Novel autogenic feeders derived from human embryonic stem cells (hESCs) support an undifferentiated status of hESCs in xeno-free culture conditions.
BACKGROUND
Clinical-grade human embryonic stem cells (hESCs) ideally should be derived and maintained in xeno-free culture conditions using defined chemicals or materials of human origin. This will reduce the possibility of xeno-derived pathogenic infection and/or unfavorable immune reaction in clinical application. The present study therefore aimed to derive autogenic feeders from hESCs and evaluate their capability to support the pluripotency of hESCs in xeno-free culture conditions.


RESULTS
H9 hESCs were cultured in media containing human serum (HS), serum replacement (SR) or KFM combination, to generate autogenic feeders (named HSdF, SRdF and KFMdF, respectively). Reverse transcription polymerase chain reaction, flow cytometry and immunofluorescence analysis using pluripotent stem cell markers, markers of early cell lineages and surface markers revealed that HSdF, SRdF and KFMdF likely belonged to different cellular subpopulations. The efficiency of the autogenic feeders in maintaining pluripotency of H9 hESCs using media containing SR, fetal bovine serum, HS or 1% HS plus various combinations of growth factors was evaluated by flow cytometric analysis of Oct4 expression. All three autogenic feeders were shown to be capable of maintaining the undifferentiated status of H9 hESCs in SR-containing media in long-term culture. When supplemented with bFGF, activin A and noggin, hESCs could also be maintained favorably on KFMdF in a medium containing 1% HS without losing their pluripotent potentials both in vitro and in vivo.


CONCLUSIONS
Novel autogenic feeders can be derived from hESCs under xeno-free conditions and they can robustly maintain the pluripotent identity of hESCs in xeno-free media containing a low concentration of HS.
Chen
Hsin-Fu
HF
Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University, Taipei, Taiwan, Republic of China.
Chuang
Ching-Yu
CY
Shieh
Yu-Kai
YK
Chang
Hao-Wei
HW
Ho
Hong-Nerng
HN
Kuo
Hung-Chih
HC
D028341:Activins / Q000494:pharmacology; D002352:Carrier Proteins / Q000494:pharmacology; D002454:Cell Differentiation; D002478:Cells, Cultured; D003470:Culture Media; D046149:Embryo Culture Techniques; D053595:Embryonic Stem Cells / Q000166:cytology*; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D039904:Pluripotent Stem Cells / Q000166:cytology*; D015262:Xenobiotics; D000099176:Noggin Protein
D028341:Activins; D002352:Carrier Proteins; D003470:Culture Media; D005346:Fibroblast Growth Factors; D015262:Xenobiotics; D000099176:Noggin Protein; C422405:activin A