Methods of enhancing stem cell differentiation into beta cells
A technology of cells and cell populations, applied in chemical instruments and methods, biochemical equipment and methods, receptors/cell surface antigens/cell surface determinants, etc.
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Embodiment 1
[0656] Example 1. Histone acetylation in hPSC-derived beta cells
[0657] A systematic approach (e.g., Arda et al. (Cell Metabolism 23:909-920, 2016)) was used to identify age-related gene expression programs in human pancreatic islets, which included (1) obtaining pancreatic tissue from children and adults; (2) developing robust and reliable cell purification methods; (3) generation of complete transcriptome and histone modification profiles; and (4) systematic analysis of islet physiology and function. Distinct patterns of histone-mediated age-related gene regulation in juvenile and adult human islets are revealed on the genome-wide histone atlas. Xu et al. (EMBO J 2014) describe decreased histone methylation of NGN3, NKX6.1 and NKX2.2 genes in endocrine progenitor cells. In NGN3 cells, H3K27me3 was deleted from NGN3, NeuroD1, and NKX6.1 elements, consistent with activation of individual genes. Haumaitre et al. (MCB 2013) reported a general decrease in HDAC expression and ...
Embodiment 2
[0658] Example 2. Inhibition of Histone Methylation and Deacetylation
[0659] NGN3 mRNA expression was studied at each step of directed differentiation described here ( Figure 5 ), and cells were observed to express NGN3 to trigger SC-β cell differentiation ( Figure 7 ). The effect of inhibition of histone methylation and deacetylation on SC-β cell differentiation was investigated ( Figure 6 ). With EZH2 inhibitor (DZNep), HDAC inhibitor (KD5170) and their combination ( Figure 6 ) to stage 5 of the directed differentiation described herein for hESC lines (D97, D114 and D241 HUES8) ( Figure 5-Figure 6 ).
[0660] Such as Figure 18 Screening of candidate inhibitors was performed as indicated. Inhibition of EZH2 or HDAC in stage 5 (D97 HUES8) increased endocrine cells and SC-β cells ( Figure 8 ). Combined inhibition of EZH2 and HDAC at stage 5 (D114 HUES8) significantly increased endocrine cells ( Figure 9-Figure 11 ). At the end of phase 5, with DZNep and KD5...
Embodiment 3
[0663] Example 3. Differentiation of Stem Cells to Pancreatic β Cells (SC-β Cells)
[0664] Such as Figure 39 As shown, two exemplary differentiation protocols v11 and v12 were tested. Both protocols are 6-stage stepwise protocols sharing similar reagents, and their first 5 stages of differentiation steps are largely based on Figure 5 The differentiation schemes shown, and their differences are as follows: (a) In the v12 scheme, 0.25 μM DMH-1 was present in the medium of stage 3 day 1 (S3d1), and at stage 3 day 1 and Activin A was present at 20 ng / mL in the medium on day 2 (S3d1-S3d2); (b) in the v11 protocol, no DMH-1 or activin A was added in stage 3; (c) in the v11 and v12 protocols , DZNep was present at 100 nM in the culture medium on days 1, 3, 5, and 7 of stage 5. Stage 6 of both the v11 and v12 protocols was performed by culturing the product cells of stage 5 in DMEM / F12 medium supplemented with 1% HAS without exogenous differentiation factors. Throughout Stage 6...
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