Polycomb group protein expression during differentiation of human embryonic stem cells into pancreatic lineage in vitro

Abstract

Background: Polycomb Group (PcG) proteins are chromatin modifiers involved in early embryonic development as well as in proliferation of adult stem cells and cancer cells. PcG proteins form large repressive complexes termed Polycomb Repressive Complexes (PRCs) of which PRC1 and PRC2 are well studied. Differentiation of human Embryonic Stem (hES) cells into insulin producing cells has been achieved to limited extent, but several aspects of differentiation remain unexplored. The PcG protein dynamics in human embryonic stem (hES) cells during differentiation into pancreatic lineage has not yet been reported. In the present study, the expression of RING1A, RING1B, BMI1, CBX2, SUZ12, EZH2, EED and JARID2 during differentiation of hES cells towards pancreatic lineage was examined.

Results: In-house derived hES cell line KIND1 was used to study expression of PcG protein upon spontaneous and directed differentiation towards pancreatic lineage. qRT-PCR analysis showed expression of gene transcripts for various lineages in spontaneously differentiated KIND1 cells, but no differentiation into pancreatic lineage was observed. Directed differentiation induced KIND1 cells grown under feeder-free conditions to transition from definitive endoderm (Day 4), primitive gut tube stage (Day 8) and pancreatic progenitors (Day 12-Day 16) as evident from expression of SOX17, PDX1 and SOX9 by qRT-PCR and Western blotting. In spontaneously differentiating KIND1 cells, RING1A and SUZ12 were upregulated at day 15, while other PcG transcripts were downregulated. qRT-PCR analysis showed transcripts of RING1B, BMI1, SUZ12, EZH2 and EED were upregulated, while RING1A and CBX2 expression remained low and JARID2 was downregulated during directed differentiation of KIND1 cells. Upregulation of BMI1, EZH2 and SUZ12 during differentiation into pancreatic lineage was also confirmed by Western blotting. Histone modifications such as H3K27 trimethylation and monoubiquitinylation of H2AK119 increased during differentiation into pancreatic lineage as seen by Western blotting.

Conclusion: Our study shows expression of PcG proteins was distinct during spontaneous and directed differentiation. Differentiation into pancreatic lineage was achieved by directed differentiation approach and was associated with increased expression of PcG proteins RING1B, BMI1, EZH2 and SUZ12 accompanied by increase in monoubiquitinylation of H2AK119 and trimethylation of H3K27.

Figure 1

Adaptation of KIND1 cells to feeder free culture system & characterization of feeder free KIND1 cells & embryoid body (EB) differentiation. (A) Bright field images of KIND1 cells cultured on HFF (a) and geltrex (b) Magnification 10X. (c) Cytogenetic analysis of KIND1 exhibiting normal 46, XX chromosome complement. (B) RT-PCR analysis of pluripotency specific gene transcripts (C) Western Blot analysis for OCT4 in undifferentiated feeder free KIND1 cell lysate loaded in triplicate with β actin as loading control. (D) Expression of representative gene transcripts of ectoderm (MAP2), mesoderm (HAND1) and endoderm (MIXL) in Day 7 and Day 15 EBs grown in suspension culture. The expression is with respect to undifferentiated KIND1 cells (set as 1). Error bars represent ± Standard Error of Mean (SEM).

Figure 2

Characterization of KIND1 cells differentiated into pancreatic lineage. (A) Expression of pluripotency associated gene transcripts (OCT4, NANOG, SOX2) during differentiation of KIND1 cells from Day 4 – Day 16. (B) Expression of E CADHERIN and N CADHERIN during differentiation in undifferentiated and Day 4 – Day 16. (C) Expression of definitive endoderm specific gene transcripts (SOX17, FOXA2, CXCR4, CERBERUS, EOMESODERMIN) in undifferentiated and Day 4 – Day 16. (D) Expression of primitive gut tube marker HNF4A in undifferentiated and Day 4 – Day 16. The expression of all gene transcripts is relative to undifferentiated KIND1 cells (set as 1). Error bars represent ± SEM, statistical significance represented as *(p <0.05).

Figure 3

Characterization of KIND1 cells differentiated into pancreatic lineage by directed differentiation approach. (A) Expression of pancreas specific gene transcripts (PDX1. SOX9, NKX6.1) in undifferentiated and Day 4 – Day 16. (B) Expression of representative gene transcripts of ectoderm (MAP2) and mesoderm (HAND1, MESP1) in undifferentiated and Day 4 – Day 16. The expression is relative to undifferentiated KIND1 cells (set as 1). Error bars represent ± SEM, statistical significance represented as *(p < 0.05) and **(p < 0.02). (C) Western Blot analysis for SOX17 protein in cell lysates from undifferentiated (UD), Day 4 (D4), Day 8 (D8), and Day 12 (D12) samples, with β ACTIN as housekeeping protein. (D) Western Blot analysis for PDX1 and SOX9 protein in cell lysates from undifferentiated (UD), Definitive Endoderm Day 4 (D4), Primitive Gut Tube Day 8 (D8), and pancreatic progenitors Day 12 (D12) - Day 16 (D16) samples, with β ACTIN as housekeeping protein.

Figure 4

Expression of PcG protein transcripts in KIND1 cells grown on HFF and feeder free KIND1 cells. qRT-PCR results show expression of (A) PRC1 group members- RING1A, RING1B, BMI1 and CBX2(B) PRC2 group members SUZ12, EZH2, EED and JARID2 in KIND1 cells cultured on human feeder fibroblasts (HFF). qRT-PCR results showing expression of (C) PRC1 group members- RING1A, RING1B, BMI1and CBX2(D) PRC2 group members SUZ12, EZH2, EED and JARID2 in feeder free KIND1 cells. The expression is with respect to highest detectable (Ct 40). Error bars represent ± SEM.

Figure 5

Characterization of Embryoid Bodies (EBs) for PcG gene transcripts. (A) Expression of PRC1 (RING1A, RING1B, BMI1and CBX2) in Day 7 and Day 15 EBs grown in suspension culture. (B) Expression of PRC2 (EZH2, SUZ12, EED and JARID 2) gene transcripts in Day 7 and Day 15 EBs grown in suspension culture. (C) Expression of PRC1 (RING1A, RING1B, BMI1and CBX2) gene transcripts at Day 10 in EBs cultured on gelatin coated dishes. (D) Expression of PRC2 (EZH2, SUZ12, EED and JARID 2) gene transcripts at Day 10 in EBs cultured on gelatin coated dishes. The expression is relative to undifferentiated KIND1 cells (set as 1). Error bars represent ± SEM, statistical significance represented as *(p < 0.05), **(p < 0.02) and ***(p < 0.001).

Figure 6

Polycomb group protein expression during directed differentiation of KIND1 cells. (A) PRC1 gene transcript (RING1A, RING1B, BMI1and CBX2) expression in undifferentiated and Day 4 – Day 16. (B) Expression of PRC2 gene transcripts (EZH2, SUZ12, EED and JARID 2) in undifferentiated and Day 4 – Day 16. The expression is relative to their expression in undifferentiated KIND1 cells (set as 1). Error bars represent ± SEM, statistical significance represented as *(p < 0.05), **(p < 0.02).

Figure 7

Polycomb group protein expression during directed differentiation of KIND1 cells. Western Blot analysis of (A) BMI1, (B) EZH2 and (C) SUZ12 proteins in undifferentiated KIND1 (UD), Day 8 (D8), Day 12 (D12) and Day 16 (D16) cell lysates during directed differentiation with β ACTIN as housekeeping protein.

Figure 8

Expression of H3K27me3 and H2AK119ub1 during directed differentiation of KIND1 cells. Western Blot analysis of (A) H2AK119ub1 (B) H3K27me3 in undifferentiated KIND1 (UD) Day 4 (D4), Day 12 (D12) and Day 16 (D16) cell lysates during directed differentiation with (C) HISTONE H3 as housekeeping protein. Densitometric analysis of (D) H2AK119ub1 and (E) H3K27me3 expression during differentiation. Error bars represent ± Standard Error of Mean, statistical significance represented as *(p < 0.05).

Figure 9

Expression of PcG gene transcripts in adult human pancreas & p19 expression during differentiation into pancreatic lineage. (A) Expression of PRC1 gene transcripts -RING1A, RING1B, BMI1and CBX2 (B) Expression of PRC2 gene transcripts -EZH2, SUZ12, EED and JARID2 in adult human pancreas. The expression is with respect to highest detectable Ct 40. (C) Expression of p19/ARF in undifferentiated and Day 4 – Day 16, the expression is relative to undifferentiated KIND1 cells (set as 1). Error bars represent ± SEM, statistical significance represented as *(p < 0.05), **(p < 0.02).