scholarly journals Acceleration of mesoderm development and expansion of hematopoietic progenitors in differentiating ES cells by the mouse Mix-like homeodomain transcription factor

Blood ◽  
2006 ◽  
Vol 107 (8) ◽  
pp. 3122-3130 ◽  
Author(s):  
Stephen Willey ◽  
Angel Ayuso-Sacido ◽  
Hailan Zhang ◽  
Stuart T. Fraser ◽  
Kenneth E. Sahr ◽  
...  
Keyword(s):  
Es Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitors ◽  
Adult Type ◽  
Mesoderm Development ◽  
Molecular Events ◽  
Organ Systems

Abstract The cellular and molecular events underlying the formation and differentiation of mesoderm to derivatives such as blood are critical to our understanding of the development and function of many tissues and organ systems. How different mesodermal populations are set aside to form specific lineages is not well understood. Although previous genetic studies in the mouse embryo have pointed to a critical role for the homeobox gene Mix-like (mMix) in gastrulation, its function in mesoderm development remains unclear. Hematopoietic defects have been identified in differentiating embryonic stem cells in which mMix was genetically inactivated. Here we show that conditional induction of mMix in embryonic stem cell–derived embryoid bodies results in the early activation of mesodermal markers prior to expression of Brachyury/T and acceleration of the mesodermal developmental program. Strikingly, increased numbers of mesodermal, hemangioblastic, and hematopoietic progenitors form in response to premature activation of mMix. Differentiation to primitive (embryonic) and definitive (adult type) blood cells proceeds normally and without an apparent bias in the representation of different hematopoietic cell fates. Therefore, the mouse Mix gene functions early in the recruitment and/or expansion of mesodermal progenitors to the hemangioblastic and hematopoietic lineages.

scholarly journals The homeobox gene Hhex regulates the earliest stages of definitive hematopoiesis

Blood ◽  
2010 ◽  
Vol 116 (8) ◽  
pp. 1254-1262 ◽  
Author(s):  
Helicia Paz ◽  
Maureen R. Lynch ◽  
Clifford W. Bogue ◽  
Judith C. Gasson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Molecular Events ◽  
G2 Phase ◽  
Definitive Hematopoiesis

Abstract The development and emergence of the hematopoietic stem cell involves a series of tightly regulated molecular events that are not well characterized. The hematopoietically expressed homeobox (Hhex) gene, a member of the homeobox gene family, is an essential regulator of embryogenesis and hematopoietic progenitor development. To investigate the role of Hhex in hematopoiesis we adapted a murine embryonic stem (ES) cell coculture system, in which ES cells can differentiate into CD41+ and CD45+ hematopoietic progenitors in vitro. Our results show that in addition to delayed hemangioblast development, Hhex−/− ES-derived progeny accumulate as CD41+ and CD41+c-kit+ cells, or the earliest definitive hematopoietic progenitors. In addition, Hhex−/− ES-derived progeny display a significantly reduced ability to develop into mature CD45+ hematopoietic cells. The observed reduction in hematopoietic maturation was accompanied by reduced proliferation, because Hhex−/− CD41+CD45−c-kit+ hematopoietic progenitors accumulated in the G2 phase of the cell cycle. Thus, Hhex is a critical regulator of hematopoietic development and is necessary for the maturation and proliferation of the earliest definitive hematopoietic progenitors.

Modulation of hematopoietic and endothelial cell differentiation from mouse embryonic stem cells by different culture conditions

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 111-114 ◽  
Author(s):  
Wen Jie Zhang ◽  
Changwon Park ◽  
Elizabeth Arentson ◽  
Kyunghee Choi
Keyword(s):  
Endothelial Cell ◽  
Cell Differentiation ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Embryoid Bodies ◽  
Vascular Endothelial ◽  
Hematopoietic Progenitors ◽  
Endothelial Cell Differentiation

Abstract Embryonic stem (ES) cells can differentiate into many different somatic cells in culture. To better correlate hematopoietic and endothelial cell differentiation of ES cells in currently available protocols, we compared fetal liver kinase-1 (Flk-1)–, stem cell leukemia (Scl)–, and vascular endothelial–cadherin (VE-cadherin)–expressing cells generated in embryoid bodies (EBs) and on OP9 cells. We report that the kinetics of Scl and Flk-1 expression were similar in EBs and OP9 cells, although Flk-1 expression was extended on OP9 cells. CD45+ and Ter-119+ cells developed more efficiently in EBs, whereas VE-cadherin+ cells developed largely on OP9 cells. Cell sorting and replating studies showed that Scl+ cells, not Flk-1+ or VE-cadherin+ cells, were enriched for primitive and definitive hematopoietic progenitors. Our studies indicate that optimal hematopoietic and endothelial cell differentiation occur in EBs and on OP9 cells, respectively. Regardless of the culture systems used, Scl is the most relevant marker for enriching primitive and definitive hematopoietic progenitors.

Endoglin Is Required for Hemangioblast Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 138-138 ◽  
Author(s):  
Rita R. Perlingeiro
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Colony Formation ◽  
Time Course ◽  
Es Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Wild Type

Abstract A critical role for endoglin (CD105) in early development has been demonstrated in mice deficient for this gene. Embryos homozygous for the endoglin mutation (eng−/−) fail to progress beyond 10.5 days postcoitum due primarily to vascular and cardiac abnormalities (Bordeau et al, 1999). Analysis of 9.5 dpc eng−/− embryos revealed abnormal vasculature and anemia of the yolk sac, suggesting that endoglin may be required for both blood and endothelial lineages. The hemangioblast, the bipotent precursor for hematopoietic and endothelial cells, can be assessed through the blast colony assay (BL-CFC) using a model system based on the in vitro differentiation of embryonic stem (ES) cells into embryoid bodies (EBs). To evaluate a role for endoglin in this early precursor, we differentiated eng−/−, eng+/−, and eng+/+ (wild-type) ES cells into EBs. At day 3 of EB differentiation, cells were disrupted and plated for blast colony formation in methylcellulose media containing vascular endothelial growth factor (VEGF), stem cell factor (SCF), and thrombopoietin (TPO). We found no difference in blast colony formation between heterozygous and wild-type ES cells. However, a significant reduction in the number of BL-CFCs was observed in eng−/− cells when compared to eng+/− or eng+/+ BL-CFCs (p < 0.001). Single eng−/−, eng+/−, and eng+/+ BL-CFCs gave rise to secondary hematopoietic colonies as well as endothelial cells, confirming their nature as hemangioblasts. These results suggest that although endoglin is required for hemangioblast development, its absence does not affect the bipotentiality of formed BL-CFCs. Since anemia was a feature of 9.5 dpc eng−/− yolk sac embryos, we also examined early erythropoiesis using the ES/EB system. For this purpose, eng−/−, eng+/−, and eng+/+ ES cells were differentiated into EBs for 4 days, at which time cells were disrupted and plated for primitive erythroid colonies (EryP) in methylcellulose media containing IL-3, IL-6, SCF, and Epo. We observed a reduction in the number of EryP colonies in eng−/− (p < 0.01) and eng+/− (p < 0.05) EBs when compared to controls (eng+/+). These results corroborate the anemia observed in vivo in the eng−/− embryos. We used RT-PCR and flow cytometry analysis to detect endoglin expression during a time course of EB differentiation. Endoglin is expressed in ES cells and disappears with differentiation. Expression re-appears at day 3 of differentiation, concomitantly with specification of the hemangioblast. Expression thereafter increases, correlating with mature endothelial cells at later time points. We did not find major differences in gene expression for Brachyury, Flk-1, Tie-2, embryonic and adult globins in a time course of EB differentiation for eng−/−, eng+/−, and eng+/+ ES cells. These data point out a role for endoglin, an ancillary receptor for several members of the transforming growth factor (TGF)-beta superfamily, in hemangioblast development.

Reprogrammed Murine Fibroblasts Differentiated into Hematopoietic Progenitors Are Able to Successfully Engraft and Repopulate the Bone Marrow

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 389-389 ◽  
Author(s):  
Zaida Alipio ◽  
Dan Xu ◽  
Jianchang Yang ◽  
Louis M. Fink ◽  
Wilson Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitors

Abstract Cellular therapy using embryonic stem cells has always been an area of great interest due to the pluripotent characteristics of stem cells. In 2006, Takahashi and Yamanaka (Cell 126, 663–676) demonstrated that somatic cells can be reprogrammed into a stem cell-like state, termed induced pluripotent stem (iPS) cells, by ectopic expression of Oct4, Sox2, Klf4 and c Myc. A later report (Nakagawa et al. Nat. Biotechnol.26:101–106, 2008) showed that iPS cells can be produced in the absence of the c Myc oncogene. We have used this latter strategy to successfully reprogram somatic cells derived from C57BL/6 mouse tail fibroblast to iPS cells. Retrovirus infected fibroblasts exhibited stem cell-like morphology by 14 days post infection. These iPS cells were then infected with a retrovirus that expressed HOXB4. Recombinant leukemia inhibitor factor (LIF) supplement was removed from media at this time and the cells allowed to differentiate into embryoid bodies. These cells were screened for specific differentiation stem cell markers, such as Oct4, Nanog, Sall4 and SSEA-1. iPS cells were converted into embryonic bodies and then infected with retroviruses expressing HOXB4. Embryoid bodies stably expressing HOXB4 were induced to hematopoietic differentiation by treatment of thrombopoietin (TPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), interferon gamma (IFNg) and fms-like tyrosine kinase (FLT3 ligand). Evaluation of iPS-derived hematopoietic cells on smears show strikingly similarity in morphology to the W4 mouse embryonic stem (ES) cells differentiated into hematopoietic cells as a control. Flow cytometry analysis of iPS-derived hematopoietic cells after 1 week exposure to cytokines revealed 7% B220+ cells (B cells), 11% Ter119+ cells (erythroid), and 13% Gr-1+ cells (granulocytes) similar to W4 ES cells. The iPS-derived hematopoietic cells were transplanted into irradiated immunodeficient mice via lateral tail vein injection. Transplantation of these iPS-derived hematopoietic progenitors tagged with GFP into irradiated SCID mice revealed that the hematopoietic progenitors were able to home to the bone marrow after 1 week of transplantation. Importantly, after 1 month, GFP+ engrafted cells remained in the bone marrow suggesting a long-term engraftment. This long term engraftment of the iPS-derived hematopoietic cells to the bone marrow constitutes an important step toward potential therapy of numerous patient-specific blood based diseases.

scholarly journals The chromosomal protein SMCHD1 regulates DNA methylation and the 2c-like state of embryonic stem cells by antagonizing TET proteins

2021 ◽  
Vol 7 (4) ◽  
pp. eabb9149
Author(s):  
Zhijun Huang ◽  
Jiyoung Yu ◽  
Wei Cui ◽  
Benjamin K. Johnson ◽  
Kyunggon Kim ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Dna Demethylation ◽  
Chromosomal Protein ◽  
Dna Hypomethylation ◽  
Tet Proteins ◽  
Target Sites ◽  
Structural Maintenance Of Chromosomes ◽  
Hinge Domain

5-Methylcytosine (5mC) oxidases, the ten-eleven translocation (TET) proteins, initiate DNA demethylation, but it is unclear how 5mC oxidation is regulated. We show that the protein SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) is found in complexes with TET proteins and negatively regulates TET activities. Removal of SMCHD1 from mouse embryonic stem (ES) cells induces DNA hypomethylation, preferentially at SMCHD1 target sites and accumulation of 5-hydroxymethylcytosine (5hmC), along with promoter demethylation and activation of the Dux double-homeobox gene. In the absence of SMCHD1, ES cells acquire a two-cell (2c) embryo–like state characterized by activation of an early embryonic transcriptome that is substantially imposed by Dux. Using Smchd1/Tet1/Tet2/Tet3 quadruple-knockout cells, we show that DNA demethylation, activation of Dux, and other genes upon SMCHD1 loss depend on TET proteins. These data identify SMCHD1 as an antagonist of the 2c-like state of ES cells and of TET-mediated DNA demethylation.

scholarly journals Regulation of Mineralocorticoid Receptor Expression during Neuronal Differentiation of Murine Embryonic Stem Cells

Endocrinology ◽  
2010 ◽  
Vol 151 (5) ◽  
pp. 2244-2254 ◽  
Author(s):  
Mathilde Munier ◽  
Geri Meduri ◽  
Say Viengchareun ◽  
Philippe Leclerc ◽  
Damien Le Menuet ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Mineralocorticoid Receptor ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Critical Role ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Receptor Expression ◽  
Neuronal Markers ◽  
Mature Neurons

Mineralocorticoid receptor (MR) plays a critical role in brain function. However, the regulatory mechanisms controlling neuronal MR expression that constitutes a key element of the hormonal response are currently unknown. Two alternative P1 and P2 promoters drive human MR gene transcription. To examine promoter activities and their regulation during neuronal differentiation and in mature neurons, we generated stably transfected recombinant murine embryonic stem cell (ES) lines, namely P1-GFP and P2-GFP, in which each promoter drove the expression of the reporter gene green fluorescent protein (GFP). An optimized protocol, using embryoid bodies and retinoic acid, permitted us to obtain a reproducible neuronal differentiation as revealed by the decrease in phosphatase alkaline activity, the concomitant appearance of morphological changes (neurites), and the increase in the expression of neuronal markers (nestin, β-tubulin III, and microtubule-associated protein-2) as demonstrated by immunocytochemistry and quantitative PCR. Using these cell-based models, we showed that MR expression increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter during neuronal differentiation, their activities increased by 7- and 5-fold, respectively, and correlated with MR expression. Finally, although progesterone and dexamethasone were ineffective, aldosterone stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of MR by small interfering RNA. In conclusion, we provide evidence for a tight transcriptional control of MR expression during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and psychiatric diseases.

X chromosome retains the memory of its parental origin in murine embryonic stem cells

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 813-821 ◽  
Author(s):  
T. Tada ◽  
M. Tada ◽  
N. Takagi
Keyword(s):  
X Chromosome ◽  
Polar Region ◽  
Biochemical Study ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Parental Origin ◽  
Visceral Endoderm ◽  
Es Cell ◽  
Preferential Inactivation

A cytogenetic and biochemical study of balloon-like cystic embryoid bodies, formed by newly established embryonic stem (ES) cell lines having a cytogenetically or genetically marked X chromosome, revealed that the paternally derived X chromosome was inactivated in the majority of cells in the yolk sac-like mural region consisting of the visceral endoderm and mesoderm. The nonrandomness was less evident in the more solid polar region containing the ectodermal vesicle, mesoderm and visceral endoderm. Since the same was true in embryoid bodies derived from ES cells at the 30th subculture generation, it was concluded that the imprinting responsible for the preferential inactivation of the paternal X chromosome that was limited to non-epiblast cells of the female mouse embryos, was stably maintained in undifferentiated ES cells. Differentiating epiblast cells should be able to erase or avoid responding to the imprint.

Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2740-2749 ◽  
Author(s):  
CD Helgason ◽  
G Sauvageau ◽  
HJ Lawrence ◽  
C Largman ◽  
RK Humphries
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Homeobox Genes ◽  
Embryoid Bodies ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Differentiation And Proliferation

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events.

scholarly journals Isolation, Characterization and Differentiation Potential of Chicken Spermatogonial Stem Cell Derived Embryoid Bodies

2016 ◽  
Vol 16 (1) ◽  
pp. 115-128 ◽  
Author(s):  
Thanh Luan Nguyen ◽  
Jae Gyu Yoo ◽  
Neelesh Sharma ◽  
Sung Woo Kim ◽  
Yong Jun Kang ◽  
...  
Keyword(s):  
Developmental Biology ◽  
Regenerative Medicine ◽  
Connexin 43 ◽  
Es Cells ◽  
Periodic Acid ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Significant Finding ◽  
Differentiation Potential

Abstract Human, murine and monkey spermatogonial stem cells (SSCs) have the capability to undergo self-renewal and differentiation into different body cell types in vitro, which are expected to serve as a powerful tool and resource for the developmental biology and regenerative medicine. We have successfully isolated and characterized the chicken SSCs from 3-day-old chicken testicular cells. The pluripotency was using Periodic Acid-Schiff (PAS ) staining or alkaline phosphatase staining, and antibodies to stage-specific embryonic antigens. In suspension culture conditions SSCs formed embryoid bodies (EBs) like embryonic stem (ES) cells. Subsequently EB differentiated into osteoblasts, adipocytes and most importantly into cardiomyocytes under induced differentiation conditions. The differentiation potential of EBs into cardiomyocyte-like cells was confirmed by using antibodies against sarcomeric α-actinin, cardiac troponin T and connexin 43. Cardiomyocytes-like cells were also confirmed by RT-PCR analysis for several cardiac cell genes like GATA-4, Nkx2-5, α-MHC, and ANF. We have successfully established an in vitro differentiation system for chicken SSCs into different body cells such as osteoblasts, adipocytes and cardiomyocytes. The most significant finding of this study is the differentiation potential of chicken SSCs into cardiomyocytes. Our findings may have implication in developmental biology and regenerative medicine by using chicken as the most potential animal model.

Differentiating Embryonic Stem (ES) Cells into Embryoid Bodies

2006 ◽  
Vol 2006 (2) ◽  
pp. pdb.prot4405 ◽  
Author(s):  
Andras Nagy ◽  
Marina Gertsenstein ◽  
Kristina Vintersten ◽  
Richard Behringer
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies
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