Hum. Reprod. Advance Access published online on June 9, 2008
Human Reproduction, doi:10.1093/humrep/den202
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Successful vitrification of human amnion-derived mesenchymal stem cells
1 Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 300 Gumi, Bundang, Seongnam, Gyeonggi 463-707, Korea 2 Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, 28 Yeongeon, Jongno, Seoul 110-744, Korea 3 Institute of Reproductive Medicine and Population, Seoul National University, 28 Yeongeon, Jongno, Seoul 110-744, Korea 4 Department of Biomedical Science and Technology, IBST, Konkuk University, Seoul 143-701, Korea 5 Department of Biotechnology, College of Natural Science, Seoul Women's University, Nowon, Seoul 139-774, Korea
6 Correspondence address. Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 300 Gumi, Bundang, Seongnam, Gyeonggi 463-707, Korea. Tel: +82-31-787-7251; Fax: +82-31-787-4054; E-mail: suhcs{at}snu.ac.kr
BACKGROUND: A cryopreservation protocol for human amnion-derived mesenchymal stem cells (HAMs) is required because these cells cannot survive for long periods in culture. The aim of this study was to determine whether vitrification is a useful freezing method for storage of HAMs.
METHODS: HAMs were cryopreserved using vitrification method. The morphology and viability of thawed HAMs was evaluated by Trypan Blue staining. The expression of several embryonic stem cell (ESC) markers was evaluated using flow cytometry, RT–PCR and immunocytochemistry. Von Kossa, Oil Red O and Alcian Blue staining were used to asses the differentiation potential of thawed HAMs.
RESULTS: The post-thawing viability of HAMs was 84.3 ± 3.2% (Mean ± SD, n = 10). The thawed HAMs showed morphological characteristics indistinguishable from the non-vitrified fresh HAMs. The expression of surface antigens (strong positive for CD44, CD49d, CD59, CD90, CD105 and HLA-ABC; weak positive for HLA-G; negative for CD31, CD34, CD45, CD106, CD117 and HLA-DR) and the expression of ESC markers [CK18, fibroblast growth factor-5, GATA-4, neural cell adhesion molecule, Nestin, Oct-4, stem cell factor, HLA-ABC, Vimentin, bone morphogenetic protein (BMP) 4, hepatocyte nuclear factor 4
(HNF-4), Pax-6, alpha-fetoprotein, Brachyury, BMP-2, TRA-1-60, stage-specific embryonic antigen (SSEA-3, SSEA-4)] were maintained in the vitrified-thawed HAMs. The thawed HAMs retained ability to differentiate into osteoblasts, adipocytes and chondrocytes under appropriate culture conditions.
CONCLUSIONS: Our results suggest that vitrification is a reliable and effective method for cryopreservation of HAMs.
Key words: vitrification/amnion/mesenchymal stem cells/cryopreservation/stem cell markers
Submitted on February 19, 2008; resubmitted on April 23, 2008; accepted on May 2, 2008.