[A preliminary study on the culture of single hematopoietic stem cell].
OBJECTIVE
To investigate the biological behavior including survival and proliferation of CD34 + CD38--Lin--cells when they are cultured at single cell level.


METHODS
Purified umbilical cord blood CD34 + CD38--Lin--cells were separated at single cell level in 96-well plates using flow cytometry for four groups: control group (CD34 + CD38--Lin--cell plus stem cell medium) , Shh group (CD34 + CD38--Lin--cell plus stem cell medium and Shh), BMP-4 group (CD34 + CD38--Lin--cell plus stem cell medium and BMP-4), Jagged-1 group (CD34 + CD38--Lin--cell plus stem cell medium and Jagged-1). Methylcellulose medium was used in the colony-forming experiment which was also in four groups as previously. The number of cells and colony-forming units in each well for the four groups was evaluated at different time points (day 1, 3, 7) with fluorescence microscopy counting method.


RESULTS
Division of single cell was observed to be amplified in all of these groups from day 3. And meanwhile, after 1-week culture, the survival rates for the treated groups were all higher than the control group (Jagged-1 group > BMP-4 group > Shh group > control), while the cell number in each well was also highest in the Jagged-1 group (Jagged-1 group > BMP-4 group > control). The number of wells with a cell number of zero was significantly fewer in all treated groups (especially the Jagged-1 group) than in the control group; meanwhile, the number of wells with a cell number higher than 17 was evidently higher in all the treated groups (especially the BMP-4 group) more than controls. Colony-forming units for erythroid (BFU-E), granulocyte (CFU-G), macrophage (CFU-M), and granulocyte macrophage (CFU-GM) were observed for all of these experimental groups, and there was no significant difference between the four experimental groups.


CONCLUSIONS
CD34 + CD38 - Lin - cell can achieve the survival, self-renewal and proliferation when cultured at single cell level, and the adding of Shh, BMP-4, and Jagged-1 can enhance such capabilities. However, CD34 + CD38 - Lin - cell can only maintain cell totipotency in its niche.
State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, CAMS and PUMC, Tianjin 300020, China.
You-Jin
YJ
Han
Jun-Yuan
JY
Qi
Lu-Gui
LG
Qiu
D051997:ADP-ribosyl Cyclase 1 / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D055415:Bone Morphogenetic Protein 4 / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D003470:Culture Media; D005312:Fetal Blood / Q000166:cytology*; D053823:Hedgehog Proteins / Q000737:chemistry; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry; D000072100:Jagged-1 Protein; D008565:Membrane Proteins / Q000737:chemistry; D000072099:Serrate-Jagged Proteins
D018952:Antigens, CD34; D055415:Bone Morphogenetic Protein 4; D002135:Calcium-Binding Proteins; D003470:Culture Media; D053823:Hedgehog Proteins; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; D000072100:Jagged-1 Protein; D008565:Membrane Proteins; D000072099:Serrate-Jagged Proteins; D051997:ADP-ribosyl Cyclase 1