Can the life span of human marrow stromal cells be prolonged by bmi-1, E6, E7, and/or telomerase without affecting cardiomyogenic differentiation?
BACKGROUND
Cell transplantation has recently been challenged to improve cardiac function of severe heart failure. Human mesenchymal stem cells (hMSCs) are multipotent cells that can be isolated from adult marrow stroma, but because of their limited life span, it is difficult to study them further. To overcome this problem, we attempted to prolong the life span of hMSCs and investigate whether the hMSCs modified with cell-cycle-associated genes can differentiate into cardiomyocytes in vitro.


METHODS
We attempted to prolong the life span of hMSCs by infecting retrovirus encoding bmi-1, human papillomavirus E6 and E7, and/or human telomerase reverse transcriptase genes. To determine whether the hMSCs with an extended life span could differentiate into cardiomyocytes, 5-azacytidine-treated hMSCs were co-cultured with fetal cardiomyocytes in vitro.


RESULTS
The established hMSCs proliferated over 150 population doublings. On day 3 of co-cultivation, the hMSCs became elongated, like myotubes, began spontaneously beating, and acquired automaticity. Their rhythm clearly differed from that of the surrounding fetal mouse cardiomyocytes. The number of beating cardiomyocytes increased until 3 weeks. hMSCs clearly exhibited differentiated cardiomyocyte phenotypes in vitro as revealed by immunocytochemistry, RT-PCR, and action potential recording.


CONCLUSIONS
The life span of hMSCs was prolonged without interfering with cardiomyogenic differentiation. hMSCs with an extended life span can be used to produce a good experimental model of cardiac cell transplantation and may serve as a highly useful cell source for cardiomyocytic transplantation.
Department of Reproductive Biology and Pathology, National Research Institute for Child Health and Development, Tokyo, Japan.
Yukiji
Y
Takeda
Taisuke
T
Mori
Hideaki
H
Imabayashi
Tohru
T
Kiyono
Satoshi
S
Gojo
Shunichirou
S
Miyoshi
Naoko
N
Hida
Makoto
M
Ita
Kaoru
K
Segawa
Satoshi
S
Ogawa
Michiie
M
Sakamoto
Shinobu
S
Nakamura
Akihiro
A
Umezawa
D000200:Action Potentials; D000368:Aged; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation; D002459:Cell Fusion; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D016922:Cellular Senescence; D004268:DNA-Binding Proteins; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D009206:Myocardium / Q000166:cytology; D032383:Myocytes, Cardiac; D009687:Nuclear Proteins / Q000235:genetics*; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D017154:Stromal Cells / Q000502:physiology; D019098:Telomerase / Q000235:genetics*; D014161:Transduction, Genetic
C489421:Bmi1 protein, mouse; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; D009687:Nuclear Proteins; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D063150:Polycomb Repressive Complex 1; D019098:Telomerase