Derivation of Hematopoietic Stem Cells from Embryonic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 223-223 ◽  
Author(s):  
Yuan Wang ◽  
Frank Yates ◽  
Eugenia Dikovskaia ◽  
Patricia Ernst ◽  
Alan J. Davidson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hox Genes ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Lymphoid Cells ◽  
Time Interval ◽  
Hematopoietic Stem

Abstract Despite the significant in vitro blood-forming potential of murine embryonic stem cells (ESCs), deriving hematopoietic stem cells (HSCs) that can reconstitute irradiated mice has proven to be challenging. Previously, we successfully engrafted lethally irradiated adult mice with ESCs engineered to ectopically express the homeodomain gene hoxB4. In engrafted animals, blood reconstitution showed a myeloid predominance, likely due to an inability to fully pattern the adult HSC from these embryonic populations. Recently, we have investigated cdx4, a caudal-related homeobox gene whose function has been linked to blood development in the zebrafish. During in vitro differentiation of murine ESCs, cdx4 is expressed during a very narrow time interval on day 3, coincident with the specification of hematopoietic mesoderm. To further characterize the function of cdx4 in mouse hematopoiesis, we have established a tetracycline-inducible murine embryonic stem cell line. When cdx4 expression is conditionally induced over a protracted period from day 2 and 6, we observe a marked enhancement of hemangioblast formation as well as significant increases in primitive and definitive hematopoietic colonies. Cdx4 acts to induce a broad array of hox genes, including a modest elevation in hoxb4. Co-expression of cdx4 and hoxb4 promotes robust expansion of hematopoietic blasts on supportive OP9 stromal cultures. When injected intravenously into lethally-irradiated mice, these cell populations provide robust radio-protection, and reconstitute high-level lymphoid-myeloid donor chimerism. Marrow from engrafted primary animals can be transplanted into irradiated secondary mice. B220+ splenic lymphoid cells and Mac-1/Gr-1+ marrow myeloid cells purified from primary and secondary mice show multiple common sites of retroviral integration, thereby proving the derivation of long-term hematopoietic stem cells from embryonic stem cells in vitro. Our data support a central role for the cdx4-hox gene pathway in specifying murine HSC development, and establish a robust system for hematopoietic reconstitution from ESCs. We have coupled techniques for generating ESCs by nuclear transfer with these methods for blood reconstitution to model the treatment of genetic disorders of the bone marrow.

scholarly journals Surface antigen phenotypes of hematopoietic stem cells from embryos and murine embryonic stem cells

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 268-278 ◽  
Author(s):  
Shannon L. McKinney-Freeman ◽  
Olaia Naveiras ◽  
Frank Yates ◽  
Sabine Loewer ◽  
Marsha Philitas ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Embryonic Stem ◽  
Hematopoietic Stem ◽  
Murine Embryonic Stem Cells ◽  
Isolation And Characterization

Abstract Surface antigens on hematopoietic stem cells (HSCs) enable prospective isolation and characterization. Here, we compare the cell-surface phenotype of hematopoietic repopulating cells from murine yolk sac, aorta-gonad-mesonephros, placenta, fetal liver, and bone marrow with that of HSCs derived from the in vitro differentiation of murine embryonic stem cells (ESC-HSCs). Whereas c-Kit marks all HSC populations, CD41, CD45, CD34, and CD150 were developmentally regulated: the earliest embryonic HSCs express CD41 and CD34 and lack CD45 and CD150, whereas more mature HSCs lack CD41 and CD34 and express CD45 and CD150. ESC-HSCs express CD41 and CD150, lack CD34, and are heterogeneous for CD45. Finally, although CD48 was absent from all in vivo HSCs examined, ESC-HSCs were heterogeneous for the expression of this molecule. This unique phenotype signifies a developmentally immature population of cells with features of both primitive and mature HSC. The prospective fractionation of ESC-HSCs will facilitate studies of HSC maturation essential for normal functional engraftment in irradiated adults.

BMP4 Favors Hematopoietic Differentiation from Murine Embryonic Stem Cells and Acts by Activation of the cdx-hox Pathway.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3613-3613
Author(s):  
Claudia Lengerke ◽  
Yuan Wang ◽  
Frank Yates ◽  
Leila Maouche-Chretien ◽  
George Q. Daley
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hox Genes ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Hematopoietic Stem ◽  
Hox Gene ◽  
Murine Embryonic Stem Cells

Abstract Cdx4 and cdx1, members of the caudal family of homeodomain-containing transcriptional regulators, are important for specifying the hematopoietic fate of mesoderm in the zebrafish. We have shown that the cdx4 gene plays a role in enhancing hematopoietic fate during in vitro differentiation of murine ESCs (Davidson et al., Nature 2003). Cdx4 induces hox genes, and genetic modification of mESCs with a combination of cdx4 and hoxb4 promotes long-term engraftment of ESC-derived HSCs in lethally irradiated primary and secondary mice (Wang et al, submitted). While cdx1 is known to be a direct target of signaling by the embryonic morphogens fgf, wnt3a, and retinoids, morphogens acting upstream of cdx4 have not yet been defined. Our goal is to determine optimal morphogen conditions for hematopoietic commitment from murine embryonic stem cells by evaluating activation of the cdx-hox pathway. We have developed quantitative RT-PCR assays for the cdx genes (cdx4, cdx1 and cdx2) and multiple hox genes as well as markers specific to hematopoietic stem cells and lineages. We have used these assays, together with a reporter line engineered to express GFP from the brachury locus (Fehling et al., Development 2003), to characterize the conditions for mesodermal induction and hematopoietic fate specification following addition of morphogens to differentiating cultures of ES cells under serum-free conditions. Among all morphogens tested (BMP4, activin, nodal, wnt3a, wnt5a, sonic hedgehog, indian hedgehog, retinoic acid), only BMP4 has been found to strongly induce CDX4 gene expression within the developing embryoid bodies, while addition of the BMP4 inhibitor noggin to serum suppressed CDX4 expression. Addition of BMP4 significantly increases the number of emerging CD41+ and CD45+ cells, the precursors of definitive hematopoietic stem cells. We are currently analyzing the functional changes following BMP4 exposure, and correlating hematopoietic maturation with changes in the Hox gene expression pattern. Analysis of the cdx-hox gene pathway provides a means of otpimizing induction of hematopoietic fate by application of embryonic morphogens.

scholarly journals B-1 lymphoid cells develop independently of Notch signaling during mouse embryonic development

2020 ◽  
Author(s):  
Nathalia Azevedo ◽  
Elisa Bertesago ◽  
Ismail Ismailoglu ◽  
Michael Kyba ◽  
Michihiro Kobayashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Development ◽  
Blood Cell ◽  
Notch Signaling ◽  
Embryonic Stem ◽  
Cell Types ◽  
Lymphoid Cells ◽  
Lineage Specification ◽  
Hematopoietic Stem

AbstractThe in vitro generation from pluripotent stem cells (PSCs) of different blood cell types, in particular those that are not replenished by hematopoietic stem cells (HSCs) like fetal-derived tissue-resident macrophages and innate-like lymphocytes, is of a particular interest. In order to succeed in this endeavor, a thorough understanding of the pathway interplay promoting lineage specification for the different blood cell types is needed. Notch signaling is essential for the HSC generation and their derivatives, but its requirement for tissue-resident immune cells is unknown. Using mouse embryonic stem cells (mESCs) to recapitulate murine embryonic development, we have studied the requirement for Notch signaling during the earliest B-lymphopoiesis and found that Rbpj-deficient mESCs are able to generate B-1 cells. Their Notch-independence was confirmed in ex vivo experiments using Rbpj-deficient embryos. In addition, we found that upregulation of Notch signaling was needed for the emergence of B-2 lymphoid cells. Taken together, these findings indicate that control of Notch signaling dosage is critical for the different B-cell lineage specification and provides pivotal information for their in vitro generation from PSCs for therapeutic applications.

Effect of NANOG overexpression on porcine embryonic development and pluripotent embryonic stem cell formation in vitro

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Gerelchimeg Bou ◽  
Shimeng Guo ◽  
Jia Guo ◽  
Zhuang Chai ◽  
Jianchao Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Embryonic Stem Cell ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Cell Clones ◽  
Morula Stage ◽  
Pluripotent Embryonic Stem Cell

Summary The efficiency of establishing pig pluripotent embryonic stem cell clones from blastocysts is still low. The transcription factor Nanog plays an important role in maintaining the pluripotency of mouse and human embryonic stem cells. Adequate activation of Nanog has been reported to increase the efficiency of establishing mouse embryonic stem cells from 3.5 day embryos. In mouse, Nanog starts to be strongly expressed as early as the morula stage, whereas in porcine NANOG starts to be strongly expressed by the late blastocyst stage. Therefore, here we investigated both the effect of expressing NANOG on porcine embryos early from the morula stage and the efficiency of porcine pluripotent embryonic stem cell clone formation. Compared with intact porcine embryos, NANOG overexpression induced a lower blastocyst rate, and did not show any advantages for embryo development and pluripotent embryonic stem cell line formation. These results indicated that, although NANOG is important pluripotent factor, NANOG overexpression is unnecessary for the initial formation of porcine pluripotent embryonic stem cell clones in vitro.

scholarly journals Stepwise Development of Hematopoietic Stem Cells from Embryonic Stem Cells

PLoS ONE ◽  
2009 ◽  
Vol 4 (3) ◽  
pp. e4820 ◽  
Author(s):  
Kenji Matsumoto ◽  
Takayuki Isagawa ◽  
Toshinobu Nishimura ◽  
Takunori Ogaeri ◽  
Koji Eto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Embryonic Stem ◽  
Hematopoietic Stem ◽  
Stepwise Development

scholarly journals Age-Associated Characteristics of Murine Hematopoietic Stem Cells

2000 ◽  
Vol 192 (9) ◽  
pp. 1273-1280 ◽  
Author(s):  
Kazuhiro Sudo ◽  
Hideo Ema ◽  
Yohei Morita ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Lymphoid Cells ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Functional Changes

Little is known of age-associated functional changes in hematopoietic stem cells (HSCs). We studied aging HSCs at the clonal level by isolating CD34−/lowc-Kit+Sca-1+ lineage marker–negative (CD34−KSL) cells from the bone marrow of C57BL/6 mice. A population of CD34−KSL cells gradually expanded as age increased. Regardless of age, these cells formed in vitro colonies with stem cell factor and interleukin (IL)-3 but not with IL-3 alone. They did not form day 12 colony-forming unit (CFU)-S, indicating that they are primitive cells with myeloid differentiation potential. An in vivo limiting dilution assay revealed that numbers of multilineage repopulating cells increased twofold from 2 to 18 mo of age within a population of CD34−KSL cells as well as among unseparated bone marrow cells. In addition, we detected another compartment of repopulating cells, which differed from HSCs, among CD34−KSL cells of 18-mo-old mice. These repopulating cells showed less differentiation potential toward lymphoid cells but retained self-renewal potential, as suggested by secondary transplantation. We propose that HSCs gradually accumulate with age, accompanied by cells with less lymphoid differentiation potential, as a result of repeated self-renewal of HSCs.

scholarly journals Gfer inhibits Jab1-mediated degradation of p27kip1 to restrict proliferation of hematopoietic stem cells

2011 ◽  
Vol 22 (8) ◽  
pp. 1312-1320 ◽  
Author(s):  
Ellen C. Teng ◽  
Lance R. Todd ◽  
Thomas J. Ribar ◽  
William Lento ◽  
Leah Dimascio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Adult Stem Cells ◽  
Kinase Inhibitor ◽  
Embryonic Stem ◽  
Mitochondrial Morphology ◽  
In Vitro Proliferation ◽  
Hematopoietic Stem

Growth factor erv1-like (Gfer) is an evolutionarily conserved sulfhydryl oxidase that is enriched in embryonic and adult stem cells and plays an essential prosurvival role in pluripotent embryonic stem cells. Here we show that knockdown (KD) of Gfer in hematopoietic stem cells (HSCs) compromises their in vivo engraftment potential and triggers a hyper-proliferative response that leads to their exhaustion. KD of Gfer in HSCs does not elicit a significant alteration of mitochondrial morphology or loss of cell viability. However, these cells possess significantly reduced levels of the cyclin-dependent kinase inhibitor p27kip1. In contrast, overexpression of Gfer in HSCs results in significantly elevated total and nuclear p27kip1. KD of Gfer results in enhanced binding of p27kip1 to its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), leading to its down-regulation. Conversely, overexpression of Gfer results in its enhanced binding to Jab1 and inhibition of the Jab1-p27kip1 interaction. Furthermore, normalization of p27kip1 in Gfer-KD HSCs rescues their in vitro proliferation deficits. Taken together, our data demonstrate the presence of a novel Gfer-Jab1-p27kip1 pathway in HSCs that functions to restrict abnormal proliferation.

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