H1 Embryonic Stem Cells Research Articles

VGLL4-TEAD1 promotes vascular smooth muscle cell differentiation from human pluripotent stem cells via TET2

The Hippo signaling pathway plays a critical role in cardiovascular development and stem cell differentiation. Using microarray profiling, we found that the Hippo pathway components vestigial-like family member 4 (VGLL4) and TEA domain transcription factor 1 (TEAD1) were upregulated during vascular smooth muscle cell (VSMC) differentiation from H1 ESCs (H1 embryonic stem cells). To further explore the role and molecular mechanisms of VGLL4 in regulating VSMC differentiation, we generated a VGLL4-knockdown H1 ESC line (heterozygous knockout) using the CRISPR/Cas9 system and found that VGLL4 knockdown inhibited VSMC specification. In contrast, overexpression of VGLL4 using the PiggyBac transposon system facilitated VSMC differentiation. We confirmed that this effect was mediated via TEAD1 and VGLL4 interaction. In addition, bioinformatics analysis revealed that Ten-eleven-translocation 2 (TET2), a DNA dioxygenase, is a target of TEAD1, and a luciferase assay further verified that TET2 is the target of the VGLL4-TEAD1 complex. Indeed, TET2 overexpression promoted VSMC marker gene expression and countered the VGLL4 knockdown-mediated inhibitory effects on VSMC differentiation. In summary, we revealed a novel role of VGLL4 in promoting VSMC differentiation from hESCs and identified TET2 as a new target of the VGLL4-TEAD1 complex, which may demethylate VSMC marker genes and facilitate VSMC differentiation. This study provides new insights into the VGLL4-TEAD1-TET2 axis in VSMC differentiation and vascular development.

Disheveled binding antagonist of β-catenin 1 interacted with β-catenin and connexin 43 in human-induced pluripotent stem cells-derived cardiomyocytes

ABSTRACT Previously, we demonstrated that the disheveled binding antagonist of β-catenin 1 (DACT1) was involved in atrial fibrillation by regulating the reorganization of connexin 43 and β-catenin in cardiomyocytes. Little is known, however, about DACT1 in human normal myocardial cells. Therefore, we used cardiomyocytes (CMs) derived from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to investigate the role of DACT1 and its connection with β-catenin and connexin 43. While the ESC-CMs and iPSC-CMs were differentiated using commercial differentiation kits, the cardiac-specific markers were detected by immunofluorescence. The expression level of DACT1 was detected using western blotting, whereas the interaction of DACT1 and connexin 43 or β-catenin was detected by immunofluorescence and co-immunoprecipitation (co-IP) assays. Both H1-CMs and SF-CMs were immunostained for cardiac-specific markers, including Troponin I, Troponin T, α-actinin, NKX2.5, and GATA6. While DACT1 was not expressed in both H1 ESCs and SF-iPSCs, it was, however, highly expressed in differentiated CMs, being also localized in the cytoplasm and the nucleus of differentiated CMs. Interestingly, the DACT1 expression in different nuclei was different in the same multinucleated cell. Moreover, DACT1 colocalized with β-catenin in both the cytoplasm and nucleus of differentiated CMs, and it also colocalized with connexin 43 in the perinuclear region and the gap junctions of differentiated CMs. Co-IP results showed that DACT1 could directly bind to β-catenin and connexin 43. Taken together, DACT1 interacted with β-catenin and connexin 43 in human-induced pluripotent stem cells-derived cardiomyocytes.

Environmentally sensitive hotspots in the methylome of the early human embryo.

In humans, DNA methylation marks inherited from gametes are largely erased following fertilisation, prior to construction of the embryonic methylome. Exploiting a natural experiment of seasonal variation including changes in diet and nutritional status in rural Gambia, we analysed three datasets covering two independent child cohorts and identified 259 CpGs showing consistent associations between season of conception (SoC) and DNA methylation. SoC effects were most apparent in early infancy, with evidence of attenuation by mid-childhood. SoC-associated CpGs were enriched for metastable epialleles, parent-of-origin-specific methylation and germline differentially methylated regions, supporting a periconceptional environmental influence. Many SoC-associated CpGs overlapped enhancers or sites of active transcription in H1 embryonic stem cells and fetal tissues. Half were influenced but not determined by measured genetic variants that were independent of SoC. Environmental 'hotspots' providing a record of environmental influence at periconception constitute a valuable resource for investigating epigenetic mechanisms linking early exposures to lifelong health and disease.

Identification of New Transcription Factors that Can Promote Pluripotent Reprogramming

BackgroundFour transcription factors, Oct4, Sox2, Klf4, and c-Myc (the Yamanka factors), can reprogram somatic cells to induced pluripotent stem cells (iPSCs). Many studies have provided a number of alternative combinations to the non-Yamanaka factors. However, it is clear that many additional transcription factors that can generate iPSCs remain to be discovered.MethodsThe chromatin accessibility and transcriptional level of human embryonic stem cells and human urine cells were compared by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to identify potential reprogramming factors. Selected transcription factors were employed to reprogram urine cells, and the reprogramming efficiency was measured. Urine-derived iPSCs were detected for pluripotency by Immunofluorescence, quantitative polymerase chain reaction, RNA sequencing and teratoma formation test. Finally, we assessed the differentiation potential of the new iPSCs to cardiomyocytes in vitro.ResultsATAC-seq and RNA-seq datasets predicted TEAD2, TEAD4 and ZIC3 as potential factors involved in urine cell reprogramming. Transfection of TEAD2, TEAD4 and ZIC3 (in the presence of Yamanaka factors) significantly improved the reprogramming efficiency of urine cells. We confirmed that the newly generated iPSCs possessed pluripotency characteristics similar to normal H1 embryonic stem cells. We also confirmed that the new iPSCs could differentiate to functional cardiomyocytes.ConclusionsIn conclusion, TEAD2, TEAD4 and ZIC3 can increase the efficiency of reprogramming human urine cells into iPSCs, and provides a new stem cell sources for the clinical application and modeling of cardiovascular disease.Graphical abstract

Splicing-associated chromatin signatures: a combinatorial and position-dependent role for histone marks in splicing definition

Alternative splicing relies on the combinatorial recruitment of splicing regulators to specific RNA binding sites. Chromatin has been shown to impact this recruitment. However, a limited number of histone marks have been studied at a global level. In this work, a machine learning approach, applied to extensive epigenomics datasets in human H1 embryonic stem cells and IMR90 foetal fibroblasts, has identified eleven chromatin modifications that differentially mark alternatively spliced exons depending on the level of exon inclusion. These marks act in a combinatorial and position-dependent way, creating characteristic splicing-associated chromatin signatures (SACS). In support of a functional role for SACS in coordinating splicing regulation, changes in the alternative splicing of SACS-marked exons between ten different cell lines correlate with changes in SACS enrichment levels and recruitment of the splicing regulators predicted by RNA motif search analysis. We propose the dynamic nature of chromatin modifications as a mechanism to rapidly fine-tune alternative splicing when necessary.

Abstract 323: A Novel Human Specific Long Noncoding RNA PG1 Controls Human Cardiogenesis via Regulating Beta-catenin Activity

Long noncoding RNAs (lncRNAs) can regulate gene expression, signaling pathway, cellular and tissue functions at various stages. Recently, accumulating evidences suggest that lncRNAs play important roles in cell fate specification and organ development. We found hundreds of lncRNAs, which show lineage-specific expressions in human embryonic stem (ES) cell-derived multipotential cardiovascular progenitor cells (MCPs), cardiomyocytes (CMs), smooth muscle cells (SMs) or endothelial cells (ECs), by using whole transcriptome sequencing. Among MCP-specific lncRNAs, we identified a novel human lncRNA, PG1, which controls cardiovascular differentiation from human pluripotent stem cells. We found PG1 could regulate the early segregation of nascent mesoderm cells towards cardiac verse hematopoietic fate. Knockout of PG1 in human induced pluripotent stem (iPS) cells and H1 ES cells by CRISPR/Cas9 significantly increased hematopoietic differentiation from pluripotent stem cells when compared with wild type controls. Furthermore, our data suggest PG1 could bind beta-catenin protein to modulate Wnt signaling activity to regulate nascent mesoderm specification. In summary, we identified a novel human specific lncRNA, PG1, which plays an essential role in early stage human heart development.

High-content screen in human pluripotent cells identifies miRNA-regulated pathways controlling pluripotency and differentiation

BackgroundBy post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs or miR) play important biological functions. H1 embryonic stem cells (hESCs) and NTera-2 embryonal carcinoma cells (ECCs) are two of the most widely used human pluripotent model cell lines, sharing several characteristics, including the expression of miRNAs associated to the pluripotent state or with differentiation. However, how each of these miRNAs functionally impacts the biological properties of these cells has not been systematically evaluated.MethodsWe investigated the effects of 31 miRNAs on NTera-2 and H1 hESCs, by transfecting miRNA mimics. Following 3–4 days of culture, cells were stained for the pluripotency marker OCT4 and the G2 cell-cycle marker Cyclin B1, and nuclei and cytoplasm were co-stained with Hoechst and Cell Mask Blue, respectively. By using automated quantitative fluorescence microscopy (i.e., high-content screening (HCS)), we obtained several morphological and marker intensity measurements, in both cell compartments, allowing the generation of a multiparametric miR-induced phenotypic profile describing changes related to proliferation, cell cycle, pluripotency, and differentiation.ResultsDespite the overall similarities between both cell types, some miRNAs elicited cell-specific effects, while some related miRNAs induced contrasting effects in the same cell. By identifying transcripts predicted to be commonly targeted by miRNAs inducing similar effects (profiles grouped by hierarchical clustering), we were able to uncover potentially modulated signaling pathways and biological processes, likely mediating the effects of the microRNAs on the distinct groups identified. Specifically, we show that miR-363 contributes to pluripotency maintenance, at least in part, by targeting NOTCH1 and PSEN1 and inhibiting Notch-induced differentiation, a mechanism that could be implicated in naïve and primed pluripotent states.ConclusionsWe present the first multiparametric high-content microRNA functional screening in human pluripotent cells. Integration of this type of data with similar data obtained from siRNA screenings (using the same HCS assay) could provide a large-scale functional approach to identify and validate microRNA-mediated regulatory mechanisms controlling pluripotency and differentiation.

Transplantation of human intestinal organoids into the mouse mesentery: A more physiologic and anatomic engraftment site

BackgroundWe previously described the development of human intestinal organoids from pluripotent stem cells, as well as their in vivo maturation when transplanted into the mouse kidney capsule. While sufficient for certain aspects of study, this model has limitations. Herein, we describe an alternative model of human intestinal organoids transplantation into the mouse mesentery. We hypothesize that efficient engraftment and marked differentiation of human intestinal organoids will be similar to our kidney model yet in a more anatomically appropriate location allowing for improved in vivo modeling. MethodsHuman intestinal organoids were generated by directed differentiation of H1 embryonic stem cells. Human intestinal organoids were then transplanted into the mesentery of immunosuppressed mice. Gross and histologic analysis of tissue was performed. ResultsHuman intestinal organoids were transplanted into the mouse mesentery and allowed to grow for 10 weeks. Mouse survival was 85%, and among the surviving mice, 82% of transplanted human intestinal organoids successfully engrafted. Upon graft harvest, transplanted HIOs were larger than in vitro human intestinal organoids (1.75 mm vs 6.27 mm, P < .0001) and grew along a vascular pedicle, allowing for interventions and reconstructive surgeries to access the human intestinal organoid lumen. Histologic analyses of transplanted human intestinal organoids confirmed the presence of major cell types, as well as stem cell activity. ConclusionsThe mouse mesentery is a viable location for the transplantation of human intestinal organoids, yielding grafts of reproducible size and quality. This improved model serves to advance functional and translational studies of human intestinal organoids.

Abstract 4316: Disruption and restoration of pioneer transcription factor hub function in bladder cancer

Abstract Urothelial bladder cancer is characterized by the failure to complete transitional epithelial-differentiation. The mechanisms underlying this failure are unknown. To address this gap in knowledge, we analyzed 2000 genes differentially expressed between normal bladder vs. bladder cancer (BLCA) (n=426, TCGA RNA sequencing). Remarkably, genes suppressed in BLCA are found in embryonic stem cells (ESC) in compacted chromatin (decreased H3K4me3, increased CpG methylation), while upregulated genes in BLCA are found in ESCs in open chromatin (H1 ESC Encode).This striking property of genes suppressed in BLCA suggested loss-of-function of pioneer transcription factor (TF) function in BLCA, as pioneer TFs access compacted chromatin to initiate the necessary remodeling for subsequent gene activation. The pioneer master TF hub containing FOXA1, GATA4/6 is highly expressed in normal bladder, and these master TFs were frequently haploinsufficient in BLCA, 32%, 56%, and 24% respectively. To better understand why target genes of FOXA1, the most expressed bladder master TF, are unremodeled and repressed, we pulled down endogenous FOXA1 from UM-UC-6 and -3 BLCA cells and used mass spectrometry and Western blot to analyze interacting coregulators, which execute the biochemical work of chromatin remodeling. These analyses revealed enrichment for corepressors, e.g., DNMT1, in the FOXA1 interactome over coactivators, e.g., ARID1A. Genetic analysis of BLCA show frequent mutation/deletion of coactivators: 43% bi-allelic ARID1A inactivation, while corepressors SMARCA5 and DNMT1 were amplified or unaltered to remain functional. Restoration of ARID1A into ARID1A haploinsufficient BLCA (UC-6) upregulated epithelial-differentiation genes and induced terminal epithelial-differentiation, downregulation of MYC and upregulation of p27 protein. To translate into therapy, we restored coactivator/corepressor balance by pharmacologic corepressor depletion: we used the clinical drug decitabine to deplete DNMT1, to compensate for genetic coactivator inactivation. Decitabine recapitulated the effects of ARID1A introduction, inducing terminal epithelial-differentiation of BLCA cells. This was also seen in vivo in a xenograft model of BLCA (UC-6). Decitabine combined with an inhibitor of its in vivo degradation, tetrahydrouridine, decreased tumor size significantly with vehicle tumor size 917.8 mm3 to treated tumor size 321.0 mm3 (p&amp;lt;0.01). In sum, genetic alterations in BLCA alter composition of a pioneer master TF hub to remove remodeling needed for transitional epithelial-differentiation. Dynamic coactivator/corepressor opposition in gene regulation, however, enables pharmacologic reduction of corepressors to compensate for genetic reduction of coactivators. Citation Format: Caroline Schuerger, Yogen Saunthararajah. Disruption and restoration of pioneer transcription factor hub function in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4316.

Predicting gene expression level by the transcription factor binding signals in human embryonic stem cells

The transcription factor (TF) binding signals play important role in the control of gene expression. In this study, to elucidate the relationship between the transcription factor binding signals and gene expression, we firstly analyze the distributions of 57 kinds of TFs’ binding signals in human H1 embryonic stem cells. Their distributions in highly and lowly expressed genes are further compared. On this basis, a statistic model of predicting gene expression level is constructed by using 57 kinds of transcription factor synthetic indexes (TFSIs). Then, the TF’s Down-regulated and Up-regulated genes are predicted and the statistics significance is estimated by one-sided Kolmogorov-Smirnov test. Based on the stepwise regression analysis, the “optimal” TFSIs are selected out, and the better results for predicting the expression level of genes with high CpG content promoters (HCPs) and low CpG content promoters (LCPs) are obtained.

More Efficient Generation of β-Globin-Expressing Erythroid Cells Using Stromal Cell-Derived Induced Pluripotent Stem Cells

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. Using traditional embryoid body methods, iPS cell-derived erythroid cells predominantly produce ε-globin and γ-globin without β-globin expression. We recently demonstrated that ES cell-derived sacs (ES sacs), known to express hemangioblast markers, allow for efficient erythroid cell generation with β-globin production, which is associated with emergence of CD34+ hematopoietic stem/progenitor cells (HSPCs) (2014 ASH). In the current study, we extend this work to evaluate erythroid cell generation using iPS cell lines generated from various sources including patients with sickle cell disease (SCD).To test our two hypotheses; (1) erythroid progenitor (EP)-derived iPS cells more efficiently differentiate to erythroid cells, and (2) stromal cell (ST)-derived iPS cells more efficiently emerge hemangioblast-like immature HSPCs which results in greater erythroid cell generation, we generated several clones of iPS cells which were derived from (1) EPs (6 clones) which were differentiated from peripheral blood mononuclear cells and (2) bone marrow STs (5 clones) in SCD patients. Transgene-free iPS cells were generated and characterized according to Merling et al. (Blood. 2013). These iPS cells and controls (2 clones of fibroblast (FB)-derived iPS cells and H1 ES cells) were used to generate ES/iPS sacs for 15 days. After a 2 day culture of ES/iPS sac-derived spherical cells on OP9 feeder cells, the suspension cells were differentiated into erythroid cells using human erythroid massive amplification culture for 13 days (Blood cells Mol Dis. 2002).Following ES/iPS sac generation, 3.5-4.8 fold greater amounts of CD34+CD45+ HSPCs emerged in both EP- and ST-derived iPS sacs, compared to FB-derived iPS sacs (p<0.01). After an additional 2 weeks of erythroid differentiation, we observed 4.5-8.7 fold greater amounts of GPA+ erythroid cells from both EP- and SC-derived iPS sacs, compared to FB-derived iPS sacs (p<0.01). Interestingly, ST-derived iPS sacs resulted in 1.4-2.0 fold greater amounts of CD34+CD45+ HSPCs and GPA+ erythroid cells (p<0.01), compared to EP-derived iPS sacs. Higher β-globin expression (21.5±4.3%) was observed by RT-qPCR in erythroid cells from ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (4.4±2.5% and 8.3±4.2%, respectively, p<0.01), which was comparable to ES sacs (23.3%). Sickle hemoglobin was detected by hemoglobin electrophoresis. The ES/iPS sac-derived erythroid cell generation was more strongly affected by cell sources (5-6 fold larger SD) than variations among iPS cell clones. These data demonstrate that ST-derived iPS sacs allow more efficient erythroid cell generation with higher β-globin production, compared to EP- and FB-derived iPS sacs.We hypothesized that ST-derived iPS sacs contain greater amounts of immature HSPCs (including hemogenic endothelium) and immature EPs (including megakaryoerythroid progenitors), since more expansion of ST-derived cells was observed during the late phase of erythroid differentiation, compared to EP- and FB-derived cells. We evaluated hemogenic endothelium markers at day 15, and observed 7.7 fold greater amounts of VEGFR+GPA- cells (p<0.01) and 1.3-1.4 fold greater amounts of CD31+CD34+ cells in ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (not detectable VEGFR+GPA- cells in EP-derived iPS sacs). Before erythroid differentiation, 3.2-16.4 fold greater amounts of GPA+CD41a+ megakaryoerythroid progenitors were observed in ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (p<0.05). In colony forming unit assays, 1.8-5.0 fold greater amounts of myeloid and erythroid colonies were observed in ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (p<0.01). These data suggest that ST-derived iPS sacs more efficiently produce immature HSPCs and immature EPs, which may result in more efficient generation of erythroid cells with β-globin production.In summary, we demonstrated that human ST-derived iPS sacs allow for more efficient erythroid cell generation with higher β-globin production, which could be caused by heightened emergence of hemogenic endothelium in ST-derived iPS sacs. Our findings should be important for in in vitro iPS cell-derived erythroid cell generation with high β-globin expression. DisclosuresWinkler:Novartis: Research Funding; GSK: Research Funding.

Dodecaploid H1 embryonic stem cells abolished pluripotency in L15F10 medium both with and without leukemia inhibitory factor

Two lines of dodecaploid H1 embryonic stem cells, 12H1 and 12H1(-) cells (mouse-originated cells), were established through polyploidization of two hexaploid H1 cells, 6H1 and 6H1(-) cells, which were cultured in L15F10 (7:3) medium with and without leukemia inhibitory factor (LIF), respectively. The G1, S, and G2/M phase fractions of 12H1 and 12H1(-) cells were almost the same as those of 6H1 and 6H1(-) cells, respectively, but the doubling time of cell proliferation was prolonged, suggesting that cell death occurred in 12H1 and 12H1(-)cells. The cell volumes of 12H1 and 12H1(-) cells were about double those of 6H1 and 6H1(-) cells, respectively. 12H1 and 12H1(-) cells showed near-negative activity of alkaline phosphatase and no ability to form teratocarcinomas in mouse abdomen, suggesting that 12H1 and 12H1(-) cells lost pluripotency. The DNA contents of 12H1 and 12H1(-) cells decayed in long-term culturing, suggesting that 12H1 and 12H1(-) cells were DNA-unstable. Possible explanations for the lost pluripotency and for the DNA decay in 12H1 and 12H1(-) cells are presented.

Different responses between diploid and tetraploid H1 embryonic stem cells appeared during long-term culturing in L15F10 medium without leukemia inhibitory factor

To examine the alteration in cellular characteristics of polyploid embryonic stem (ES) cells during long-term culturing without leukemia inhibitory factor (LIF), mouse diploid and tetraploid H-1 (ES) cells (2H1 and 4H1 cells, respectively) were cultured without LIF for approximately 5 months. 2H1 and 4H1 cells were adapted to the medium without LIF by decreasing the concentration for several passages, and they were denoted as 2H1(⁻) and 4H1(⁻) cells, respectively. DNA content of 4H1(⁻) cells decreased gradually in the early stage, increased abruptly in the second stage, and then was maintained for a long time. 4H1(⁻) cells exhibited longer doubling time and equivalent phase fraction compared with those of 2H1(⁻) cells. The G₁ phase fractions of 2H1(⁻) and 4H1(⁻) cells were increased compared with that of 2H1 cells. Cellular morphology and pluripotency were maintained in 4H1(⁻) cells but not in 2H1(⁻) cells. 2H1(⁻) cells showed a cell population consisting of several kinds of cells, and they lost alkaline phosphatase activity, suggesting that the cells had differentiated. 4H1(⁻) cells, however, exhibited alkaline phosphatase activity and formed teratocarcinoma in mouse abdomen, suggesting that the cells maintained their pluripotency in the medium without LIF.

Cells Extract from Fetal Liver Promotes the Hematopoietic Differentiation of Human Embryonic Stem Cells

Here, we have now developed a new inducing system to promote the differentiation of human stem cells (hESCs) toward hematopoietic lineages by the treatment with cells extract of human fetal liver tissue (hFLT). The embryoid bodies (EBs) obtained from human H1 embryonic stem cells were exposed to buffer, hFLT cells extract, heated hFLT cell extract, and cell extract of human liver cells lines-LO2. Then, the feature of EBs in different groups was characterized by real-time RT-PCR and colony-forming assays. The results showed the treatment by hFLT cells extract could activate the hematopoietic genes expression and improve the capacity for hematopoietic progenitor development of hEBs. After that, we cocultured hFLT extract treated hEBs on the hFLSCs (human fetal liver stromal cells) feeder to differentiate them into hematopoietic cells. As a control, untreated hEBs were cocultured on hFLSCs feeder with cytokines. The feature of induced cells from hEBs was characterized by flow cytometry, Wright-Giemsa staining, and colony-forming assays. The results demonstrated that hFLT cells extract was capable of inducing hEBs into hematopoietic cells and combing it with hFLSCs feeder could largely promote hematopoietic differentiation of hESCs. This method may supply a new way to substitute the cytokines required in hematopoietic induction of hESCs.

Large-scale production of embryonic red blood cells from human embryonic stem cells

To develop a method to produce in culture large number of erythroid cells from human embryonic stem cells. Human H1 embryonic stem cells were differentiated into hematopoietic cells by coculture with a human fetal liver cell line, and the resulting CD34-positive cells were expanded in vitro in liquid culture using a three-step method. The erythroid cells produced were then analyzed by light microscopy and flow cytometry. Globin expression was characterized by quantitative reverse-transcriptase polymerase chain reaction and by high-performance liquid chromatography. CD34-positive cells produced from human embryonic stem cells could be efficiently differentiated into erythroid cells in liquid culture leading to a more than 5000-fold increase in cell number. The erythroid cells produced are similar to primitive erythroid cells present in the yolk sac of early human embryos and did not enucleate. They are fully hemoglobinized and express a mixture of embryonic and fetal globins but no beta-globin. We have developed an experimental protocol to produce large numbers of primitive erythroid cells starting from undifferentiated human embryonic stem cells. As the earliest human erythroid cells, the nucleated primitive erythroblasts, are not very well characterized because experimental material at this stage of development is very difficult to obtain, this system should prove useful to answer a number of experimental questions regarding the biology of these cells. In addition, production of mature red blood cells from human embryonic stem cells is of great potential practical importance because it could eventually become an alternate source of cell for transfusion.