Abstract 356: Scl Represses Cardiogenesis via Distant Enhancers during Hemogenic Endothelium Specification

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Tonis Org ◽  
Dan Duan ◽  
Roberto Ferrari ◽  
Amelie Montel-Hagen ◽  
Ben Van Handel ◽  
...  
Keyword(s):  
Target Genes ◽  
Es Cells ◽  
Bhlh Transcription Factor ◽  
Hemogenic Endothelium ◽  
Master Regulator ◽  
Critical Function ◽  
Hematopoietic Stem ◽  
Mesoderm Development ◽  
Chip Sequencing ◽  
Mouse Es Cells

Understanding the mechanisms directing mesoderm specification holds a great potential to advance the development of cell-based therapies for cardiovascular and blood disorders. The bHLH transcription factor Scl is known as the master regulator of the hematopoietic fate. We recently discovered that, in addition to its critical function in promoting the establishment of hemogenic endothelium during hematopoietic stem/progenitor cell (HS/PC) development, Scl is also required to repress cardiomyogenesis in endothelium in hematopoietic tissues and endocardium in the heart. However, the mechanisms for the cardiac repression have remained unknown. Using ChIP-sequencing and microarray analysis of Flk+ mesoderm differentiated from mouse ES cells, we show that Scl both directly activates a broad gene regulatory network required for hemogenic endothelium and HS/PC development (e.g. Runx1, cMyb, Lyl1, Mef2C, Sox7 etc.), and directly represses transcriptional regulators required for cardiogenesis (e.g. Gata4, Gata6, Myocd, etc.) and mesoderm development (Eomes, Mixl1, Etv2, etc.). Repression of cardiac and mesodermal programs occurs during a short developmental window through Scl binding to distant enhancers, while binding to hematopoietic regulators extends throughout HS/PC and red blood cell development and encompasses both distant and proximal binding sites. We also discovered that, surprisingly, Scl complex partners Gata 1 and 2 are dispensable for hematopoietic vs. cardiac specification and Scl binding to majority of its target genes. Nevertheless, Gata factors co-operate with Scl to activate selected transcription factors that facilitate HS/PC emergence from hemogenic endothelium. These results denote Scl as a true master regulator of hematopoietic vs. cardiac fate choice and suggest a mechanism by which lineage-specific bHLH factors direct the divergence of competing fates.

Latent Cardiogenic Potential in Endocardium and Hemogenic Endothelium Revealed in the Absence of Scl/tal1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2362-2362
Author(s):  
Amelie Montel-Hagen ◽  
Ben Van Handel ◽  
Roberto Ferrari ◽  
Rajkumar Sasidharan ◽  
Tonis Org ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Lineage Tracing ◽  
Bhlh Transcription Factor ◽  
Wild Type ◽  
Transcriptional Program ◽  
Hemogenic Endothelium ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 2362 The endothelium in embryonic and extraembryonic hematopoietic tissues has the capacity to generate hematopoietic stem and progenitor cells (HS/PC). However, it is unknown how this unique endothelium is specified. Microarray analysis of endothelial cells from hematopoietic tissues of embryos deficient for the bHLH transcription factor Scl/tal1 revealed that Scl establishes a robust hematopoietic transcriptional program in the endothelium. Surprisingly, lack of Scl also induced an unexpected fate switching of the prospective hemogenic endothelium to the cardiac lineage. Scl deficient embryos displayed a dramatic upregulation of cardiac transcription factors and structural proteins within the yolk sac vasculature, resulting in the generation of spontaneously beating cardiomyocytes. Ectopic cardiac potential in Scl deficient embryos arose from endothelial-derived CD31+Pdgfrα+ cardiogenic progenitor cells (CPCs), which were present in all sites of HS/PC generation. Analysis of Runx1-deficient embryos revealed, that although Runx1 acts downstream of Scl during the emergence of definitive HS/PCs, it is not required for the suppression of the cardiac fate in the endothelium. The only wild type tissue that contained CD31+Pdgfrα+ putative CPCs was the heart, and this population was greatly expanded in Scl deficient embryos. Strikingly, endocardium in Scl−/− hearts also activated a robust cardiomyogenic transcriptional program and generated Troponin T+ cardiomyocytes both in vivo and in vitro. Although CD31+Pdgfrα+ CPCs from wild type hearts did not generate readily beating cells in culture, they produced cells expressing endothelial, smooth muscle and cardiomyocyte specific genes, implying multipotentiality of this novel CPC population. Furthermore, CD31+Pdgfrα+ CPCs were greatly reduced in Isl1−/− hearts, which fail to generate functional, multipotential CPCs. Lineage tracing using VE-cadherin Cre Rosa-YFP mouse strain demonstrated that, in addition to generating HS/PCs in hematopoietic tissues, endothelial cells are also the cell of origin for CD31+Pdgfrα+ CPCs in the heart. Together, these data suggest a broader role for embryonic endothelium as a potential source of tissue-specific stem and progenitor cells and implicate Scl/tal1 as an important regulator of endothelial fate choice. Disclosures: No relevant conflicts of interest to declare.

LSD1 Plays an Important Role in GATA Switch during Erythroid Differentiation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4846-4846
Author(s):  
Yue Jin ◽  
Yidi Guo ◽  
Dongxue Liang ◽  
Yue Li ◽  
Zhe Li ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Es Cells ◽  
Regulatory Element ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Mouse Es Cells ◽  
Murine Erythroleukemia

Abstract GATA factors play important role in hematopoiesis. In particular, GATA2 is critical for maintenance of hematopoietic stem and progenitor cells (HS/PCs) and GATA1 is required for erythropoiesis. GATA1 and GATA2 are expressed in reciprocal patterns during erythroid differentiation. It was shown that GATA1 occupied the -2.8Kb regulatory element and mediated repression of the GATA2 promoter in terminally differentiating erythroid cells. However, the detailed molecular mechanisms that control the enhancer/promoter activities of the GATA2 gene remain to be elucidated. In this report, we found that LSD1 and TAL1 co-localize at GATA2 1S promoter through ChIP and double-ChIP assays in murine erythroleukemia (MEL) cells. To further test whether LSD1 and its mediated H3K4 demethylation is important for repression of the GATA2 gene during erythroid differentiation, we silenced LSD1 expression in both MEL cells and mouse ES cells using retrovirus mediated shRNA knockdown and induced them to differentiate into erythroid cells with DMSO and EPO, respectively. GATA2 expression was elevated while the level of GATA1 was repressed by RT-qPCR. Furthermore, consistent with the GATA witch hypothesis, ChIP analysis revealed that the levels of H3K4me2 were increased at the GATA2 1S promoter.  In addition, knock-down of LSD1 in MEL cells results in inhibition of erythroid cell differenciation and attenuation of MEL cell proliferation and survival. Thus, our data reveal that LSD1 involved in control of terminal erythroid differentiation by regulating GATA switch. The LSD1 histone demethylase complex may be recruited to the GATA2 1S promoter by interacting with TAL1. The H3K4 demethylation activity of LSD1 leads to downregulation of the active H3K4m2 mark at the GATA2 promoter that alters chromatin structure and represses transcription of the GATA2 genes. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells Via Transcription Factor Conversion of Hemogenic Endothelium

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 371-371
Author(s):  
Ryohichi Sugimura ◽  
Areum Han ◽  
Deepak Jha ◽  
Yi-Fen Lu ◽  
Jeremy A Goettel ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Progenitor Cells ◽  
Pluripotent Stem Cells ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Functional Characterization ◽  
Hemogenic Endothelium ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract A variety of tissues can be differentiated from pluripotent stem cells (PSCs) in vitro through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors (TFs). Despite considerable effort, neither approach has yielded functional human hematopoietic stem cells (HSCs). Building upon recent evidence that HSCs derive from definitive hemogenic endothelium (HE), we performed morphogen-directed differentiation of human PSCs into HE followed by screening of 26 candidate HSC-specifying TFs for the capacity to promote multi-lineage hematopoietic engraftment in irradiated immune deficient murine hosts. From genomic PCR of engrafted cells, we recovered seven TFs (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1) that were sufficient to convert HE into hematopoietic stem and progenitor cells (HSPCs) that engraft GLY-A+ erythrocytes, CD33+ myeloid, CD15+ CD31+ neutrophils, CD19+ IgM+ B and CD3+ T cells in primary and secondary murine recipients for 12-14 weeks. Limiting dilution analysis indicated that the frequency of repopulating cells generated by this method was 1 in 4,707-15,029, lower than the frequency in CD34+ cord blood cells (1 in 1,819-5,173). Functional characterization of terminally differentiated cells demonstrated features of definitive erythropoiesis (expression of adult beta globin and enucleation). Engrafted neutrophils responded to cytokine stimuli by activation of myeloperoxidase. Human IgM and IgG could be detected in the serum of engrafted mice, and titers of ovalbumin specific antibody increased in response to protein immunization, indicating boostable immunity. T-cells responded to PMA/Ionomycin stimuli by activation of IFNγ, and sequencing of the T cell receptor revealed a broad clonotype diversity. Proviral integration analysis demonstrated derivation of myeloid and lymphoid progeny from common clones in secondary animals, indicating generation of self-renewing, multipotential HSC-like cells from PSCs. Mechanistically, the seven TFs induced HOXA target genes (LMO2, SOX4, MEIS1 and ID2); upregulated expression of homing-related genes (CXCR4, VLA5 and S1PR1); and enhanced the endothelial to hematopoietic transition (EHT), as indicated by a 2.4-fold induction of a RUNX1c-reporter. Our combined approach of morphogen-driven differentiation and TF-mediated cell fate conversion produced HSPCs from PSCs that hold promise for modeling hematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ten-Eleven Translocation 1 (Tet1) Is Regulated by O-Linked N-Acetylglucosamine Transferase (Ogt) for Target Gene Repression in Mouse Embryonic Stem Cells

2013 ◽  
Vol 288 (29) ◽  
pp. 20776-20784 ◽  
Author(s):  
Feng-Tao Shi ◽  
Hyeung Kim ◽  
Weisi Lu ◽  
Quanyuan He ◽  
Dan Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Target Genes ◽  
Es Cells ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Mouse Es Cells ◽  
Chromatin Regulators ◽  
Glcnac Transferase

As a member of the Tet (Ten-eleven translocation) family proteins that can convert 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5hmC), Tet1 has been implicated in regulating global DNA demethylation and gene expression. Tet1 is highly expressed in embryonic stem (ES) cells and appears primarily to repress developmental genes for maintaining pluripotency. To understand how Tet1 may regulate gene expression, we conducted large scale immunoprecipitation followed by mass spectrometry of endogenous Tet1 in mouse ES cells. We found that Tet1 could interact with multiple chromatin regulators, including Sin3A and NuRD complexes. In addition, we showed that Tet1 could also interact with the O-GlcNAc transferase (Ogt) and be O-GlcNAcylated. Depletion of Ogt led to reduced Tet1 and 5hmC levels on Tet1-target genes, whereas ectopic expression of wild-type but not enzymatically inactive Ogt increased Tet1 levels. Mutation of the putative O-GlcNAcylation site on Tet1 led to decreased O-GlcNAcylation and level of the Tet1 protein. Our results suggest that O-GlcNAcylation can positively regulate Tet1 protein concentration and indicate that Tet1-mediated 5hmC modification and target repression is controlled by Ogt.

scholarly journals Thrombopoietin (TPO) Induces Hematopoietic Differentiation of Mouse ES Cells Via HIF-1 Alpha-Dependent Activation of a BMP4 Autoregulatory Loop

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1186-1186
Author(s):  
Azadeh Zahabi ◽  
Tatsuya Morishima ◽  
Andri Pramono ◽  
Dan Lan ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Es Cells ◽  
Embryonic Stem ◽  
Efficient Production ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem

Abstract Understanding the molecular mechanisms underlying hematopoietic differentiation of embryonic stem (ES) cells may help to ascertain the optimal conditions for the production of hematopoietic cells as a source for transplantation or experimental use. Previously, we found that patients with congenital amegakaryocytic thrombocytopenia (CAMT), who develop pancytopenia early after birth, harbor mutations within the thrombopoietin (TPO) receptor, c-mpl. This knowledge, together with observations in vitro and in animal models in vivo, suggests that TPO/c-mpl signaling promotes early hematopoiesis. However, the downstream mechanisms underlying TPO signaling are not fully elucidated. Here, we describe for the first time a direct connection between the TPO and bone morphogenetic protein 4 (BMP4) signaling pathways in the hematopoietic fate decision of ES cells. BMP4 is a classical morphogen and a well-known inducer of early hematopoietic differentiation of ES cells. Treatment of ES cells with TPO induced the autocrine production of BMP4 by ES cells with concomitant upregulation of the BMP receptor, BMPR1A, phosphorylation of Smad1, 5, and 8 and activation of the specific target genes, Id1, 2, and 3, and Msx1 and 2. This was mediated by TPO-dependent binding of the HIF-1α transcription factor to the BMP4 gene promoter, resulting in further activation of the BMP4-autoregulatory loop. Treatment of ES cells with the BMP antagonist noggin substantially reduced TPO-dependent hematopoietic differentiation of ES cell. Taken together, our findings contribute to the understanding the mechanisms of hematopoietic differentiaiton of ES cells and might help to establish new methods for the efficient production of hematopoietic stem cells in vitro. Disclosures No relevant conflicts of interest to declare.

Induction and Patterning of Pre-Hemato-Vascular Mesoderm by the Mouse Mix Homeodomain Protein.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 565-565
Author(s):  
Margaret H. Baron ◽  
Stephen Willey ◽  
Kenneth Sahr ◽  
Hailan Zhang ◽  
Kevin Balbi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Target Genes ◽  
Es Cells ◽  
Ectopic Expression ◽  
Embryoid Bodies ◽  
Homeodomain Protein ◽  
Stem Cell Markers ◽  
Es Cell ◽  
Hematopoietic Stem

Abstract Members of the Xenopus and zebrafish Mix/Bix family of paired class homeodomain proteins play determining roles in both mesoderm and endoderm development and are induced by members of the TGFbeta/BMP family of signaling molecules. A single Mix gene has been identified in mouse, humans and chick. Prior to gastrulation, the mouse Mix (mMix) gene is expressed in the visceral endoderm and later in the primitive streak and nascent mesoderm, where it overlaps, in part, with T. Mix expression in ES-derived embryoid bodies is early and transient, overlapping partially with Flk1 activation around the time of formation of hemangioblasts. Both mMix mRNA and protein are found in a FACS-sorted population of T+Flk1+ cells from ES cell-derived embryoid bodies (EBs) which contains hemangioblasts. A complex embryonic lethal phenotype has been reported for Mix deficient embryos, including defects in allantoic (vascular) and cardiogenesis. Mesoderm forms in these embryos but is not patterned properly. Embryonic lethality occurs around E10.5–11, presumably as a result of the cardiovascular defects. We have generated inducible ES cell lines in which expression of Mix protein is responsive to doxycycline. Ectopic expression of Mix in EBs results in premature, enhanced expression of hemangioblast, angioblast and hematopoietic stem cell markers (mRNA and FACS analyses) and increased formation of stem/progenitor cells in clonogenic assays in methylcellulose. Together, the expression analyses, knockout phenotype, and gain-of-function studies in ES cells suggest that mMix functions early in induction and patterning of mesoderm, including formation of hematopoietic and endothelial lineages. Potential mMix target genes are being identified by microarray analyses of the inducible Mix ES lines. To examine mMix activities in vivo, we have generated null and conditional mMix knockout mice from several independently targeted ES cell lines. Analysis of these animals is in progress. Like Xenopus Mix.1, mouse Mix may represent an important connection between the TGFbeta/BMP pathway and hematopoietic/vascular development in the embryo.

LSD1-Mediated Epigenetic Modification Is Important for TAL1 Function

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4757-4757
Author(s):  
Xin Hu ◽  
Xingguo Li ◽  
River Ybarra ◽  
Kristell Valverde ◽  
Xueqi Fu ◽  
...  
Keyword(s):  
Target Genes ◽  
Protein Complexes ◽  
Epigenetic Modification ◽  
Es Cells ◽  
Gene Promoter ◽  
Erythroid Cell ◽  
Hematopoietic Stem ◽  
Target Gene Promoter ◽  
Cell Growth And Differentiation ◽  
Erythroid Cell Differentiation

Abstract TAL1/SCL is critical for normal and abnormal hematopoiesis by regulating hematopoietic stem/progenitor cell growth and differentiation. However, it is still unclear how its transcriptional activities are controlled during hematopoiesis. Here, we undertook the biochemical isolation of TAL1-associated protein complexes in erythroleukemia cells and showed that TAL1 interacts with histone demethylase LSD1 complexes containing LSD1, CoREST, HDAC1 and HDAC2. Interestingly, although TAL1 specifically colocalizes with LSD1 at the target gene promoter p4.2 in undifferentiated MEL cells, the recruitment of LSD1 is decreased at the p4.2 promoter upon induced MEL differentiation indicating that LSD1 may differentially regulate TAL1 target genes during differentiation. The siRNA-mediated knockdown of LSD1 in MEL and ES cells resulted in the derepression of p4.2 by increasing dimeH3K4 at their promoter region, respectively. Finally, we demonstrated that TAL1-associated LSD1 complexes, H3K4 demethylase, and histone deacetylase activities are coordinately regulated during erythroid cell differentiation. Thus, the data suggest that LSD1 mediated epigenetic modification may affect hematopoiesis and leukemogenesis through its association with the lineage-specific transcription factor TAL1.

Expression Cloning of Genes Enabling Erythropoietin -Independent Erythropoiesis in Vitro.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3608-3608
Author(s):  
Tomoko Yokoo ◽  
Ryo Kurita ◽  
Atsushi Takahashi ◽  
Michiyo Okada ◽  
Hirotaka Kawano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Apoptotic Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Expression Cloning ◽  
Hematopoietic Stem

Abstract Abstract 3608 Poster Board III-544 Erythropoietin (EPO) is essential during both ontogeny and the course of erythropoiesis. In primitive (yolk sac) erythropoiesis, however, the role of EPO is not fully understood. Elucidation of such role in primitive erythropoiesis would be very helpful for the development of ex vivo red blood cell expansion system from embryonic stem (ES) cells. Recently, we reported the establishment of the ex vivo induction of hematopoietic stem cells from common marmoset ES cells using lentiviral-Tal1/Scl gene transfer in the absence of any stromal cells (Stem Cells 24: 2014-2022, 2006). This method should also be applicable to both of human ES cells and induced pluripotential stem (iPS) cells, but the efficiency will be very critical for its clinical application. In the present study, we proceed further to find out unknown factors which accelerate ex vivo proliferation and differentiation of erythroid cells, constructed the human fetal liver cDNA expression lentiviral library (Mol Cell Biochem 319: 181-187, 2008) and screened for cDNAs which confer EPO independency to an EPO-dependent cell line, UT-7/Epo ( Blood 82: 456-464, 1993). Among twenty-two candidate genes cloned after screening of 6×10∧5 cDNA, we particularly focused on two full-length genes, ribosomal protein L11 (RPL11) and retinol dehydrogenase 11 (RDH11). Two candidate gene-transduced-UT-7/Epo cells, respectively named LV-RPL11 and LV-RDH11, showed complete EPO-independent survival and proliferation, increased expression of fetal γ-globin, and decreased expression of adult β-globin compared with parental UT-7/Epo cells in the presence of EPO. Cell cycle and apoptosis analyses showed decreased apoptotic cell death and increased S/G2/M cells in LV-RPL11 and LV-RDH11 cells compared with UT-7/Epo cells in the absence of Epo. Moreover, STAT5 phosphorylation and upregulation of its target genes, c-Myc, cyclin D and Pim, were observed in LV-RPL11, LV-RDH11 cells in the absence of EPO. In conclusion, the findings suggest that RPL11 and RDH11 play a role in EPO-independent erythropoiesis and might be applicable to ex vivo expansion of red blood cells from ES/iPS cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Defining Essential Enhancer for Pluripotent stem cells using Features Oriented CRISPR-Cas9 Screen

2019 ◽  
Author(s):  
Hao Fei Wang ◽  
Tushar Warrier ◽  
Chadi EL Farran ◽  
Zheng Zihao ◽  
Qiao Rui Xing ◽  
...  
Keyword(s):  
Cell Fate ◽  
Target Genes ◽  
Es Cells ◽  
Functional Characterization ◽  
Regulatory Elements ◽  
Downstream Target ◽  
Mouse Es Cells ◽  
Cellular Processes ◽  
Stat3 Signaling ◽  
Stat3 Signaling Pathway

ABSTRACTCis Regulatory Elements (CREs) regulate the expression of the genes in their genomic neighborhoods and influence cellular processes such as cell-fate maintenance and differentiation. To date, there remain major gaps in the functional characterization of CREs and the identification of its target genes in the cellular native environment. In this study, we performed a Features Oriented CRISPR Utilized Systematic (FOCUS) screen of OCT4-bound CREs using CRISPR/Cas9 to identify functional enhancers important for pluripotency maintenance in mouse ES cells. From the initial 235 candidates tested, 16 CREs were identified to be essential stem cell enhancers. Using RNA-seq and genomic 4C-seq, we further uncovered a complex network of candidate CREs and their downstream target genes, which supports the growth and self-renewal of mESCs. Notably, an essential enhancer, CRE111, and its target, Lrrc31, form the important switch to modulate the LIF-JAK1-STAT3 signaling pathway.

Developmental Potentials of Human Embryonic Stem Cells Lacking PIG-A and GPI-Anchored Proteins.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1314-1314
Author(s):  
Guibin Chen ◽  
Zhaohui Ye ◽  
Xiaobing Yu ◽  
Robert A. Brodsky ◽  
Linzhao Cheng
Keyword(s):  
Stem Cells ◽  
Cell Surface ◽  
Es Cells ◽  
Surface Expression ◽  
Embryoid Bodies ◽  
Cell Surface Expression ◽  
Hematopoietic Stem ◽  
Body Formation ◽  
Mouse Es Cells ◽  
Hes Cells

Abstract Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal disorder of hematopoietic stem cells (HSCs) acquiring mutations in the PIG-A gene. Its product (PIG-A protein) is required for biosynthesis of dozens of cell surface proteins to be linked to glycosyl-phosphatidyl-inositol (GPI) molecule and anchored on plasma membrane. The underlying mechanisms of PIG-A mutant clonal dominance in PNH patients and the close relationship of PNH to other bone marrow failure diseases (aplastic anemia and myelodysplasia syndrome) and leukemia remain unknown. Establishing a mutation of PIG-A gene in human HSCs from healthy donors remains unfeasible due to the current inability to expand and select clonal HSCs in culture. Although mouse models have been generated by disrupting conditionally the mouse Pig-a gene, the existing Pig-a null mice indeed lacking GPI-APs in blood cells did not replicate faithfully PNH pathological symptoms. To create a human cell-based, prospective experimental system to investigate the effects of PIG-A/GPI-AP deficiency and pathophysiology of PNH, we made mutated hES cells lacking GPI-APs. FACS analysis revealed that two independent hES clones lack cell-surface expression of CD55 and CD59, as well as CD90/Thy and Cripto that are preferentially expressed in undifferentiated hES cells. However, the cell-surface expression of these GPI-APs can be restored by a lentiviral vector inducibly expressing the PIG-A cDNA. Like mouse ES cells, lack of PIG-A/GPI-APs did not affect the growth of undifferentiated hES cells in culture. Unlike mouse ES cells, however, PIG-A/GPI-AP deficient hES cells formed embryoid bodies normally in culture. RT-PCR analysis of marker gene expression indicated that commitment to the 3 (somatic) germ layers appeared normal within embryonic bodies from either the mutated or wildtype hES cells. However, formation of extra-embryonic cells such as trophoblasts from the PIG-A/GPI-AP deficient hES cells is defective in both embryoid body formation and BMP4-induced assays. Expression of trophoectoderm-specific genes such as hCGalpha could not be induced in PIG-A/GPI-AP deficient hES cells upon BMP4 induced trophoectodermal differentiation. The induction of other trophoectoderm markers such as hCGbeta and CDX2 was also significantly reduced after the BMP4 treatment. The lack of tropho-ectoderm was further confirmed by lacking of hCG hormone production. The defect in trophoectoderm differentiation from the PIG-A/GPI-AP deficient hES cells was restored by the expression of the PIG-A cDNA in the mutated hES cells. For somatic cell differentiation, we are currently examining the effects of PIG-A/GPI-AP deficiency beyond the initial differentiation commitment during embryonic body formation stage. Along mesodermal and hematopoietic differentiation, we found that the PIG-A/GPI-AP deficiency in hES cells had little effect on the formation of CD34+ cells, a precursor cell population for human hematopoietic and endothelial lineages. We are currently examining the effects of PIG-A/GPI-AP deficiency on properties of hematopoietic cells derived from the mutated and normal hES cells. This study represents one of first cases that hES cells may provide a prospective research model to investigate genetic and developmental basis of human diseases.

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