CONCLUSIONES Y RECOMENDACIONES 6.1 CONCLUSIONES

Here, I have examined the capacity of haploid ES cells to reprogram somatic cells towards pluripotency using cell fusion. Previously my host laboratory showed that diploid ES cells are capable of reprogramming diploid somatic cells using cell fusion system (Pereira et al., 2008; Piccolo et al., 2011a). Other studies have demonstrated that tetraploid fibroblast fused with diploid ES cells do not undergo reprogramming towards pluripotency but instead reprogram ES cells towards fibroblast (Kruglova et al., 2010). This suggests that ploidy may be important for the direction of reprogramming outcomes. To understand the mechanism behind these observations I examined how ploidy may affect reprogramming.

Here, I have demonstrated that haploid ES cells can reprogram somatic cells towards pluripotency with a lower efficiency as compared to diploid ES cells (Figure 4-3). This confirmed the assumption that ploidy may be important for reprogramming towards pluripotency mediated by cell fusion. Single cell analysis has revealed that transcript level within a cell population varies in between cells. Transcript and protein level may vary within the haploid ES cells population leading to a population of haploid ES cells more prone to reprogram somatic cells towards pluripotency. In order to ascertain this, several clones could be derived from the original haploid ES cell population and cell fusion could be performed with each of them to assess the range of cellular reprogramming potential of haploid ES cells. Another cause of the low reprogramming potential of haploid ES cells could be that the kinetics of reprogramming towards pluripotency is slower in haploid ES cells. This was assessed with the generation of hybrid cells from haploid ES cells fusions and compared with hybrid cells from diploid ES cells fusions. The result revealed that even at a later time point haploid ES cells still have a lower reprogramming potential than diploid ES cells (Figure 4-4). Low reprogramming of haploid ES cells could also be due to their sizes. Haploid ES cells are much smaller than diploid ES cells. their volume is 2.5 time smaller compared to diploid ES cells. The same observation was done on their nucleus (Anton Wutz, personal communication).

Finally, I assessed whether low ploidy could also lead to low protein level in haploid ES cells compared to diploid ES cells. I showed that that haploid ES cells have less total RNA and protein than diploid ES cells. This result is in agreement with previous work reviewed in (Marguerat and Bähler, 2012). Specifically, the level of crucial pluripotency associated factors, such as Oct4 and Nanog is reduced in haploid ES cells compared to diploid ES cells (Figure 4-5). Previous study in iPS showed evidence that reprogramming efficiency was regulated by the ectopic level of Oct4, Sox2, Klf4 and c-Myc (OSKM) (Sridharan et al., 2009). Polo et al. 2012 (Polo et al., 2012) corroborated this hypothesis by genetically engineering fibroblast to express a higher dose of transcription factors (OSKM), which led to an increase in the efficiency/kinetics of pluripotent reprogramming. They also noticed that the protein levels of those four factors were reduced in cells blocked at an intermediate states compared to cells which were able to be reprogrammed towards pluripotency. A similar process could explain the low reprogramming potential of haploid ES cells, where a minimum amount of pluripotency-associated proteins is required to reprogram somatic cells towards pluripotency. To assess this, I overexpressed Nanog in haploid ES cells and showed that the low reprogramming potential of haploid ES cells can be rescued by overexpression of Nanog in haploid ES cells (Figure 4-7). This result is in agreement with previous studies which stated that Nanog overexpression enhances the efficiency of reprogramming (Silva et al., 2009; Theunissen et al., 2011). As previously mentioned in the results, Nanog directly targets Esrrb which activates the regulation of Oct4. Besides Nanog and Oct4 are part of the core transcription factors network that regulates pluripotency (Rodda et al., 2005). Therefore, the increase in reprogramming potential in haploid ES cells overexpressing Nanog could be due to the increase in protein level of Oct4 and Esrrb as well. To assess whether Nanog overexpression partially rescue the low reprogramming of haploid ES cell at a single cell level, clonal population of haploid ES cells overexpressing Nanog should be derived. Those clone would then be fused with somatic cells to determine the reprogramming potential of each NanogOE clonal population. This will allow me to determine whether the frequency of reprogramming is equivalent between clones. Further studies will be required to explore whether overexpression of other transcription factors such as Oct4 have the same effect as supplementing with Nanog. It would be interesting to overexpress Nanog in haploid ES cells at the same level as it is expressed in diploid ES cells.

It would be important to investigate whether this low level of proteins is also present in other haploid ES cells line and is not just a specificity of this particular cell line. At the moment, there is only one other haploid ES cell line available (Elling et al., 2011). Preliminary data performed on this new cell line shows that the total protein level as well as the protein level of Oct4 and Nanog is also lower in this cell line suggesting that the low amount of proteins is a feature of haploid ES cells in general. More cell fusion experiments need to be done on this new haploid ES cell line to see whether the reprogramming efficiency is also low compared to diploid ES cells.

Chapter 5

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VISUALIZING MITOTIC CHROMATIN BY HIGH-RESOLUTION