GP2-enriched pancreatic progenitors give rise to functional beta cells in vivo and eliminate the risk of teratoma formation

Abstract

Human pluripotent stem cell (hPSC)-derived pancreatic progenitors (PPs) can be differentiated into beta-like cells in vitro and in vivo and therefore have therapeutic potential for type 1 diabetes (T1D) treatment. However, the purity of PPs varies across different hPSC lines, differentiation protocols, and laboratories. The uncommitted cells may give rise to non-pancreatic endodermal, mesodermal, or ectodermal derivatives in vivo, hampering the safety of hPSC-derived PPs for clinical applications and their differentiation efficiency in research settings. Recently, proteomics and transcriptomics analyses identified glycoprotein 2 (GP2) as a PP-specific cell surface marker. The GP2-enriched PPs generate higher percentages of beta-like cells in vitro, but their potential in vivo remains to be elucidated. Here, we demonstrate that the GP2-enriched-PPs give rise to all pancreatic cells in vivo, including functional beta-like cells. Remarkably, GP2 enrichment eliminates the risk of teratomas, which establishes GP2 sorting as an effective method for PP purification and safe pancreatic differentiation.

Keywords: beta cells; cell replacement therapy; cell surface marker; ectoderm; endoderm; human embryonic stem cells; mesoderm; pancreatic progenitors; teratoma; type 1 diabetes.

Graphical abstract

Figure 1

Enrichment of GP2⁺ cells in hESC-derived PPs (A) Schematic of the hESC differentiation to pancreatic progenitors (PPs). (B–D) Representative flow cytometry plots and quantification of NKX6-1/PDX1 co-expression in hESC-H1- (B) or H9- (C) derived PPs at the end of stage 4 (D). (E–G) Representative flow cytometry plots and quantification of GP2 expression in H1- (E) or H9- (F) derived PPs at the end of stage 4 (G). (H1, n = 6 independent experiments; H9, n = 3 independent experiments; unpaired Student’s t test; error bars represent SEM). (H) Schematic of GP2 enrichment using MACS. (I, J, L, and M) Representative flow cytometry plots and quantification of the percentage of GP2-expressing cells in not sorted (NS), flow-through (FT), and GP2-enriched (GP2⁺) fractions of H1- (I and J) or H9- (L and M) derived PPs. (K and N) Mean fluorescence intensity (MFI) over isotype control in NS, FT, and GP2⁺ fractions of H1- (K) or H9- (N) derived PPs. (H1, n = 6 independent experiments; H9, n = 3 independent experiments; one-way ANOVA analysis with Tukey’s multiple comparisons; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; error bars represent SEM). See also Figure S1.

Figure 2

Transplantation of GP2-enriched PPs prevents teratoma formation (A) Schematic of RFP-H1 differentiation to RFP-PPs followed by GP2 enrichment using MACS. (B) Live imaging of mice transplanted subcutaneously with NS, FT, and GP2⁺ cell populations, at weeks 1, 4, and 12 post-transplantation. See also Figure S2. (C) Representative image of outgrowths visible with the naked eye in the mouse transplanted with NS cells (dashed line). No outgrowths detected in mice transplanted with GP2⁺ cells. (D–I) Representative images of NS-derived grafts stained with H&E (D), toluidine blue (E), SOX2 (F), FOXA2 (G), CDX2 (H), and NKX2-1 (F) at week 15 post-transplantation ([D] scale bar is 100 μm; [F] scale bar is 200 μm; and [E, G–I] scale bar is 400 μm). See also Figure S3. (J) Percentage of NS-, FT-, and GP2⁺-recipient mice that generated teratomas/outgrowths (NS, n = 15 mice from six independent experiments; FT, n = 17 mice from six independent experiments; and GP2⁺, n = 11 mice from six independent experiments).

Figure 3

GP2-enriched PPs have lower expression of endoderm-derived non-pancreatic cell markers (A–C) RT-PCR quantification of SOX2, NKX2-1, and CDX2 normalized to RPL-19 housekeeping gene, in H1-derived NS, FT, and GP2⁺ PP fractions (n = 3 independent experiments, NS, FT, and GP2⁺; n = 1, human stomach, lung, and intestine; one-way ANOVA analysis with Tukey’s multiple comparisons; ^∗p < 0.05, ^∗∗∗p < 0.01; error bars represent SEM). (D–G) Representative flow cytometry plots indicating SOX2 and CDX2 expression in NS, GP2⁻, and GP2⁺ populations and quantification of cell percentages (n = 7 independent experiments; one-way ANOVA analysis with Tukey’s multiple comparisons; ^∗p < 0.05, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, error bars represent SEM). See also Figures S4 and S5.

Figure 4

GP2-enriched PPs are less proliferative and give rise to smaller grafts with lower CDX2⁺ and PRSS1⁺ cells (A–D) Representative images and quantification of H&E and KU80 staining of the non-teratoma grafts retrieved from mice 15 weeks post-transplantation (scale bar is 400 μm; n = 3 independent experiments; one-way ANOVA analysis with Tukey’s multiple comparisons; ^∗p < 0.05, ^∗∗p < 0.01; error bars are SEM). (E and F) RT-PCR quantification of MKI67 and TOP2A normalized to RPL19 housekeeping gene in NS, FT, and GP2⁺ populations before transplantation. (n = 3 independent experiments; one-way ANOVA analysis with Tukey’s multiple comparisons; ^∗p < 0.05, ^∗∗p < 0.01; error bars represent SEM). (G and H) Representative flow cytometry plots indicating KI67 expression in NS, GP2⁻, and GP2⁺ populations (n = 7 independent experiments; one-way ANOVA analysis with Tukey’s multiple comparisons; ^∗p < 0.05, error bars represent SEM). (I–Q) Representative immunostaining staining of grafts at week 15 post-transplantation, indicating the presence of cells expressing CDX2 (intestinal marker), NKX2-1 (lung marker), chromogranin A (CHGA, pancreatic endocrine marker), cytokeratin 19 (KRT19, ductal cell marker), and trypsin (PRSS1, acinar cell marker) (scale bar is 200μm), and quantification of cell numbers (n = 3 independent experiments; one-way ANOVA analysis with Tukey’s multiple comparisons; ^∗p < 0.05; error bars represent SEM). (R) Ratio of endocrine to acinar cells (n = 3 independent experiments; error bars are SEM).

Figure 5

GP2⁺-enriched PPs give rise to functional beta cells in vivo (A and B) Glucose-stimulated C-peptide secretion assay at 15 weeks post-transplantation of H1- (A) or H9- (B) derived NS, FT, and GP2⁺ fractions ([A] NS, n = 15 mice from 6 independent experiments; FT, n = 14 mice from six independent experiments; GP2⁺, n = 9 mice from 6 independent experiments; [B] NS, n = 6 mice from three independent experiments; FT, n = 7 mice from 3 independent experiments; GP2⁺, n = 4 mice from three independent experiments; two-way ANOVA analysis with Tukey’s multiple comparisons; ^∗p < 0.05, ^∗∗p < 0.01; error bars represent SEM). (C and D) Representative immunostaining staining of grafts without teratoma retrieved from mice at week 15 post-transplantation indicating the presence of cells expressing C-peptide (Cp, beta cell marker), glucagon (GCG, alpha cell marker), somatostatin (SST, delta cell marker), and SLC18A1 (enterochromaffin cell marker). DAPI marks cell nuclei. (Scale bar is 200μm).