Evaluating KIND1 human embryonic stem cell-derived pancreatic progenitors to ameliorate streptozotocin-induced diabetes in mice

Background & objectives: Diabetes is a global disease burden. Various stem cell types are being explored to serve as an alternative source of islets. This study was conducted to evaluate the ability of in-house developed human embryonic stem (hES) cells-derived pancreatic progenitors to ameliorate diabetic symptoms in mice. Methods: Pancreatic progenitors were packed in macro-capsules and transplanted into six male Swiss mice and four mice were taken as controls. Thirty days post-transplantation, diabetes was induced by streptozotocin treatment. Mice were then followed up for >100 days and body weight and blood glucose levels were regularly monitored. Results: Control mice lost weight, maintained high glucose levels and did not survive beyond 40 days, whereas transplanted group maintained body weight and four of the six mice had lowered blood glucose levels. About five-fold increase was observed in human C-peptide levels in the recipients of progenitor transplants as compared to diabetic control. Interpretation & conclusions: The beneficial effect of transplanted cells was not long-lasting. Further studies are required to critically evaluate and compare the potential of endogenous pluripotent stem cells and hES cells-derived progenitors before moving from bench to the bedside

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

Abstract Human embryonic (hES) stem cells are widely used as an in vitro model to understand global genetic and epigenetic changes that occur during early embryonic development. In-house derived hES cells (KIND1) were subjected to directed differentiation into cardiovascular progenitors (D12) and beating cardiomyocytes (D20). Transcriptome profiling of undifferentiated (D0) and differentiated (D12 and 20) cells was undertaken by microarray analysis. ChIP and sequential ChIP were employed to study role of transcription factor NR2F2 during hES cells differentiation. Microarray profiling showed that an alteration of about 1400 and 1900 transcripts occurred on D12 and D20 respectively compared to D0 whereas only 19 genes were altered between D12 and D20. This was found associated with corresponding expression pattern of chromatin remodelers, histone modifiers, miRNAs and lncRNAs marking the formation of progenitors and cardiomyocytes on D12 and D20 respectively. ChIP sequencing and sequential ChIP revealed the binding of NR2F2 with polycomb group member EZH2 and pluripotent factor OCT4 indicating its crucial involvement in cardiac differentiation. The study provides a detailed insight into genetic and epigenetic changes associated with hES cells differentiation into cardiac cells and a role for NR2F2 is deciphered for the first time to down-regulate OCT-4 via EZH2 during cardiac differentiation

Additional file 2: of Transcriptional activator DOT1L putatively regulates human embryonic stem cell differentiation into the cardiac lineage

Characterization of cardiac differentiation of HES3 cells by quantitative real-time PCR (qRT-PCR). Expression of transcripts representing pluripotency (OCT4), cardiac mesoderm (MESP1), cardiac progenitors (NKX2.5, MEF2C), and cardiomyocytes (CTNT) at days 0, 12, and 20 during 20 days of cardiac differentiation. Note OCT-4 expression in undifferentiated cells is downregulated as the cells initiate differentiation. Early cardiac markers detected on day 12 and mature markers upregulated on day 20. Similar changes in transcripts expression observed when KIND1 cells were differentiated into cardiac cells as described earlier [43]. Error bars represent ÂąSEM. (PDF 410 kb

Additional file 5: of Transcriptional activator DOT1L putatively regulates human embryonic stem cell differentiation into the cardiac lineage

Dystrophin gene expression during cardiac differentiation of KIND1 hES cells on days 0, 12, and 20 during cardiac differentiation of KIND1 hES cell line. Expression of Dystrophin increased in cardiac progenitors and cardiomyocytes compared to undifferentiated KIND1 cells. Results in agreement with earlier reports in DOT1L conditional knockout mice heart concluding Dystrophin as a direct target of DOT1L [35]. Error bars represent ¹SEM. (PDF 329 kb

Additional file 1: of Transcriptional activator DOT1L putatively regulates human embryonic stem cell differentiation into the cardiac lineage

Brightfield images of HES3 hES cells during directed differentiation into cardiac lineage. Differentiation results in distinct morphological changes leading to increased compaction among the cells as differentiation proceeds from day 0 to day 20. Similar changes observed when KIND1 cells were differentiated into cardiac cells as described earlier [43]. Magnification 10×. (PDF 554 kb

Additional file 4: of Transcriptional activator DOT1L putatively regulates human embryonic stem cell differentiation into the cardiac lineage

ChIP sequencing in KIND1 and HES3 cells during cardiac differentiation visualized by Integrated Genome Viewer shows binding profile of H3K79me2 modification across genes HNF4A, LEFTY1, NOGGIN, NQO1, OTX2, and NPTX2 in KIND1 (green) and HES3 (red) cells at days 0, 12, and 20 of cardiac differentiation. (PDF 548 kb

Additional file 3: of Transcriptional activator DOT1L putatively regulates human embryonic stem cell differentiation into the cardiac lineage

Characterization of cardiac differentiation of HES3 cells by immunofluorescence studies. Expression of NKX2.5 (A) and CTNT (B) on days 12 and 20 observed by immunofluorescence. (A) Distinct nuclear expression of NKX2.5 observed and (B) CTNT cell surface expression. Similar changes observed when KIND1 cells were differentiated into cardiac cells as described earlier [43]. Counterstaining using DAPI. Magnifications 20×. (PDF 450 kb

Additional file 6: of Transcriptional activator DOT1L putatively regulates human embryonic stem cell differentiation into the cardiac lineage

ChIP sequencing of occupancy of H3K79me2 on DMD gene during cardiac differentiation of KIND1 and HES3 cells showing occupancy of H3K79me2 methylation mark brought about by DOT1L on DMD gene during cardiac differentiation. Results clearly show significant peaks representing the DOT1L specific methylation mark on days 12 and 20 as compared to day 0 suggestive of its activation by DOT1L during cardiac differentiation in vitro. (PDF 614 kb