Expression of early transcription factors Oct-4, Sox-2 and Nanog by porcine umbilical cord (PUC) matrix cells.
BACKGROUND
Three transcription factors that are expressed at high levels in embryonic stem cells (ESCs) are Nanog, Oct-4 and Sox-2. These transcription factors regulate the expression of other genes during development and are found at high levels in the pluripotent cells of the inner cell mass. The downregulation of these three transcription factors correlates with the loss of pluripotency and self-renewal, and the beginning of subsequent differentiation steps. The roles of Nanog, Oct-4 and Sox-2 have not been fully elucidated. They are important in embryonic development and maintenance of pluripotency in ESCs. We studied the expression of these transcription factors in porcine umbilical cord (PUC) matrix cells.


METHODS
Cells were isolated from Wharton's jelly of porcine umbilical cords (PUC) and histochemically assayed for the presence of alkaline phosphatase and the presence of Nanog, Oct-4 and Sox-2 mRNA and protein. PCR amplicons were sequenced and compared with known sequences. The synthesis of Oct-4 and Nanog protein was analyzed using immunocytochemistry. FACS analysis was utilized to evaluate Hoechst 33342 dye-stained cells.


RESULTS
PUC isolates were maintained in culture and formed colonies that express alkaline phosphatase. FACS analysis revealed a side population of Hoechst dye-excluding cells, the Hoechst exclusion was verapamil sensitive. Quantitative and non-quantitative RT-PCR reactions revealed expression of Nanog, Oct-4 and Sox-2 in day 15 embryonic discs, PUC cell isolates and porcine fibroblasts. Immunocytochemical analysis detected Nanog immunoreactivity in PUC cell nuclei, and faint labeling in fibroblasts. Oct-4 immunoreactivity was detected in the nuclei of some PUC cells, but not in fibroblasts.


CONCLUSIONS
Cells isolated from PUC express three transcription factors found in pluripotent stem cell markers both at the mRNA and protein level. The presence of these transcription factors, along with the other characteristics of PUC cells such as their colony-forming ability, Hoechst dye-excluding side population and alkaline phosphatase expression, suggests that PUC cells have properties of primitive pluripotent stem cells. Furthermore, PUC cells are an easily and inexpensively obtained source of stem cells that are not hampered by the ethical or legal issues associated with ESCs. In addition, these cells can be cryogenically stored and expanded.
Carlin
Ryan
R
Dept of Anatomy & Physiology, Kansas State University, Manhattan, KS, USA. rcarlin@vet.k-state.edu
Davis
Duane
D
Weiss
Mark
M
Schultz
Bruce
B
Troyer
Deryl
D
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics*; D005260:Female; D005347:Fibroblasts / Q000737:chemistry; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D024002:HMGB Proteins / Q000032:analysis / Q000235:genetics*; D018398:Homeodomain Proteins / Q000032:analysis / Q000235:genetics*; D007150:Immunohistochemistry; D008297:Male; D050814:Octamer Transcription Factor-3 / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors; D013234:Stem Cells / Q000737:chemistry / Q000201:enzymology; D013552:Swine; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics*; D014470:Umbilical Cord / Q000737:chemistry* / Q000166:cytology
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D024002:HMGB Proteins; D018398:Homeodomain Proteins; D050814:Octamer Transcription Factor-3; D012333:RNA, Messenger; D055748:SOXB1 Transcription Factors; D014157:Transcription Factors; D000469:Alkaline Phosphatase