Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells.
OBJECTIVE
To compare the hematopoietic support provided by telomerized human mesenchymal stem cells (MSCs) and telomerized MSC-derived stromal cells.


METHODS
We transfected the human telomerase catalytic subunit (hTERT) gene into primary MSCs to establish hTERT-transduced MSCs (hTERT-MSCs). Stromal induction of hTERT-MSCs was performed by replacing the culture medium with Dexter-type culture medium. Hematopoietic support was examined by coculture with cord blood CD34(+) cells.


RESULTS
The hTERT-MSCs were morphologically identical with the primary MSCs and expressed surface antigens including CD105, CD73, and CD166. hTERT-MSCs showed a similar doubling time as primary MSCs and continued to proliferate to over 80 population doublings (PD), although the primary MSCs underwent crisis in vitro at 16 PD. The osteogenic, chondrogenic, adipogenic, neurogenic, and stromal differentiation potential of hTERT-MSCs were maintained up to at least 40 PD. The degree of expansion of CD34(+) cells and total number of colony-forming units in culture (CFU-C) upon 12-day coculture with the hTERT-MSC-derived stromal cells were nearly the same as those upon 12-day coculture with hTERT-MSCs (CD34, 33.0-fold+/-2.8-fold vs 36.1-fold+/-1.7-fold of the initial cell number; CFUs, 344.4-fold+/-62.5-fold vs 239.3-fold+/-87.0-fold; CFU-mix, 368.4-fold+/-113.7-fold vs 341.3-fold+/-234.3-fold). However, on day 18 of coculture, the number of cobblestone areas (CA) observed beneath the stromal cells was 15 times higher than that beneath hTERT-MSCs (CA, 146.9+/-54.6 vs 9.4+/-8.1, p<0.01).


CONCLUSIONS
Stromal induction of hTERT-MSCs exclusively enhanced the support of CA formation provided by hTERT-MSCs. Our human hTERT-MSCs will be useful for elucidating the mechanism of the formation of CAs.
Kobune
Masayoshi
M
Dept. of Molecular Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Kawano
Yutaka
Y
Ito
Yoshinori
Y
Chiba
Hiroki
H
Nakamura
Kiminori
K
Tsuda
Hajime
H
Sasaki
Katsunori
K
Dehari
Hironari
H
Uchida
Hiroaki
H
Honmou
Osamu
O
Takahashi
Sho
S
Bizen
Akiko
A
Takimoto
Rishu
R
Matsunaga
Takuya
T
Kato
Junji
J
Kato
Kazunori
K
Houkin
Kiyohiro
K
Niitsu
Yoshiro
Y
Hamada
Hirofumi
H
D000328:Adult; D015703:Antigens, CD / Q000032:analysis; D002454:Cell Differentiation; D002478:Cells, Cultured / Q000166:cytology; D018920:Coculture Techniques; D003470:Culture Media / Q000494:pharmacology; D004268:DNA-Binding Proteins; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D008648:Mesoderm / Q000166:cytology*; D039902:Multipotent Stem Cells / Q000166:cytology*; D017154:Stromal Cells / Q000166:cytology; D019098:Telomerase / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D015703:Antigens, CD; D003470:Culture Media; D004268:DNA-Binding Proteins; D019098:Telomerase