In vitro differentiation of c-myb−/− ES cells reveals that the colony forming capacity of unilineage macrophage precursors and myeloid progenitor commitment are c-Myb independent

Abstract We previously described a zebrafish mutant, frascati (frs), which exhibits profound hypochromic anemia and erythroid maturation arrest due to defects in mitochondrial iron uptake. Through positional cloning, we showed that the frs gene encodes a novel member of the vertebrate mitochondrial solute carrier family (SLC25), mitoferrin (mfrn, slc25a37). Mfrn, which is highly expressed in fetal and adult hematopoietic tissues of zebrafish and mouse, functions as the major mitochondrial iron importer essential for heme biosynthesis in vertebrate erythroblasts (Shaw GC, et al. 2006 Nature 440:96–100). To study the function of Mfrn in mammalian organisms, we identified an embryonic stem (ES) cell clone that harbors a gene trap b-geo cassette in intron 1 that inactivates the Mfrn locus. Homozygous disruption of the Mfrn locus results in embryonic lethality at E11.5 from profound anemia due to a failure of primitive erythropoiesis, confirming the requirement of Mfrn in mammalian development . Circumventing the embryonic lethality, we generated Mfrn−/− ES cells to study the role of Mfrn in definitive erythropoiesis by in vitro differentiation of embryoid bodies and mixed chimera assays. Mfrn−/− ES cells were defective in promoting the growth, differentiation, and hemoglobinization of both primitive and definitive erythroblasts by in vitro differentiation of embryoid bodies. In mixed chimera studies, Mfrn−/− ES cells failed to contribute to the erythroid compartment of adult mosaic mice, whereas measurable contribution of Mfrn−/− donor cells could be assayed in the non-erythroid, leukocyte compartment. Transcriptome microarray analysis, using the mouse Affymetrix GeneChip and the custom IronChip, revealed unexpected down-regulation of transcripts for heme-biosynthetic enzymes in Mfrn−/− erythroblasts. The block in protoprophyrin synthesis, as well as mitochondrial heme synthesis, could be partially rescued by the addition of aminolevulinic acid (ALA) to Mfrn−/− erythroblasts in vitro. Our data demonstrate that mitochondrial iron homeostasis, working through the Mfrn iron importer, coordinately regulates the synthetic pathways for porphyrin and heme in developing mammalian erythroblasts.

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Thrombopoietin Enhances Proliferation and Differentiation of Murine Yolk Sac Erythroid Progenitors

Blood ◽

10.1182/blood.v89.4.1207 ◽

1997 ◽

Vol 89 (4) ◽

pp. 1207-1213 ◽

Cited By ~ 21

Author(s):

Takumi Era ◽

Tomomi Takahashi ◽

Katsuya Sakai ◽

Kazuo Kawamura ◽

Toru Nakano

Keyword(s):

Colony Formation ◽

Es Cells ◽

Embryonic Stem ◽

Yolk Sac ◽

In Vitro Differentiation ◽

Hematopoietic Progenitors ◽

Erythroid Progenitors ◽

Proliferation And Differentiation ◽

Differentiation Status

Abstract Thrombopoietin (TPO), the ligand for the receptor proto-oncogene c-Mpl, has been cloned and shown to be the critical regulator of proliferation and differentiation of megakaryocytic lineage. Initially, TPO was not considered to have the activity on hematopoietic lineages other than megakaryocytes. Recently, however, TPO was reported to enhance the in vitro erythroid colony formation from human bone marrow (BM) CD34+ progenitors or from mouse BM cells in combination with other cytokines. We examined the effects of TPO on the colony formation of hematopoietic progenitors in mouse yolk sac. TPO remarkably enhanced proliferation and differentiation of erythroid-lineage cells in the presence of erythropoietin (Epo). This effect was observed even in the absence of Epo. Compared with adult BM, yolk sac turned out to have relatively abundant erythroid and erythro-megakaryocytic progenitors, which responded to TPO and Epo stimulation. TPO similarly stimulated erythroid colony formation from in vitro differentiation-induced mouse embryonic stem (ES) cells whose hematopoietic differentiation status was similar to that of yolk sac. These findings help to understand the biology of hematopoietic progenitors of the early phase of hematopoiesis. Yolk sac cells or in vitro differentiation-induced ES cells would be good sources to analyze the TPO function on erythropoiesis.

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In vitro differentiation of mouse ES cells into hepatocytes with coagulation factors VIII and IX expression profiles

Science in China Series C Life Sciences ◽

10.1007/s11427-006-0259-3 ◽

2006 ◽

Vol 49 (3) ◽

pp. 259-264 ◽

Cited By ~ 3

Author(s):

Ying Meng ◽

Shaoliang Huang ◽

Jun Min ◽

Zhongmin Guo

Keyword(s):

Expression Profiles ◽

Es Cells ◽

Coagulation Factors ◽

In Vitro Differentiation ◽

Mouse Es Cells ◽

Viii And Ix

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In Vitro Differentiation of Mouse ES Cells into Hematopoietic, Endothelial, and Osteoblastic Cell Lineages: The Possibility of In Vitro Organogenesis

Methods in Enzymology - Differentiation of Embryonic Stem Cells ◽

10.1016/s0076-6879(03)65007-x ◽

2003 ◽

pp. 98-114 ◽

Cited By ~ 9

Author(s):

Motokazu Tsuneto ◽

Toshiyuki Yamane ◽

Hiromi Okuyama ◽

Hidetoshi Yamazaki ◽

Shin-Ichi Hayashi

Keyword(s):

Es Cells ◽

Osteoblastic Cell ◽

In Vitro Differentiation ◽

In Vitro Organogenesis ◽

Cell Lineages ◽

Mouse Es Cells

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Quantitative RT-PCR assays show Xist RNA levels are low in mouse female adult tissue, embryos and embryoid bodies

Development ◽

10.1242/dev.120.12.3529 ◽

1994 ◽

Vol 120 (12) ◽

pp. 3529-3536 ◽

Cited By ~ 3

Author(s):

C.H. Buzin ◽

J.R. Mann ◽

J. Singer-Sam

Keyword(s):

Es Cells ◽

Embryoid Bodies ◽

Es Cell ◽

In Vitro Differentiation ◽

Rt Pcr ◽

Adult Tissue ◽

Female Adult ◽

Adult Kidney ◽

Xist Rna

We have investigated expression of the Xist gene in mouse female adult kidney, embryos and embryonic stem (ES) cells undergoing in vitro differentiation as embryoid bodies. Using the quantitative RT-PCR single nucleotide primer extension (SNuPE) assay, we found that the amount of Xist RNA in adult kidney of three mouse strains was less than approximately 2000 transcripts per cell, with only modest differences between strains carrying different Xce alleles. Female embryos 7.5 days post coitum had the same number of Xist transcripts per cell as isogenic adult tissue. Using quantitative oligonucleotide hybridization assays after RT-PCR, we investigated Xist expression in ES lines heterozygous at the Pgk-1 and Xist loci. We found that, while in most (XX) ES lines Xist RNA levels increased during embryoid body formation, the levels seen were less than 10% those found in adult female kidney. In addition, we found that the allelic ratio of Xist transcripts from reciprocal (XX) ES cell lines differentiating in vitro was identical to that of isogenic 10.5 to 11.5 day female embryos. These latter results suggest that there is no pattern of preferential paternal imprinting during days 1 to 9 of in vitro differentiation of ES cells. However, the influence of the Xce locus on the randomness of X-inactivation in embryos seems to operate also in ES cell lines. Our overall conclusion is that the low levels of Xist RNA in female kidney, embryos and differentiating (XX) ES cells are compatible only with models that do not require Xist RNA to cover the entire inactive X chromosome.

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