Improved efficiency and pace of generating induced pluripotent stem cells from human adult and fetal fibroblasts.
It was reported recently that human fibroblasts can be reprogrammed into a pluripotent state that resembles that of human embryonic stem (hES) cells. This was achieved by ectopic expression of four genes followed by culture on mouse embryonic fibroblast (MEF) feeders under a condition favoring hES cell growth. However, the efficiency of generating human induced pluripotent stem (iPS) cells is low, especially for postnatal human fibroblasts. We started supplementing with an additional gene or bioactive molecules to increase the efficiency of generating iPS cells from human adult as well as fetal fibroblasts. We report here that adding SV40 large T antigen (T) to either set of the four reprogramming genes previously used enhanced the efficiency by 23-70-fold from both human adult and fetal fibroblasts. Discernible hES-like colonies also emerged 1-2 weeks earlier if T was added. With the improved efficiency, we succeeded in replacing MEFs with immortalized human feeder cells that we previously established for optimal hES cell growth. We further characterized individually picked hES-like colonies after expansion (up to 24 passages). The majority of them expressed various undifferentiated hES markers. Some but not all the hES-like clones can be induced to differentiate into the derivatives of the three embryonic germ layers in both teratoma formation and embryoid body (EB) formation assays. These pluripotent clones also differentiated into trophoblasts after EB formation or bone morphogenetic protein 4 induction as classic hES cells. Using this improved approach, we also generated hES-like cells from homozygous fibroblasts containing the sickle cell anemia mutation Hemoglobin Sickle. Disclosure of potential conflicts of interest is found at the end of this article.
Mali
Prashant
P
Stem Cell Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Broadway Research Building, Room 747, 733 North Broadway, Baltimore, Maryland 21205, USA.
Ye
Zhaohui
Z
Hommond
Holly H
HH
Yu
Xiaobing
X
Lin
Jeff
J
Chen
Guibin
G
Zou
Jizhong
J
Cheng
Linzhao
L
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D003470:Culture Media / Q000378:metabolism; D046149:Embryo Culture Techniques; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006720:Homozygote; D006801:Humans; D007621:Karyotyping; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D009154:Mutation; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D019076:Transgenes
D000952:Antigens, Polyomavirus Transforming; D003470:Culture Media