PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
 
Mol Cell Biol. 1993 January; 13(1): 473–486.
PMCID: PMC358927

Hematopoietic commitment during embryonic stem cell differentiation in culture.

Abstract

We report that embryonic stem cells efficiently undergo differentiation in vitro to mesoderm and hematopoietic cells and that this in vitro system recapitulates days 6.5 to 7.5 of mouse hematopoietic development. Embryonic stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of differentiation, and by day 6, more than 85% of EBs contain such cells. A comparative reverse transcriptase-mediated polymerase chain reaction profile of marker genes for primitive endoderm (collagen alpha IV) and mesoderm (Brachyury) indicates that both cell types are present in the developing EBs as well in normal embryos prior to the onset of hematopoiesis. GATA-1, GATA-3, and vav are expressed in both the EBs and embryos just prior to and/or during the early onset of hematopoiesis, indicating that they could play a role in the early stages of hematopoietic development both in vivo and in vitro. The initial stages of hematopoietic development within the EBs occur in the absence of added growth factors and are not significantly influenced by the addition of a broad spectrum of factors, including interleukin-3 (IL-3), IL-1, IL-6, IL-11, erythropoietin, and Kit ligand. At days 10 and 14 of differentiation, EB hematopoiesis is significantly enhanced by the addition of both Kit ligand and IL-11 to the cultures. Kinetic analysis indicates that hematopoietic precursors develop within the EBs in an ordered pattern. Precursors of the primitive erythroid lineage appear first, approximately 24 h before precursors of the macrophage and definitive erythroid lineages. Bipotential neutrophil/macrophage and multilineage precursors appear next, and precursors of the mast cell lineage develop last. The kinetics of precursor development, as well as the growth factor responsiveness of these early cells, is similar to that found in the yolk sac and early fetal liver, indicating that the onset of hematopoiesis within the EBs parallels that found in the embryo.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (3.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Adams JM, Houston H, Allen J, Lints T, Harvey R. The hematopoietically expressed vav proto-oncogene shares homology with the dbl GDP-GTP exchange factor, the bcr gene and a yeast gene (CDC24) involved in cytoskeletal organization. Oncogene. 1992 Apr;7(4):611–618. [PubMed]
  • Adamson ED, Ayers SE. The localization and synthesis of some collagen types in developing mouse embryos. Cell. 1979 Apr;16(4):953–965. [PubMed]
  • Azoulay M, Webb CG, Sachs L. Control of hematopoietic cell growth regulators during mouse fetal development. Mol Cell Biol. 1987 Sep;7(9):3361–3364. [PMC free article] [PubMed]
  • Begley CG, Aplan PD, Denning SM, Haynes BF, Waldmann TA, Kirsch IR. The gene SCL is expressed during early hematopoiesis and encodes a differentiation-related DNA-binding motif. Proc Natl Acad Sci U S A. 1989 Dec;86(24):10128–10132. [PubMed]
  • Begley CG, Visvader J, Green AR, Aplan PD, Metcalf D, Kirsch IR, Gough NM. Molecular cloning and chromosomal localization of the murine homolog of the human helix-loop-helix gene SCL. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):869–873. [PubMed]
  • Boltz-Nitulescu G, Wiltschke C, Holzinger C, Fellinger A, Scheiner O, Gessl A, Förster O. Differentiation of rat bone marrow cells into macrophages under the influence of mouse L929 cell supernatant. J Leukoc Biol. 1987 Jan;41(1):83–91. [PubMed]
  • Brotherton TW, Chui DH, Gauldie J, Patterson M. Hemoglobin ontogeny during normal mouse fetal development. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2853–2857. [PubMed]
  • Burkert U, von Rüden T, Wagner EF. Early fetal hematopoietic development from in vitro differentiated embryonic stem cells. New Biol. 1991 Jul;3(7):698–708. [PubMed]
  • Chelly J, Concordet JP, Kaplan JC, Kahn A. Illegitimate transcription: transcription of any gene in any cell type. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2617–2621. [PubMed]
  • Chelly J, Kaplan JC, Maire P, Gautron S, Kahn A. Transcription of the dystrophin gene in human muscle and non-muscle tissue. Nature. 1988 Jun 30;333(6176):858–860. [PubMed]
  • Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. [PubMed]
  • Choppin J, Casadevall N, Lacombe C, Wendling F, Goldwasser E, Berger R, Tambourin P, Varet B. Production of erythropoietin by cloned malignant murine erythroid cells. Exp Hematol. 1985 Aug;13(7):610–615. [PubMed]
  • Cline MJ, Moore MA. Embryonic origin of the mouse macrophage. Blood. 1972 Jun;39(6):842–849. [PubMed]
  • Coppola J, Bryant S, Koda T, Conway D, Barbacid M. Mechanism of activation of the vav protooncogene. Cell Growth Differ. 1991 Feb;2(2):95–105. [PubMed]
  • Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol. 1985 Jun;87:27–45. [PubMed]
  • Dyson PJ, Poirier F, Watson RJ. Expression of c-myb in embryonal carcinoma cells and embryonal stem cells. Differentiation. 1989 Oct;42(1):24–27. [PubMed]
  • Dziadek MA, Andrews GK. Tissue specificity of alpha-fetoprotein messenger RNA expression during mouse embryogenesis. EMBO J. 1983;2(4):549–554. [PubMed]
  • Flanagan JG, Chan DC, Leder P. Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the Sld mutant. Cell. 1991 Mar 8;64(5):1025–1035. [PubMed]
  • Froesch ER, Schmid C, Schwander J, Zapf J. Actions of insulin-like growth factors. Annu Rev Physiol. 1985;47:443–467. [PubMed]
  • Gabbianelli M, Sargiacomo M, Pelosi E, Testa U, Isacchi G, Peschle C. "Pure" human hematopoietic progenitors: permissive action of basic fibroblast growth factor. Science. 1990 Sep 28;249(4976):1561–1564. [PubMed]
  • Gorman DM, Itoh N, Kitamura T, Schreurs J, Yonehara S, Yahara I, Arai K, Miyajima A. Cloning and expression of a gene encoding an interleukin 3 receptor-like protein: identification of another member of the cytokine receptor gene family. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5459–5463. [PubMed]
  • Haar JL, Ackerman GA. Ultrastructural changes in mouse yolk sac associated with the initiation of vitelline circulation. Anat Rec. 1971 Aug;170(4):437–455. [PubMed]
  • Hara T, Miyajima A. Two distinct functional high affinity receptors for mouse interleukin-3 (IL-3). EMBO J. 1992 May;11(5):1875–1884. [PubMed]
  • Heberlein C, Fischer KD, Stoffel M, Nowock J, Ford A, Tessmer U, Stocking C. The gene for erythropoietin receptor is expressed in multipotential hematopoietic and embryonal stem cells: evidence for differentiation stage-specific regulation. Mol Cell Biol. 1992 Apr;12(4):1815–1826. [PMC free article] [PubMed]
  • Herrmann BG, Labeit S, Poustka A, King TR, Lehrach H. Cloning of the T gene required in mesoderm formation in the mouse. Nature. 1990 Feb 15;343(6259):617–622. [PubMed]
  • Hogan BL, Taylor A, Adamson E. Cell interactions modulate embryonal carcinoma cell differentiation into parietal or visceral endoderm. Nature. 1981 May 21;291(5812):235–237. [PubMed]
  • Ikuta K, Weissman IL. Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1502–1506. [PubMed]
  • Itoh N, Yonehara S, Schreurs J, Gorman DM, Maruyama K, Ishii A, Yahara I, Arai K, Miyajima A. Cloning of an interleukin-3 receptor gene: a member of a distinct receptor gene family. Science. 1990 Jan 19;247(4940):324–327. [PubMed]
  • Johnson GR, Barker DC. Erythroid progenitor cells and stimulating factors during murine embryonic and fetal development. Exp Hematol. 1985 Mar;13(3):200–208. [PubMed]
  • Johnson GR, Metcalf D. Sources and nature of granulocyte-macrophage colony stimulating factor in fetal mice. Exp Hematol. 1978 Mar;6(3):327–335. [PubMed]
  • Karasuyama H, Melchers F. Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur J Immunol. 1988 Jan;18(1):97–104. [PubMed]
  • Katzav S, Martin-Zanca D, Barbacid M. vav, a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells. EMBO J. 1989 Aug;8(8):2283–2290. [PubMed]
  • Ho IC, Vorhees P, Marin N, Oakley BK, Tsai SF, Orkin SH, Leiden JM. Human GATA-3: a lineage-restricted transcription factor that regulates the expression of the T cell receptor alpha gene. EMBO J. 1991 May;10(5):1187–1192. [PubMed]
  • Labastie MC, Thiery JP, Le Douarin NM. Mouse yolk sac and intraembryonic tissues produce factors able to elicit differentiation of erythroid burst-forming units and colony-forming units, respectively. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1453–1456. [PubMed]
  • Lindenbaum MH, Grosveld F. An in vitro globin gene switching model based on differentiated embryonic stem cells. Genes Dev. 1990 Dec;4(12A):2075–2085. [PubMed]
  • Lüscher B, Eisenman RN. New light on Myc and Myb. Part II. Myb. Genes Dev. 1990 Dec;4(12B):2235–2241. [PubMed]
  • Matsui Y, Zsebo KM, Hogan BL. Embryonic expression of a haematopoietic growth factor encoded by the Sl locus and the ligand for c-kit. Nature. 1990 Oct 18;347(6294):667–669. [PubMed]
  • Miura Y, Wilt FH. Tissue interaction and the formation of the first erythroblasts of the chick embryo. Dev Biol. 1969 Feb;19(2):201–211. [PubMed]
  • Moore MA, Metcalf D. Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo. Br J Haematol. 1970 Mar;18(3):279–296. [PubMed]
  • Mortensen RM, Conner DA, Chao S, Geisterfer-Lowrance AA, Seidman JG. Production of homozygous mutant ES cells with a single targeting construct. Mol Cell Biol. 1992 May;12(5):2391–2395. [PMC free article] [PubMed]
  • Mucenski ML, McLain K, Kier AB, Swerdlow SH, Schreiner CM, Miller TA, Pietryga DW, Scott WJ, Jr, Potter SS. A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell. 1991 May 17;65(4):677–689. [PubMed]
  • Murre C, McCaw PS, Vaessin H, Caudy M, Jan LY, Jan YN, Cabrera CV, Buskin JN, Hauschka SD, Lassar AB, et al. Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell. 1989 Aug 11;58(3):537–544. [PubMed]
  • Nicola NA. Hemopoietic cell growth factors and their receptors. Annu Rev Biochem. 1989;58:45–77. [PubMed]
  • Nocka K, Majumder S, Chabot B, Ray P, Cervone M, Bernstein A, Besmer P. Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice--evidence for an impaired c-kit kinase in mutant mice. Genes Dev. 1989 Jun;3(6):816–826. [PubMed]
  • Oohira A, Wight TN, McPherson J, Bornstein P. Biochemical and ultrastructural studies of proteoheparan sulfates synthesized by PYS-2, a basement membrane-producing cell line. J Cell Biol. 1982 Feb;92(2):357–367. [PMC free article] [PubMed]
  • Papayannopoulou T, Kurachi S, Brice M, Nakamoto B, Stamatoyannopoulos G. Asynchronous synthesis of HbF and HbA during erythroblast maturation. II. Studies of G gamma, A gamma, and beta chain synthesis in individual erythroid clones from neonatal and adult BFU-E cultures. Blood. 1981 Mar;57(3):531–536. [PubMed]
  • Paul SR, Bennett F, Calvetti JA, Kelleher K, Wood CR, O'Hara RM, Jr, Leary AC, Sibley B, Clark SC, Williams DA, et al. Molecular cloning of a cDNA encoding interleukin 11, a stromal cell-derived lymphopoietic and hematopoietic cytokine. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7512–7516. [PubMed]
  • Pevny L, Simon MC, Robertson E, Klein WH, Tsai SF, D'Agati V, Orkin SH, Costantini F. Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature. 1991 Jan 17;349(6306):257–260. [PubMed]
  • Quesniaux VF, Clark SC, Turner K, Fagg B. Interleukin-11 stimulates multiple phases of erythropoiesis in vitro. Blood. 1992 Sep 1;80(5):1218–1223. [PubMed]
  • Righetti PG, Gianazza E, Gianni AM, Comi P, Giglioni B, Ottolenghi S, Secchi C, Rossi-Bernardi L. Human globin chain separation by isoelectric focusing. J Biochem Biophys Methods. 1979;1(1):45–57. [PubMed]
  • Robertson E, Bradley A, Kuehn M, Evans M. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature. 1986 Oct 2;323(6087):445–448. [PubMed]
  • Schmitt RM, Bruyns E, Snodgrass HR. Hematopoietic development of embryonic stem cells in vitro: cytokine and receptor gene expression. Genes Dev. 1991 May;5(5):728–740. [PubMed]
  • Simon MC, Pevny L, Wiles MV, Keller G, Costantini F, Orkin SH. Rescue of erythroid development in gene targeted GATA-1- mouse embryonic stem cells. Nat Genet. 1992 May;1(2):92–98. [PubMed]
  • Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, Rogers D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature. 1988 Dec 15;336(6200):688–690. [PubMed]
  • Whitelaw E, Tsai SF, Hogben P, Orkin SH. Regulated expression of globin chains and the erythroid transcription factor GATA-1 during erythropoiesis in the developing mouse. Mol Cell Biol. 1990 Dec;10(12):6596–6606. [PMC free article] [PubMed]
  • Wiles MV, Keller G. Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development. 1991 Feb;111(2):259–267. [PubMed]
  • Wilkinson DG, Bhatt S, Herrmann BG. Expression pattern of the mouse T gene and its role in mesoderm formation. Nature. 1990 Feb 15;343(6259):657–659. [PubMed]
  • Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature. 1988 Dec 15;336(6200):684–687. [PubMed]
  • WILT FH. ERYTHROPOIESIS IN THE CHICK EMBRYO: THE ROLE OF ENDODERM. Science. 1965 Mar 26;147(3665):1588–1590. [PubMed]
  • Witte ON. Steel locus defines new multipotent growth factor. Cell. 1990 Oct 5;63(1):5–6. [PubMed]
  • Wong PM, Chung SW, Chui DH, Eaves CJ. Properties of the earliest clonogenic hemopoietic precursors to appear in the developing murine yolk sac. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3851–3854. [PubMed]
  • Wong PM, Chung SW, Eaves CJ, Chui DH. Ontogeny of the mouse hemopoietic system. Prog Clin Biol Res. 1985;193:17–28. [PubMed]
  • Wong PM, Chung SW, Reicheld SM, Chui DH. Hemoglobin switching during murine embryonic development: evidence for two populations of embryonic erythropoietic progenitor cells. Blood. 1986 Mar;67(3):716–721. [PubMed]
  • Ymer S, Tucker WQ, Sanderson CJ, Hapel AJ, Campbell HD, Young IG. Constitutive synthesis of interleukin-3 by leukaemia cell line WEHI-3B is due to retroviral insertion near the gene. Nature. 1985 Sep 19;317(6034):255–258. [PubMed]
  • Zhang XK, Wang Z, Lee A, Huang DP, Chiu JF. Differential expression of cellular oncogenes during rat liver development. Cancer Lett. 1988 Aug 15;41(2):147–155. [PubMed]
  • Zon LI, Mather C, Burgess S, Bolce ME, Harland RM, Orkin SH. Expression of GATA-binding proteins during embryonic development in Xenopus laevis. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10642–10646. [PubMed]
  • Zsebo KM, Williams DA, Geissler EN, Broudy VC, Martin FH, Atkins HL, Hsu RY, Birkett NC, Okino KH, Murdock DC, et al. Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell. 1990 Oct 5;63(1):213–224. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)