Isolation and culture of human embryonic AGM derived HSPCs in hematopoietic culture systems created by AGM stromal cells.
- Author:
Bei-Yan WU
1
;
Shao-Liang HUANG
;
Hui-Qin CHEN
;
Xu-Chao ZHANG
Author Information
1. Department of Pediatrics, The First Hospital, Shantou University Medical College, Shantou 515041, Guangdong Province, China.
- Publication Type:Journal Article
- MeSH:
Aorta;
cytology;
Cell Culture Techniques;
methods;
Cell Separation;
Cells, Cultured;
Coculture Techniques;
Fetal Blood;
cytology;
Gonads;
cytology;
Hematopoietic Stem Cells;
cytology;
Humans;
Mesonephros;
cytology;
Stromal Cells;
cytology;
physiology
- From:
Journal of Experimental Hematology
2008;16(3):579-583
- CountryChina
- Language:Chinese
-
Abstract:
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.
