Refinement of Culture Conditions for Maintenance of Undifferentiated Equine Umbilical Cord Blood Stem Cells

Umbilical cord blood (UCB) was collected from Thoroughbred mares at foaling, and stem cells were isolated. UCB stem cells were cultured in growth media (GM), GM + Fibroblast Growth Factor 2, GM + Flt3/Thrombopoietin/c-Kit ligand, or conditioned media. Population doubling times (PDTs) were calculated. UCB stem cells cultured in standard GM exhibited faster PDTs (30.07 ± 1.59 hours) than those cultured in any treatment media. PDT was further accelerated by cultivation in GM on fibronectin (26.99 ± 1.57 hours)-, collagen I (26.21 ± 1.18 hours)-, or gelatin (27.32 ± 1.50 hours)-coated surfaces. Time in culture increased PDT regardless of media or substrata conditions. Because Oct4, nanog, KLF4, Sox2, and c-myc are implicated in embryonic stem cell pluripotency, total RNA was harvested from UCB stem cells as well as mouse ES cells and analyzed by reverse-transcription polymerase chain reaction. Both populations expressed all transcripts. The persistence of Oct4, nanog, and Sox2 expression in UCB stem cells was monitored over time in culture. Oct4 was detected throughout the duration of the experiment. Sox2 and nanog expression was lost with time in culture. Cells cultured on protein matrices maintained nanog expression longer than those on uncoated plasticware. These phenotypes remain unaffected by the type of culture matrix and FGF2. However, the plasticity markers are lost with serial passage.