Validating expression of beta cell maturation-associated genes in human pancreas development

The identification of genes associated with human pancreatic beta cell maturation could stimulate a better understanding of normal human islet development and function, be informative for improving stem cell-derived islet (SC-islet) differentiation, and facilitate the sorting of more mature beta cells from a pool of differentiated cells. While several candidate factors to mark beta cell maturation have been identified, much of the data supporting these markers come from animal models or differentiated SC-islets. One such marker is Urocortin-3 (UCN3). In this study, we provide evidence that UCN3 is expressed in human fetal islets well before the acquisition of functional maturation. When SC-islets expressing significant levels of UCN3 were generated, the cells did not exhibit glucose-stimulated insulin secretion, indicating that UCN3 expression is not correlated with functional maturation in these cells. We utilized our tissue bank and SC-islet resources to test an array of other candidate maturation-associated genes, and identified CHGB, G6PC2, FAM159B, GLUT1, IAPP and ENTPD3 as markers with expression patterns that correlate developmentally with the onset of functional maturation in human beta cells. We also find that human beta cell expression of ERO1LB, HDAC9, KLF9, and ZNT8 does not change between fetal and adult stages.


Supplemental Fig. 2 | Assessment of SC-islets generated in Protocols A and B
Immunofluorescent staining for insulin (green, top), glucagon (red, top), Nkx6.1 (red,bottom) and Pdx1 (green, bottom) for SC-islets from Protocol A (A) and Protocol B (B). Quantification of the percentage of cell area that is positive for insulin (C) and glucagon (D) in SC-islets dervided from Protocols A and B, and the percentage of insulin-positive area that is double positive for glucagon in SC-islets from Protocols A and B, and human fetal pancreas (HFP) (E). QPCR gene expression data for characteristic islet genes in undifferentiated cells (H1 cells, normalized to 1 for each gene), Protocol A, Protocol B and adult human islets (AHI) (F). Statistical comparisons are relative to AHI.

Supplemental Fig. 3 | ITGA1 immunostaining in human islets in situ, during isolation, and during culture
Immunofluorescent staining of ITGA1 in islets at different stages of isolation and culture, from two different islet isolations (A, Donor A and B, Donor B). (A) Islets from donor A were stained before isolation (in situ) (a), collected during isolation (b), and collected after 2 days of culture (c). (B) Islets from donor B were stained before isolation (in situ) (a), collected during isolation (b), and collected after 7 days of culture (c).

Supplemental Fig. 4 | Additional immunofluorescent staining of fetal and adult human islets
Immunofluorescent staining of candidate human beta cell maturation markers reveals no change in beta cell-specific expression of the markers CHGA (A), ZNT8 (B), HDAC9 (C), ERO1B (D), KLF9 (E) between human fetal and adult pancreas sections. Individual channel images are the same magnification as the larger merged images. Scale bars = 50 microns.

Supplemental Fig. 5 | MAFA expression is low in Protocol B SC-islets.
To validate the accuracy of the MAFA antibody and our findings that MAFA is expressed in human fetal beta cells, Protocol B SC-islets were stained for MAFA (red), demonstrating very low MAFA protein expression which correlates with gene expression data for these cells.

Supplemental Fig. 6 | Nuclear SIX2 expression correlates with beta cell maturation.
Nuclear expression of SIX2 is low in human fetal beta cells and significantly increased in adult beta cells (A). Nuclear expression of SIX2 is high in HFP grafts after an additional 32 weeks of in vivo-maturation (B). Representative images (N=3 donors per group).

Supplemental Fig. 7 | Expression of glucose transporters GLUT1 and GLUT2
QPCR-based analysis of gene expression of glucose transporters SLC2A1 (GLUT1) and SLC2A2 (GLUT2) in adult human islets ("AHI"), human fetal pancreas ("HFP") and Protocols A ("A") and B ("B"). A) Gene expression was normalized to beta-actin within the same sample, then normalized to undifferentiated H1 cells and presented as a ratio of gene expression over CHGA to normalize for differences in endocrine mass between the various samples. B) Gene expression was normalized to beta-actin within the same sample, then normalized to AHI. Statistics relative to AHI are represented in black font, relative to HFP are represented in red font, relative to Protocol A are represented in light blue font, and relative to Protocol B are represented in purple font.