Mesodermal Commitment Research Articles

Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells.

BackgroundDuchenne muscular dystrophy (DMD) is a devastating X-linked recessive genetic myopathy. DMD physiopathology is still not fully understood and a prenatal onset is suspected but difficult to address.MethodsThe bone morphogenetic protein 4 (BMP4) is a critical signaling molecule involved in mesoderm commitment. Human induced pluripotent stem cells (hiPSCs) from DMD and healthy individuals and human embryonic stem cells (hESCs) treated with BMP4 allowed us to model the early steps of myogenesis in normal and DMD contexts.ResultsUnexpectedly, 72h following BMP4 treatment, a new long DMD transcript was detected in all tested hiPSCs and hESCs, at levels similar to that found in adult skeletal muscle. This novel transcript named “Dp412e” has a specific untranslated first exon which is conserved only in a sub-group of anthropoids including human. The corresponding novel dystrophin protein of 412-kiloDalton (kDa), characterized by an N-terminal-truncated actin-binding domain, was detected in normal BMP4-treated hiPSCs/hESCs and in embryoid bodies. Finally, using a phosphorodiamidate morpholino oligomer (PMO) targeting the DMD exon 53, we demonstrated the feasibility of exon skipping validation with this BMP4-inducible hiPSCs model.ConclusionsIn this study, the use of hiPSCs to analyze early phases of human development in normal and DMD contexts has led to the discovery of an embryonic 412 kDa dystrophin isoform. Deciphering the regulation process(es) and the function(s) associated to this new isoform can contribute to a better understanding of the DMD physiopathology and potential developmental defects. Moreover, the simple and robust BMP4-inducible model highlighted here, providing large amount of a long DMD transcript and the corresponding protein in only 3 days, is already well-adapted to high-throughput and high-content screening approaches. Therefore, availability of this powerful cell platform can accelerate the development, validation and improvement of DMD genetic therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0062-6) contains supplementary material, which is available to authorized users.

Arsenic trioxide alters the differentiation of mouse embryonic stem cell into cardiomyocytes.

Chronic arsenic exposure is associated with increased morbidity and mortality for cardiovascular diseases. Arsenic increases myocardial infarction mortality in young adulthood, suggesting that exposure during foetal life correlates with cardiac alterations emerging later. Here, we investigated the mechanisms of arsenic trioxide (ATO) cardiomyocytes disruption during their differentiation from mouse embryonic stem cells. Throughout 15 days of differentiation in the presence of ATO (0.1, 0.5, 1.0 μM) we analysed: the expression of i) marker genes of mesoderm (day 4), myofibrillogenic commitment (day 7) and post-natal-like cardiomyocytes (day 15); ii) sarcomeric proteins and their organisation; iii) Connexin 43 and iv) the kinematics contractile properties of syncytia. The higher the dose used, the earlier the stage of differentiation affected (mesoderm commitment, 1.0 μM). At 0.5 or 1.0 μM the expression of cardiomyocyte marker genes is altered. Even at 0.1 μM, ATO leads to reduction and skewed ratio of sarcomeric proteins and to a rarefied distribution of Connexin 43 cardiac junctions. These alterations contribute to the dysruption of the sarcomere and syncytium organisation and to the impairment of kinematic parameters of cardiomyocyte function. This study contributes insights into the mechanistic comprehension of cardiac diseases caused by in utero arsenic exposure.

Identification of novel long noncoding RNAs underlying vertebrate cardiovascular development.

Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here, we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development. By relying on human pluripotent stem cell differentiation models, we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells. RNA sequencing led to the generation of large data sets that serve as a gene expression roadmap highlighting gene expression changes during human pluripotent cell differentiation. Stage-specific analyses led to the identification of 3 previously uncharacterized lncRNAs, TERMINATOR, ALIEN, and PUNISHER, specifically expressed in undifferentiated pluripotent stem cells, cardiovascular progenitors, and differentiated endothelial cells, respectively. Functional characterization, including localization studies, dynamic expression analyses, epigenetic modification monitoring, and knockdown experiments in lower vertebrates, as well as murine embryos and human cells, confirmed a critical role for each lncRNA specific for each analyzed developmental stage. We have identified and functionally characterized 3 novel lncRNAs involved in vertebrate and human cardiovascular development, and we provide a comprehensive transcriptomic roadmap that sheds new light on the molecular mechanisms underlying human embryonic development, mesodermal commitment, and cardiovascular specification.

Wnt signaling and tbx16 form a bistable switch to commit bipotential progenitors to mesoderm.

Anterior to posterior growth of the vertebrate body is fueled by a posteriorly located population of bipotential neuro-mesodermal progenitor cells. These progenitors have a limited rate of proliferation and their maintenance is crucial for completion of the anterior-posterior axis. How they leave the progenitor state and commit to differentiation is largely unknown, in part because widespread modulation of factors essential for this process causes organism-wide effects. Using a novel assay, we show that zebrafish Tbx16 (Spadetail) is capable of advancing mesodermal differentiation cell-autonomously. Tbx16 locks cells into the mesodermal state by not only activating downstream mesodermal genes, but also by repressing bipotential progenitor genes, in part through a direct repression of sox2. We demonstrate that tbx16 is activated as cells move from an intermediate Wnt environment to a high Wnt environment, and show that Wnt signaling activates the tbx16 promoter. Importantly, high-level Wnt signaling is able to accelerate mesodermal differentiation cell-autonomously, just as we observe with Tbx16. Finally, because our assay for mesodermal commitment is quantitative we are able to show that the acceleration of mesodermal differentiation is surprisingly incomplete, implicating a potential separation of cell movement and differentiation during this process. Together, our data suggest a model in which high levels of Wnt signaling induce a transition to mesoderm by directly activating tbx16, which in turn acts to irreversibly flip a bistable switch, leading to maintenance of the mesodermal fate and repression of the bipotential progenitor state, even as cells leave the initial high-Wnt environment.

MiRNA-1 and miRNA-133a are involved in early commitment of pluripotent stem cells and demonstrate antagonistic roles in the regulation of cardiac differentiation.

miRNA-1 (miR-1) and miRNA-133a (miR-133a) are muscle-specific miRNAs that play an important role in heart development and physiopathology. Although both miRNAs have been broadly studied during cardiogenesis, the mechanisms by which miR-1 and miR-133a could influence linage commitment in pluripotent stem cells remain poorly characterized. In this study we analysed the regulation of miR-1 and miR-133a expression during pluripotent stem cell differentiation [P19.CL6 cells; embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] and investigated their role in DMSO and embryoid body (EB)-mediated mesodermal and cardiac differentiation by gain- and loss-of-function studies, as well as in vivo, by the induction of teratomas. Gene expression analysis revealed that miR-1 and miR-133a are upregulated during cardiac differentiation of P19.CL6 cells, and also during ESC and iPSC EB differentiation. Forced overexpression of both miRNAs promoted mesodermal commitment and a concomitant decrease in the expression of neural differentiation markers. Moreover, overexpression of miR-1 enhanced the cardiac differentiation of P19.CL6, while miR-133a reduced it with respect to control cells. Teratoma formation experiments with P19.CL6 cells confirmed the influence of miR-1 and miR-133a during in vivo differentiation. Finally, inhibition of both miRNAs during P19.CL6 cardiac differentiation had opposite results to their overexpression. In conclusion, gene regulation involving miR-1 and miR-133a controls the mesodermal and cardiac fate of pluripotent stem cells. Copyright © 2014 John Wiley & Sons, Ltd.

Histone deacetylase 1 and 3 regulate the mesodermal lineage commitment of mouse embryonic stem cells.

The important role of histone acetylation alteration has become increasingly recognized in mesodermal lineage differentiation and development. However, the contribution of individual histone deacetylases (HDACs) to mesoderm specification remains poorly understood. In this report, we found that trichostatin A (TSA), an inhibitor of histone deacetylase (HDACi), could induce early differentiation of embryonic stem cells (ESCs) and promote mesodermal lineage differentiation. Further analysis showed that the expression levels of HDAC1 and 3 are decreased gradually during ESCs differentiation. Ectopic expression of HDAC1 or 3 significantly inhibited differentiation into the mesodermal lineage. By contrast, loss of either HDAC1 or 3 enhanced the mesodermal differentiation of ESCs. Additionally, we demonstrated that the activity of HDAC1 and 3 is indeed required for the regulation of mesoderm gene expression. Furthermore, HDAC1 and 3 were found to interact physically with the T-box transcription factor T/Bry, which is critical for mesodermal lineage commitment. These findings indicate a key mechanism for the specific role of HDAC1 and 3 in mammalian mesoderm specification.

Microscale technologies for regulating human stem cell differentiation.

During development and regeneration, tissues emerge from coordinated sequences of stem cell renewal, specialization, and assembly that are orchestrated by cascades of regulatory factors. This complex in vivo milieu, while necessary to fully recapitulate biology and to properly engineer progenitor cells, is difficult to replicate in vitro. We are just starting to fully realize the importance of the entire context of cell microenvironment-the other cells, three-dimensional matrix, molecular and physical signals. Bioengineered environments that combine tissue-specific transport and signaling are critical to study cellular responses at biologically relevant scales and in settings predictive of human condition. We therefore developed microbioreactors that couple the application of fast dynamic changes in environmental signals with versatile, high-throughput operation and imaging capability. Our base device is a microfluidic platform with an array of microwells containing cells or tissue constructs that are exposed to stable concentration gradients. Mathematical modeling of flow and mass transport can predict the shape of these gradients and the kinetic changes in local concentrations. A single platform, the size of a microscope slide, contains up to 120 biological samples. As an example of application, we describe studies of cell fate specification and mesodermal lineage commitment in human embryonic stem cells and induced pluripotent stem cells. The embryoid bodies formed from these cells were subjected to single and multiple concentration gradients of Wnt3a, Activin A, bone morphogenic protein 4 (BMP4), and their inhibitors, and the gene expression profiles were correlated to the concentration gradients of morphogens to identify the exact conditions for mesodermal differentiation.

BMP4 Promotes EMT and Mesodermal Commitment in Human Embryonic Stem Cells via SLUG and MSX2

Bone morphogenetic proteins (BMPs) initiate differentiation in human embryonic stem cells (hESCs) but the exact mechanisms have not been fully elucidated. We demonstrate here that SLUG and MSX2, transcription factors involved in epithelial-mesenchymal transitions, essential features of gastrulation in development and tumor progression, are important mediators of BMP4-induced differentiation in hESCs. Phosphorylated Smad1/5/8 colocalized with the SLUG protein at the edges of hESC colonies where differentiation takes place. The upregulation of the BMP target SLUG was direct as shown by the binding of phosphorylated Smad1/5/8 to its promoter, which interrupted the formation of adhesion proteins, resulting in migration. Knockdown of SLUG by short hairpin RNA blocked these changes, confirming an important role for SLUG in BMP-mediated mesodermal differentiation. Furthermore, BMP4-induced MSX2 expression leads to mesoderm formation and then preferential differentiation toward the cardiovascular lineage.

Snail1-dependent control of embryonic stem cell pluripotency and lineage commitment

Embryonic stem cells (ESCs) exhibit the dual properties of self-renewal and pluripotency as well as the ability to undergo differentiation that gives rise to all three germ layers. Wnt family members can both promote ESC maintenance and trigger differentiation while also controlling the expression of Snail1, a zinc-finger transcriptional repressor. Snail1 has been linked to events ranging from cell cycle regulation and cell survival to epithelial-mesenchymal transition (EMT) and gastrulation, but its role in self-renewal, pluripotency or lineage commitment in ESCs remains undefined. Here we demonstrate using isogenic pairs of conditional knockout mouse ESCs, that Snail1 exerts Wnt- and EMT independent control over the stem cell transcriptome without affecting self-renewal or pluripotency-associated functions. By contrast, during ESC differentiation, an endogenous Wnt-mediated burst in Snail1 expression regulates neuroectodermal fate while playing a required role in epiblast stem cell exit and the consequent lineage fate decisions that define mesoderm commitment.

Retinoic Acid Is a Negative Regulator In Hematopoietic Development and Definitive Hematopoietic Progenitor Maintenance

Retinoic acid (RA) is a key morphogen involved in the establishment of various cell-fates in the developing embryo. The establishment of certain tissues, including mesodermal derivates such as the somites and heart depends on restricted exposure to RA during embryonic development, however, in regard to the possible involvement of RA for the emergence of blood in the human embryo very little is known. By using an inhibitor of cellular RA synthesis (DEAB) together with our human iPS-to-blood differentiation system we report enhanced generation of human hematopoietic progenitors cells (CD43/45+,CD34+), and early progenitor cells with an adult hematopoietic stem cell phenotype (CD43/45+,CD34+,CD38,CD90+,CD45RA-) possessing myeloid and lymphoid differentiation capacity. RA inhibition increased the output of these early progenitor cells by 2.7-fold (p-value: 0,006, n=6) compared to DMSO control, and increased the number of clonogenic progenitors (CFUs) by 2-fold (p-value: 0,022, n=6). This improvement is consistent when using both a human ES line (Hues3) and a human iPS-line (RB9-CB1, generated from cord blood). RA inhibition also increased the number of iPS-derived blood progenitors capable of differentiating into the lymphoid lineage, generating phenotypic T-lineage and natural killer cells after sub-culture on OP9-DL1 stroma. Conversely, and in support of our findings, directly adding RA was found to severely decrease the blood generation efficiency, with the early progenitor fraction significantly decreased even at low levels of RA exposure. In order to assess how the RA inhibition led to the increase in early progenitors, whether by directing pluripotent stem cell differentiation of mesodermal precursors towards blood, or by inferring an early progenitor cell maintenance and/or expansion, we performed the following analyses. Firstly, the loss of clonogenic progenitors was reduced 1.6 fold with extended culture in the presence of the inhibitor. Secondly, within the iPS derived blood (CD43/45+) fraction of the cultures, RA inhibition increased the frequency of the CD34+,CD38-,CD90+,CD45RA- compartment by 2.1-fold (p-value: 0,021, n=6). Similarly when comparing the CFU-forming capacity from equal numbers of sorted hematopoietic progenitors (CD43/45+,CD34+), cells generated with the inhibitor demonstrated 2-fold higher CFU frequency suggesting increased preservation of CFU-forming cells within the CD34+ progenitor fraction. Together this suggests that progenitor maintenance is improved with the inhibitor, however, the contribution to the total fold expansion seen with RA inhibition cannot be attributed solely to the increased maintenance of early progenitor cells in the culture. Using multi-timepoint RT-qPCR of samples obtained during the differentiation protocol, we analysed expression of key developmental genes and saw improved commitment of the differentiating culture towards mesoderm (BRACHYURY), decreased amounts of anterior lateral plate/cardiogenic mesoderm (NKX2.5), and enhanced expression of genes known to be relevant for definitive hematopoiesis and hemogenic endothelium (RUNX1, FLK1, APLNR, PDGFRa). In addition, preliminary data show that RA inhibition increases the frequency of the CD34hi,CD90hi,CD43neg population observed at day 8, recently described as the precursor population of definitive hematopoiesis, indicating that RA inhibition confers an early positive effect on the developmental commitment towards the hematopoietic mesoderm. Together, our data indicate that modulation of retinoic acid signaling improves blood generation from human pluripotent stem cell lines both by improving the development towards the hematopoietic mesoderm by favoring mesodermal blood precursor commitment, and by preserving the immature progenitor population from differentiation. Therefore, we propose that RA is a negative regulator of mesodermal formation and the specification towards hematopoietic precursors, and that it promotes the exhaustion of definitive hematopoietic progenitors. This suggests the possibility that blood evolutionarily formed in a region of the mesoderm where RA exposure from neighboring tissues, such as RA producing ectoderm, was restricted. Therefore, using small molecules to inhibit RA signaling is a novel approach to improve the generation of human blood in vitro. Disclosures:Rönn:Primorigen Biosciences: Co-inventor on pending patent. No financial ties., Co-inventor on pending patent. No financial ties. Patents & Royalties. Guibentif:Primorigen Biosciences: Co-inventor on pending patent. No financial ties., Co-inventor on pending patent. No financial ties. Patents & Royalties. Garcia:Primorigen Biosciences: Employment. Woods:Primorigen Biosciences: Co-inventor on pending patent., Co-inventor on pending patent. Patents & Royalties, Membership on an entity's Board of Directors or advisory committees.

Post-transcriptional mechanisms contribute to Etv2 repression during vascular development

etv2 is an endothelial-specific ETS transcription factor that is essential for vascular differentiation and morphogenesis in vertebrates. While recent data suggest that Etv2 is dynamically regulated during vascular development, little is known about the mechanisms involved in this process. Here, we find that etv2 transcript and protein expression are highly dynamic during zebrafish vascular development, with both apparent during early somitogenesis and subsequently down-regulated as development proceeds. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis stages, but not later, caused severe vascular defects, suggesting a specific role in early commitment of lateral mesoderm to the endothelial linage. Accordingly, Etv2-overexpressing cells showed an enhanced ability to commit to endothelial lineages in mosaic embryos. We further find that the etv2 3' untranslated region (UTR) is capable of repressing an endothelial autonomous transgene and contains binding sites for members of the let-7 family of microRNAs. Ectopic expression of let-7a could repress the etv2 3'UTR in sensor assays and was also able to block endogenous Etv2 protein expression, leading to concomitant reduction of endothelial genes. Finally, we observed that Etv2 protein levels persisted in maternal-zygotic dicer1 mutant embryos, suggesting that microRNAs contribute to its repression during vascular development. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is then down-regulated, in part through post-transcriptional repression by microRNAs, to allow normal vascular development.

Jmjd3 Controls Mesodermal and Cardiovascular Differentiation of Embryonic Stem Cells

The developmental role of the H3K27 demethylases Jmjd3, especially its epigenetic regulation at target genes in response to upstream developmental signaling, is unclear. To determine the role of Jmjd3 during mesoderm and cardiovascular lineage commitment. Ablation of Jmjd3 in mouse embryonic stem cells does not affect the maintenance of pluripotency and self-renewal but compromised mesoderm and subsequent endothelial and cardiac differentiation. Jmjd3 reduces H3K27me3 marks at the Brachyury promoter and facilitates the recruitment of β-catenin, which is critical for Wnt signal-induced mesoderm differentiation. These data demonstrate that Jmjd3 is required for mesoderm differentiation and cardiovascular lineage commitment.

Profiling of transcriptional and epigenetic changes during directed endothelial differentiation of human embryonic stem cells identifies FOXA2 as a marker of early mesoderm commitment

IntroductionDifferentiation of vascular endothelial cells (ECs) in clinically relevant numbers for injection into ischaemic areas could offer therapeutic potential in the treatment of cardiovascular conditions, including myocardial infarction, peripheral vascular disease and stroke. While we and others have demonstrated successful generation of functional endothelial-like cells from human embryonic stem cells (hESCs), little is understood regarding the complex transcriptional and epigenetic changes that occur during differentiation, in particular during early commitment to a mesodermal lineage.MethodsWe performed the first gene expression microarray study of hESCs undergoing directed differentiation to ECs using a monolayer-based, feeder-free and serum-free protocol. Microarray results were confirmed by quantitative RT-PCR and immunocytochemistry, and chromatin immunoprecipitation (ChIP)-PCR analysis was utilised to determine the bivalent status of differentially expressed genes.ResultsWe identified 22 transcription factors specific to early mesoderm commitment. Among these factors, FOXA2 was observed to be the most significantly differentially expressed at the hESC–EC day 2 timepoint. ChIP-PCR analysis revealed that the FOXA2 transcription start site is bivalently marked with histone modifications for both gene activation (H3K4me3) and repression (H3K27me3) in hESCs, suggesting the transcription factor may be a key regulator of hESC differentiation.ConclusionThis enhanced knowledge of the lineage commitment process will help improve the design of directed differentiation protocols, increasing the yield of endothelial-like cells for regenerative medicine therapies in cardiovascular disease.

Essential and Unexpected Role of Yin Yang 1 to Promote Mesodermal Cardiac Differentiation

Cardiogenesis is regulated by a complex interplay between transcription factors. However, little is known about how these interactions regulate the transition from mesodermal precursors to cardiac progenitor cells (CPCs). To identify novel regulators of mesodermal cardiac lineage commitment. We performed a bioinformatic-based transcription factor binding site analysis on upstream promoter regions of genes that are enriched in embryonic stem cell-derived CPCs. From 32 candidate transcription factors screened, we found that Yin Yang 1 (YY1), a repressor of sarcomeric gene expression, is present in CPCs in vivo. Interestingly, we uncovered the ability of YY1 to transcriptionally activate Nkx2.5, a key marker of early cardiogenic commitment. YY1 regulates Nkx2.5 expression via a 2.1-kb cardiac-specific enhancer as demonstrated by in vitro luciferase-based assays, in vivo chromatin immunoprecipitation, and genome-wide sequencing analysis. Furthermore, the ability of YY1 to activate Nkx2.5 expression depends on its cooperative interaction with Gata4 at a nearby chromatin. Cardiac mesoderm-specific loss-of-function of YY1 resulted in early embryonic lethality. This was corroborated in vitro by embryonic stem cell-based assays in which we showed that the overexpression of YY1 enhanced the cardiogenic differentiation of embryonic stem cells into CPCs. These results demonstrate an essential and unexpected role for YY1 to promote cardiogenesis as a transcriptional activator of Nkx2.5 and other CPC-enriched genes.

The L-type Ca2+ channels blocker nifedipine represses mesodermal fate determination in murine embryonic stem cells.

Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca2+ channels of different cell types. Moreover, influx of Ca2+ through L-type Ca2+ channels (LTCCs) activates Ca2+ signaling pathways, which in turn contribute to numerous cellular processes. Although LTCCs are expressed in undifferentiated cells, very little is known about its contributions to the transcriptional regulation of mesodermal and cardiac genes. This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage in vitro. The murine embryonic stem (ES, cell line D3) and induced pluripotent stem (iPS, cell clone 09) cells were differentiated into enhanced green fluorescence protein (EGFP) expressing spontaneously beating cardiomyocytes (CMs). Early treatment of differentiating cells with 10 µM nifedipine led to a significant inhibition of the cardiac mesoderm formation and cardiac lineage commitment as revealed by gene regulation analysis. This was accompanied by the inhibition of spontaneously occurring Ca2+ transient and reduction of LTCCs current density (I CaL) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca2+ channel α1C (CaV1.2) subunits. Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function. Our data indicate that nifedipine blocks the determined path of pluripotent stem cells to cardiomyogenesis by inhibition of mesodermal commitment at early stages of differentiation, thus the proper upkeep Ca2+ concentration and pathways are essentially required for cardiac gene expression, differentiation and function.

Microfluidic bioreactor for dynamic regulation of early mesodermal commitment in human pluripotent stem cells

During development and regeneration, tissues emerge from coordinated sequences of stem cell renewal, specialization and assembly that are orchestrated by cascades of regulatory signals. The complex and dynamic in vivo milieu cannot be replicated using standard in vitro techniques. Microscale technologies now offer potential for conducting highly controllable and sophisticated experiments at biologically relevant scales, with real-time insights into cellular responses. We developed a microbioreactor providing time sequences of space-resolved gradients of multiple molecular factors in three-dimensional (3D) cell culture settings, along with a versatile, high-throughput operation and imaging compatibility. A single microbioreactor yields up to 120 data points, corresponding to 15 replicates of a gradient with 8 concentration levels. Embryoid bodies (EBs) obtained from human embryonic and induced pluripotent stem cells (hESC, hiPSC) were exposed to concentration gradients of Wnt3a, Activin A, BMP4 and their inhibitors, to get new insights into the early-stage fate specification and mesodermal lineage commitment. We were able to evaluate the initiation of mesodermal induction by measuring and correlating the gene expression profiles to the concentration gradients of mesoderm-inducing morphogens. We propose that the microbioreactor systems combining spatial and temporal gradients of molecular and physical factors to hESC and hiPSC cultures can form a basis for predictable in vitro models of development and disease.

RUNX1a Enhances Hematopoietic Lineage Commitment From Human Embryonic Stem Cells and Inducible Pluripotent Stem Cells

Abstract 347Both human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are pluripotent stem cells (hPSCs) with potential to differentiate into all types of somatic cells. Patients suffering from blood disorders can be cured with hematopoietic cell transplantations (HCT). Technical advancements in hPSC production and handling have revolutionized their potential applications in regenerative medicine and provided enormous hope for patients who may need HCT. hiPSCs derived from autologous cells could provide unlimited leukocyte antigen matched blood cells on a patient-specific basis. A remaining hurdle in this process remains the need for efficient and effective generation of specific blood cells from hPSCs for therapeutic use. Transcription factors play key roles in regulating maintenance, expansion, and differentiation of blood cells from hPSCs. Studies have shown that transcription factor RUNX1 is required for the formation of definitive blood cells. There are several alternatively spliced isoforms of the RUNX1 protein, including the shortest form RUNX1a and two longer forms RUNX1b and RUNX1c. Based on known properties of RUNX1 proteins, we hypothesized that RUNX1a promotes the production of therapeutic hematopoietic stem cells from hPSCs. By employing ectopic expression of RUNX1a on different human ESC and iPSC lines (H9, BC1, iCB5) under a defined hematopoietic differentiation system, we aimed to identify function of RUNX1a on lineage commitment and molecular mechanisms of RUNX1 activity in differentiation of PSCs to hematopoietic cells. We demonstrated that expression of endogenous RUNX1a parallels lineage commitment and hematopoietic emergence from hPSCs. During differentiation process RUNX1a enhanced the expression of several mesoderm and hematopoietic differentiation related factors, including KDR, SCL, GATA2, and PU.1. In addition, over-expression of RUNX1a in embryoid bodies (EBs) showed more efficient and earlier emergence of typical sac structures, which predicts cell lineage commitment and germ layer development at the early stage of EB differentiation. At day 7, EBs derived from hPSCs was dissociated into single cells for flow cytometry analysis. The mean frequency of CD31+CD34+CD45− and total CD34+ cells with hemato-endothelial cell features are 35.1% and 67.1% from RUNX1a-overexpressing EBs, and 8.7% and 24.1% from vector control EBs. Immunohistochemistry analysis of EBs at day 9 of differentiation confirmed that expression of RUNX1a accelerated mesoderm commitment and emergence of hemato-endothelial precursors. Flow cytometry analysis on EBs collected at days 9, 11, 13 showed that ectopic RUNX1a induced a robust increase in the frequency of hematopoietic progenitor cells in all hPSC lines examined. At Day 9, RUNX1a-overexpressing EBs generated 48.5% CD43+CD45+ cells, 45.1% CD34+CD45+ cells, and 8.5 folds higher CD43+ cells than vector EBs. Later at Day 13, 80% CD45+ and 75% CD43+/CD34+CD45+ hematopoietic stem/progenitor cells (HSPCs) achieved from dissociated EBs. In liquid culture, RUNX1a HSPC showed strong expansion and high percentage of CD235a+CD45− (20%) and CD71+CD235a+ (16%), markers for erythroid populations. Flow cytometry and western blots on RUNX1a-EB formed colonies showed significantly higher β-globin production than that of the vector, suggesting expression of RUNX1a in HSPC enhanced definitive hematopoiesis. RUNX1a-hPSCs derived HSPCs possess self-renewal capability and are capable of differentiating into multi-lineages ex vivo. Furthermore HSPCs generated from RUNX1a-EBs possessed the capacity of interacting with surrogate niche and showed long-term repopulation ability under LTC-IC (Long-Term Culture-Initiating Cell Assay) condition. Colonies generated from HSPC of RUNX1a-EBs after 3 week bulk LTC-IC culture showed 300 folds higher than vector control. RUNX1a-hPSCs derived CD34+CD45+ cells could maintain a non-adherent population in ouldCD45+ sEBsND THIS SENTENCE5 week culture on stromal cell M210. In summary we identified that RUNX1a enhances derivation of definitive hematopoietic cells from human PSCs. Our study provides an important and useful system to enhance specificity and efficiency of generating functional blood cells and further differentiated cells from human PSCs, which may provide valuable source for future clinical applications in patients with hematologic disorders. Disclosures:No relevant conflicts of interest to declare.

Polymeric Fibrous Matrices for Substrate-Mediated Human Embryonic Stem Cell Lineage Differentiation

Chitosan-based fibrous matrices are prepared to mimic the ECM architecture and elucidate substrate-mediated hESC differentiation due to topographical scale and anisotropy without exogenic morphogens. Fibrous matrices support fewer pluripotent hESCs than films but enable topography-mediated hESC differentiation. Matrices composed of 400 nm and 1.1 µm diameter fibers support increased expression of neural markers indicative of ectodermal commitment while matrices of 200 nm diameter fibers increase expression of osteogenic and hepatic markers indicative of endodermal and mesodermal commitment. The fibrous-mediated hESC differentiation highlights the significant implication of tailored ECM-like substrates for hESC-based therapies.

Heart Cells with Regenerative Potential from Pediatric Patients with End Stage Heart Failure: A Translatable Method to Enrich and Propagate

Background. Human cardiac-derived progenitor cells (hCPCs) have shown promise in treating heart failure (HF) in adults. The purpose of this study was to describe derivation of hCPCs from pediatric patients with end-stage HF. Methods. At surgery, discarded right atrial tissues (hAA) were obtained from HF patients (n = 25; hAA-CHF). Minced tissues were suspended in complete (serum-containing) DMEM. Cells were selected for their tissue migration and expression of stem cell factor receptor (hc-kit). Characterization of hc-kitpositive cells included immunohistochemical screening with a panel of monoclonal antibodies. Results. Cells, including phase-bright cells identified as hc-kitpositive, spontaneously emigrated from hAA-CHF in suspended explant cultures (SEC) after Day 7. When cocultured with tissue, emigrated hc-kitpositive cells proliferated, first as loosely attached clones and later as multicellular clusters. At Day 21~5% of cells were hc-kitpositive. Between Days 14 and 28 hc-kitpositive cells exhibited mesodermal commitment (GATA-4positive and NKX2.5positive); then after Day 28 cardiac lineages (flk-1positive, smooth muscle actinpositive, troponin-Ipositive, and myosin light chainpositive). Conclusions. C-kitpositive hCPCs can be derived from atrial tissue of pediatric patients with end-stage HF. SEC is a novel culture method for derivation of migratory hc-kitpositive cells that favors clinical translation by reducing the need for exogenously added factors to expand hCPCs in vitro.

Smad1 signaling restricts hematopoietic potential after promoting hemangioblast commitment

AbstractBone morphogenetic protein (BMP) signaling regulates embryonic hematopoiesis via receptor-mediated activation of downstream SMAD proteins, including SMAD1. In previous work, we showed that Smad1 expression is sufficient to enhance commitment of mesoderm to hemangioblast fate. We also found indirect evidence to support a subsequent repressive function for Smad1 in hematopoiesis. To test this hypothesis directly, we developed a novel system allowing temporal control of Smad1 levels by conditional knockdown in embryonic stem cell derivatives. Depletion of Smad1 in embryoid body cultures before hemangioblast commitment limits hematopoietic potential because of a block in mesoderm development. Conversely, when Smad1 is depleted in FlK1+ mesoderm, at a stage after hemangioblast commitment, the pool of hematopoietic progenitors is expanded. This involves enhanced expression levels for genes specific to hematopoiesis, including Gata1, Runx1 and Eklf, rather than factors required for earlier specification of the hemangioblast. The phenotype correlates with increased nuclear SMAD2 activity, indicating molecular cross-regulation between the BMP and TGF-β signaling pathways. Consistent with this mechanism, hematopoiesis was enhanced when Smad2 was directly expressed during this same developmental window. Therefore, this study reveals a temporally defined function for Smad1 in restricting the expansion of early hematopoietic progenitors.