TY - JOUR
T1 - Bcl-xL enhances single-cell survival and expansion of human embryonic stem cells without affecting self-renewal
AU - Bai, Hao
AU - Chen, Kang
AU - Gao, Yong Xing
AU - Arzigian, Melanie
AU - Xie, Yin Liang
AU - Malcosky, Christopher
AU - Yang, Yong Guang
AU - Wu, Wen Shu
AU - Wang, Zack Z.
N1 - Funding Information:
This work was supported by NIH NCRR 2P20RR018789 , NIH/RC1 Challenge Grant RC1 HL100117 , and American Society of Hematology Faculty Award (ZW). Additional technical core support was provided by NIH Center of Excellence in Stem Cell Biology and Regenerative Medicine core facilities (2P20RR18789) and Center of Excellence in Vascular Biology core facilities (2P20RR15555).
PY - 2012/1
Y1 - 2012/1
N2 - Robust expansion and genetic manipulation of human embryonic stem cells (hESCs) and induced-pluripotent stem (iPS) cells are limited by poor cell survival after enzymatic dissociation into single cells. Although inhibition of apoptosis is implicated for the single-cell survival of hESCs, the protective role of attenuation of apoptosis in hESC survival has not been elucidated. Bcl-xL is one of several anti-apoptotic proteins, which are members of the Bcl-2 family of proteins. Using an inducible system, we ectopically expressed Bcl-xL gene in hESCs, and found a significant increase of hESC colonies in the single-cell suspension cultures. Overexpression of Bcl-xL in hESCs decreased apoptotic caspase-3 + cells, suggesting attenuation of apoptosis in hESCs. Without altering the kinetics of pluripotent gene expression, the efficiency to generate embryoid bodies (EBs) in vitro and the formation of teratoma in vivo were significantly increased in Bcl-xL-overexpressing hESCs after single-cell dissociation. Interestingly, the number and size of hESC colonies from cluster cultures were not affected by Bcl-xL overexpression. Several genes of extracellular matrix and adhesion molecules were upregulated by Bcl-xL in hESCs without single-cell dissociation, suggesting that Bcl-xL regulates adhesion molecular expression independent of cell dissociation. In addition, the gene expressions of FAS and several TNF signaling mediators were downregulated by Bcl-xL.These data support a model in which Bcl-xL promotes cell survival and increases cloning efficiency of dissociated hESCs without altering hESC self-renewal by i) attenuation of apoptosis, and ii) upregulation of adhesion molecules to facilitate cell-cell or cell-matrix interactions.
AB - Robust expansion and genetic manipulation of human embryonic stem cells (hESCs) and induced-pluripotent stem (iPS) cells are limited by poor cell survival after enzymatic dissociation into single cells. Although inhibition of apoptosis is implicated for the single-cell survival of hESCs, the protective role of attenuation of apoptosis in hESC survival has not been elucidated. Bcl-xL is one of several anti-apoptotic proteins, which are members of the Bcl-2 family of proteins. Using an inducible system, we ectopically expressed Bcl-xL gene in hESCs, and found a significant increase of hESC colonies in the single-cell suspension cultures. Overexpression of Bcl-xL in hESCs decreased apoptotic caspase-3 + cells, suggesting attenuation of apoptosis in hESCs. Without altering the kinetics of pluripotent gene expression, the efficiency to generate embryoid bodies (EBs) in vitro and the formation of teratoma in vivo were significantly increased in Bcl-xL-overexpressing hESCs after single-cell dissociation. Interestingly, the number and size of hESC colonies from cluster cultures were not affected by Bcl-xL overexpression. Several genes of extracellular matrix and adhesion molecules were upregulated by Bcl-xL in hESCs without single-cell dissociation, suggesting that Bcl-xL regulates adhesion molecular expression independent of cell dissociation. In addition, the gene expressions of FAS and several TNF signaling mediators were downregulated by Bcl-xL.These data support a model in which Bcl-xL promotes cell survival and increases cloning efficiency of dissociated hESCs without altering hESC self-renewal by i) attenuation of apoptosis, and ii) upregulation of adhesion molecules to facilitate cell-cell or cell-matrix interactions.
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U2 - 10.1016/j.scr.2011.08.002
DO - 10.1016/j.scr.2011.08.002
M3 - Article
C2 - 22099018
AN - SCOPUS:80052592682
SN - 1873-5061
VL - 8
SP - 26
EP - 37
JO - Stem Cell Research
JF - Stem Cell Research
IS - 1
ER -