H9 Human Embryonic Stem Cells Research Articles

HAND factors regulate cardiac lineage commitment and differentiation from human pluripotent stem cells

BackgroundTranscription factors HAND1 and HAND2 (HAND1/2) play significant roles in cardiac organogenesis. Abnormal expression and deficiency of HAND1/2 result in severe cardiac defects. However, the function and mechanism of HAND1/2 in regulating human early cardiac lineage commitment and differentiation are still unclear.MethodsWith NKX2.5eGFP H9 human embryonic stem cells (hESCs), we established single and double knockout cell lines for HAND1 and HAND2, respectively, whose cardiomyocyte differentiation efficiency could be monitored by assessing NKX2.5-eGFP+ cells with flow cytometry. The expression of specific markers for heart fields and cardiomyocyte subtypes was examined by quantitative PCR, western blot and immunofluorescence staining. Microelectrode array and whole-cell patch clamp were performed to determine the electrophysiological characteristics of differentiated cardiomyocytes. The transcriptomic changes of HAND knockout cells were revealed by RNA sequencing. The HAND1/2 target genes were identified and validated experimentally by integrating with HAND1/2 chromatin immunoprecipitation sequencing data.ResultsEither HAND1 or HAND2 knockout did not affect the cardiomyocyte differentiation kinetics, whereas depletion of HAND1/2 resulted in delayed differentiation onset. HAND1 knockout biased cardiac mesoderm toward second heart field progenitors at the expense of first heart field progenitors, leading to increased expression of atrial and outflow tract cardiomyocyte markers, which was further confirmed by the appearance of atrial-like action potentials. By contrast, HAND2 knockout cardiomyocytes had reduced expression of atrial cardiomyocyte markers and displayed ventricular-like action potentials. HAND1/2-deficient hESCs were more inclined to second heart field lineage and its derived cardiomyocytes with atrial-like action potentials than HAND1 single knockout during differentiation. Further mechanistic investigations suggested TBX5 as one of the downstream targets of HAND1/2, whose overexpression partially restored the abnormal cardiomyocyte differentiation in HAND1/2-deficient hESCs.ConclusionsHAND1/2 have specific and redundant roles in cardiac lineage commitment and differentiation. These findings not only reveal the essential function of HAND1/2 in cardiac organogenesis, but also provide important information on the pathogenesis of HAND1/2 deficiency-related congenital heart diseases, which could potentially lead to new therapeutic strategies.

Generation of a gene-edited H9 embryonic stem cell line carrying a DOX-inducible NGN2 expression cassette in the CLYBL locus

The pro-neural transcription factor neurogenin-2 (NGN2) possesses the ability to rapidly and effectively transform stem cells into fully operational neurons. Here we report the successful generation of a modified H9 human embryonic H9 stem cell line containing a doxycycline (DOX) inducible NGN2 expression construct featuring a floxed Blasticidin/mApple selection module in the safe-harbor locus CLYBL. This cell line retains its pluripotent state in the absence of DOX, yet readily transitions into a neuronal state upon DOX introduction.

COL1A1 regulates the apoptosis of embryonic stem cells by mediating the PITX1/TBX4 signaling.

The purpose of this study is to explore the regulatory function of COL1A1 against the apoptosis of embryonic stem cells (ESCs) and the potential function in congenital talipes equinovarus (CTEV). Muscle tissues were collected from 20 children with CTEV and 20 children without CTEV, followed by detecting the expression of COL1A1 using the RT-PCR method. COL1A1 was knocked down in H1 and H9 human ESCs using the RNA interference technology, followed by determining the level of COL1A1, PITX1, TBX4, HOXD10, Fas, FasL, and Bax using the Western blotting assay. COL1A1 was found markedly upregulated in muscle tissues of CTEV children. In H1 and H9 human ESCs, compared to the empty vector, COL1A1, PITX1, TBX4, HOXD10, Fas, FasL, and Bax were found notably downregulated after transfected with the siRNA targeting COL1A1. COL1A1 induced the apoptosis of ESCs by mediating the PITX1/TBX4 signaling and might be a potential target for treating CTEV.

Profiling mitochondria-polyribosome lncRNAs associated with pluripotency

Pluripotent stem cells (PSCs) provide unlimited resources for regenerative medicine because of their potential for self-renewal and differentiation into many different cell types. The pluripotency of these PSCs is dynamically regulated at multiple cellular organelle levels. To delineate the factors that coordinate this inter-organelle crosstalk, we profiled those long non-coding RNAs (lncRNAs) that may participate in the regulation of multiple cellular organelles in PSCs. We have developed a unique strand-specific RNA-seq dataset of lncRNAs that may interact with mitochondria (mtlncRNAs) and polyribosomes (prlncRNAs). Among the lncRNAs differentially expressed between induced pluripotent stem cells (iPSCs), fibroblasts, and positive control H9 human embryonic stem cells, we identified 11 prlncRNAs related to stem cell reprogramming and exit from pluripotency. In conjunction with the total RNA-seq data, this dataset provides a valuable resource to examine the role of lncRNAs in pluripotency, particularly for studies investigating the inter-organelle crosstalk network involved in germ cell development and human reproduction.

Human embryonic stem cells secrete macrophage migration inhibitory factor: A novel finding.

Macrophage migration inhibitory factor (MIF) is expressed in a variety of cells and participates in important biological mechanisms. However, few studies have reported whether MIF is expressed in human Embryonic stem cells (ESCs) and its effect on human ESCs. Two human ESCs cell lines, H1 and H9 were used. The expression of MIF and its receptors CD74, CD44, CXCR2, CXCR4 and CXCR7 were detected by an immunofluorescence assay, RT-qPCR and western blotting, respectively. The autocrine level of MIF was measured via enzyme-linked immunosorbent assay. The interaction between MIF and its main receptor was investigated by co-immunoprecipitation and confocal immunofluorescence microscopy. Finally, the effect of MIF on the proliferation and survival of human ESCs was preliminarily explored by incubating cells with exogenous MIF, MIF competitive ligand CXCL12 and MIF classic inhibitor ISO-1. We reported that MIF was highly expressed in H1 and H9 human ESCs. MIF was positively expressed in the cytoplasm, cell membrane and culture medium. Several surprising results emerge. The autosecreted concentration of MIF was 22 ng/mL, which was significantly higher than 2 ng/mL-6 ng/mL in normal human serum, and this was independent of cell culture time and cell number. Human ESCs mainly expressed the MIF receptors CXCR2 and CXCR7 rather than the classical receptor CD74. The protein receptor that interacts with MIF on human embryonic stem cells is CXCR7, and no evidence of interaction with CXCR2 was found. We found no evidence that MIF supports the proliferation and survival of human embryonic stem cells. In conclusion, we first found that MIF was highly expressed in human ESCs and at the same time highly expressed in associated receptors, suggesting that MIF mainly acts in an autocrine form in human ESCs.

Temporal Profiling of Epitranscriptomic Modulators during Osteogenic Differentiation of Human Embryonic Stem Cells.

Osteogenesis is modulated by multiple regulatory networks. Recent studies showed that RNA modifications and their reader, writer, and eraser (RWE) proteins are involved in regulating various biological processes. Few studies, however, were conducted to investigate the functions of RNA modifications and their RWE proteins in osteogenesis. By using LC-MS/MS in parallel-reaction monitoring (PRM) mode, we performed a comprehensive quantitative assessment of 154 epitranscriptomic RWE proteins throughout the entire time course of osteogenic differentiation in H9 human embryonic stem cells (ESCs). We found that approximately half of the 127 detected RWE proteins were down-regulated during osteogenic differentiation, and they included mainly proteins involved in RNA methylation and pseudouridylation. Protein-protein interaction (PPI) network analysis unveiled significant associations between the down-regulated epitranscriptomic RWE proteins and osteogenesis-related proteins. Gene set enrichment analysis (GSEA) of publicly available RNA-seq data obtained from osteogenesis imperfecta patients suggested a potential role of METTL1 in osteogenesis through the cytokine network. Together, this is the first targeted profiling of epitranscriptomic RWE proteins during osteogenic differentiation of human ESCs, and our work unveiled potential regulatory roles of these proteins in osteogenesis. LC-MS/MS data were deposited on ProteomeXchange (PXD039249).

Targeted Proteomic Profiling Revealed Roles of Small GTPases during Osteogenic Differentiation.

The small GTPase superfamily of proteins are crucial for numerous cellular processes, including early development. The roles of these proteins in osteogenic differentiation, however, remained poorly explored. In this study, we employed a high-throughput targeted proteomic method, relying on scheduled liquid chromatography-multiple-reaction monitoring (LC-MRM) coupled with synthetic stable isotope-labeled peptides, to interrogate systematically the temporal responses of the entire small GTPase proteome during the course of osteogenic differentiation of H9 human embryonic stem cells. Our results demonstrated that the method offers high quantification accuracy, reproducibility, and throughput. In addition, the quantification results revealed altered expression of a large number of small GTPases accompanied with osteogenic differentiation, especially those involved with autophagy. We also documented a previously unrecognized role of KRAS in osteogenesis, where it regulates the accumulation of extracellular matrix for mineralization through attenuating the activity of secreted matrix metalloproteinase 9 (MMP9). Together, this study represents a novel application of a state-of-the-art analytical method, i.e., targeted quantitative proteomics, for revealing the progressive reprogramming of the small GTPase proteome during osteogenic differentiation of human embryonic stem cells, and our results revealed KRAS as a new regulator for osteogenesis.

DUX4 double whammy: The transcription factor that causes a rare muscular dystrophy also kills the precursors of the human nose.

SMCHD1 mutations cause congenital arhinia (absent nose) and a muscular dystrophy called FSHD2. In FSHD2, loss of SMCHD1 repressive activity causes expression of double homeobox 4 (DUX4) in muscle tissue, where it is toxic. Studies of arhinia patients suggest a primary defect in nasal placode cells (human nose progenitors). Here, we show that upon SMCHD1 ablation, DUX4 becomes derepressed in H9 human embryonic stem cells (hESCs) as they differentiate toward a placode cell fate, triggering cell death. Arhinia and FSHD2 patient-derived induced pluripotent stem cells (iPSCs) express DUX4 when converted to placode cells and demonstrate variable degrees of cell death, suggesting an environmental disease modifier. HSV-1 may be one such modifier as herpesvirus infection amplifies DUX4 expression in SMCHD1 KO hESC and patient iPSC. These studies suggest that arhinia, like FSHD2, is due to compromised SMCHD1 repressive activity in a cell-specific context and provide evidence for an environmental modifier.

The fidelity of transcription in human cells.

To determine the error rate of transcription in human cells, we analyzed the transcriptome of H1 human embryonic stem cells with a circle-sequencing approach that allows for high-fidelity sequencing of the transcriptome. These experiments identified approximately 100,000 errors distributed over every major RNA species in human cells. Our results indicate that different RNA species display different error rates, suggesting that human cells prioritize the fidelity of some RNAs over others. Cross-referencing the errors that we detected with various genetic and epigenetic features of the human genome revealed that the invivo error rate in human cells changes along the length of a transcript and is further modified by genetic context, repetitive elements, epigenetic markers, and the speed of transcription. Our experiments further suggest that BRCA1, a DNA repair protein implicated in breast cancer, has a previously unknown role in the suppression of transcription errors. Finally, we analyzed the distribution of transcription errors in multiple tissues of a new mouse model and found that they occur preferentially in neurons, compared to other cell types. These observations lend additional weight to the idea that transcription errors play a key role in the progression of various neurological disorders, including Alzheimer's disease.

Generation of a human embryonic stem cell line (SKLRMe004-A) carrying NKX2.5-EGFP and TBX5-Tdtomato dual fluorescent reporters

TBX5 and NKX2-5 are two important transcription factors regulating cardiomyocytes commitment. In this study, we generated a dual fluorescent reporter cell line based on H9 human embryonic stem cells (hESCs) and called it H9-NKX2.5GFP/TBX5Td-T2A-18, which could be short for T2A-18. When T2A-18 was induced towards a cardiomyocyte fate, EGFP and Tdtomato fluorescences were observed, indicating the expression of NKX2.5 and TBX5, respectively. Meanwhile, T2A had normal karyotype and could maintain main characteristics of wildtype H9. Therefore, T2A-18 could be used as a tool to help us study the mechanism of cardiomyocytes specification from hESCs.

MODL-25. ANALYSIS OF TUMOR CELL HETEROGENEITY IN A HUMAN NEURAL PROGENITOR-BASED MODEL OF GLIOBLASTOMA REVEALS NEURODEVELOPMENTAL PROGRAMS AND CYTOSKELETAL DYNAMICS

Abstract Glioblastoma (GBM) is the most common primary malignant neoplasm with poor survival despite treatment. Developing effective therapies remains challenging due to intratumoral heterogeneity, which drives therapeutic resistance and recurrence. To understand how genetic events alter the epigenome to enhance clonal fitness, we developed an isogenic human neural progenitor cell (NPC)-based model of the proneural (PRO) GBM subtype. We introduced TERT promoter (TERTp) C228T and gain-of-function TP53 R248Q mutations in H1 human embryonic stem cells. Wildtype (WT) cells, single TERTp, and double TERTp/TP53 mutants underwent differentiation to NPCs. Lentiviral transduction of double mutants with PDGFRA D842V resulted in triple mutant PRO NPCs. Bulk and single cell transcriptomics of our model system revealed hundreds of gene expression changes with increased mesoderm and human GBM mesenchymal (MES) subtype signatures in single TERTp and double TERTp/TP53 mutants, versus WT cells. TERTp mutation increased telomerase expression and activity, conferring proliferative advantage and immortalization in NPCs and astrocytes. Additionally, TERT expression was further increased in TERTp/TP53 mutants. Surprisingly, triple mutant PRO NPCs, versus WT, displayed < 100 differentially expressed genes, associated with neurodevelopmental and dynamic cytoskeletal processes. Evolution analyses using gene counts signature and splicing dynamics revealed a developmental trajectory model from WT to additive mutants to PRO NPCs. Only triple mutant PRO NPCs formed tumors after intracranial injection in athymic nude mice, with mean survival of 100 days. Tumors presented histopathological features of GBM, and single cell transcriptomic analyses revealed evolution from immune-interacting to both neural progenitor- and neuronal-like subpopulations, with similar cell cycling signatures. Transcription factor genes related to WNT signaling and lineage commitment as well as glial and neuronal cytoskeletal genes exhibited epigenetic selection in vivo, signatures also observed in PRO NPCs in vitro. Our model thus provides opportunity for dissection of epigenetic and functional mechanisms underlying serial mutations during PRO tumor evolution.

Single-Cell Transcriptomic Profiling of Human Retinal Organoids Revealed a Role of IGF1-PHLDA1 Axis in Photoreceptor Precursor Specification.

Cone and rod photoreceptors in the retina convert light to electrical signals which are transmitted to the visual cortex of the brain. Abnormal photoreceptor development and degeneration results in blindness. So far, the mechanism that controls photoreceptor specification and its subsequent fate bifurcation remain elusive. To trace and enrich the human photoreceptor lineage, we first engineered H9 human embryonic stem cell (hESC) reporter line by fusing EGFP to endogenous BLIMP1 using CRISPR/CAS9 gene-editing technology, and then used the cell line to generate 3D retinal organoids. Following EGFP-based cell sorting, single-cell RNA-sequencing was conducted via 10x Genomics Chromium system, and the data were analyzed using Seurat. Immunofluorescence combined with lentivirus-mediated knockdown and overexpression experiments were used as validation approaches. Single-cell transcriptomic profiling revealed that retinal progenitor cells were temporally programmed to differentiate to cone and rod sequentially. We identified PHLDA1 as a novel regulator of photoreceptor specification. PHLDA1 mediated the effects of IGF1 through IGF1R, and inhibited AKT phosphorylation during photoreceptor development. Our data established a transcriptomic cell atlas of the human photoreceptor lineage, and identified IGF1-PHLDA1 axis to regulate human photoreceptor development.

The balance between NANOG and SOX17 mediated by TET proteins regulates specification of human primordial germ cell fate

BackgroundHuman primordial germ cells (hPGCs) initiate from the early post-implantation embryo at week 2–3 and undergo epigenetic reprogramming during development. However, the regulatory mechanism of DNA methylation during hPGC specification is still largely unknown due to the difficulties in analyzing early human embryos. Using an in vitro model of hPGC induction, we found a novel function of TET proteins and NANOG in the hPGC specification which was different from that discovered in mice.MethodsUsing the CRISPR–Cas9 system, we generated a set of TET1, TET2 and TET3 knockout H1 human embryonic stem cell (hESC) lines bearing a BLIMP1-2A-mKate2 reporter. We determined the global mRNA transcription and DNA methylation profiles of pluripotent cells and induced hPGC-like cells (hPGCLCs) by RNA-seq and whole-genome bisulfite sequencing (WGBS) to reveal the involved signaling pathways after TET proteins knockout. ChIP-qPCR was performed to verify the binding of TET and NANOG proteins in the SOX17 promoter. Real-time quantitative PCR, western blot and immunofluorescence were performed to measure gene expression at mRNA and protein levels. The efficiency of hPGC induction was evaluated by FACS.ResultsIn humans, TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) impaired the NODAL signaling pathway and impeded hPGC specification in vitro, while the hyperactivated NODAL signaling pathway led to gastrulation failure when Tet proteins were inactivated in mouse. Specifically, TET proteins stimulated SOX17 through the NODAL signaling pathway and directly regulates NANOG expression at the onset of hPGCLCs induction. Notably, NANOG could bind to SOX17 promoter to regulate its expression in hPGCLCs specification. Furthermore, in TKO hESCs, DNMT3B-mediated hypermethylation of the NODAL signaling-related genes and NANOG/SOX17 promoters repressed their activation and inhibited hPGCLC induction. Knockout of DNMT3B in TKO hESCs partially restored NODAL signaling and NANOG/SOX17 expression, and rescued hPGCLC induction.ConclusionOur results show that TETs-mediated oxidation of 5-methylcytosine modulates the NODAL signaling pathway and its downstream genes, NANOG and SOX17, by promoting demethylation in opposition to DNMT3B-mediated methylation, suggesting that the epigenetic balance of DNA methylation and demethylation in key genes plays a fundamental role in early hPGC specification.

Appropriate Exogenous Expression Stoichiometry of GATA4 as an Important Factor for Cardiac Reprogramming of Human Dermal Fibroblasts.

Reprogramming of human dermal fibroblasts (HDFs) into induced cardiomyocyte-like cells (iCMs) represents a promising strategy for human cardiac regeneration. Different cocktails of cardiac transcription factors can convert HDFs into iCMs, although with low efficiency and immature phenotype. Here, GATA4, MEF2C, TBX5, MESP1, and MYOCD (GMTMeMy for short) were used to reprogram HDFs by retrovirus infection. We found that the exogenous expression stoichiometry of GATA4 (GATA4 stoichiometry) significantly affected reprogramming efficiency. When 1/8 dosage of GATA4 virus (GATA4 dosage) plus MTMeMy was used, the reprogramming efficiency was obviously improved compared with average pooled virus encoding each factor, which measured, by the expression level of cardiac genes, the percentage of cardiac troponin T and alpha-cardiac myosin heavy-chain immunopositive cells and the numbers of iCMs showing calcium oscillation or beating synchronously in co-culture with mouse CMs. In addition, we prepared conditioned maintenance medium (CMM) by CM differentiation of H9 human embryonic stem cell line. We found that compared with traditional maintenance medium (TMM), CMM made iCMs show well-organized sarcomere formation and characteristic calcium oscillation wave earlier. These findings demonstrated that appropriate GATA4 stoichiometry was essential for cardiac reprogramming and some components in CMM were important for maturation of iCMs.

Loss of POGZ alters neural differentiation of human embryonic stem cells

POGZ is a pogo transposable element derived protein with multiple zinc finger domains. Many de novo loss-of-function (LoF) variants of the POGZ gene are associated with autism and other neurodevelopmental disorders. However, the role of POGZ in human cortical development remains poorly understood. Here we generated multiple POGZ LoF lines in H9 human embryonic stem cells (hESCs) using CRISPR/CAS9 genome editing. These lines were then differentiated into neural structures, similar to those found in early to mid-fetal human brain, a critical developmental stage for studying disease mechanisms of neurodevelopmental disorders. We found that the loss of POGZ reduced neural stem cell proliferation in excitatory cortex-patterned neural rosettes, structures analogous to the cortical ventricular zone in human fetal brain. As a result, fewer intermediate progenitor cells and early born neurons were generated. In addition, neuronal migration from the apical center to the basal surface of neural rosettes was perturbed due to the loss of POGZ. Furthermore, cortical-like excitatory neurons derived from multiple POGZ homozygous knockout lines exhibited a more simplified dendritic architecture compared to wild type lines. Our findings demonstrate how POGZ regulates early neurodevelopment in the context of human cells, and provide further understanding of the cellular pathogenesis of neurodevelopmental disorders associated with POGZ variants.

Monoclonal antibody K312-based depletion of pluripotent cells from differentiated stem cell progeny prevents teratoma formation

Human pluripotent stem cells (PSCs) have been utilized as a promising source in regenerative medicine. However, the risk of teratoma formation that comes with residual undifferentiated PSCs in differentiated cell populations is most concerning in the clinical use of PSC derivatives. Here, we report that a monoclonal antibody (mAb) targeting PSCs could distinguish undifferentiated PSCs, with potential teratoma-forming activity, from differentiated PSC progeny. A panel of hybridomas generated from mouse immunization with H9 human embryonic stem cells (hESCs) was screened for ESC-specific binding using flow cytometry. A novel mAb, K312, was selected considering its high stem cell-binding activity, and this mAb could bind to several human induced pluripotent stem cells and PSC lines. Cell-binding activity of K312 was markedly decreased as hESCs were differentiated into embryoid bodies or by retinoic acid treatment. In addition, a cell population negatively isolated from undifferentiated or differentiated H9 hESCs via K312 targeting showed a significantly reduced expression of pluripotency markers, including Oct4 and Nanog. Furthermore, K312-based depletion of pluripotent cells from differentiated PSC progeny completely prevented teratoma formation. Therefore, our findings suggest that K312 is utilizable in improving stem cell transplantation safety by specifically distinguishing residual undifferentiated PSCs.

In vitro culture at 39\xa0\xb0C during hepatic maturation of human ES cells facilitates hepatocyte-like cell functions

Hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) offer an alternative to primary hepatocytes commonly used for drug screenings and toxicological tests. However, these cells do not have hepatic functions comparable to those of hepatocytes in vivo due to insufficient hepatic differentiation. Here we showed that the hepatic functions of hPSC-HLCs were facilitated by applying physiological liver temperatures during hepatic differentiation. We identified the optimal temperature by treating HLCs derived from H9 human embryonic stem cells (hESC-HLCs) at 39 °C; the 42 °C treatment caused significantly greater cell death than the 39 °C treatment. We confirmed the improvement of hepatic functions, such as albumin secretion, cytochrome P450 3A activity, and collagen production, without severe cell damage. In combination with existing hepatic differentiation protocols, the method proposed here may further improve hepatic functions for hPSCs and lead to the realization of drug discovery efforts and drug toxicological tests.

Percutaneous Intracoronary Delivery of Plasma Extracellular Vesicles Protects the Myocardium Against Ischemia-Reperfusion Injury in Canis.

[Figure: see text].

Single Nucleus RNA Sequence (snRNAseq) Analysis of the Spectrum of Trophoblast Lineages Generated From Human Pluripotent Stem Cells in vitro.

One model to study the emergence of the human trophoblast (TB) has been the exposure of pluripotent stem cells to bone morphogenetic protein 4 (BMP4) in presence of inhibitors of ACTIVIN/TGFB; A83–01 and FGF2; PD173074 (BAP), which generates a mixture of cytotrophoblast, syncytiotrophoblast, and cells with similarities to extravillous trophoblast. Here, H1 human embryonic stem cells were BAP-exposed under two O2 conditions (20% and 5%, respectively). At day 8, single nuclei RNA sequencing was used for transcriptomics analysis, thereby allowing profiling of fragile syncytial structures as well as the more resilient mononucleated cells. Following cluster analysis, two major groupings, one comprised of five (2,4,6,7,8) and the second of three (1,3,5) clusters were evident, all of which displayed recognized TB markers. Of these, two (2 and 3) weakly resembled extravillous trophoblast, two (5 and 6) strongly carried the hallmark transcripts of syncytiotrophoblast, while the remaining five were likely different kinds of mononucleated cytotrophoblast. We suggest that the two populations of nuclei within syncytiotrophoblast may have arisen from fusion events involving two distinct species of precursor cells. The number of differentially expressed genes between O2 conditions varied among the clusters, and the number of genes upregulated in cells cultured under 5% O2 was highest in syncytiotrophoblast cluster 6. In summary, the BAP model reveals an unexpectedly complex picture of trophoblast lineage emergence that will need to be resolved further in time-course studies.

GATA2 Is Essential for Endothelial-to-Hematopoietic Transition but Does Not Affect Formation of Hemogenic Endothelium

The formation of blood cells from hemogenic endothelium (HE) is a key element of leading to establishment of the hematopoietic system during embryogenesis. It has become increasingly clear that HE represents a distinct lineage of vascular endothelium with a capacity to undergo endothelial-to-hematopoietic transition (EHT). However, the processes guiding HE specification and EHT are poorly understood. The GATA2 transcription factor is of particular interest since it is critical for development of the entire hematopoietic system including hematopoietic stem cells (HSCs) during embryogenesis. Although GATA2 knockout has been shown to abrogate formation of intraortic hematopoietic clusters, it is not clear whether GATA2 is required for HE formation, EHT per se, or if it controls HE specification and EHT in a non-cell autonomous manner. To study the function of GATA2 during hematopoietic development, we engineered a GATA2 knockout H1 human embryonic stem cell (hESC) carrying doxycycline (DOX)-inducible GATA2 transgene. We demonstrated that GATA2 deficiency dramatically impaired definitive and primitive hematopoiesis from hESCs. To define the stage of development affected by GATA2, we treated iG2-/- and iG2+/+ hESCs with DOX in stepwise fashion, and evaluated the stage of development at which GATA2 induction has the most profound effect on hematopoiesis. We found that DOX treatment has the most dramatic effect on production of CD43+ cells and CFC potential when performed on days 3-4 or 4-5 of differentiation. In contrast, DOX treatment on days 0-2 suppressed differentiation, while treatment on days 5-6 has little to no effect. Since formation of HE and EHT in our system occurs during days 4-5 of differentiation, i.e. when we see the most dramatic effect of DOX treatment, we concluded that GATA2 may be important for HE formation or EHT. Flow cytometric analysis revealed that formation of VE-cadherin+CD43-CD73- HE, including DLL4+ arterial HE subset, and non-HE was not affected in iG2-/- hESCs, or following GATA2 induction with DOX. Next, we isolated iG2-/- HE from day 4 of differentiation and assessed how DOX treatment affected blood formation. In the absence of DOX, iG2-/-HE produced very few blood cells, while DOX treatment restored blood formation from HE. No significant difference was found in proliferation and in apoptosis of hematopoietic progenitors following EHT, regardless of the presence of GATA2, implying the specific effect of GATA2 on EHT.To define molecular pathways which involved in EHT impairment in iG2-/- cells, we performed RNAseq analysis of HE in DOX- and DOX+ conditions. Gene set enrichment analysis (GSEA) detected significant upregulation of sets of genes responding to TGFβ stimulation in iG2-/- HE on day 1 of secondary culture without DOX as compared to DOX treated cells, thereby suggesting that upregulation of target genes of TGFβ signaling may contribute to EHT impairment in GATA2-deficient cells. Indeed, treatment of iG2-/- HE with TGFβ-inhibitor significantly increased EHT and CD43+ hematopoietic progenitor production with CFU-G and GM potential. Overall, our studies suggest that GATA2 is dispensable for specification of HE, but is essential for EHT and proper hematopoietic specification. In addition, inhibiting TGFβ signaling partially restored the GATA2-dependent EHT in the absence of GATA2. DisclosuresNo relevant conflicts of interest to declare.