The X Chromosome Status Of Female Rabbit Embryonic Stem Cells

The deficiency of X-inactive specific transcript (Xist) on the inactive X chromosome affects the behavior of female ES cells (Embryonic stem cells) and iPS cells (iuduced pluripotent stem cells, iPS cells). However, X chromosome instability has not been identified in other species.The rabbit is a classical experimeatal animal. Previously established rabbit ES cells and rabbit iPS cells exhibited flat colony morphology and bFGF dependence and displayed many characteristics that were similar to those of human ES cells. Although rabbit ES cells have been previously derived in several laboratories, little is known about the X chromosome status of female rabbit ES cells and its correlation with pluripotency.In the present study, we investigated the X chromosome status of three female rabbit ES cell lines and found variations in X chromosome inactivation in early passages. Two of the rabbit ES cell lines B28-6and NJ12can be defined as class Ⅱ because they display a heterochromatic Xi with Xist RNA coating and H3K27me3foci. In contrast, the third cell line NJ10lacked Xist expression and Xist RNA coating. However, unlike the XaXa state, this cell line displayed H3K27me3foci at early passage (P8), did not express Xist during differentiation, thus indicated as Xist-deficient XiXa. Moreover, the H3K27me3foci would further lose in passaging and yield an eroded Xi (P12). Therefore, this cell line is considered to be class Ⅲ. We further found that the class Ⅲ rabbit ES cells exhibited relatively high expression levels of some oncogenes, orthologous genes of which were reported to be highly expressed in class Ⅲ human iPS cells. The class Ⅲ rabbit ES cells also showed poor differentiation in vivo compared to class Ⅱ and male rabbit ES cells. Furthermore, we found that the class Ⅲ rabbit ES cells could be converted into a mESC-like pluripotent state by overexpressing the reprogramming factors: Oct4, Sox2, Klf4and c-Myc. We examined the colony morphology, colony formation ability, transcriptional changes and the activation of signaling pathways associated with pluripotency. We found that the induced rabbit ES cells are more like mouse ES cells, which therefore defined as mESC-like rabbit ES cells. We then confirmed that the mESC-like rabbit ES cells re-obtain two active X chromosomes, meanwhile, the oncogene expression is repressed and the impaired differentiation ability in vivo is rescued.This experiments first indicated that the X chromosome instability and the impact of Xist-deficiency in Xi might be conserved between human and rabbit. These data also revealed that forced expression of reprogramming factors under optimized culture condition can reprogram the rabbit ES cells to mESC-like state which is XaXa state. Futher these data may be informative to other groups who will attempt to derive fully pluripotent rabbit ES cells or rabbit iPS cells.