotent state which may have important downstream effects on differentiation, specifically towards the PGC lineage. This knowledge contributes to our limited understanding of the true nature of the human pluripotent state in vitro. This research is supported by the Concerted Research Actions funding from Bijzonder Onderzoeksfonds University Ghent (BOF GOA 01G01112).The authors declare no conflict of interest.

Ectopic expression of recipient CD47 inhibits mouse macrophage-mediated immune rejection against human stem cell transplants.

Like conventional transplants, immunosuppression is required to facilitate survival and function of human embryonic stem cell (hESC) derivatives after implantation into xenogeneic recipients. We have previously reported that T cells alone are sufficient to reject allogeneic murine ESC derivatives; and strategies that inhibit T-cell activation, including coreceptor and costimulation blockade, prevent hESC derivatives from being rejected. This study aimed to investigate, in addition to T cells, whether macrophages contribute to transplant rejection of hESC xenografts with nonobese diabetic (NOD)/SCID mice that lack functional T and B cells but have macrophages. We show that acute rejection against hESC-derived endothelial cells (hESC-ECs) was mediated, to some degree, by infiltrating macrophages that phagocytosed them. Transgenic expression of murine CD47 on cell surface of hESC-ECs mitigates macrophage-mediated phagocytosis and improves their survival after transplantation. Our results highlight that innate immune cells, such as macrophages, can reject hESC derivatives, raising concern against the use of NOD/SCID as transplant recipients for testing in vivo function of hESC-derived tissues. Augmenting CD47 signaling promotes survival and function of hESC derivatives after xenogeneic transplantation.-Leung, C. S., Li, J., Xu, F., Wong, A. S. L., Lui, K. O. Ectopic expression of recipient CD47 inhibits mouse macrophage-mediated immune rejection against human stem cell transplants.

States and Origins of Mammalian Embryonic Pluripotency In Vivo and in a Dish.

Mouse embryonic stem cells (ESC), derived from preimplantation embryos in 1981, defined mammalian pluripotency for many decades. However, after the derivation of human ESC in 1998, comparative studies showed that different types of pluripotency exist in early embryos and that these can be captured in vitro under various culture conditions. Over the past decade much has been learned about the key signaling pathways, growth factor requirements, and transcription factor profiles of pluripotent cells in embryos, allowing improvement of derivation and culture conditions for novel pluripotent stem cell types. More recently, studies using single-cell transcriptomics of embryos from different species provided an unprecedented level of resolution of cellular interactions and cell fate decisions that are informing new ways to understand the emergence of pluripotency in different organisms. These new approaches enhance knowledge of species differences during early embryogenesis and will be instrumental for improving methodologies for generating intra- and interspecies chimeric animals using pluripotent stem cells. Here, we discuss the recent developments in our understanding of early embryogenesis in different mammalian species.

Matrix Topographical Cue-Mediated Myogenic Differentiation of Human Embryonic Stem Cell Derivatives.

Biomaterials varying in physical properties, chemical composition and biofunctionalities can be used as powerful tools to regulate skeletal muscle-specific cellular behaviors, including myogenic differentiation of progenitor cells. Biomaterials with defined topographical cues (e.g., patterned substrates) can mediate cellular alignment of progenitor cells and improve myogenic differentiation. In this study, we employed soft lithography techniques to create substrates with microtopographical cues and used these substrates to study the effect of matrix topographical cues on myogenic differentiation of human embryonic stem cell (hESC)-derived mesodermal progenitor cells expressing platelet-derived growth factor receptor alpha (PDGFRA). Our results show that the majority (>80%) of PDGFRA+ cells on micropatterned polydimethylsiloxane (PDMS) substrates were aligned along the direction of the microgrooves and underwent robust myogenic differentiation compared to those on non-patterned surfaces. Matrix topography-mediated alignment of the mononucleated cells promoted their fusion resulting in mainly (~86%–93%) multinucleated myotube formation. Furthermore, when implanted, the cells on the micropatterned substrates showed enhanced in vivo survival (>5–7 times) and engraftment (>4–6 times) in cardiotoxin-injured tibialis anterior (TA) muscles of NOD/SCID mice compared to cells cultured on corresponding non-patterned substrates.

Metabolic plasticity complements the unique nature and demands of distinct pluripotency states.

Pluripotency, the ability to differentiate into all somatic cell types, is a pertinent feature of embryonic stem cells (ESCs). However, during early ontogenesis, in vivo , this capacity only exists transiently (1,2). Within this window of development, extracellular signals governing embryogenesis grant pluripotency a dynamic nature, while molecular changes in cellular identity occur in preparation for further lineage specification (2). By precisely coordinating the cellular microenvironment and embryonic derivation stage, ESCs can be stabilized in distinct states of pluripotency, in vitro . Conventional derivation of human ESCs (hESCs) from the inner cell mass (ICM) of the pre-implantation blastocyst gives rise to stem cells, which are markedly different from mouse ESCs (mESCs). In vitro , hESCs originate from a post-ICM intermediate, a transient epiblast-like structure (3,4). As such, hESCs adopt the distinct primed state of pluripotency, sharing more similarities with mouse epiblast stem cells (mEpiSCs) derived from post-implantation stage embryos (5). Conversely, mESCs, adopt the naive state, which constitutes the functional in vitro equivalent of the pre-implantation epiblast (5).

The Molecular Karyotype of 25 Clinical-Grade Human Embryonic Stem Cell Lines.

The application of human embryonic stem cell (hESC) derivatives to regenerative medicine is now becoming a reality. Although the vast majority of hESC lines have been derived for research purposes only, about 50 lines have been established under Good Manufacturing Practice (GMP) conditions. Cell types differentiated from these designated lines may be used as a cell therapy to treat macular degeneration, Parkinson’s, Huntington’s, diabetes, osteoarthritis and other degenerative conditions. It is essential to know the genetic stability of the hESC lines before progressing to clinical trials. We evaluated the molecular karyotype of 25 clinical-grade hESC lines by whole-genome single nucleotide polymorphism (SNP) array analysis. A total of 15 unique copy number variations (CNVs) greater than 100 kb were detected, most of which were found to be naturally occurring in the human population and none were associated with culture adaptation. In addition, three copy-neutral loss of heterozygosity (CN-LOH) regions greater than 1 Mb were observed and all were relatively small and interstitial suggesting they did not arise in culture. The large number of available clinical-grade hESC lines with defined molecular karyotypes provides a substantial starting platform from which the development of pre-clinical and clinical trials in regenerative medicine can be realised.

Histone Demethylase Expression Enhances Human Somatic Cell Nuclear Transfer Efficiency and Promotes Derivation of Pluripotent Stem Cells

The extremely low efficiency of human embryonic stem cell (hESC) derivation using somatic cell nuclear transfer (SCNT) limits its potential application. Blastocyst formation from human SCNT embryos occurs at a low rate and with only some oocyte donors. We previously showed in mice that reduction of histone H3 lysine 9 trimethylation (H3K9me3) through ectopic expression of the H3K9me3 demethylase Kdm4d greatly improves SCNT embryo development. Here we show that overexpression of a related H3K9me3 demethylase KDM4A improves human SCNT, and that, as in mice, H3K9me3 in the human somatic cell genome is an SCNT reprogramming barrier. Overexpression of KDM4A significantly improves the blastocyst formation rate in human SCNT embryos by facilitating transcriptional reprogramming, allowing efficient derivation of SCNT-derived ESCs using adult Age-related Macular Degeneration (AMD) patient somatic nuclei donors. This conserved mechanistic insight has potential applications for improving SCNT in a variety of contexts, including regenerative medicine.

HLA Class I Depleted hESC as a Source of Hypoimmunogenic Cells for Tissue Engineering Applications.

Rapidly improving protocols for the derivation of autologous cells from stem cell sources is a welcome development. However, there are many circumstances when off-the-shelf universally immunocompatible cells may be needed. Embryonic stem cells (ESCs) provide a unique opportunity to modify the original source of differentiated cells to minimize their rejection by nonautologous hosts. Immune rejection of nonautologous human embryonic stem cell (hESC) derivatives can be reduced by downregulating human leukocyte antigen (HLA) class I molecules, without affecting the ability of these cells to differentiate into specific lineages. Beta-2-microglobulin (B2M) expression was decreased by lentiviral transduction using human anti-HLA class I light-chain B2M short hairpin RNA. mRNA levels of B2M were decreased by 90% in a RUES2-modified hESC line, as determined by quantitative real time-polymerase chain reaction analysis. The transduced cells were selected under puromycin pressure and maintained in an undifferentiated state. The latter was confirmed by Oct4 and Nanog expression, and by the formation of characteristic round-shaped colonies. B2M downregulation led to diminished HLA-I expression on the cell surface, as determined by flow cytometry. When used as target cells in a mixed lymphocyte reaction assay, transduced hESCs and their differentiated derivatives did not stimulate allogeneic T-cell proliferation. Using a cardiac differentiation protocol, transduced hESCs formed a confluent layer of cardiac myocytes and maintained a low level of B2M expression. Transduced hESCs were also successfully differentiated into a hepatic lineage, validating their capacity to differentiate into multiple lineages. HLA-I depletion does not preclude hESC differentiation into cardiac or hepatic lineages. This methodology can be used to engineer tissue from nonautologous hESC sources with improved immunocompatibility.

Application Of Small Molecules Favoring Naïve Pluripotency during Human Embryonic Stem Cell Derivation.

In mice, inhibition of both the fibroblast growth factor (FGF) mitogen-activated protein kinase kinase/extracellular-signal regulated kinase (MEK/Erk) and the Wnt signaling inhibitor glycogen synthase-3β (GSK3β) enables the derivation of mouse embryonic stem cells (mESCs) from nonpermissive strains in the presence of leukemia inhibitory factor (LIF). Whereas mESCs are in an uncommitted naïve state, human embryonic stem cells (hESCs) represent a more advanced state, denoted as primed pluripotency. This burdens hESCs with a series of characteristics, which, in contrast to naïve ESCs, makes them not ideal for key applications such as cell-based clinical therapies and human disease modeling. In this study, different small molecule combinations were applied during human ESC derivation. Hereby, we aimed to sustain the naïve pluripotent state, by interfering with various key signaling pathways. First, we tested several combinations on existing, 2i (PD0325901 and CHIR99021)-derived mESCs. All combinations were shown to be equally adequate to sustain the expression of naïve pluripotency markers. Second, these conditions were tested during hESC derivation. Overall, the best results were observed in the presence of medium supplemented with 2i, LIF, and the noncanonical Wnt signaling agonist Wnt5A, alone and combined with epinephrine. In these conditions, outgrowths repeatedly showed an ESC progenitor-like morphology, starting from day 3. Culturing these "progenitor cells" did not result in stable, naïve hESC lines in the current conditions. Although Wnt5A could not promote naïve hESC derivation, we found that it was sustaining the conversion of established hESCs toward a more naïve state. Future work should aim to distinct the effects of the various culture formulations, including our Wnt5A-supplemented medium, reported to promote stable naïve pluripotency in hESCs.

Well-defined differentiation of hESC-derived hemangioblasts by embryoid body formation without enzymatic treatment.

Human hemangioblasts exist only during the early embryonic developmental stage thereby limiting the adult cellular source from which to obtain such cells for study. To overcome this, hemangioblast studies have focused on utilizing human embryonic stem cell (hESC) derivatives but current methods are cell-line dependent. Single cell dissociation of a hESC colony quickly led to cell death in most hESC lines due to enzyme treatment which, in turn, reduced induction potential and hemangioblast differentiation efficiency. Therefore, we sought to effectively improve the process of cell dissociation that is adaptable to various hESC lines and increase the initial induction potential of embryoid body (hEB). As a result, we determined an effective cell dissociation method through a comparison study involving various reagents which demonstrated successful dissociation regardless of cell line and enhanced hemangioblast differentiation efficiency.

OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells.

ABSTRACTMedulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is currently divided into four subtypes based on different genomic alterations, gene expression profiles and response to treatment: WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. This extensive heterogeneity has made it difficult to assess the functional relevance of genes to malignant progression. For example, expression of the transcription factor Orthodenticle homeobox2 (OTX2) is frequently dysregulated in multiple MB variants; however, its role may be subtype specific. We recently demonstrated that neural precursors derived from transformed human embryonic stem cells (trans-hENs), but not their normal counterparts (hENs), resemble Groups 3 and 4 MB in vitro and in vivo. Here, we tested the utility of this model system as a means of dissecting the role of OTX2 in MB using gain- and loss-of-function studies in hENs and trans-hENs, respectively. Parallel experiments with MB cells revealed that OTX2 exerts inhibitory effects on hEN and SHH MB cells by regulating growth, self-renewal and migration in vitro and tumor growth in vivo. This was accompanied by decreased expression of pluripotent genes, such as SOX2, and was supported by overexpression of SOX2 in OTX2+ SHH MB and hENs that resulted in significant rescue of self-renewal and cell migration. By contrast, OTX2 is oncogenic and promotes self-renewal of trans-hENs and Groups 3 and 4 MB independent of pluripotent gene expression. Our results demonstrate a novel role for OTX2 in self-renewal and migration of hENs and MB cells and reveal a cell-context-dependent link between OTX2 and pluripotent genes. Our study underscores the value of human embryonic stem cell derivatives as alternatives to cell lines and heterogeneous patient samples for investigating the contribution of key developmental regulators to MB progression.

Biomimetic Material-Assisted Delivery of Human Embryonic Stem Cell Derivatives for Enhanced In Vivo Survival and Engraftment.

The ability of human embryonic stem cells (hESCs) and their derivatives to differentiate and contribute to tissue repair has enormous potential to treat various debilitating diseases. However, improving the in vivo viability and function of the transplanted cells, a key determinant of translating cell-based therapies to the clinic, remains a daunting task. Here, we develop a hybrid biomaterial consisting of hyaluronic acid (HA) grafted with 6-aminocaproic acid moieties (HA-6ACA) to improve cell delivery and their subsequent in vivo function using skeletal muscle as a model system. Our findings show that the biomimetic material-assisted delivery of hESC-derived myogenic progenitor cells into cardiotoxin-injured skeletal muscles of NOD/SCID mice significantly promotes survival and engraftment of transplanted cells in a dose-dependent manner. The donor cells were found to contribute to the regeneration of damaged muscle fibers and to the satellite cell (muscle specific stem cells) compartment. Such biomimetic cell delivery vehicles that are cost-effective and easy-to-synthesize could play a key role in improving the outcomes of other stem cell-based therapies.

DNA methylation dynamics of the human preimplantation embryo.

In mammals, cytosine methylation is predominantly restricted to CpG dinucleotides and stably distributed across the genome, with local, cell type-specific regulation directed by DNA binding factors1-3. This comparatively static landscape dramatically contrasts the events of fertilization, where the paternal genome is globally reprogrammed. Paternal genome demethylation includes the majority of CpGs, though methylation is maintained at several notable features4-7. While these dynamics have been extensively characterized in the mouse, only limited observations are available in other mammals, and direct measurements are required to understand the extent to which early embryonic landscapes are conserved8-10. We present genome-scale DNA methylation maps of human preimplantation development and embryonic stem cell (ESC) derivation, confirming a transient state of global hypomethylation that includes most CpGs, while sites of persistent maintenance are primarily restricted to gene bodies. While most features share similar dynamics to mouse, maternally contributed methylation is divergently targeted to species-specific sets of CpG island (CGI) promoters that extend beyond known Imprint Control Regions (ICRs). Retrotransposon regulation is also highly diverse and transitions from maternally to embryonically expressed, species-specific elements. Together, our data confirm that paternal genome demethylation is a general attribute of early mammalian development that is characterized by distinct modes of epigenetic regulation.

Immunosuppressive Agents Modulate Function, Growth, and Survival of Cardiomyocytes and Endothelial Cells Derived from Human Embryonic Stem Cells

Cardiac cell replacement therapy by using human embryonic stem cell (hESC) derivatives remains a potential approach to regenerate myocardium. The major hurdles to clinical application of this technology are immunogenicity and post-transplantation cell death. Here we examined the effects of calcineurin-targeting immunosuppressants cyclosporine A (CsA) and FK506, as well as rapamycin and a selective inhibitor of calcineurin-binding downstream nuclear factor of activated T-cell (NFAT) transcription factor VIVIT on the proliferative activity, function, and survival of hESC-derived cardiomyocytes (hESC-CM) and endothelial cells (hESC-EC) in culture. As shown by automated microscopy, treatments with CsA, FK506, and rapamycin all decreased proliferation, reducing the percentage of hESC-CM and hESC-EC with the mitotic marker Ki67(+) by as much as 60% and 74%, respectively. Administration of the cell permeable analogue 11R-VIVIT protein did not modulate their proliferative activity. All immunosuppressants reversed the proapoptotic effect of chelerythrine in hESC-CM demonstrating an inhibitory role of calcineurin/NFAT and mammalian target of rapamycin (mTOR) pathways in hESC-CM survival (using apoptotic marker caspase-3), whereas the protection was less obvious in hESC-EC exposed to H2O2. Immunosuppressants did not affect cell viability in hESC-EC. Our results show that immunosuppressants reduce proliferation, while offsetting cell loss to a smaller extent by reduction in apoptosis of hESC-CM. Immunosuppressant therapy would be compatible with stem cell transplantation, but the resulting reduction in graft expansion capabilities would potentially necessitate implantation of increased cell numbers when immunosuppressants are given. The effects of NFAT-binding immunosuppressant molecules, which do not affect hESC-CM proliferation, may point the way forward for new classes of compounds better suited to cell implantation.

Treatment of human embryos with the TGF inhibitor SB431542 increases epiblast proliferation and permits successful human embryonic stem cell derivation

Is there an effect of the TGFβ inhibitor SB431542 (SB) on the epiblast compartment of human blastocysts, and does it affect subsequent human embryonic stem cell (hESC) derivation? SB increases the mean number of NANOG-positive cells in the inner cell mass (ICM), and allows for subsequent hESC derivation. It is known that inhibition of TGFβ by SB has a positive effect on mouse ESC self-renewal, while active TGFβ signalling is needed for self-renewal of primed ESC. From December 2011 until March 2012, 263 donated spare embryos were used from patients who had undergone IVF/ICSI in our centre. Donated human embryos were cultured in the presence of SB or Activin A, and immunocytochemistry was performed on Day 6 blastocysts for NANOG and GATA6. Moreover, blastocysts were used for the derivation of hESC, with or without exposure to SB. Immunocytochemistry revealed a significantly higher number of NANOG-positive ICM cells in the SB group compared with the control (12.0 ± 5.9 versus 6.1 ± 4.7), while no difference was observed in the Activin A group compared with other groups (6.7 ± 3.7). The number of GATA6-positive ICM cells did not differ between the SB, Activin A and control group (8.8 ± 4.3, 8.0 ± 4.6 and 7.2 ± 4.0, respectively). Blocking TGFβ signalling did not prevent subsequent hESC line derivation. The number of human blastocysts available for this study was too low to reveal if the observed increase in NANOG-positive epiblast cells after exposure to SB affected the efficiency of hESC derivation (12.5% compared with 16.7%). This work can contribute to the derivation of naive hESC lines in the future. M.V.d.J. is holder of a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT, grant number SB093128), Belgium. G.D. and this research are supported by the Research Foundation Flanders (FWO), grant number FWO-3G062910) and a Concerted Research Actions funding from BOF (Bijzonder Onderzoeksfonds University Ghent, grant number BOF GOA 01G01112). S.M.C.d. S.L. is supported by the Netherlands Organization of Scientific Research (NWO) (ASPASIA 015.007.037) and the Interuniversity Attraction Poles (PAI) (no. P7/07). P.D.S. is holder of a fundamental clinical research mandate by the FWO. We would like to thank Ferring Company (Aalst, Belgium) for financial support of this study. The authors do not have any competing interests to declare. Not applicable.