Transient Over-Expression of HOX-Regulating Genes CDX2 and MLL during Human Embryoid Body Differentiation Greatly Augments the Generation of Multipotent Hematopoietic Progenitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1243-1243
Author(s):  
Michal A. Levine ◽  
Christopher Roxbury ◽  
Elias T. Zambidis
Keyword(s):  
Stem Cell ◽  
Embryonic Development ◽  
Transient Expression ◽  
Embryoid Body ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Development ◽  
Rna Molecules ◽  
Over Expression ◽  
Definitive Hematopoiesis

Abstract Homeobox (HOX) genes play critical roles in normal anterior-posterior patterning of embryonic development, and in hematopoietic stem cell (HSC) development. Conversely, dysregulated expressions of HOX-regulating factors such as CDX2 (Caudal) and MLL (Mixed Lineage Leukemia) are directly linked to development of acute leukemia. Although CDX family (e.g. cdx4) and mll factors play important roles in murine HSC development, their role in normal human embryonic blood development is obscure. The role of CDX genes (e.g., CDX1, CDX2, CDX4), expressed exclusively during embryonic development, is difficult to evaluate in human hematopoietic development, since fetal tissue is difficult to obtain. Our group has developed a human embryonic stem cell (hESC) differentiation system that recapitulates the 2nd-6th gestational weeks of human yolk sac (YS) development, and initiates from an embryonic hemangioblastic progenitor of primitive and definitive hematopoiesis. The role of HOX-regulating genes (and also HOX-regulating microRNAs (miRNAs), e.g., mir196) that regulate the earliest stages of human hematopoietic development can therefore be studied directly in vitro using our hESC model. We tested the effects of pulsatile, transient over-expression of HOX-regulating factors and miRNAs on the generation of primitive and definitive hematopoeitic progenitors during human embryoid body (hEB) differentiation. Since expression of HOX-regulating genes and miRNAs follow temporal, transient expression patterns during normal embryonic, and also hEB development, we developed a methodology that allows similar transient expression of DNA and RNA molecules at multiple time points of advancing hEB differentiation. This method, termed whole embryoid body (WEB) nucleofection was optimized using GFP-expressing DNA constructs, GFP-silencing siRNA, and also miRNA molecules within intact, whole hEB. WEB nucleofection allowed expression in 15–90% of day 4–9 hEB cells without disrupting their three-dimensional structural integrity, and with minimal toxicity. GFP-nucleofected day 5–13 hEB demonstrated peak expression levels at 48 hrs post-nucleofection, and expression was sustained for approximately one week. A FITC-labeled dsRNA oligonucleotide, was used to demonstrate that the efficiency of WEB nucleofection with RNA molecules approached ∼90%. WEB nucleofection was utilized to transiently over-express CDX2 and MLL constructs within intact, developing hEBs, and the effects on generation of hEB-derived primitive and definitive hematopoiesis were assayed by colony-forming cell (CFC), and FACS analysis. CDX2 and MLL-nucleofected hEB each produced 5-10X greater amounts of multipotent, mixed CFU, in comparison to controls. Moreover, MLL-nucleofected hEB demonstrated a bias toward development of definitive erythroid progenitors. Hematopoietic regulation by over-expression or inhibition of miRNAs implicated in HOX regulation (e.g. mir-196, mir-10) is also currently being evaluated by WEB nucleofection. Our ability to specifically control multiple combinations of transgenic DNA, siRNA or miRNA molecules, temporally and spatially during hEB differentiation, provides novel opportunities to manipulate the CDX-HOX axis for generating and expanding multi-potent hematopoietic progenitors from hESC. The role of HOX-regulating factors and miRNAs involved in regulating the earliest steps of human hematopoietic commitment can now be directly evaluated.

Transient Over-Expression of HOX-Regulating Genes CDX2 and MLL during Human Embryoid Body Differentiation Greatly Augments the Generation of Multipotent Hematopoietic Progenitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3745.5-3745.5
Author(s):  
Michal A. Levine ◽  
Christopher Roxbury ◽  
Elias T. Zambidis
Keyword(s):  
Stem Cell ◽  
Embryonic Development ◽  
Transient Expression ◽  
Embryoid Body ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Development ◽  
Rna Molecules ◽  
Over Expression ◽  
Definitive Hematopoiesis

Abstract Homeobox (HOX) genes play critical roles in normal anterior-posterior patterning of embryonic development, and in hematopoietic stem cell (HSC) development. Conversely, dysregulated expressions of HOX-regulating factors such as CDX2 (Caudal) and MLL (Mixed Lineage Leukemia) are directly linked to development of acute leukemia. Although CDX family (e.g. cdx4) and mll factors play important roles in murine HSC development, their role in normal human embryonic blood development is obscure. The role of CDX genes (e.g., CDX1, CDX2, CDX4), expressed exclusively during embryonic development, is difficult to evaluate in human hematopoietic development, since fetal tissue is difficult to obtain. Our group has developed a human embryonic stem cell (hESC) differentiation system that recapitulates the 2nd–6th gestational weeks of human yolk sac (YS) development, and initiates from an embryonic hemangioblastic progenitor of primitive and definitive hematopoiesis. The role of HOX-regulating genes (and also HOX-regulating microRNAs (miRNAs), e.g., mir196) that regulate the earliest stages of human hematopoietic development can therefore be studied directly in vitro using our hESC model. We tested the effects of pulsatile, transient over-expression of HOX-regulating factors and miRNAs on the generation of primitive and definitive hematopoeitic progenitors during human embryoid body (hEB) differentiation. Since expression of HOX-regulating genes and miRNAs follow temporal, transient expression patterns during normal embryonic, and also hEB development, we developed a methodology that allows similar transient expression of DNA and RNA molecules at multiple time points of advancing hEB differentiation. This method, termed whole embryoid body (WEB) nucleofection was optimized using GFP-expressing DNA constructs, GFP-silencing siRNA, and also miRNA molecules within intact, whole hEB. WEB nucleofection allowed expression in 15–90% of day 4–9 hEB cells without disrupting their three-dimensional structural integrity, and with minimal toxicity. GFP-nucleofected day 5–13 hEB demonstrated peak expression levels at 48 hrs post-nucleofection, and expression was sustained for approximately one week. A FITC-labeled dsRNA oligonucleotide, was used to demonstrate that the efficiency of WEB nucleofection with RNA molecules approached ∼90%. WEB nucleofection was utilized to transiently over-express CDX2 and MLL constructs within intact, developing hEBs, and the effects on generation of hEB-derived primitive and definitive hematopoiesis were assayed by colony-forming cell (CFC), and FACS analysis. CDX2 and MLL-nucleofected hEB each produced 5-10X greater amounts of multipotent, mixed CFU, in comparison to controls. Moreover, MLL-nucleofected hEB demonstrated a bias toward development of definitive erythroid progenitors. Hematopoietic regulation by over-expression or inhibition of miRNAs implicated in HOX regulation (e.g. mir-196, mir-10) is also currently being evaluated by WEB nucleofection. Our ability to specifically control multiple combinations of transgenic DNA, siRNA or miRNA molecules, temporally and spatially during hEB differentiation, provides novel opportunities to manipulate the CDX-HOX axis for generating and expanding multi-potent hematopoietic progenitors from hESC. The role of HOX-regulating factors and miRNAs involved in regulating the earliest steps of human hematopoietic commitment can now be directly evaluated.

Abstract 052: Epicardial-derived Adrenomedullin Drives Cardiac Hyperplasia During Embryogenesis

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Sarah E Wetzel-Strong ◽  
Manyu Li ◽  
Toshio Nishikimi ◽  
Kathleen M Caron
Keyword(s):  
Embryonic Development ◽  
Heart Development ◽  
Cardiac Development ◽  
Lymphatic Vessels ◽  
Heart Weight ◽  
Cardiovascular Development ◽  
Proliferating Cells ◽  
Over Expression ◽  
Knockout Animals

The multi-functional peptide adrenomedullin ( Adm = gene, AM = protein) plays important roles in embryonic development and disease. Previous studies demonstrated that Adm knockout mice die at embryonic day 13.5 with small, disorganized hearts and hypoplastic lymphatic vessels, highlighting the importance of this peptide in normal cardiovascular development. Since Adm knockout animals are embryonic lethal, our goal was to generate and characterize a novel model of Adm over-expression to study the role of Adm during development and disease processes. Through gene targeting techniques, we generated a novel mouse model of Adm over-expression, abbreviated as Adm hi/hi . When we assessed gene expression of Adm from 10 different tissues, we found Adm hi/hi mice express 3- to 15-fold more Adm than wildtype littermates. Additionally, peptide levels of AM in lung and kidney, as well as circulating plasma levels of AM were elevated 3-fold over wildtype mice, indicating a functional increase in AM. Our initial analysis revealed that adult Adm hi/hi mice have larger heart weight to body weight ratios than wildtype littermates (4.93±0.23 vs. 5.96±0.29, n = 11-12). We found that compared to wildtype, Adm hi/hi embryos have more proliferating cells during heart development (14.46±1.11 vs. 31.97±2.84, n=4), indicating that hyperplasia drives Adm hi/hi heart enlargement. By crossing the Adm hi/hi line to different tissue-specific Cre lines, we were able to excise the stabilizing bovine growth hormone 3’UTR, thereby returning Adm expression levels back to wildtype in cells with active Cre recombinase. Using this approach, we identified the epicardium as a major source of AM during cardiac development. In conclusion, we found that AM derived primarily from the epicardium drives cardiac hyperplasia during embryonic development resulting in persistent, enlarged hearts of adult Adm hi/hi mice. Since our Adm hi/hi mice recapitulate the 3-fold plasma elevation of AM observed during human disease, this mouse line will be a useful tool for studying the role of elevated AM during disease.

scholarly journals Dicer Ablations in Bone Marrow Niche Impair Hematopoietic Progenitor/Stem Cells and Induce Myelodysplasia in Young Mice but Are Dispensable for Adult Hematopoiesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1196-1196 ◽  
Author(s):  
Bijender Kumar ◽  
Mayra Garcia ◽  
Guido Marcucci ◽  
Ching-Cheng Chen
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Embryonic Development ◽  
Bone Marrow Niche ◽  
Hematopoietic Progenitors ◽  
Mirna Processing ◽  
Adult Mice ◽  
Hsc Niche ◽  
Young Mice

Abstract MicroRNAs (miRNAs) regulate hematopoietic cell fate and their global down-regulation by Dicer1 deletion promotes tumorigenesis in a cancer-cell-autonomous manner (Kumar M.S. et al, 2007). Raajimakers MH et al. (2010) using neonatal Osterix specific dicer deletion showed altered hematopoiesis and developed myelodysplasia. However, there is no study illustrating the role of the ablation of bone marrow (BM) niche specific miRNA processing machinery in the adult mice. Since expression and functions of different mesenchymal and osteoprogenitors vary from embryonic development to adulthood, studying the dicer ablation in adult mice may provide more insight about the role of miRNA processing in adult mice niche. Here we investigate whether adult Osterix expressing cells play a similar role in the HSC niche compared to fetal Osterix expressing cells. We crossed Osx-GFP-tTA-Cre recombinase mice with mice with floxed Dicer1 allele. Crossing generated Osx- GFP-tTA-Cre+Dicerfl/+ (OCDfl/+control) and Osx-GFP- tTA-Cre+ Dicerfl/fl (OCDfl/fl mutant) mice. Osx-GFP-tTA-Cre expression was either activated during embryonic development (young dicer KO) or suppressed using tetracycline until mice were 6 weeks of age (adult dicer KO). We found young dicer KO mice had reduced weight (p=0.0031), leukopenia, anemia, reduced mature CD19+B220- B lymphocytes (p=0.0034) and increased CD11b+Gr- monocytes and CD11b+Gr+ neutrophils (p=0.02 and p=0.04 respectively) in peripheral blood compared to OCDfl/+ control aged littler mates. The leucocytes and platelets showed dysplastic changes suggestive of myelodysplasia and had extra-medullary hematopoiesis. Adult dice KO, on the other hand, show no leukemia development 6 months after Cre activation. The number of BM hematopoietic progenitors (Lin-Sca1+ c-Kit+ cells, LSK) and long term hematopoietic stem cells (LT-HSCs, LSK CD150+CD48+ cells) in young dicer KO mice were significantly reduced compared to age matched control (OCDfl/+ control) mice. We observed increased Annexin V positive LSK, LT-HSCs and megakaryocytes erythroid progenitors (MEP) in the young dicer KO mice indicating increased apoptosis. Adult dicer KO mice didn't have significant changes in apoptosis in different hematopoietic progenitors. In young dicer KO mice, BM derived LSK and LT-HSCs showed increased cycling (SG2M phase, p=0.0133) and less quiescenece (Go phase, p=0.013). However LT-HSC from adult dicer KO didn't show any difference in cell cycling (p=0.18 and 0.09 respectively). Together these results indicate that while Osterix expressing cells in fetal and young mice give rise to a variety of HSC niche supporting cells the adult expression is limited to more mature osteoblast that are not absolutely essential for HSC maintenance. Our study provides the rationale for further exploration of the complexity in hierarchy of activity within niche constituting mesenchymal stroma progenitors and their role in different developmental stages to maintain hematopoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Expression of a Knocked-In AML1-ETO Leukemia Gene Inhibits the Establishment of Normal Definitive Hematopoiesis and Directly Generates Dysplastic Hematopoietic Progenitors

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3134-3143 ◽  
Author(s):  
Tsukasa Okuda ◽  
Zhongling Cai ◽  
Shouli Yang ◽  
Noel Lenny ◽  
Chuhl-joo Lyu ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Hematopoietic Progenitors ◽  
Self Renewal ◽  
Retroviral Transduction ◽  
Acute Myelogenous ◽  
Immortalized Cell ◽  
Adult Bone ◽  
Definitive Hematopoiesis

Abstract The t(8;21)-encoded AML1-ETO chimeric product is believed to be causally involved in up to 15% of acute myelogenous leukemias through an as yet unknown mechanism. To directly investigate the role of AML1-ETO in leukemogenesis, we used gene targeting to create anAML1-ETO “knock-in” allele that mimics the t(8;21). Unexpectedly, embryos heterozygous for AML1-ETO(AML1-ETO/+) died around E13.5 from a complete absence of normal fetal liver–derived definitive hematopoiesis and lethal hemorrhages. This phenotype was similar to that seen following homozygous disruption of either AML1 orCBFβ. However, in contrast to AML1- or CBFβ-deficient embryos, fetal livers from AML1-ETO/+ embryos contained dysplastic multilineage hematopoietic progenitors that had an abnormally high self-renewal capacity in vitro. To further document the role of AML1-ETO in these growth abnormalities, we used retroviral transduction to express AML1-ETO in murine adult bone marrow–derived hematopoietic progenitors. AML1-ETO–expressing cells were again found to have an increased self-renewal capacity and could be readily established into immortalized cell lines in vitro. Taken together, these studies suggest that AML1-ETO not only neutralizes the normal biologic activity of AML1 but also directly induces aberrant hematopoietic cell proliferation.

Runx1 is required for zebrafish blood and vessel development and expression of a human RUNX1-CBF2T1 transgene advances a model for studies of leukemogenesis

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 2015-2030 ◽  
Author(s):  
Maggie L. Kalev-Zylinska ◽  
Julia A. Horsfield ◽  
Maria Vega C. Flores ◽  
John H. Postlethwait ◽  
Maria R. Vitas ◽  
...  
Keyword(s):  
Transient Expression ◽  
Normal Function ◽  
Model System ◽  
Zebrafish Embryos ◽  
Lateral Plate ◽  
Vessel Development ◽  
Morpholino Oligonucleotides ◽  
Antisense Morpholino Oligonucleotides ◽  
Definitive Hematopoiesis

RUNX1/AML1/CBFA2 is essential for definitive hematopoiesis, and chromosomal translocations affecting RUNX1 are frequently involved in human leukemias. Consequently, the normal function of RUNX1 and its involvement in leukemogenesis remain subject to intensive research. To further elucidate the role of RUNX1 in hematopoiesis, we cloned the zebrafish ortholog (runx1) and analyzed its function using this model system. Zebrafish runx1 is expressed in hematopoietic and neuronal cells during early embryogenesis. runx1 expression in the lateral plate mesoderm co-localizes with the hematopoietic transcription factor scl, and expression of runx1 is markedly reduced in the zebrafish mutants spadetail and cloche. Transient expression of runx1 in cloche embryos resulted in partial rescue of the hematopoietic defect. Depletion of Runx1 with antisense morpholino oligonucleotides abrogated the development of both blood and vessels, as demonstrated by loss of circulation, incomplete development of vasculature and the accumulation of immature hematopoietic precursors. The block in definitive hematopoiesis is similar to that observed in Runx1 knockout mice, implying that zebrafish Runx1 has a function equivalent to that in mammals. Our data suggest that zebrafish Runx1 functions in both blood and vessel development at the hemangioblast level, and contributes to both primitive and definitive hematopoiesis. Depletion of Runx1 also caused aberrant axonogenesis and abnormal distribution of Rohon-Beard cells, providing the first functional evidence of a role for vertebrate Runx1 in neuropoiesis.To provide a base for examining the role of Runx1 in leukemogenesis, we investigated the effects of transient expression of a human RUNX1-CBF2T1 transgene [product of the t(8;21) translocation in acute myeloid leukemia] in zebrafish embryos. Expression of RUNX1-CBF2T1 caused disruption of normal hematopoiesis, aberrant circulation, internal hemorrhages and cellular dysplasia. These defects reproduce those observed in Runx1-depleted zebrafish embryos and RUNX1-CBF2T1 knock-in mice. The phenotype obtained with transient expression of RUNX1-CBF2T1 validates the zebrafish as a model system to study t(8;21)-mediated leukemogenesis.

FGF-2 Supplementation Enhances Hematopoietic Differentiation of Rhesus ESC’s.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1667-1667
Author(s):  
Aimen Shaaban ◽  
Lasya Gaur ◽  
James A. Thomson ◽  
Deepika Rajesh
Keyword(s):  
Embryoid Body ◽  
Critical Role ◽  
Embryonic Stem ◽  
Cell Types ◽  
Large Animal ◽  
Hematopoietic Differentiation ◽  
Fgf Signaling ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Development ◽  
Close Phylogenetic Relationship

Abstract Progress toward clinical application of embryonic stem cells (ESC) derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model such as the rhesus macaque. However, in contrast to human ESC’s (hESC’s), efforts to induce conclusive hematopoietic differentiation from rhesus ESC’s (rESC’s) have been unsuccessful. Despite their close phylogenetic relationship, subtle differences exist between the hematopoietic differentiation of rESC’s and hESC’s. We recently reported that although rESC’s have the potential for hematopoietic differentiation; they exhibit an arrest at the hematoendothelial precursor stage of hematopoietic development in culture conditions developed for hESC’s. One possible difference may be in the requirement for fibroblast growth factor (FGF) signaling. Despite documentation of its contribution to the maintenance ESC’s in an undifferentiated state, the role for FGF-2 in the hematopoietic differentiation of hESC’s and rESC’s has not been similarly examined. Given its critical role for the formation and subsequent hematopoietic differentiation of murine ESC-derived hemangioblasts, we wondered if enhanced hematopoietic differentiation from rESC’s could be achieved by culture supplementation with FGF-2. To answer this question, undifferentiated rESC’s were subjected to embryoid body (EB) differentiation with daily FGF-2 supplementation of the cytokine-rich media. Cultures were analyzed by flow cytometry after 16 days of EB culture. We found that the FGF-2 supplemented cultures appeared more robust with an overall higher numbers of cells. More importantly, a dramatic expansion of hematoendothelial precursors (Flk1hi+ VE-cadherin- CD45−), committed hematopoietic progenitors (CD34+CD45+Lin−), and hematopoietic cells (CD45+) was seen in FGF-2 supplemented cultures when compared to controls. These effects were consistent in two separate lines of rESC’s (R420 and R456). Next we wondered if the observed effect of FGF-2 on hematopoietic development was concentration-dependent. Therefore, we compared serial increases in FGF-2 concentration (0, 10, 50 and 100 ng/ml) of the EB differentiation media and found the effect to be concentration-dependent. From these results, we conclude that FGF-2 appears to play a critical role in the hematopoietic differentiation of rESC’s. Both the development of hematoendothelial precursors and the differentiation of committed hematopoietic cell types are augmented. To study this further, the significance of FGF signaling at various stages of rESC-derived hematopoietic differentiation must be evaluated. A better understanding of the requirements for FGF-2 in EB development will likely lead to improved protocols for the production of human and rhesus ESC-derived hematopoietic progenitors.

scholarly journals Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures

Blood ◽  
2006 ◽  
Vol 108 (6) ◽  
pp. 2095-2105 ◽  
Author(s):  
Maxim A. Vodyanik ◽  
James A. Thomson ◽  
Igor I. Slukvin
Keyword(s):  
Stromal Cells ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Surface Expression ◽  
Embryonic Stem Cell Differentiation ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Development ◽  
B Lymphoid ◽  
Definitive Hematopoiesis

AbstractDuring hematopoietic differentiation of human embryonic stem cells (hESCs), early hematopoietic progenitors arise along with endothelial cells within the CD34+ population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays, their phenotype has not been defined. Here, using hESC differentiation in coculture with OP9 stromal cells, we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45, persisted on differentiating hematopoietic cells, and reliably separated the hematopoietic CD34+ population from CD34+CD43–CD31+KDR+ endothelial and CD34+CD43–CD31–KDR– mesenchymal cells. Furthermore, we demonstrated that the first-appearing CD34+CD43+CD235a+CD41a+/–CD45– cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34+CD43+CD41a–CD235a–CD45– cells. These cells were negative for lineage-specific markers (Lin–), expressed KDR, VE-cadherin, and CD105 endothelial proteins, and expressed GATA-2, GATA-3, RUNX1, C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34+CD43+CD45–Lin– cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34+CD43+CD45+Lin– cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3highGATA3lowRUNX1lowPU1highMPOhighIL7RAhigh gene expression profile.

TET2 Favors Mesoderm and Hematopoietic Differentiation in Human Embryonic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2418-2418
Author(s):  
Barbara da Costa Reis Monte Mor ◽  
Thierry Langlois ◽  
Nathalie Droin ◽  
Elodie Pronier ◽  
Jean-Pierre Le Couédic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Conflicts Of Interest ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Development ◽  
Neuronal Genes ◽  
Hes Cells

Abstract Abstract 2418 TET2 belongs to the TET family proteins that catalyze 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (hmc) and plays an important role in normal and malignant adult hematopoiesis. The role of TET2 in human hematopoietic development remains unknown. Here we show that TET2 is expressed at low level in human embryonic stem (hES) cell lines and that its expression increases during hematopoietic differentiation in three different hES. TET2 knockdown does not modify hmc level and pluripotent properties of ES cells. However TET2 depletion by two different shRNA skewed differentiation into neuroectoderm at the expense of endoderm and mesoderm. This was associated with a decrease or an increase in promoter methylation of neuroectoderm and meso/endoderm genes, respectively during hES specification. Subsequently, we observed a decrease in hematopoietic progenitors (CD34+CD43+) and their cloning capacities due to a marked increase in apoptosis. Alteration of hematopoietic differentiation was coupled with a profound alteration in gene expression with up and down regulated genes including the abnormal expression of neuronal genes in hematopoietic cells. Thus our results suggest that TET2 regulates embryonic development by inhibiting neuroectoderm specification and enabling hematopoietic differentiation in hES cells. Disclosures: No relevant conflicts of interest to declare.

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