Hematopoietic differentiation of embryonic stem cells: an in vitro model to study gene regulation during megakaryocytopoiesis.
We are interested in the regulation of the tissue specificity of the megakaryocyte-specific platelet glycoprotein IIb gene. The murine embryonic stem (ES) cells are able to differentiate into erythroid, mast and granulomonocytic cells in appropriate culture conditions. Our goal is to optimize the production of myeloid cells including megakaryocytes (MKs) by ES cells. We have found that coculture with MS-5 stromal cells and the presence of a cocktail of hematopoietic growth factors (HGFs) [stem cell factor, interleukin 3 (IL-3), IL-6, IL-11, G-CSF and erythropoietin] had a high synergistic activity on differentiation of ES cells into pure and MK-containing myeloid colonies from day 12 embryoid bodies. Thrombopoietin increased the number of MKs only when added to the HGF cocktail in the presence of MS-5 cells. Interestingly, many MKs exhibited a "hairy" appearance evocative of pseudopodial proplatelet formation. Expression of genes specific for the megakaryocytic lineage, GPIIb, PF4, mpl and GPIIIa, was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) during differentiation of ES cells, and their relative time course was evaluated. This demonstrates that optimized culture conditions for the differentiation of ES cells into the MK lineage provide a useful tool for the study of the regulation of expression of genes during megakaryocytopoiesis.
Uzan
G
G
CEA, Laboratoire d'Hématologie, INSERM U217, Grenoble, France.
Prandini
M H
MH
Rosa
J P
JP
Berthier
R
R
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D019070:Cell Lineage; D018920:Coculture Techniques; D004622:Embryo, Mammalian / Q000378:metabolism; D004921:Erythropoietin / Q000494:pharmacology; D005786:Gene Expression Regulation; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D006801:Humans; D017370:Interleukin-11 / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019089:Stem Cell Factor / Q000494:pharmacology; D017154:Stromal Cells / Q000378:metabolism; D013997:Time Factors
D017370:Interleukin-11; D007377:Interleukin-3; D015850:Interleukin-6; D019089:Stem Cell Factor; D004921:Erythropoietin; D016179:Granulocyte Colony-Stimulating Factor