Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization

Abstract

Although isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts were used, a mechanical method of passaging led to better cell growth than passaging by trypsin digestion. We also found that exogenous supplementation with leukemia inhibitory factor maintained the embryonic stem cell-like cells in an undifferentiated state, whereas addition of stem cell factor resulted in their differentiation. Our findings provide an experimental basis for the establishment of an effective culture system for bovine embryonic stem cells.

Establishment of bovine embryonic stem cells after knockdown of CDX2

“…After a relative long-term in culture, CDX2-KD bESCs stably maintained monolayer, but morphology of control bESCs started to form cystic structures, and accompanied up-regulation of CDX2 . Cystic structures and cytoplasmic lipid inclusions are the typical feature of trophoblasts that usually emerge during culturing ESCs of domesticated ungulates, ever used the most stringent immuno-surgery method to isolate ICM192425. In normal mouse ESCs, the trophoblast lineage was induced by ectopic expression of TEAD4 , GATA3 and CDX2 .…”

Derivation and Characterization of Bovine Induced Pluripotent Stem Cells by Transposon-Mediated Reprogramming

“…The availability of bovine pluripotent stem cells could facilitate the application of advanced reproductive technologies in this species. Numerous attempts to establish bovine ESC lines have been undertaken; however, they have had limited success (Gong et al, 2010;Jin et al, 2012;Maruotti et al, 2012;Saito et al, 1992;Saito et al, 2003;Solter et al, 2000). Most ESC-like cultures proliferated slowly, lost pluripotency markers, and ceased to divide at early passages (Cao et al, 2009;Malaver-Ortega et al, 2012;Maruotti et al, 2012;Munoz et al, 2008;Saito et al, 1992).…”

Microarray analysis of embryo-derived bovine pluripotent cells: The vulnerable state of bovine embryonic stem cells

“…However, the efficiency of in vivo differentiation is extremely low and teratoma formation was induced abnormally [14]. Many previous studies in the literature have also revealed similar problems [48, 57, 58], even in bovine iPSCs [35]. …”