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Prepublished online as a Blood First Edition Paper on September 5, 2002; DOI 10.1182/blood-2002-04-1268.
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Blood, 15 January 2003, Vol. 101, No. 2, pp. 532-540
HEMATOPOIESIS
Ex vivo expansion of human umbilical cord hematopoietic
progenitor cells using a coculture system with human telomerase
catalytic subunit (hTERT)-transfected human stromal
cells
Yutaka Kawano,
Masayoshi Kobune,
Miki Yamaguchi,
Kiminori Nakamura,
Yoshinori Ito,
Katsunori Sasaki,
Sho Takahashi,
Takafumi Nakamura,
Hiroki Chiba,
Tsutomu Sato,
Takuya Matsunaga,
Hiroshi Azuma,
Kenji Ikebuchi,
Hisami Ikeda,
Junji Kato,
Yoshiro Niitsu, and
Hirofumi Hamada
From the Department of Molecular Medicine, Sapporo
Medical University, Japan; Fourth Department of Internal
Medicine, Sapporo Medical University, Japan; Hokkaido Red
Cross Blood Center, Sapporo, Japan; and Department of
Biochemistry, Tokyo Medical University, Tokyo, Japan.
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.
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