A requirement for Notch1 distinguishes 2 phases of definitive hematopoiesis during development

Abstract Notch1 is known to play a critical role in regulating fates in numerous cell types, including those of the hematopoietic lineage. Multiple defects exhibited by Notch1-deficient embryos confound the determination of Notch1 function in early hematopoietic development in vivo. To overcome this limitation, we examined the developmental potential of Notch1–/– embryonic stem (ES) cells by in vitro differentiation and by in vivo chimera analysis. Notch1 was found to affect primitive erythropoiesis differentially during ES cell differentiation and in vivo, and this result reflected an important difference in the regulation of Notch1 expression during ES cell differentiation relative to the developing mouse embryo. Notch1 was dispensable for the onset of definitive hematopoiesis both in vitro and in vivo in that Notch1–/– definitive progenitors could be detected in differentiating ES cells as well as in the yolk sac and early fetal liver of chimeric mice. Despite the fact that Notch1–/– cells can give rise to multiple types of definitive progenitors in early development, Notch1–/– cells failed to contribute to long-term definitive hematopoiesis past the early fetal liver stage in the context of a wild-type environment in chimeric mice. Thus, Notch1 is required, in a cell-autonomous manner, for the establishment of long-term, definitive hematopoietic stem cells (HSCs).

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GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

Blood ◽

10.1182/blood.v92.11.4108 ◽

1998 ◽

Vol 92 (11) ◽

pp. 4108-4118 ◽

Cited By ~ 54

Author(s):

Naruyoshi Suwabe ◽

Satoru Takahashi ◽

Toru Nakano ◽

Masayuki Yamamoto

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Erythroid Cells ◽

Es Cell ◽

Globin Mrna ◽

Differentiated Cells ◽

Vitro Analysis ◽

Definitive Hematopoiesis

Abstract Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

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Hematopoietic Repopulation, In Vivo, with Genetically Defined Clones Derived from HOXB4 Expressing ES-Cells.

Blood ◽

10.1182/blood.v106.11.196.196 ◽

2005 ◽

Vol 106 (11) ◽

pp. 196-196

Author(s):

Sandra Pilat ◽

Sebastian Carotta ◽

Bernhard Schiedlmeier ◽

Kenji Kamino ◽

Andreas Mairhofer ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Es Cells ◽

Es Cell ◽

Somatic Gene Therapy ◽

Hematopoietic System ◽

Somatic Gene

Abstract In the context of somatic gene therapy of the hematopoietic system, transplantation of molecularly defined and, hence, “safe” clones would be highly desirable. However, techniques which allow gene targeting, subsequent in vitro selection and clonal expansion are only available for embryonic stem (ES) cells. After in vitro differentiation, some of their progeny cells are capable of mediating long term hematopoietic repopulation after transplantation into immunodeficient recipient mice, in vivo. This is especially efficient when the homeodomain transcription factor HOXB4 is ectopically expressed (1). We have recently shown that HOXB4-ES-cell derivatives behave similar to bone marrow cells also expressing this transcription factor ectopically, both in vitro and in vivo (2). Here we demonstrate that long term repopulation (>6 months) in Rag2(−/−)γ C(−/−) mice can be achieved with ES-cell derived hematopoietic cells (ES-HCs) obtained from single, molecularly characterized ES-clones, in which the insertion sites of the retroviral expression vector had been defined. Clones expressing HOXB4 above a certain level showed a high extent of chimerism in the bone marrow of transplanted mice (average 75%; range 45–95%, n=4) whereas ES-HC clones expressing lower levels only repopulated with very low efficiency (average 2.5% chimerism, range 1–4%, n=6 mice). These results suggest that the capability of long-term repopulation, in vivo, is highly dependent on the expression levels of HOXB4 in the transplanted clones. Only mice reconstituted with ES-HC clones expressing high amounts of HOXB4 and thus showing substantial chimerism, recapitulated the morphohistological phenotype observed in polyclonally reconstituted mice. This included the bias towards myelopoiesis, “benign” myeloid proliferation in spleen and the incompatibility of HOXB4 expression with T-cell poiesis (2). In summary, we demonstrate that repopulation of the hematopoietic system can be achieved with preselected clones of genetically manipulated stem cells in which a) the insertion site of the retroviral (gene therapy) vector has been characterized prior to transplantation and b) in which ectopic HOXB4 has to be expressed above a certain threshold level. Thus, ES cells carry the potential for performing safe somatic gene therapy when using integrating gene therapy vectors. Nevertheless, advanced cell therapy will certainly require the expression of HOXB4 in a regulated manner to avoid unwanted effects such as disturbed lineage differentiation.

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Identification and Characterization of Hemoangiogenic Progenitors during Cynomolgus Monkey ES Cell Differentiation.

Blood ◽

10.1182/blood.v104.11.3222.3222 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3222-3222

Author(s):

Katsutsugu Umeda ◽

Toshio Heike ◽

Momoko Yoshimoto ◽

Mitsutaka Shiota ◽

Feng Ma ◽

...

Keyword(s):

Cell Differentiation ◽

Es Cells ◽

Close Association ◽

Growth Factor Receptor ◽

Intermediate Stage ◽

Surface Markers ◽

Es Cell ◽

Cd34 Cells

Abstract Previous reports about the close association of development of hematopoietic (HCs) and endothelial cells (ECs) have suggested the presence of the hemangioblast, a putative common precursor of both lineage cells in mouse. The presence of putative ancestors is suggested by the fact that both HCs and ECs share common markers such as vascular endothelial growth factor receptor (VEGFR)-2, CD34, and CD31. The precise analysis about the relationship of HCs and ECs in human embryogenesis, however, remains unclear because of obstacle including the ethical restrictions on experiments using their embryo. To further understand the mechanisms that regulate the differentiation of HCs and ECs in humans, it is currently necessary to employ primate (human and monkey) ES cells. In this study, we identified the intermediate-stage cells with HC and EC differentiation potentials from nonhuman primate ES cells by coculture with OP9 stromal cells. Sequential FACS analysis demonstrated that during in vitro HC and EC differentiation, the expression of common surface markers such as CD34 and CD31 could first be detected, followed by the expression of lineage-specific markers (CD45, CD41 and VE-cadherin) in order. Sequential FACS and immunostaining analyses showed that both lineage developments concomitantly occurred, which were preceded by emergence of two hemoangiogenic progenitors, VEGFR-2high CD34− and VEGFR-2high CD34+ cells. While both VEGFR-2 high cells could differentiate into primitive and definitive HCs as well as ECs, VEGFR-2high CD34+ cells possessed higher hemoangiogenic potential than VEGFR-2high CD34− cells. In contrast, HC and EC production cluster was rare in the VEGFR-2low or − fractions. During HC and EC differentiation, most of VEGFR-2high cells first expressed CD34, and then diverged into both lineage cells at the VEGFR-2high CD34+ stage. We found that the VEGFR-2high cells can generate both HCs and ECs at the single cell level, which strongly supports the existence of hemangioblasts in primates. To our knowledge, this is the first time that hemangioblasts have been shown to be present during primate ES cell differentiation. Furthermore, differentiation pathway into HC and EC lineages can be defined by surface markers, which facilitates further investigations in vitro and in vivo.

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GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

Blood ◽

10.1182/blood.v92.11.4108.423k29_4108_4118 ◽

1998 ◽

Vol 92 (11) ◽

pp. 4108-4118 ◽

Cited By ~ 4

Author(s):

Naruyoshi Suwabe ◽

Satoru Takahashi ◽

Toru Nakano ◽

Masayuki Yamamoto

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Erythroid Cells ◽

Es Cell ◽

Globin Mrna ◽

Differentiated Cells ◽

Vitro Analysis ◽

Definitive Hematopoiesis

Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

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VWRPY motif–dependent and –independent roles of AML1/Runx1 transcription factor in murine hematopoietic development

Blood ◽

10.1182/blood-2003-06-2109 ◽

2004 ◽

Vol 103 (2) ◽

pp. 562-570 ◽

Cited By ~ 37

Author(s):

Motohiro Nishimura ◽

Yoko Fukushima-Nakase ◽

Yasuko Fujita ◽

Mitsushige Nakao ◽

Shogo Toda ◽

...

Keyword(s):

Transcription Factor ◽

Es Cells ◽

Embryonic Stem ◽

Es Cell ◽

Thymocyte Development ◽

Hematopoietic Development ◽

Cell Clones ◽

Definitive Hematopoiesis

Abstract AML1/Runx1 is a frequent target of leukemia-associated gene aberration, and it encodes a transcription factor essential for definitive hematopoiesis. We previously reported that the AML1 molecules with trans-activation subdomains retained can rescue in vitro hematopoietic defects of AML1-deficient mouse embryonic stem (ES) cells when expressed by using a knock-in approach. Extending this notion to in vivo conditions, we found that the knock-in ES cell clones with AML1 mutants, which retain trans-activation subdomains but lack C-terminal repression subdomains including the conserved VWRPY motif, contribute to hematopoietic tissues in chimera mice. We also found that germline mice homozygous for the mutated AML1 allele, which lacks the VWRPY motif, exhibit a minimal effect on hematopoietic development, as was observed in control knock-in mice with full-length AML1. On the other hand, reduced cell numbers and deviant CD4 expression were observed during early T-lymphoid ontogeny in the VWRPY-deficient mice, whereas the contribution to the thymus by the corresponding ES cell clones was inadequate. These findings demonstrate that AML1 with its trans-activating subdomains is essential and sufficient for hematopoietic development in the context of the entire mouse. In addition, its trans-repression activity, depending on the C-terminal VWRPY motif, plays a role in early thymocyte development.

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Hemogenic and nonhemogenic endothelium can be distinguished by the activity of fetal liver kinase (Flk)–1promoter/enhancer during mouse embryogenesis

Blood ◽

10.1182/blood-2002-02-0655 ◽

2003 ◽

Vol 101 (3) ◽

pp. 886-893 ◽

Cited By ~ 49

Author(s):

Hideyo Hirai ◽

Minetaro Ogawa ◽

Norio Suzuki ◽

Masayuki Yamamoto ◽

Georg Breier ◽

...

Keyword(s):

Molecular Mechanisms ◽

Fetal Liver ◽

Es Cells ◽

Embryonic Stem ◽

Specific Gene ◽

Vascular Endothelial ◽

Myb Genes ◽

Definitive Hematopoiesis

Abstract Accumulating evidence in various species has suggested that the origin of definitive hematopoiesis is associated with a special subset of endothelial cells (ECs) that maintain the potential to give rise to hematopoietic cells (HPCs). In this study, we demonstrated that a combination of 5′-flanking region and 3′ portion of the first intron of the Flk-1 gene (Flk-1 p/e) that has been implicated in endothelium-specific gene expression distinguishes prospectively the EC that has lost hemogenic activity. We assessed the activity of this Flk-1 p/e by embryonic stem (ES) cell differentiation culture and transgenic mice by using theGFP gene conjugated to this unit. The expression ofGFP differed from that of the endogenous Flk-1gene in that it is active in undifferentiated ES cells and inactive in Flk-1+ lateral mesoderm. Flk-1 p/e becomes active after generation of vascular endothelial (VE)–cadherin+ ECs. Emergence of GFP− ECs preceded that of GFP+ ECs, and, finally, most ECs expressed GFP both in vitro and in vivo. Cell sorting experiments demonstrated that only GFP− ECs could give rise to HPCs and preferentially expressed Runx1 and c-Myb genes that are required for the definitive hematopoiesis. Integration of both GFP+ and GFP− ECs was observed in the dorsal aorta, but cell clusters appeared associated only to GFP−ECs. These results indicate that activation of Flk-1 p/e is associated with a process that excludes HPC potential from the EC differentiation pathway and will be useful for investigating molecular mechanisms underlying the divergence of endothelial and hematopoietic lineages.

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ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients

Development ◽

10.1242/dev.118.4.1343 ◽

1993 ◽

Vol 118 (4) ◽

pp. 1343-1351

Author(s):

A.M. Muller ◽

E.A. Dzierzak

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Es Cells ◽

Embryonic Stem ◽

Mouse Development ◽

Hematopoietic Stem ◽

Developmental Potential

While hematopoietic stem cells from adult and fetal stages of murine development are capable of long term reconstitution of all mature blood lineages in vivo, embryonic hematopoietic stem cell repopulation in vivo has proved difficult. It is thought that there are many fewer hematopoietic stem cells in the embryo than in the fetal/adult stages of mouse development and that these cells possess a different developmental potential. One source of such cells are embryonic stem (ES) cells which can differentiate into most mature blood lineages in vitro. We have therefore used transplantation of differentiated ES cells to assess the hematopoietic potential of embryonic hematopoietic cells in vivo. We demonstrate here that precursors obtained from in vitro cultures of normal ES cells can contribute only to restricted and limited hematopoiesis in a mouse without leading to tumour formation. Repopulation occurs for greater than 6.5 months at levels ranging from 0.1% to 6% in B and T cell lineages in peripheral blood. In contrast to in vitro colony data demonstrating the myeloid lineage developmental potential of ES cells, no donor-derived myeloid repopulation was observed in CFU-S assays and no macrophage and mast cells were found in long term repopulated recipients. Thus, the hematopoietic potential of ES cells in vivo is limited to low levels of repopulation and is restricted to the lymphoid lineage.

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Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽

10.1242/dev.116.supplement.157 ◽

1992 ◽

Vol 116 (Supplement) ◽

pp. 157-165 ◽

Cited By ~ 20

Author(s):

R. S. P. Beddington ◽

P. Rashbass ◽

V. Wilson

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Primitive Streak ◽

Coat Colour ◽

Es Cell ◽

Wild Type ◽

Mesoderm Cell ◽

Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

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Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes

Development ◽

10.1242/dev.125.12.2273 ◽

1998 ◽

Vol 125 (12) ◽

pp. 2273-2282 ◽

Cited By ~ 10

Author(s):

W. Dean ◽

L. Bowden ◽

A. Aitchison ◽

J. Klose ◽

T. Moore ◽

...

Keyword(s):

Developmental Stages ◽

Es Cells ◽

Embryonic Stem ◽

Upstream Region ◽

Imprinted Genes ◽

Epigenetic Alterations ◽

Es Cell ◽

Biallelic Expression

In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

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Early Maturation of T-Cell Progenitors in the Absence of Glucocorticoids

Blood ◽

10.1182/blood.v94.8.2819.420k12_2819_2826 ◽

1999 ◽

Vol 94 (8) ◽

pp. 2819-2826 ◽

Cited By ~ 12

Author(s):

Rosa Sacedón ◽

Angeles Vicente ◽

Alberto Varas ◽

Eva Jiménez ◽

Juan José Muñoz ◽

...

Keyword(s):

Cell Differentiation ◽

T Cell ◽

Glucocorticoid Receptors ◽

Fetal Liver ◽

Cell Receptor ◽

T Cell Differentiation ◽

Pregnant Rats ◽

Early Maturation

In the present work, we demonstrated that both fetal liver and thymic T-cell precursors express glucocorticoid receptors (GRs) indirectly suggesting a role for glucocorticoids (GCs) in the earliest events of T-cell differentiation. To evaluate this issue, we analyzed the thymic ontogeny in the progeny of adrenalectomized pregnant rats (Adx fetuses), an in vivo experimental model, which ensures the absence of circulating GCs until the establishment of the fetal hypothalamus-pituitary-adrenal (HPA) axis. In the absence of maternal GCs, T-cell development was significantly accelerated, the process being reversed by in vivo GC replacement. Mature single positive thymocytes (both CD4 and CD8) appeared in 16-day old fetal Adx thymus when in the control fetuses, most thymocytes still remained in the double-negative (DN) CD4−CD8− cell compartment. In addition, emigration of T-cell receptor (TcR)β positive cells to the spleen also occurred earlier in Adx fetuses than in control ones. In vitro recolonization of cultured deoxiguanosine-treated mouse fetal thymus lobes with 13-day-old fetal liver cell suspensions from both Adx and control fetuses demonstrated changes in the developmental capabilities of fetal liver T-cell precursors from embryos grown in the absence of GCs. Furthermore, a precocious lymphoid colonization of the thymic primordium from Adx fetuses was evidenced by ultrastructural analysis of both Adx and Sham early thymus. Both findings accounted for the accelerated T-cell differentiation observed in Adx fetuses. Together, these results support a role for GCs not only in the thymic cell death, but also in the early steps of T-cell differentiation.

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