MAFA and T3 Drive Maturation of Both Fetal Human Islets and Insulin-Producing Cells Differentiated From hESC

Abstract

Context: Human embryonic stem cells (hESCs) differentiated toward β-cells and fetal human pancreatic islet cells resemble each other transcriptionally and are characterized by immaturity with a lack of glucose responsiveness, low levels of insulin content, and impaired proinsulin-to-insulin processing. However, their response to stimuli that promote functionality have not been compared.

Objective: The objective of the study was to evaluate the effects of our previous strategies for functional maturation developed in rodents in these two human models of β-cell immaturity and compare their responses. Design, Settings, Participants, and Interventions: In proof-of-principle experiments using either adenoviral-mediated overexpression of V-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA) or the physiologically driven path via thyroid hormone (T3) and human fetal islet-like cluster (ICC) functional maturity was evaluated. Then the effects of T3 were evaluated upon the functional maturation of hESCs differentiated toward β-cells.

Main outcome measures: Functional maturation was evaluated by the following parameters: glucose responsiveness, insulin content, expression of the mature β-cell transcription factor MAFA, and proinsulin-to-insulin processing.

Results: ICCs responded positively to MAFA overexpression and T3 treatment as assessed by two different maturation parameters: increased insulin secretion at 16.8 mM glucose and increased proinsulin-to-insulin processing. In hESCs differentiated toward β-cells, T3 enhanced MAFA expression, increased insulin content (probably mediated by the increased MAFA), and increased insulin secretion at 16.8 mM glucose.

Conclusion: T3 is a useful in vitro stimulus to promote human β-cell maturation as shown in both human fetal ICCs and differentiated hESCs. The degree of maturation induced varied in the two models, possibly due to the different developmental status at the beginning of the study.

Figure 1.

Midgestation (19–21 wk) HFP had many insulin-positive cells as singlets or small clusters associated with ductal cells (A and B) and with glucagon-positive cells (C). D, Key β-cell genes had significantly lower expression in fetal ICCs than in adult human islets by qPCR. Dotted line indicates the adult levels (n = 4 for Pdx1 [*, P = .01]; n = 9 for insulin [*, P = .000 003], and Mafa [*, P = .05]) with an unpaired t test. E, At this age, insulin-positive cells were NKX6.1 positive (arrows) and SOX9 negative. Ngn3-positive cells (arrow) were still present in ductal structures. β-Cells from fetal human pancreas had lower levels of PDX1 (F) and MAFA (G) (arrows) when compared with adult pancreas (19 wk fetal human pancreas, representative of two blocks each from two HFP). Magnification bar, 50 μm, except in panel E for PDX1 and SOX9 in which the magnification bar was 25 μm.

Figure 2.

Functional effects of adenoviral-mediated overexpression of MAFA in ICCs. A, Insulin secretion at 2.6 mM glucose. B, Increased insulin secretion to 16.8 mM glucose. C, Increased insulin content. There was no change in proinsulin secretion (D) or proinsulin to insulin ratio (E). F, Key β-cell genes had increased expression as measured by qPCR and compared with untreated samples (dotted line). For panels A–E, n = 12 pancreas of 19–22 weeks' gestation; connector lines represent experimental and control data for individual pancreases; for panel F, n = 3–4 of 20 weeks' gestation. Nonparametric statistics were used for panels B and C; paired t test was used for the others.

Figure 3.

T₃ effects on ICC function and gene expression. A, Insulin secretion at 2.6 mM glucose. At 16.8 mM glucose, insulin secretion was increased (B). there was no change in insulin content (C) or proinsulin secreted at 16.8 mM glucose (D). The proinsulin to insulin ratio (E) is shown, with nonsignificant changes in proinsulin (F) cellular content. G, T₃ effects on gene expression of ICCs as measured by qPCR and compared with untreated samples (dotted line) (n = 8 human fetal pancreas of 20–22 wk gestation); connector lines represent experimental and control data for individual pancreases. *, P ≤ .05, nonparametric statistics used for panels C and E and a Student t test for the others. C, control.

Figure 4.

hESCs after differentiation express transcription factors characteristic of endocrine progenitors and some important β-cell-specific transcription factors but not MAFA. Stage 1 (A) represents cells that are differentiated to definitive endoderm and stage 4 (B) further differentiated to pancreatic endoderm but before exposure to T₃. Magnification bar, 100 μm. C, Scatter plots of mRNA levels of different genes before the differentiation protocol (d0) and at the end of stage 4 (d12). Individual points represent independent samples from a single experiment and are expressed in relation to housekeeping gene TBP. D, Representative pictures of cells at the end of the differentiation at stage 5 (control conditions) immunostained for CGA, NKX 6.1, glucagon (GCG), C-peptide (CPEP), and 4′,6-diamino-2-phenylindole (DAPI) to counterstain nuclei. E, FACS quantification at the end of stage 4. F, FACS quantification at the end of stage 5.

Figure 5.

T₃ effects on hESC-derived cell function and gene expression. At the end of stage 5, T₃ (100 nM)-treated cells were assessed for insulin secretion at 2.6 mM glucose (A), at 16.8 mM glucose (B), insulin cell content (C), proinsulin (D), and proinsulin to insulin ratio (E). For functional experiments, n = 7–8 samples from three independent experiments. F, qPCR was used to evaluate the expression of the insulin gene and other key β-cell genes (dotted line, control = 1). All data are expressed as fold change to untreated control samples. For gene expression, n = 5–9 samples from two to four independent experiments and normalized to levels of TBP as an endogenous control. Student t test was used for all panels. C, control.

Figure 6.

ICCs obtained at different gestational ages respond differently to MAFA overexpression. mRNA of insulin (A) and PDX1 (B) as well as insulin secretion at 16.8 mM glucose (C) increased in an age-dependent manner after adenovirally mediated MAFA overexpression. Each dot represents an independent sample from independent experiments.