Sequential analysis of alpha- and beta-globin gene expression during erythropoietic differentiation from primate embryonic stem cells.
The temporal pattern of embryonic, fetal, and adult globin expression in the alpha (zeta --> alpha) and beta (epsilon --> gamma and gamma --> beta) clusters were quantitatively analyzed at the transcriptional and translational levels in erythrocytes induced from primate embryonic stem cells in vitro. When vascular endothelial growth factor receptor-2(high) CD34(+) cells were harvested and reseeded onto OP9 stromal cells, two-wave erythropoiesis occurred sequentially. Immunostaining and real-time reverse transcription-polymerase chain reaction analyses of floating mature erythrocytes revealed that globin switches occurred in parallel with the erythropoietic transition. Colony-forming assays showed replacement of primitive clonogenic progenitor cells with definitive cells during culturing. A decline in embryonic zeta- and epsilon-globin expression at the translational level occurred in individual definitive erythroid progenitors. Expression of beta-globin in individual definitive erythroid progenitors was upregulated in the presence of OP9 stromal cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of the globin switch in humans.
Umeda
Katsutsugu
K
Department of Pediatrics, Graduate School of Medicine, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Heike
Toshio
T
Nakata-Hizume
Mami
M
Niwa
Akira
A
Arai
Masato
M
Shinoda
Gen
G
Ma
Feng
F
Suemori
Hirofumi
H
Luo
Hong Yuan
HY
Chui
David H K
DH
Torii
Ryuzo
R
Shibuya
Masabumi
M
Nakatsuji
Norio
N
Nakahata
Tatsutoshi
T
D000818:Animals; D018952:Antigens, CD34 / Q000276:immunology; D018920:Coculture Techniques; D053595:Embryonic Stem Cells / Q000166:cytology*; D004912:Erythrocytes / Q000166:cytology; D004920:Erythropoiesis / Q000235:genetics*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D005914:Globins / Q000235:genetics*; D006801:Humans; D008252:Macaca fascicularis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
D018952:Antigens, CD34; D012333:RNA, Messenger; D005914:Globins; D040301:Vascular Endothelial Growth Factor Receptor-2