Bone marrow stromal cells transduced with human hemangiopoietin gene support hematopoiesis in vitro.
OBJECTIVE
The aim of this study was to construct a eukaryotic expression vector containing human hemangiopoietin (hHAPO) gene and express it in mouse bone marrow stromal cell line HESS-5, then support hematopoiesis in vitro with gene-modified HESS-5 (hHAPO-HESS-5).


METHODS
The polymerase chain reaction (PCR) products of HAPO were digested with BamHI and BgII. Then the HAPO gene segment obtained was again cloned into pIRES2-EGFP to construct recombinant eukaryotic expression vector HAPO-pIRES2-EGFP. The recombinant vector was identified by enzyme digestion analysis, PCR, and sequencing. HESS-5 cells were transformed by recombinant vector and positive clones were selected with G418. The expression of HAPO gene in the transformed cells was detected by studying EGFP expression, reverse transcription (RT)-PCR, and Western-blotting analysis. Support of human hematopoiesis by hHAPO-HESS-5 cells was evaluated in co-culture experiments with human CD34+ cells.


RESULTS
Enzyme digestion analysis and sequencing showed that the target gene had been cloned into the recombinant vector. The expression of HAPO gene in the transformed stromal cells was demonstrated by fluoro-microscopy and RT-PCR analysis. HAPO protein was also detected in the supernatant of hHAPO-HESS-5 by Western blot analysis. As expected, stably transfected hHAPO-HESS-5 cells significantly increased in both relative and absolute numbers of CD34+ cells after 14 days of culture. The PKH26 study demonstrated that cell division was faster in CD34+ cells co-cultured with hHAPO-HESS-5 cells than in cells cocultured with vector-HESS-5 cells. The hHAPO-HESS-5 cells also supported human hematopoiesis in vitro more efficiently than did control vector-HESS-5 cells.


CONCLUSIONS
A recombinant eukaryotic expression vector has been constructed and expressed successfully in transformed cells. The hHAPO-HESS-5 cells support rapid generation of primitive progenitor cells and maintain reconstituting ability of hematopoietic stem cells in vitro. Therefore, it would be possible to use stromal cells expressing HAPO gene as seed cells in the bone marrow transplantation.
Xu
Zhen-shu
ZS
TEDA Life and Technology Research Center, State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China.
Liu
Yong-Jun
YJ
Lv
Lu-lu
LL
Han
Zhi-Bo
ZB
He
Rui
R
Lu
Shi-hong
SH
Wang
Tong
T
Xu
Bin
B
Chen
Zhi-zhe
ZZ
Han
Zhong Chao
ZC
D042682:Angiopoietins / Q000235:genetics*; D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology*; D016026:Bone Marrow Transplantation; D002460:Cell Line; D018920:Coculture Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006410:Hematopoiesis / Q000502:physiology*; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction; D011509:Proteoglycans / Q000235:genetics* / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology; D017154:Stromal Cells / Q000166:cytology*; D000099158:Proteoglycan 4
D042682:Angiopoietins; D018952:Antigens, CD34; D049452:Green Fluorescent Proteins; D011509:Proteoglycans; C419197:PRG4 protein, human; D000099158:Proteoglycan 4