Establishment of human pluripotent stem cell-derived pancreatic β-like cells in the mouse pancreas

Abstract

Type 1 diabetes is characterized by autoimmune destruction of β cells located in pancreatic islets. However, tractable in vivo models of human pancreatic β cells have been limited. Here, we generated xenogeneic human pancreatic β-like cells in the mouse pancreas by orthotopic transplantation of stem cell-derived β (SC-β) cells into the pancreas of neonatal mice. The engrafted β-like cells expressed β cell transcription factors and markers associated with functional maturity. Engrafted human cells recruited mouse endothelial cells, suggesting functional integration. Human insulin was detected in the blood circulation of transplanted mice for months after transplantation and increased upon glucose stimulation. In addition to β-like cells, human cells expressing markers for other endocrine pancreas cell types, acinar cells, and pancreatic ductal cells were identified in the pancreata of transplanted mice, indicating that this approach allows studying other human pancreatic cell types in the mouse pancreas. Our results demonstrate that orthotopic transplantation of human SC-β cells into neonatal mice is an experimental platform that allows the generation of mice with human pancreatic β-like cells in the endogenous niche.

Keywords: beta cells; diabetes; human pluripotent stem cells; humanized mice.

Fig. 1.

Transplantation of SC-β cells into neonatal mice leads to efficient functional engraftment of human pancreatic β-like cells. (A) Cells at different stages of pancreatic β-like cell differentiation were transplanted into the pancreatic regions of developing embryos in utero (Top) or in neonatal pups (Bottom). (B) Survival of mice transplanted in utero and postnatally. Neonatal denotes orthotopic transplantation into neonatal mice. Kidney capsule denotes transplantation under the kidney capsule of adult mice. (C) Human insulin levels from plasma samples of recipient mice were tested with human insulin-specific ELISA at 7 wk posttransplantation. The percentage of mice showing positive human insulin (>0.15 μIU/mL) was plotted as gray bars, and levels of human insulin were plotted as green dots. Error bars show SEM. N (below the bars) indicates the total number of mice analyzed. (D) Microscopic analysis (Left and Center Left) and immunohistochemistry with anti-GFP (Center Right, IHC-GFP) or anti-human C-peptide (Right, IHC-human C-peptide) antibodies of pancreata of control mice (CTL; Top) and NSG mice transplanted with SC-β cells differentiated from HUES8-GFP cells at the neonatal stage (Bottom). (White scale bar: 2 mm; black scale bar: 20 μm.) (E) Percentage of mice showing fluorescent human cells in mouse pancreata. All analyses in D and E were performed on 6-mo-old mice. (F) Flow cytometry analysis of GFP-positive cells from dissociated pancreata of CTL (Left) or 6-mo-old mice transplanted with SC-β cells differentiated from HUES8-GFP cells at the neonatal stage (Center). (Right) Percentage of GFP-positive cells was quantified. Error bars show SEM. **P < 0.01 (two-tailed t test). (G) Quantitative PCR analysis of human mitochondrial DNA in total DNA samples extracted from different organs of mice engrafted with human cells into the pancreas. Dashed green, blue, and red lines show relative quantification (RQ) signal from one human cell in 10³, 10⁴, and 10⁵ mouse cells, respectively, from the standard curve. ***P < 0.001 (one-way ANOVA).

Fig. 2.

Characterization of engrafted human β-like cells in the mouse pancreas. (A) Representative fluorescent micrographs of anti-human C-peptide staining (red) and anti-GFP staining (green) in control (CTL) mouse pancreas (Top) and in the pancreas of 6-mo-old transplanted mice (Bottom). (B) Representative fluorescent micrographs of anti-human C-peptide (red) and antiglucagon antibody (green) staining in pancreatic tissue from CTL (Top) and a 6-mo-old mouse engrafted with human β-like cells into the pancreas (Bottom). (C) Fluorescent micrographs showing coexpression of C-peptide and insulin in human β-like cells engrafted in the mouse pancreas (Bottom), but not in β cells in the CTL mouse (Top). (D) Expression of β cell transcription factors PDX1, NKX2.2, NKX6.1, and ISL1 in β-like cells engrafted into the mouse pancreas. (Scale bars: 10 μm.)

Fig. 3.

Vascularization of engrafted human cells. (A) Representative fluorescent micrographs of anti-CD31 (green), antiinsulin (red), and anti-human C-peptide (magenta) staining in pancreatic islets of a control (CTL) mouse (Left) and of a transplanted mouse (Right). (B) Quantification of CD31 foci per mouse β cells (gray bar) or per human β-like cells (black bar) was plotted (n = 3 mice), and the total numbers of analyzed mouse β cells and human β-like cells are labeled. (C) Endothelial cells in the engrafted regions are of mouse origin, as indicated by the lack of expression of GFP in CD31-expressing cells. All analyses were performed on samples from 6-mo-old mice. n.s., not significant (t test).

Fig. 4.

Human SC-β cells transplanted under the mouse kidney capsule. (A, Left and Center Left) Microscopy analysis of green fluorescence signal in control (CTL) kidney (Top) or kidney transplanted with SC-β differentiated from HUES8-GFP cells from 7.5-mo-old mice (6 mo after transplantation to 1.5-mo-old mice) (Bottom). (A, Center Right and Right) Immunohistochemistry analysis with an anti-GFP antibody (IHC-GFP) or an anti-human C-peptide antibody (IHC-human C-peptide) of samples from the CTL mouse (Top) and the transplanted mouse (Bottom). (B) Representative fluorescent micrographs showing the presence of GFP- and C-peptide–expressing human cells in transplanted kidney (Bottom), but not in CTL (Top). (C) Representative fluorescent micrographs showing C-peptide–expressing cells engrafted under the kidney capsule did not express glucagon. (D) Expression of transcription factors PDX1, NKX2.2, NKX6.1, and ISL1 in engrafted C-peptide–expressing β-like cells under the kidney capsule. (Scale bars: 10 μm.)

Fig. 5.

Functional characterization of human β-like cells engrafted into the mouse pancreas. (A, Left) Experimental design to compare the same number of pancreatic β-like cells transplanted under the kidney capsule or into the neonatal pancreas. (A, Right) Comparison of human insulin in mouse blood from the two transplantation methods. N (below the bars) indicates the total number of mice analyzed. (B) Ratio of mice showing detectable human insulin in the blood circulation 1 mo after kidney capsular transplantation or neonatal orthotopic transplantation. N (below the bars) indicates the total number of mice analyzed. (C) GSIS measurements at different time posttransplantation in mice orthotopically transplanted with human pancreatic β-like cells at the neonatal stage. After a 16-h fasting period, mouse plasma samples were collected (fasting insulin levels shown by black dots), glucose solution was injected i.p. (2 g per 1 kg of body weight), and plasma was collected 30 min postglucose injection (postglucose stimulation, shown by gray squares). n.s., not significant (t test). *P < 0.05 (paired t test).