Ex vivo expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subunit (hTERT)-transfected human stromal cells.
We developed a new human stromal cell line that could expand human hematopoietic progenitor/stem cells. Primary human bone marrow stromal cells were infected with retrovirus containing the human telomerase catalytic subunit (hTERT) gene, resulting in increased population doubling and the acquisition of cell immortalization. Characteristics of the hTERT-transduced stromal (hTERT-stromal) cells were identical with those of the primary stromal cells in terms of morphologic appearance and expression of surface antigens. Human cord blood (CB) CD34(+) cells were expanded by coculture with primary stromal or hTERT-stromal cells in the presence of stem cell factor, thrombopoietin, and Flk-2/Flt-3 ligand under serum-free condition. The degree of expansion of CD34(+) cells and total number of colony-forming units in culture (CFU-Cs) after 2 weeks' coculture with the hTERT-stromal cells were nearly the same as those after 2 weeks' coculture with primary stromal cells (CD34(+) cells, 118-fold +/- 8-fold versus 117-fold +/- 13-fold; CFU-Cs, 71-fold +/- 5-fold versus 67-fold +/- 5-fold of initial cell number). CB expansion on hTERT-stromal cells occurred at a similar rate through 7 weeks. In contrast, the rate of CB expansion on primary stromal cells had drastically declined at 7 weeks. In nonobese diabetic/severe combined immunodeficiency (SCID) mice, the degree of engraftment of SCID-repopulating cells that had been cocultured with hTERT-stromal cells for 4 weeks was significantly higher than that of precocultured CB cells. These results indicate that this hTERT-stromal cell line could be useful for ex vivo expansion of hematopoietic progenitor/stem cells and for analyzing the microenvironment of human bone marrow.
Kawano
Yutaka
Y
Department of Molecular Medicine, Sapporo Medical University, Japan.
Kobune
Masayoshi
M
Yamaguchi
Miki
M
Nakamura
Kiminori
K
Ito
Yoshinori
Y
Sasaki
Katsunori
K
Takahashi
Sho
S
Nakamura
Takafumi
T
Chiba
Hiroki
H
Sato
Tsutomu
T
Matsunaga
Takuya
T
Azuma
Hiroshi
H
Ikebuchi
Kenji
K
Ikeda
Hisami
H
Kato
Junji
J
Niitsu
Yoshiro
Y
Hamada
Hirofumi
H
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000235:genetics / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D018920:Coculture Techniques; D004268:DNA-Binding Proteins; D005312:Fetal Blood / Q000166:cytology; D006410:Hematopoiesis / Q000187:drug effects; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism*; D019098:Telomerase / Q000235:genetics* / Q000494:pharmacology; D014162:Transfection; D014183:Transplantation, Heterologous; D018631:ras Proteins / Q000235:genetics / Q000494:pharmacology
D000957:Antigens, Viral, Tumor; D004268:DNA-Binding Proteins; D019098:Telomerase; D018631:ras Proteins