[Isolation and culture of human embryonic AGM derived HSPCs in hematopoietic culture systems created by AGM stromal cells].
This study was purposed to isolate human embryonic AGM derived HSPCs and investigate the effect of AGM stromal cells on AGM-derived HSPCs. Immunohistochemical sections of human AGM tissue were investigated for CD34, Flk-1 and VEGF expression. Human AGM-derived single cells were isolated and seeded onto pre-treated feeder of human AGM stromal cells (hAGMS3 and hAGMS4) by direct contact and non-contact co-culture in Transwell culture system. Growth characteristics of HSPCs with cobblestone area-forming cells (CAFCs) were observed and number of cobblestone area (CA) was counted. Indirect immunofluorescent assay was used to detect CD34 and Flk-1 expression on the surface of suspended cells as well as CAFCs in contact co-culture system. The cells after culture for 2 weeks were collected from both contact and non-contact co-culture systems for CFU assay. The result showed that hematopoietic cells in AGM tissue expressed CD34 and Flk-1. Both of the hematopoietic culture systems could produce CFCs. Nevertheless, direct contact co-culture produced CD34(+)Flk-1(+) CAFC and more CFUs than those from indirect non-contact culture (hAGMS3 system: 1647 +/- 194 vs 389 +/- 31, p < 0.05; hAGMS4 system: 1586 +/- 75 vs 432 +/- 35, p < 0.05). It is concluded that there were CD34(+)Flk-1(+) HSCs in human embryonic AGM region. The hematopoietic co-culture systems composed of AGM-derived HSPCs and AGM stromal cells are successfully established, both direct contact and Transwell non-contact co-culture can expand AGM-derived definitive HSPCs. Cell-cell contact between AGM-derived HSPCs and AGM stromal cells are of most importance to maintain and expand AGM-HSPCs.
Department of Pediatrics, The First Hospital, Shantou University Medical College, Shantou 515041, Guangdong Province, China.
Bei-Yan
BY
Wu
Shao-Liang
SL
Huang
Hui-Qin
HQ
Chen
Xu-Chao
XC
Zhang
D001011:Aorta / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation; D002478:Cells, Cultured; D018920:Coculture Techniques; D005312:Fetal Blood / Q000166:cytology*; D006066:Gonads / Q000166:cytology*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D008650:Mesonephros / Q000166:cytology*; D017154:Stromal Cells / Q000166:cytology / Q000502:physiology*