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

Abstract

BackgroundPolycomb 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.ResultsIn-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.ConclusionOur 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.

Genetic and Epigenetic Profiling Reveals EZH2-mediated Down Regulation of OCT-4 Involves NR2F2 during Cardiac Differentiation of Human Embryonic Stem Cells

“…Importance of PcG proteins for differentiation of ES cells and not in their self-renewal was identified when the ES cells deficient for PRC2 proteins (SUZ12, EZH2, JARID2) could still be maintained in their undifferentiated state in vitro 14 , 15 . Dynamics of PcG proteins during differentiation of human ES cells into the three lineages was for the first time reported by our group 16 , 17 . Transcripts of both PRC1 and PRC2 were found up regulated during endodermal differentiation while during ectodermal differentiation only PRC1 transcripts were found to be up regulated.…”

Activin/Smad2-induced Histone H3 Lys-27 Trimethylation (H3K27me3) Reduction Is Crucial to Initiate Mesendoderm Differentiation of Human Embryonic Stem Cells

“…These observations demonstrate an essential role of PRC2 in early mouse development. Accumulating evidence also suggests that PRC2 plays an important role in ESC differentiation toward mesoderm and endoderm lineages (8,17,18).…”

Stem cells, progenitors & regenerative medicine: A retrospection

“…Initial work from our group resulted in the derivation of two hES cell lines KIND1 and KIND-2 3 , we studied their propensity 36 , adapted both the cell lines to feeder-free conditions and established directed differentiation protocols to make pancreatic 37 and tripotent cardiovascular progenitors 38 . At present, pre-clinical evaluation of safety, efficacy and feasibility of these progenitors is being studied in animal models.…”