Expression analysis of pluripotency factors in the undifferentiated porcine inner cell mass and epiblast during in vitro culture.
Limited understanding of the importance of known pluripotency factors in pig embryonic stem cells (ESC) impedes the establishment and validation of porcine ESC lines. This study evaluated the expression of known mouse ESC and human ESC (hESC) pluripotency markers in in vivo inner cell mass (ICM) and in vitro-cultured undifferentiated porcine epiblast cells isolated from 8-day porcine blastocysts, primary cultures of epiblast-derived neuroprogenitor cells, and endoderm cells. The expression profile of common pluripotency markers (POU domain 5 transcript factor 1, SRY-box containing gene 2, and Nanog homeobox), species-specific markers, ESC-associated factors, and differentiation markers was evaluated. The mRNA of uncultured ICMs, cultured epiblast cells, epiblast-derived neuroprogenitor cells, and endoderm cells was amplified prior to expression analysis of candidate genes by real-time RT-PCR. ESC factors whose expression correlated best with the undifferentiated epiblast state were identified by comparative mRNA expression analysis between porcine epiblast-derived somatic cell lines, fetal fibroblasts, and adult tissues. Across tissue types Nanog homeobox exhibited ubiquitous expression, whereas POU domain 5 transcript factor 1, teratocarcinoma-derived growth factor 1, and RNA exonuclease homolog 1 transcript expression was restricted primarily to undifferentiated epiblasts. Our results suggested that expression of pluripotency markers in undifferentiated pig epiblast cells more closely resembled that observed in hESC. Expression alterations of ESC-associated factors in epiblast cells were also observed during in vitro culture. Our data demonstrate the potential use of some pluripotency factors as markers of porcine epiblast stem cells and indicate that the in vitro environment may influence the cultured epiblast's developmental state.
Biotechnology and Germplasm Laboratory, USDA Agricultural Research Service, Beltsville, Maryland 20705, USA. lblomberg@anri.barc.usda.gov
Le Ann
LA
Blomberg
Lori L
LL
Schreier
Neil C
NC
Talbot
D000818:Animals; D053624:Blastocyst Inner Cell Mass / Q000378:metabolism*; D002454:Cell Differentiation / Q000235:genetics*; D046149:Embryo Culture Techniques; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D005855:Germ Layers / Q000378:metabolism*; D008954:Models, Biological; D009474:Neurons / Q000502:physiology; D039904:Pluripotent Stem Cells / Q000378:metabolism; D013552:Swine