PRC2 group proteins have been shown to be dispensable for pluripotency; however they are required during differentiation shown by both in vivo and in vitro studies [32, 39]. Though importance of PcG proteins during differentiation is known, their expression during differentiation of hES cells into pancreatic lineage has not yet been evaluated. To achieve this, we differentiated KIND1 via spontaneous differentiation and directed differentiation. EBs showed higher expression of endoderm and mesoderm gene transcript while lower expression of ectoderm gene transcripts was seen. However gene transcripts for PDX1, SOX9 and NKX6.1 were not detected in differentiated EBs. Hence, KIND1 cells were differentiated by directed differentiation which involves sequential addition of cytokines to generate cells of pancreatic lineage, and this necessitated culturing KIND1 cells under feeder free conditions. The PcG gene transcripts profile of feeder-free KIND1 cells was similar to KIND1 cells cultured on HFF cells.
Upregulation of pluripotency controlling gene NANOG on day 4 indicated that it may be required for endoderm differentiation, in contrast to its role in maintaining undifferentiated state. Previous reports have also shown that these transcription factors are involved in lineage specification and differentiation [40–42]. Gene transcripts of ectodermal marker (MAP2), cardiac mesoderm marker (MESP1) were seen at low level during pancreatic differentiation, indicating differentiation of KIND1 cells primarily into endoderm lineage. Pancreas specific gene transcripts PDX1, SOX9 and NKX6.1 were expressed during later stages of differentiation (day12-day16) which was also confirmed by Western blotting, indicating the differentiation of KIND1 cells into cells of pancreatic lineage by directed differentiation.
Once the differentiation of KIND1 into pancreatic lineage was achieved, we studied the expression of PcG proteins. BMI1 has been shown to be important for self renewal and differentiation of neural stem cells and hematopoietic stem cells. BMI1 represses the expression of cell cycle inhibitors like p21, p19 and p16, leading to proliferation of stem cells [25, 26, 43, 44]. BMI1 was found to be highly expressed during directed differentiation of KIND1 cells into pancreatic lineage and this is being reported for the first time. We found the increased expression of BMI1 led to decrease in expression of p19/CDKN2D transcript. CBX2 and RING1A gene transcript levels remain almost unaltered during differentiation, thus it suggests that CBX2 and RING1A may not play important role during endoderm differentiation. RING1B transcript was significantly upregulated during differentiation compared to undifferentiated KIND1 cells. We found the increased RING1B and BMI1 led to increase in H2AK119ub1 levels during differentiation. Total adult human pancreatic RNA also showed higher expression of BMI1 than RING1A, RING1B and CBX2 thus highlighting role of BMI1 in differentiated endodermal cells.
In mice EZH2, SUZ12, EED and JARID2 knockout results in embryonic lethality [11, 22, 45, 46]. Our data shows that EZH2, EED and SUZ12 transcript expression increased compared to undifferentiated KIND1 cells during differentiation. On the other hand, JARID 2 levels decreased steadily during both directed and spontaneous differentiation, similar to results obtained by earlier groups [7, 34]. Studies carried out earlier have shown EZH2, EED decrease upon differentiation of mouse ES cells [6, 47, 48]. Also, previous reports that studied expression of PcG proteins in differentiated embryonic stem cells, have used spontaneous differentiation approach to induce differentiation [8, 9, 48, 49]. Van Arensbergen et al.  reported that pancreatic progenitors have high levels of H3K27me3 which gives them plasticity to differentiate either to acinar or beta cell. PRC2 proteins EED, SUZ12 and EZH2 are actively involved in H3K27me3 modification [6, 7]. Results of the present study show that SUZ12, EZH2 and EED were upregulated during differentiation of KIND1 cells into pancreatic cells along with an increase in the H3K27me3 levels. The expression profile of PRC2 proteins at the transcript level observed in adult human pancreas is similar to that seen in KIND1 cells differentiated into pancreatic lineage.
The expression of RING1B, BMI1, EZH2, EED, and SUZ12 gene transcripts during spontaneous and directed differentiated hES cells indicates that changes in PcG expression is not a generalized phenomenon observed during differentiation. The increase in expression of PcG proteins correlated with increase in trimethylation of histone H3K27 and monoubiquitinylation at H2AK119. Understanding the dynamics of polycomb group proteins during hES cell differentiation may help to design better differentiation strategies in future.