Efficient and repetitive production of hematopoietic and endothelial cells from feeder-free monolayer culture system of primate embryonic stem cells.
We have established an innovative culture system for the efficient differentiation of hematopoietic and endothelial cells from primate embryonic stem (ES) cells without feeder cells, embryoid bodies, or cell-sorting processes. After several days' culture in murine stromal OP9-conditioned medium supplemented with a cytokine cocktail on collagen-coated dishes, ES cells differentiated into a very unique population of cells with a finger-like appearance. These finger-like cells were positive for mesodermal and/or hemangioblastic markers of kinase insert domain receptor (KDR) and T-cell acute lymphocytic leukemia 1 (TAL1), and produced large amounts of protein tyrosine phosphatase, receptor type, C-positive hematopoietic cells. These hematopoietic cells showed the morphology of immature hematopoietic cells, formed blast cell colonies with high efficiency, and were positive for CD34 antigen, KDR, TAL1, and GATA binding protein 1, suggesting that these blast cells are equivalent to the multipotent hematopoietic progenitor cells. Moreover, they produced functional macrophages in murine stromal MS-5-conditioned medium and primitive erythroblasts in the presence of erythropoietin. The finger-like cells, putative mesodermal progenitors and/or hemangioblasts, actively proliferated and repetitively produced hematopoietic cells as long as they were maintained on the original dish. By contrast, the majority of the finger-like cells differentiated into endothelial cells with specific markers and specific functions after transfer to fresh dishes, indicating that conditions established in the original dish supported the proliferation and hematopoietic differentiation of the finger-like cells. Our method provides a highly controllable culture protocol for repetitive production of hematopoietic and endothelial cells from feeder-free monolayer cultivation of primate ES cells.
Zhang
Hong
H
Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo 162-8655, Japan.
Saeki
Kumiko
K
Kimura
Akiko
A
Saeki
Koichi
K
Nakahara
Masako
M
Doshi
Masaru
M
Kondo
Yasushi
Y
Nakano
Toru
T
Yuo
Akira
A
D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology; D004921:Erythropoietin / Q000494:pharmacology; D050982:GATA1 Transcription Factor / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology; D008252:Macaca fascicularis / Q000196:embryology*; D008264:Macrophages / Q000166:cytology / Q000502:physiology; D051379:Mice; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D029961:Zebrafish Proteins / Q000378:metabolism
D018952:Antigens, CD34; D051792:Basic Helix-Loop-Helix Proteins; D015415:Biomarkers; D004921:Erythropoietin; D050982:GATA1 Transcription Factor; D011518:Proto-Oncogene Proteins; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D040301:Vascular Endothelial Growth Factor Receptor-2; D029961:Zebrafish Proteins; C489366:Tal1 protein, mouse; C494691:gata1a protein, zebrafish