제   목 No Expression of Non-Human Sialic Acid in Human Embryonic Stem Cell Lines Cultured and Derived in Xeno-Free Culture Condition.
등록일 2015/12/18 조회수 381 첨부파일
2012 The Society for the Study of Reproduction.

Jeoung Eun Lee, Hye-Yoon Park, Myung Sun Shim, Yuri Kim, Sung-Geum Lee, and Dong Ryul Lee

Abstract
Recent advances in stem cell science offer great promise to treat incurable diseases in the near future, and human embryonic stem cells (hESCs) are the most attractive candidate for tissue and cell therapy by their self-renewal and pluripotency. For clinical application, hESCs should not have any risk of infection and immune-reaction caused by animal pathogens, viruses, and/or animal substances in cell culture. Specially, it is known that the use of animal-derived components in media brings about the corporation and expression of non-human sialic acid (Neu5Gc) on cultured hESCs, and it may cause an immune response in the body. Therefore, it would be the best way to establish and maintain hESCs under xeno-free culture condition, but there is no report of derivation of hESC lines in xeno-free condition. To establish the hESC culture condition suitable for future clinical use, we developed xeno-free culture condition with human feeder and humanized serum substitute, and verified the expression of Neu5Gc on hESCs cultured in this condition. Finally, we tested if the xeno-free culture condition could support hESC derivation. For xeno-free culture condition, we used human endometrial cells (hEndo) as feeder cells which were cultured primarily in DMEM+10% human serum. CELLStart was used for humanized coating material, and hESCs were cultured in DMEM/F12+15% Knockout SR XenoFree (SR-XF)+4ng/ml hrbFGF. In order to test our xeno-free culture condition, we transferred CHA-hES15 cells, maintained in mEF+SR, to xeno-free condition and CHA-hES15 was characterized by karyotype, RT-PCR and immunostaining after more than 20 passages. Also, the expression of Neu5Gc on hESCs was analyzed by immunostaining and western blot. CHA-hES15 was well maintained in xeno-free condition with no change of karyotype and hES characters. Also, we found that Neu5Gc was not detected on hESCs cultured in xeno-free conditions. For derivation of hESC, we used donated frozen human embryos under informed consent and IRB approval. Thawed embryos were cultured to the blastocyst stage and then cultured in xeno-free culture condition after removal of zona pellucida using Acid Tyrode solution. Trophoectoderm was removed mechanically after seeding onto hEndo. Four hESC lines such as CHA-hES39, 40, 41 and 42, were successfully derived from 13 blastocysts and maintained over 35 passages in xeno-free culture condition. CHA-hES39, 40, 41 and 42 had 46,XX, 46,XY, 46,XX, and 47,XY,+5 karyotype, respectively, and characterized by expression of hESC-specific surface antigens and gene expression. In the present study, we demonstrated that hESCs were maintained successfully in our xeno-free culture condition, and then the expression of Neu5Gc on hESCs could be removed. Furthermore, we successfully derived and maintained 4 hESC lines under this xeno-free condition. Our xeno-free culture condition might be useful for establishment and maintenance of clinical grade hESCs or other pluripotent stem cells for clinical application.