[The in vitro isolation, culture and transfection of human fetal epidermal stem cells].
OBJECTIVE
To explore the in vitro methods of isolation and culture of human fetal epidermal stem cells (HFESCs) and the feasibility of the cultured cells as the target cells for gene transfection.


METHODS
The HFESCs were isolated by means of type IV collagen rapid adhering method. The culture medium for HFESCs was prepared according to that for human fetal fibroblasts. The cultured cells were identified by immunohistochemistry staining of keratin-19 and integrin-beta1, cell cycle analysis and clone forming rate determination. Then the cultured cells were gene transfected in vitro by liposome mediating method in which eukaryon expression vector pcDNA3.1/VEGF165 containing vascular endothelial growth factor 165 (VEGF165) were transfected into cultured cells, or by virus vector mediating method in which recombinant adenovirus accompanied vector (raav) containing green fluorescent protein (GFP) (raav/GFP) were transfected into the cultured cells, respectively. The results of in vitro gene transfection of HFESCs were observed by immunohistochemisty staining and fluorescence microscope.


RESULTS
HFESCs grew well and formed large clones with higher cloning efficiency and higher ratio of G1 cells than keratinocytes. The cultured cells were strongly positive with immunohistochemistry staining of keratin-19 and integrin-beta1. After being gene-transfected by pcDNA3.1/VEGF165, the VEGF165 of HFESCs showed positive immunohistochemistry staining property, while the HFESCs transfected by raav/GFP exhibited strong fluorescence.


CONCLUSIONS
HFESCs could be isolated and cultured in vitro by means of rapid adherence to type IV collagen. It seemed feasible that HFESCs were gene transfected with liposome or adeno-associated virus as the vector.
Ding
Guo-Bin
GB
Department of Burns, Xijing Hospital, The Fourth Military Medical University, Xi'an 710033. Shaanxi Province, P.R. China.
Chen
Bi
B
Han
Jun-Tao
JT
Tang
Chao-Wu
CW
Wang
Bo-Tao
BT
D002448:Cell Adhesion; D002453:Cell Cycle / Q000502:physiology; D002478:Cells, Cultured; D016228:Endothelial Growth Factors / Q000235:genetics / Q000378:metabolism*; D004817:Epidermis; D005333:Fetus; D016193:G1 Phase; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D019012:Integrin beta1 / Q000032:analysis; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D015603:Keratinocytes / Q000166:cytology; D007633:Keratins / Q000032:analysis; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008222:Lymphokines / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D013234:Stem Cells / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D019012:Integrin beta1; D036341:Intercellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008222:Lymphokines; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D049452:Green Fluorescent Proteins; D007633:Keratins