Year 3 & NCE
Induced pluripotent stem cells (iPS) can be produced from virtually any somatic cell by the overexpression of a few transcription factors, a process termed “nuclear reprogramming”. However, the generation of iPS is slow (2 weeks) and the frequency of somatic cells which undergo successful reprogramming is very low (0.1-1%). At present, the molecular mechanisms underlying reprogramming are not well understood. This is in large part due to an inability to analyze early stages of reprogramming at the molecular level in populations which are heterogeneous or where cell numbers are limiting. We hypothesized that the inefficiency of reprogramming to iPS is due to as yet unidentified molecular regulators or pathways critical to the early onset of reprogramming.
In order to study the molecular mechanisms of reprogramming, a different experimental system was needed; one with a highly efficient, rapid onset of reprogramming. Our previous research (Bhutani et al, Nature 2010) showed the development of a synchronous, high efficiency, rapid reprogramming approach consisting of heterokaryons (interspecies multinucleate fused cells). In these multinucleate cells, activation of human pluripotency genes such as Oct4 and Nanog occurs rapidly (24hrs) and efficiently (70% of single heterokaryons). During the first year of funding, our results demonstrated that reprogramming toward pluripotency in heterokaryons is fast and efficient and involves active DNA demethylation since there is no cell division or DNA replication. In addition, we showed that the AID enzyme, known for its role in generating antibody diversity in B cells, is a key component for reprogramming human somatic cells towards pluripotency.
Now during our third year of funding, we have both demonstrated the requirement of AID for iPS generation but also the ability of AID to increase iPS generation by roughly two fold. Moreover, because we had reasoned that global RNA-sequencing of heterokaryons would provide us with further insight into the early reprogramming process, we now have optimized methodologies which allow us to accomplish this aim and have performed global RNA-seq at 6hr, day 1, day 2, and day 3 post-heterokaryon formation. Through this analysis we have now identified a secreted factor identified via RNA sequencing in Heterokaryons that can substitute for one of the key iPS reprogramming factors, c-myc. The substitution of myc by a secreted factor allows for the generation of safer patient derived iPS cells by relieving the need for viral integration of the potent oncogene c-myc.
In sum, the speed and efficiency of reprogramming in the heterokaryon system provides a means to identify critical transcription factors and cellular pathways involved in early reprogramming. Our research with heterokaryons enables mechanistic insights into the process of nuclear reprograming which are not possible to identify using iPS.