Reprogramming ofXistagainst the pluripotent state in fusion hybrids

Abstract

The fusion of somatic cells with pluripotent cells results in the generation of pluripotent hybrid cells. Because the `memory' of somatic cells seems to be erased during fusion-induced reprogramming, genetic reprogramming is thought to be a largely unidirectional process. Here we show that fusion-induced reprogramming, which brings about the formation of pluripotent hybrids, does not always follow a unidirectional route. Xist is a unique gene in that it is reprogrammed to the state of somatic cells in fusion-induced pluripotent hybrids. In hybrids formed from the cell fusion of embryonal carcinoma cells (ECCs) with male neural stem cells (mNSCs), the Xist gene was found to be reprogrammed to the somatic cell state, whereas the pluripotency-related and tissue-specific marker genes were reprogrammed to the pluripotent cell state. Specifically, Xist is not expressed in hybrids, because the `memory' of the somatic cell has been retained (i.e. mNSCs do not exhibit Xist expression) and that of the pluripotent cell erased (i.e. inactivation of the partially active Xist gene of ECCs, complete methylation of the Xist region). The latter phenomenon is induced by male, but not by female, NSCs.

Using heterokaryons to understand pluripotency and reprogramming

“…Collectively, these data suggest that stem cell lines derived from primitive cells that emerge at early stages of development possess powerful and often dominant reprogramming capabilities. Whether reprogramming is necessarily uni-or bidirectional has been the subject of much recent [32,33] and past debate [8,29,34], although it seems reasonable to assume that the eventual outcome depends on the relative abundance of certain factors, for example, Nanog [35], as well as the 'compatibility' of the parental cell types and culture conditions used to select the hybrids. Future studies will need to address these uncertainties, for example, by examining the impact of nuclear 'dosage' on lineage conversion ( figure 3) and by assessing the reprogramming outcomes using cell-cycle-stage-enriched ES and target cells.…”

Kinetics of reprogramming in cell fusion hybrids

“…Since EC cells share many of the key pluripotent characteristics with ES and EC cells can provide a readily amenable alternative source for reprogramming (Do et al, 2009a;Flasza et al, 2003;Mise et al, 1996). Moreover, mouse EC cells can reprogram human somatic cells into pluripotent state, indicating that reprogramming factors can cross-act through another species (Flasza et al, 2003).…”

“…This phenomenon is also observed when two types of pluripotent cells are fused. After fusion of ES and F9 EC cells, EC-like and ESlike hybrid cells were generated and the EC-like cells rarely differentiated into neural cells, otherwise, ES-like hybrid cells easily differentiate into neural cells (Do et al, 2009a).…”

“…The 'memory' of somatic cells is-almost like a dogmaconsidered to be erased by fusion with pluripotent cells during fusion-induced pluripotential reprogramming. Silva et al, for example, mentioned that fusion-induced reprogramming is a unidirectional process resulting in an ES cell phenotype without other viable cell states (Silva et al, 2006 (Do et al, 2009a).…”

“…One of the distinct differences of pluripotent cells vs. somatic cells (including multipotent somatic stem cells), regardless of sex, is the absence of an inactive X chromosome (Xi) (Do et al, 2009a;Lee, 2005). Therefore, pluripotent cells only contain active X chromosome (Xa); male cells have one and female cells have two Xa.…”