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Isolation and characterization of embryonic stem‐like cells derived from in vivo‐produced cat blastocysts
Author(s) -
Yu Xianfeng,
Jin Guangzhen,
Yin Xijun,
Cho Sujin,
Jeon Jintae,
Lee Sangsuk,
Kong Ilkeun
Publication year - 2008
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.20867
Subject(s) - biology , embryonic stem cell , inner cell mass , embryoid body , fetal bovine serum , microbiology and biotechnology , stem cell , cell culture , alkaline phosphatase , cellular differentiation , blastocyst , embryo , adult stem cell , embryogenesis , biochemistry , genetics , gene , enzyme
Embryonic stem (ES)‐like cells were isolated from in vivo‐produced cat embryos. Total of 101 blastocysts were collected from female cats. The inner cell mass (ICM) were mechanically isolated and cultured on mitomycin‐C‐treated cat embryonic fibroblast feeder layers in medium supplemented with knockout™ Serum Replacement (KSR‐medium) or fetal bovine serum (FBS‐medium). Putative ES‐like cell colonies developed in both KSR‐ and FBS‐medium conditions, but formed domed and flat colonies, respectively. ICM cell attachment and ES‐like cell colony formation were significantly higher in KSR‐medium, but subsequent cell proliferation was significantly lower than in FBS‐medium. For passaging, 32 and 18 colonies in KSR‐ and FBS‐medium were separated by enzymatic dissociation or mechanical disaggregation. Enzymatic dissociation resulted in cell differentiation; however, mechanical disaggregation generated cells that remained undifferentiated over more than four passages and yielded two cat ES‐like cell lines that continued to grow for up to eight passages in FBS‐medium. These cells had typical stem cell morphology, expressed high levels of alkaline phosphatase activity, and were positive for the ES cell‐markers Oct‐4, stage‐specific embryonic antigen‐1 (SSEA‐1), SSEA‐3, and SSEA‐4. These cells formed embryoid bodies (EBs) in suspension culture after extended suspension culture. When simple EBs were cultured on tissue culture plates, they differentiated into several cell types, including epithelium‐like and neuron‐like cells. In addition, EBs were positive for mesoderm marker, desmin. After prolonged in vitro culture, some colonies spontaneously differentiated into beating myocardiocytes, and were positive for alpha‐actinin. These observations indicate that cat ES‐like cells were successfully isolated and characterized from in vivo‐produced blastocysts. Mol. Reprod. Dev. 75: 1426–1432, 2008. © 2008 Wiley‐Liss, Inc.