Human embryonic stem cell (hESC)-derived pancreatic α- and β-cells can be used to develop cell replacement therapies to treat diabetes. However, recent published differentiation protocols yield varying amounts of α- and β-cells amid heterogeneous cell populations. To visualize and isolate hESC-derived α- and β-cells, we generated a GLUCAGON-2A-mScarlet and INSULIN-2A-EGFP dual fluorescent reporter (INSEGFPGCGmScarlet) hESC line using CRISPR/Cas9. We established robust expression of EGFP and mScarlet fluorescent proteins in insulin- and glucagon-expressing cells, respectively, without compromising the differentiation or function of these cells. We also showed that the insulin- and glucagon-expressing bihormonal population at the maturing endocrine cell stage (stage 6) of our pancreatic islet differentiation lose insulin expression over time, while maintaining an α-like expression profile, suggesting these bihormonal cells are cell-autonomously fated to become α-like cells. We also demonstrated this cell line can be used to monitor hESC-derived insulin- and glucagon-expressing cells, and hESC-derived islet morphology in vivo, by transplanting them into the anterior chamber of the eye in mice. Together, the INSEGFPGCGmScarlet hESC line provides an efficient strategy for tracking populations of hESC-derived β- and α-like cells.

Article Highlights

  • Differentiation protocols used to generate stem cell–derived islet cells yield heterogenous cell populations.

  • We generated a human embryonic stem cell line that reports insulin- and glucagon-expressing cells in vitro and in vivo without altering their differentiation or function.

  • We showed some insulin- and glucagon-expressing bihormonal cells are cell-autonomously fated to become α-like cells.

  • This reporter cell line can be used to further study and improve stem cell–derived islet differentiation and transplantation.

Pancreatic β- and α-cells secrete hormones insulin and glucagon, respectively, to regulate glucose homeostasis in the blood. These cells organize into clusters during pancreas development, forming the islets of Langerhans. People living with type 1 diabetes (T1D) lack sufficient β-cells, resulting in elevated blood glucose levels that can lead to life-threatening complications. Thus, it is critical for these people to rely on exogenous insulin to manage their disease long term. Transplantation of deceased donor human islets has been shown to effectively restore regulated insulin production in people living with T1D, allowing patients to become independent of exogenous insulin (1,2). However, there is a scarce supply of donor islets, and the quality of these islets is variable, limiting the accessibility of this treatment (3,4). Therefore, human embryonic stem cells (hESCs) have been proposed to serve as an unlimited and reliable source of pancreatic β- and α-cells for the treatment of diabetes (5).

hESC lines engineered to report insulin using a fluorescent protein have proved to be an efficient tool used to further improve the generation of pancreatic β-cells in vitro (6–8). These fluorescent protein–expressing cells can easily be visualized using a benchtop fluorescence microscope, used in high-throughput screens, or isolated using FACS for downstream analyses (9). However, recently published differentiation protocols generate heterogeneous cultures, with varying amounts of insulin-expressing cells, glucagon-expressing cells, and insulin- and glucagon-expressing bihormonal cells (7,10–12). Thus, an insulin single reporter hESC line would not be able to differentiate between these monohormonal and bihormonal cells.

To visualize and isolate monohormonal and bihormonal cell populations during differentiation, we generated an insulin and glucagon dual fluorescent reporter hESC line using CRISPR/Cas9. Specifically, we knocked in added-on 2A-EGFP and 2A-mScarlet expression cassettes downstream of the INS and GCG loci of the H1/WA01 hESC line. To validate the cell line, we showed EGFP and mScarlet expression report insulin and glucagon expression, respectively. We confirmed the differentiation and function of these reporter-derived cells were not affected by the insertion of the expression cassettes. To demonstrate this cell line can be used to enrich β- and α-like cells, we sorted cells based on fluorescent reporter expression and found distinct populations that expressed key β- and α-cell markers. Interestingly, we also found the insulin- and glucagon-expressing cell population at the maturing endocrine cell stage more closely resembles that of α-cells than β-cells. To test whether this population will further differentiate into monohormonal glucagon-expressing α-cells, we reaggregated these bihormonal cells for further culture and found that the majority of these cells lose insulin expression over time. These findings suggest these bihormonal cells represent a transient population that is cell-autonomously fated to become monohormonal α-like cells. Finally, we transplanted mature pancreatic endocrine cells derived from this cell line into the anterior chamber of the eye (ACE) in mice and demonstrated this model can be used to monitor insulin- and glucagon-expressing cells in vivo.

Cell Culture

Stem cells were maintained as previously described (13). Briefly, undifferentiated H1 (WA01; XY) hESCs were maintained in mTeSR Plus (STEMCELL Technologies Inc.) at 5% CO2, 37°C. Cells were split every 4 days using ReLeSR (STEMCELL Technologies Inc.), and were plated at 1 × 106 and 2 × 106 cells on Geltrex (Gibco, 1:100 DMEM/F12; Thermo Fisher Scientific)-coated 60-mm and 10-cm tissue culture plates, respectively, with 10 μmol/L Y-27632 (STEMCELL Technologies Inc.).

CRISPR/Cas9 Knock-In

The CRISPR/Cas9 system used to generate the INSEGFPGCGmScarlet hESC line was as previously described (8,13,14). The pCCC CRISPR/Cas9 plasmids were generated to express guide RNA (5′-TGCAACTAGACGCAGCCCGC-3′) and (5′-AACATCACCTGCTAGCCACG-3′) for targeting the 3′ end of the endogenous insulin (INS) and glucagon (GCG) coding regions, respectively. The homology arms of the selected CRISPR clones were genotyped and subsequently sequenced to confirm the fidelity of recombination (Supplementary Fig. 1A). For a list of genotyping primers used, see Supplementary Table 1. Clones with the insertions were then tested for common chromosomal abnormalities using the hPSC Genetic Analysis Kit (STEMCELL Technologies Inc.).

hESC Differentiation

The protocol used to differentiate the hESC lines was based on previously published protocols (7,10,11,13). Briefly, hESCs were dissociated using Accutase (STEMCELL Technologies Inc.) for 5 min at 37°C and washed using Dulbecco’s PBS with calcium and magnesium (Gibco). One million cells/mL was seeded in a total of 5.5 mL mTesr Plus containing 10 μmol/L Y-27632 in each well of non–tissue culture-treated six-well plates (Greiner Bio-One). The plates were placed on a shaker (25 mm orbit) (Celtron; Infors HT) set at 94 rpm at 37°C, 5% CO2 overnight. The 3D spheroids were washed using Dulbecco’s PBS with calcium and magnesium and replaced with differentiation media. For detailed formulations of differentiation media at each stage, see Supplementary Table 2.

Flow Cytometry and FACS

For flow cytometry analysis, spheroids were washed with PBS and dissociated using Accutase (STEMCELL Technologies Inc.) for 8 min at 37°C. Dissociated cells were centrifuged for 5 min at 200g and resuspended in Fixable Viability Dye eFluor 780 (1:1,000 in PBS; Thermo Fisher Scientific) for 30 min at 22°C. Cells were subsequently centrifuged for 5 min at 200g and resuspended in 4% paraformaldehyde (PFA) (Thermo Scientific Chemicals) for 30 min at 22°C. Cells were then centrifuged for 5 min at 200g and resuspended in PBS for long-term storage. On the day of analysis, cells were permeabilized using 0.5% triton X-100 (Thermo Fisher Scientific) and stained with conjugated antibodies for 1 h on ice: mouse anti-C-peptide antibody conjugated with Alexa Fluor 647 (BD Biosciences) and mouse antiglucagon antibody conjugated with Bv421 (BD Biosciences). Cells were analyzed on a BD LSR II using unstained and single-stain controls, and the data were processed using FlowJo. To sort mScarlet-EGFPlo, mScarlet-EGFPhi, mScarlet+ EGFP, and mScarlet+EGFPhi populations, spheroids were rinsed with PBS and dissociated using Accumax for 8 min at 37°C. Dissociated cells were resuspended in 2% FBS (HyClone; Thermo Fisher Scientific) in PBS with 10 μmol/L Y-27632 on ice and sorted into stage 7 media with 10 μmol/L Y-27632 using the BD FACS Aria or Fusion. To reaggregate cells, 300,000 and 1 million unsorted or sorted cells were transferred to Aggrewell-800 and -400 plates, respectively (STEMCELL Technologies Inc.), containing stage 7 media with 10 μmol/L Y-27632 for 60 h before further culture in six-well plate format or transplantation.

Islet Transplantation and Noninvasive In Vivo Imaging in Mice

The transplant experiments were approved by the University of British Columbia Animal Care Committee (Vancouver, BC, Canada; A20-0017) and were performed as previously described (15). NOD SCID γ (NSG) mice (JAX 005557) were purchased from the Jackson Laboratory. The site of transplantation was the ACE. Female nondiabetic 15- to 18-week-old NSG mice were used for the transplant study. Seventy islet clusters isolated from wild-type mice were transplanted for the control group, and 70 INSEGFPGCGmScarlet-derived mature pancreatic endocrine clusters were transplanted for the experimental group. Blood glucose measurements and samples were collected via the saphenous vein. For fast-refeed studies, mice were fasted for 12 h and challenged with 2 g/kg glucose via intraperitoneal (i.p.) injection. Human C-peptide measurements were collected 30 min post-i.p. injection, and total glucagon measurements were collected post–12-h fast. In vivo imaging was performed on anesthetized mice using a Leica SP8 confocal microscope fifty days post-transplant; a focal point image was taken with Pinhole 1.0. using 16× objective.

Gene Expression Analyses

RNA extraction and RT quantitative PCR (RT-PCR) using sorted cells was performed as previously described (16), and gene expression was normalized to the housekeeping gene TBP. For the list of TAQMAN primers used, see Supplementary Table 3. NanoString gene expression analysis was performed as previously described (13). Fifty thousand sorted cells were resuspended in 100 μL Buffer RLT (Qiagen) containing 1% β-mercaptoethanol (Sigma-Aldrich). The prepared sample cartridge was read on a NanoString nCounter SPRINT profiler and analyzed using nSolver 4.0 for relative gene expression. Data were normalized to six housekeeping genes: B2M, GAPDH, GUSB, HPRT1, POLR2A, and TBP. For the list of target gene sequences, see Supplementary Table 4.

Immunohistochemical Analyses

Briefly, 100–200 spheroids were fixed in 4% PFA for 30 min at 22°C, and embedded in 2% agarose. The agarose containing the spheroids was then dehydrated and embedded into paraffin before generating 5-μm sections. Sections were rehydrated, heated in antigen retrieval buffer (0.433% v/v citraconic anhydride 98% in dH2O; Alfa Aesar), and blocked for 30 min in 5% v/v horse serum in PBS. The sections were stained with primary antibodies diluted in 5% v/v horse serum overnight at 4°C: mouse antiglucagon antibody (1:1,000; Sigma-Aldrich), guinea pig anti-insulin (1:500; Agilent), rat anti-RFP (1:1,000; ChromoTek), mouse anti-GLP-1 (1:250; Abcam), rabbit antiproglucagon (1:1,000; Abcam), and rabbit anti-GFP (1:500; MBL). Then, the sections were stained with secondary antibody for 1 h at 22°C in the dark: anti-mouse Cy3 (1:250; Jackson ImmunoResearch), anti-guinea pig Alexa Fluor 594 (1:250; Jackson ImmunoResearch), anti-rat Rhodamine Red X (1:250; Jackson ImmunoResearch), anti-rabbit FITC (1:450; Jackson ImmunoResearch), and TO-PRO-3 (1:10,000; Thermo Fisher Scientific). Images were taken using a 20× oil immersion and a 10× dry objective on a Leica TCS SP8 confocal microscope.

For ACE explant samples, the mice were euthanized via CO2 inhalation and cardiac puncture. The mice were then perfused with 5 mL PBS and, subsequently, 30 mL 4% PFA before the eyes were dissected and further fixed at 4°C for 2 h. The fixed eyes were then treated using sucrose solutions and embedded in frozen blocks with embedding compound (Epredia). The frozen blocks were sectioned into 16-μm sections. Sections were permeabilized using 0.1% Triton X-100 solution and stained as described above.

Hormone Secretion Assay

Fifty size-matched spheroids were used per treatment and preincubated in 500 μL Krebs-ringer bicarbonate HEPES supplemented with 2.8 mmol/L d-glucose or 5 mmol/L d-glucose (Sigma-Aldrich) for 1 h for glucose-stimulated insulin secretion and glucose-mediated glucagon secretion assays, respectively. After preincubation, the spheroids were transferred into wells containing 250 μL of the treatment solution and incubated for 1 h at 5% CO2, 37°C. For glucose-stimulated insulin secretion, the treatments solutions were 250 μL Krebs-ringer bicarbonate HEPES that contained 2.8 mmol/L d-glucose, 16 mmol/L d-glucose, or 16 mmol/L d-glucose with 30 mmol/L KCl. For glucagon secretion, the treatment solutions contained 5 mmol/L d-glucose, 1 mmol/L d-glucose, or 1 mmol/L d-glucose with 30 mmol/L arginine. After the incubation, the supernatants were centrifuged at 5,000g for 10 min and collected. The spheroids were then transferred into 500 μL acid ethanol (1 mol/L HCl in 70% ethanol) for measuring total hormone content. Hormone levels in the supernatant were detected using the human C-peptide ELISA (Alpco) or the human glucagon ELISA (Mercodia) kits.

Western Blot

hESC-derived cells or human islets were lysed in nonreducing sample buffer with protease inhibitors (Roche) and analyzed by Western blot as previously described (17,18). Fifty micrograms of cell lysate was separated by SDS-PAGE and blotted. The blot was then blocked using 5% milk powder in Tris-buffered saline with 0.1% Tween and probed using primary antibodies, rabbit anti-proglucagon (1:1,000; Abcam) and mouse anti-GAPDH (1:100,000; Sigma-Aldrich), overnight at 4°C. The membrane was then probed with horseradish peroxidase–conjugated secondary antibodies, goat anti-rabbit and anti-mouse (1:1,000; Jackson ImmunoResearch), and visualized using Luminata Crescendo Western horseradish peroxidase substrate (EMD Milipore).

Statistical Analyses

Statistical analyses were performed using Prism 9 (GraphPad Software). All data are presented as mean ± SEM. For stem cell differentiations, a biological replicate was considered to be one differentiation; experiments were repeated with at least three biological replicates. Data were analyzed using either Student t test, one-way ANOVA, or two-way ANOVA followed by a Dunnett post hoc test for multiple comparisons where appropriate. P < 0.05 was considered statistically significant.

Generation of the INSEGFPGCGmScarlet hESC Reporter Line Using CRISPR/Cas9

We generated a reporter hESC line using CRISPR/Cas9-mediated knock-in to identify and isolate insulin- and glucagon-expressing cells in hESC-derived pancreatic endocrine cell cultures. We first developed the single knock-in INSEGFP hESC line by inserting a 2A-EGFP expression cassette into exon 3 of the endogenous INS gene in the H1 hESC line (Fig. 1Aand Supplementary Fig. 1A) (8). We specifically replaced the stop codon of the INS gene to prevent disruptions to insulin processing in INS-expressing cells (19). The 2A sequence that promotes ribosomal skipping was used to generate a bicistronic reporter system that allows equimolar protein expression of insulin and EGFP (19,20). The double knock-in INSEGFPGCGmScarlet hESC line was generated using the INSEGFP hESC line in a similar manner. We introduced a 2A-mScarlet expression cassette downstream of the glucagon (GCG) gene using the INSEGFP Clone #26 hESC line, replacing the STOP codon of the endogenous GCG gene (Fig. 1Band Supplementary Fig. 1A).

Figure 1

Generation of INSEGFP and INSEGFPGCGmScarlet reporter hESC lines using CRISPR/Cas9. A and B: Schematic overview of the CRISPR/Cas9 targeting strategy to knock in (A) a 2A-EGFP expression cassette onto exon 3 of the INS gene and (B) a 2A-mScarlet expression cassette onto exon 6 of the GCG gene of the wild-type H1 hESC line. C: Differentiated spheroids derived from the INSEGFP and INSEGFPGCGmScarlet reporter cell lines expressing EGFP (green) and mScarlet (red) at different stages of pancreatic endocrine cell differentiation. Images were obtained on a Leica SP8 confocal microscope. Scale bars = 200 μm.

Figure 1

Generation of INSEGFP and INSEGFPGCGmScarlet reporter hESC lines using CRISPR/Cas9. A and B: Schematic overview of the CRISPR/Cas9 targeting strategy to knock in (A) a 2A-EGFP expression cassette onto exon 3 of the INS gene and (B) a 2A-mScarlet expression cassette onto exon 6 of the GCG gene of the wild-type H1 hESC line. C: Differentiated spheroids derived from the INSEGFP and INSEGFPGCGmScarlet reporter cell lines expressing EGFP (green) and mScarlet (red) at different stages of pancreatic endocrine cell differentiation. Images were obtained on a Leica SP8 confocal microscope. Scale bars = 200 μm.

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INSEGFPGCGmScarlet Reporter Line Reports INS and GCG in hESC-Derived Pancreatic Endocrine Cells Without Affecting Their Differentiation Capacity

To ensure the genomic modifications did not affect the differentiation of hESC-derived pancreatic endocrine cells, we followed the differentiation of pancreatic endocrine cells derived from the single and double knock-in hESC lines. We observed the morphology of the spheroids derived from both genetically modified cell lines remained comparable to those generated using previously published differentiation protocols (Supplementary Fig. 1BD). We also found EGFP and mScarlet reporters were expressed when INS and GCG were expected to be expressed, namely, starting at the endocrine progenitor cell stage (stage 5), and persisting throughout the maturation process of pancreatic endocrine cells (Fig. 1C). Of our total culture, we found 2.6% ± 0.8% expressed mScarlet only (mScarlet+), 23.9% ± 3.5% expressed both EGFP and mScarlet (EGFP+ mScarlet+), and 35.1% ± 3.8% expressed EGFP only (EGFP+) at the maturing endocrine cell stage (stage 6 day 25) using the INSEGFPGCGmScarlet Clone E7 (n = 3), which was comparable to the differentiation efficiencies of two other INSEGFPGCGmScarlet clones (Supplementary Fig. 2A).

To determine whether the fluorescent reporter proteins report INS and GCG expression, maturing endocrine cells derived from the knock-in hESC lines were immunostained for these hormones and their corresponding fluorescent proteins. We observed insulin and EGFP coexpression in the single and double knock-in lines, indicating EGFP reported insulin hormone expression (Fig. 2A). Flow cytometry analysis also showed 96.5% ± 1.1% and 97.7% ± 0.4% C-peptide-expressing cells also expressed EGFP for the INSEGFP and INSEGFPGCGmScarlet cell lines, respectively, at the maturing endocrine cell stage (stage 6 day 2) (Fig. 2C and Supplementary Fig. 2B). Similarly, we observed glucagon and mScarlet coexpression in the double knock-in line and the mScarlet reporter efficiency was 83.9% ± 4.8%, demonstrating mScarlet reported glucagon hormone expression (Fig. 2B and D and Supplementary Fig. 2C). Flow analysis further demonstrated the EGFP-expressing cell population that did not express C-peptide at the maturing pancreatic endocrine stage (stage 6 day 21) was significantly reduced by the mature pancreatic endocrine cell stage (stage 7 day 45) (Supplementary Fig. 2B and C). In addition, similar analyses also showed most of the mScarlet-expressing cells coexpressed glucagon by this stage of our differentiation (Supplementary Fig. 2D and E), suggesting the mScarlet-expressing cells that did not express glucagon were possibly pancreatic endocrine progenitors fated to become α-like cells.

Figure 2

EGFP and mScarlet expression report insulin and glucagon hormone expression. A: Immunostaining of EGFP (green), insulin (gray), and DAPI (blue) of maturing endocrine cells (stage 6 day 21) derived from the INSEGFP and INSEGFPGCGmScarlet reporter cell lines; n = 3; scale bars = 50 μm. B: Immunostaining of mScarlet (red), glucagon (gray), and DAPI (blue) of maturing endocrine cells (stage 6 day 21) derived from the INSEGFPGCGmScarlet reporter cell line; n = 3; scale bars = 50 μm. C and D: Quantitative flow cytometry analysis of the reporter efficiency of maturing endocrine cells (stage 6 day 21) derived from the H1 parental, INSEGFP, and INSEGFPGCGmScarlet reporter cell lines, measuring percentage of (C) EGFP+ cells of C-peptide+ cells and (D) mScarlet+ cells of glucagon+ cells; n = 3–5; error bars report ±SEM.

Figure 2

EGFP and mScarlet expression report insulin and glucagon hormone expression. A: Immunostaining of EGFP (green), insulin (gray), and DAPI (blue) of maturing endocrine cells (stage 6 day 21) derived from the INSEGFP and INSEGFPGCGmScarlet reporter cell lines; n = 3; scale bars = 50 μm. B: Immunostaining of mScarlet (red), glucagon (gray), and DAPI (blue) of maturing endocrine cells (stage 6 day 21) derived from the INSEGFPGCGmScarlet reporter cell line; n = 3; scale bars = 50 μm. C and D: Quantitative flow cytometry analysis of the reporter efficiency of maturing endocrine cells (stage 6 day 21) derived from the H1 parental, INSEGFP, and INSEGFPGCGmScarlet reporter cell lines, measuring percentage of (C) EGFP+ cells of C-peptide+ cells and (D) mScarlet+ cells of glucagon+ cells; n = 3–5; error bars report ±SEM.

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To confirm that cells that express mScarlet but not the corresponding hormone were immature, we probed for proglucagon expression in INSEGFPGCGmScarlet-derived maturing pancreatic endocrine cells and found almost all mScarlet-expressing cells coexpressed proglucagon (Supplementary Fig. 3A and B). We then demonstrated bihormonal cells expressed higher transcript levels of prohormone convertase PCSK2 and lower transcript levels of PCSK1 compared with the other fluorescent populations, suggesting proglucagon was processed mainly into glucagon (Fig. 3Fand Supplementary Fig. 3C). Since immature α-cells have been shown to also express incretin hormone glucagon-like peptide 1 (GLP-1) due to higher expression of PCSK1 in immature cells compared with their mature counterpart, we also checked for expression of GLP-1 in our differentiation (21). We showed some mScarlet-expressing cells coexpressed GLP-1 at this stage, further suggesting there were immature glucagon-expressing cells in our differentiation at stage 6 day 28 (Supplementary Fig. 3D). Together, EGFP and mScarlet report expression of insulin and glucagon in these knock-in cell lines. These data also suggest the insertion of the expression cassettes does not affect the differentiation, morphology, or hormone expression of the hESC-derived pancreatic endocrine cell clusters.

Figure 3

INSEGFPGCGmScarlet-derived maturing endocrine cells can be used to enrich populations with distinct transcriptional profiles using FACS. A: FACS plot of maturing pancreatic endocrine cells derived from the INSEGFPGCGmScarlet reporter cell line (stage 6 day 25). BI: Transcript expression of (B) INS, (C) GCG, (D) NKX6-1, (E) ARX, (F) PCSK1, (G) CHGA, (H) PAX4, and (I) SLC30A8 in FACS-sorted maturing pancreatic endocrine cells (stage 6 day 25); n = 6; ns, P ≤ 0.1234; *P ≤ 0.0332; **P ≤ 0.0021; ***P ≤ 0.0002, one-way ANOVA; error bars report ±SEM.

Figure 3

INSEGFPGCGmScarlet-derived maturing endocrine cells can be used to enrich populations with distinct transcriptional profiles using FACS. A: FACS plot of maturing pancreatic endocrine cells derived from the INSEGFPGCGmScarlet reporter cell line (stage 6 day 25). BI: Transcript expression of (B) INS, (C) GCG, (D) NKX6-1, (E) ARX, (F) PCSK1, (G) CHGA, (H) PAX4, and (I) SLC30A8 in FACS-sorted maturing pancreatic endocrine cells (stage 6 day 25); n = 6; ns, P ≤ 0.1234; *P ≤ 0.0332; **P ≤ 0.0021; ***P ≤ 0.0002, one-way ANOVA; error bars report ±SEM.

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INSEGFPGCGmScarlet Reporter Line Can Enrich for β- and α-Like Cell Populations

To determine whether the fluorescent reporter proteins can be used to effectively isolate β- and α-like cell populations, we sorted distinct fluorescent cell populations using FACS for gene expression analysis using NanoString (Fig. 3A). The EGFP-expressing population was split into two groups, cells with high (EGFPhi) and low (EGFPlo) fluorescence intensities, to determine whether reporter fluorescence intensity can be used to isolate transcriptionally distinct populations. To better compare between fluorescent populations, only mScarlet-expressing cells that also express high levels of EGFP were included in the double-positive population (EGFPhimScarlet+). We defined the gating of our populations more stringently so as to enrich for more pure cell populations. Our NanoString analysis showed the transcriptomic profile of EGFPhimScarlet+ and EGFPhi populations more closely resembled primary α- and β-cells within our cultures, as they expressed higher levels of the endocrine hormones INS and GCG, the pan-endocrine marker CHGA, and the hormone secretion machinery SLC30A8 compared with the two other populations. Furthermore, we found the EGFPhimScarlet+ cells adopted a more α-like expression profile with higher expression levels of key α-cell regulator ARX and lower levels of key β-cell regulator NKX6-1 compared with the EGFPhi population, despite expressing similar levels of INS (Fig. 3B–E). On the other hand, the EGFPhi population was more β-like, as it expressed key β-cell regulators such as NKX6-1, PCSK1, and PAX4, at significantly higher levels than the EGFPhimScarlet+ population (Fig. 3D, F, and H).

To determine whether EGFPhimScarlet+ cells were fated to become α-like cells, this cell population was sorted and reaggregated at the maturing endocrine cell stage (Fig. 4A). Confocal imaging and flow cytometry analyses showed there were significantly less EGFPhimScarlet+ cells over time in unsorted differentiation cultures (Figs. 4,B and 5,B). In addition, we found the sorted and reaggregated EGFPhimScarlet+ cell population became mostly mScarlet+, while the sorted and reaggregated EGFPhi population remained mostly EGFP+ (Fig. 4B). RT-PCR analysis further demonstrated the sorted and reaggregated EGFPhimScarlet+ cells expressed lower levels of INS and higher levels of GCG, ARX, and IRX2 compared with the EGFPhi cells, suggesting EGFPhimScarlet+ cells were fated to become α-like cells (Fig. 4C, F-H) with our pancreatic endocrine cell differentiation protocol. Longitudinal flow analysis further showed EGFP expression was progressively downregulated in the mScarlet-expressing cell population over the maturation period, showcasing the ability of our cell line to track the transition of bihormonal cells into α-like cells (Fig. 4I).

Figure 4

EGFPhimScarlet+ sorted and reaggregated cells are fated to become α-like cells. A: Schematic of experimental methodology. B: Unsorted, and sorted and reaggregated, EGFPhimScarlet+ and EGFPhi populations 5 days postsort; n = 3; scale bars = 50 μm. C–H: Transcript analysis of (C) INS, (D) NKX6-1, (E) PCSK1, (F) GCG, (G) ARX, and (H) IRX2 of FACS-sorted and reaggregated maturing endocrine cells derived from INSEGFPGCGmScarlet reporter cell line; n = 3–4; *P ≤ 0.0332; **P ≤ 0.0021; ***P ≤ 0.0002, unpaired t test; error bars report ±SEM. I: Histogram plots illustrating INS-EGFP expression in mScarlet-expressing cells at stage 7 days 30, 37, 45, and 50 of in vitro differentiation.

Figure 4

EGFPhimScarlet+ sorted and reaggregated cells are fated to become α-like cells. A: Schematic of experimental methodology. B: Unsorted, and sorted and reaggregated, EGFPhimScarlet+ and EGFPhi populations 5 days postsort; n = 3; scale bars = 50 μm. C–H: Transcript analysis of (C) INS, (D) NKX6-1, (E) PCSK1, (F) GCG, (G) ARX, and (H) IRX2 of FACS-sorted and reaggregated maturing endocrine cells derived from INSEGFPGCGmScarlet reporter cell line; n = 3–4; *P ≤ 0.0332; **P ≤ 0.0021; ***P ≤ 0.0002, unpaired t test; error bars report ±SEM. I: Histogram plots illustrating INS-EGFP expression in mScarlet-expressing cells at stage 7 days 30, 37, 45, and 50 of in vitro differentiation.

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Figure 5

Targeting the INS and GCG loci does not affect the function of hESC-derived pancreatic β- and α-like cells. A: Mature pancreatic endocrine cells (stage 7 days 44 and 45) derived from the INSEGFP and INSEGFPGCGmScarlet reporter cell lines. Images were obtained on a Leica SP8 confocal microscope. Scale bars = 200 μm. B: Differentiation efficiency of mature pancreatic endocrine cells (stage 7 days 45–55) derived from INSEGFP and INSEGFPGCGmScarlet reporter cell lines determined using flow cytometry analysis; n = 3–4; error bars report ±SEM. C: Glucose-stimulated insulin secretion assay using mature pancreatic endocrine cells (stage 7 days 50–60) derived from the H1 parental and INSEGFPGCGmScarlet reporter cell lines; n = 7–10; *P ≤ 0.0332; **P ≤ 0.0021, two-way ANOVA; error bars report ±SEM. D: Glucose-mediated glucagon secretion assay using mature pancreatic endocrine cells (stage 7 days 45–55) derived from the H1 parental and INSEGFPGCGmScarlet reporter cell lines; n = 3; *P ≤ 0.0332, two-way ANOVA; error bars report ±SEM.

Figure 5

Targeting the INS and GCG loci does not affect the function of hESC-derived pancreatic β- and α-like cells. A: Mature pancreatic endocrine cells (stage 7 days 44 and 45) derived from the INSEGFP and INSEGFPGCGmScarlet reporter cell lines. Images were obtained on a Leica SP8 confocal microscope. Scale bars = 200 μm. B: Differentiation efficiency of mature pancreatic endocrine cells (stage 7 days 45–55) derived from INSEGFP and INSEGFPGCGmScarlet reporter cell lines determined using flow cytometry analysis; n = 3–4; error bars report ±SEM. C: Glucose-stimulated insulin secretion assay using mature pancreatic endocrine cells (stage 7 days 50–60) derived from the H1 parental and INSEGFPGCGmScarlet reporter cell lines; n = 7–10; *P ≤ 0.0332; **P ≤ 0.0021, two-way ANOVA; error bars report ±SEM. D: Glucose-mediated glucagon secretion assay using mature pancreatic endocrine cells (stage 7 days 45–55) derived from the H1 parental and INSEGFPGCGmScarlet reporter cell lines; n = 3; *P ≤ 0.0332, two-way ANOVA; error bars report ±SEM.

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INSEGFPGCGmScarlet Reporter Line Can Differentiate Into Functionally Mature Pancreatic Endocrine Cells

To determine whether the genetic modifications affect the functional maturation of hESC-derived pancreatic endocrine cells, we differentiated the INSEGFP and INSEGFPGCGmScarlet cell lines into mature pancreatic endocrine cells (Fig. 5A). At this stage, we observed 9.6% ± 1.9% and 8.4% ± 1.2% of total events expressed EGFP only using INSEGFP and INSEGFPGCGmScarlet cell lines (n = 3), respectively, and 36.4% ± 2.7% expressed mScarlet only using the INSEGFPGCGmScarlet cell line (n = 3) (Fig. 5B). To test whether these cells were functionally mature, we performed insulin and glucagon secretion tests on mature pancreatic endocrine cells generated using the reporter hESC lines and compared the results to cells generated using the H1 parental cell line. We found C-peptide secretion was stimulated at high glucose concentrations in cells derived from both control H1 and the INSEGFPGCGmScarlet cell lines (Fig. 5C). In addition, these cell lines secreted comparable levels of glucagon (Fig. 5D). Next, we sorted and reaggregated INSEGFPGCGmScarlet-derived mature pancreatic endocrine cells but did not see any improvements in glucose-mediated hormone secretion (Supplementary Fig. 4). Notably, glucose-stimulated insulin secretion was lost following dissociation and reaggregation (compare Fig. 5C and Supplementary Fig. 4E). To investigate potential reasons behind this loss of function, we performed immunostaining on sorted and reaggregated EGFP-expressing cells after 7 days of culture. We found some cells lose EGFP and insulin expression over time (Supplementary Fig. 4BD). Some of these noninsulin-expressing cells expressed somatostatin or SLC18A1, which are markers of δ-cells and enterochromaffin cells, respectively. Similar cells have been observed in stem cell–derived islet differentiations (10,22–27). Somatostatin is known to inhibit insulin secretion, so the presence of, presumably immature, δ-like cells in these reaggregates may have reduced glucose-stimulated insulin secretion in vitro (28,29). Furthermore, Veres et al. (26) demonstrated stem cell–derived islets with fewer enterochromaffin-like cells have improved glucose-stimulated insulin secretion in vitro; thus, the presence of enterochromaffin-like cells may have impaired insulin secretion. Altogether, these data suggest the genetic modifications do not affect hormone secretion and maturation of hESC-derived pancreatic endocrine cells.

INSEGFPGCGmScarlet Reporter Line Allows for In Vivo Monitoring of Insulin- and Glucagon-Expressing Cells

To determine whether INSEGFPGCGmScarlet-derived pancreatic endocrine cells can regulate glucose homeostasis in vivo, we transplanted pancreatic endocrine cells (stage 7 day 53) derived from INSEGFPGCGmScarlet cells into ACE of nondiabetic NSG mice. Mice that received the INSEGFPGCGmScarlet islet and mouse islet transplants showed comparable blood glucose before and after 12-h fast, and 30 min following a 2g/kg glucose challenge (Fig. 6A). Human plasma C-peptide was detectable 30 min post–glucose injection in mice that received the INSEGFPGCGmScarlet transplants (Fig. 6B). It was particularly interesting to observe a twofold increase in plasma human C-peptide levels from 50 to 58 days post-transplant (n = 3), suggesting continuing maturation of hESC-derived insulin-expressing cells in vivo. Total glucagon levels post–12-h fast were comparable between mice that received INSEGFPGCGmScarlet and those that received primary mouse islet transplants at both 50 and 58 days post-transplant, likely due to the fact that glucagon ELISAs do not distinguish between glucagon from the two species (Fig. 6C).

Figure 6

Fluorescent proteins continue to report insulin and glucagon after transplant into the mouse eye. A: Blood glucose levels before fast, after 12-h fast, and 30 min after refeed of NSG mice 2 months post-transplant. Control mice were transplanted with mouse islets, and experimental mice were transplanted with INSEGFPGCGmScarlet-derived mature pancreatic endocrine cells (stage 7 day 53); n = 3. B: Human plasma C-peptide levels (solid line) and blood glucose levels (dashed line) of mice with transplantations post–glucose i.p. injection; n = 3; ***P ≤ 0.0002, two-way ANOVA. C: Total plasma glucagon levels (solid line) and blood glucose levels (dashed line) of mice with transplantations post–12-h fast; n = 3. D: In vivo imaging of mature pancreatic endocrine cell reaggregates (stage 7 day 53) derived from INSEGFPGCGmScarlet reporter cell line transplanted into the mouse eye. Images were obtained on a Leica SP8 confocal microscope. Scale bars = 100 μm. E and F: Immunostaining of (E) insulin (gray), EGFP (green), and nuclei (blue) and (F) glucagon (gray), mScarlet (red), and nuclei (blue) of mature pancreatic endocrine cell reaggregates (stage 7 day 53) derived from INSEGFPGCGmScarlet reporter cell line transplanted into the mouse eye. Images were obtained on a Leica SP8 confocal microscope. Scale bars = 100 μm.

Figure 6

Fluorescent proteins continue to report insulin and glucagon after transplant into the mouse eye. A: Blood glucose levels before fast, after 12-h fast, and 30 min after refeed of NSG mice 2 months post-transplant. Control mice were transplanted with mouse islets, and experimental mice were transplanted with INSEGFPGCGmScarlet-derived mature pancreatic endocrine cells (stage 7 day 53); n = 3. B: Human plasma C-peptide levels (solid line) and blood glucose levels (dashed line) of mice with transplantations post–glucose i.p. injection; n = 3; ***P ≤ 0.0002, two-way ANOVA. C: Total plasma glucagon levels (solid line) and blood glucose levels (dashed line) of mice with transplantations post–12-h fast; n = 3. D: In vivo imaging of mature pancreatic endocrine cell reaggregates (stage 7 day 53) derived from INSEGFPGCGmScarlet reporter cell line transplanted into the mouse eye. Images were obtained on a Leica SP8 confocal microscope. Scale bars = 100 μm. E and F: Immunostaining of (E) insulin (gray), EGFP (green), and nuclei (blue) and (F) glucagon (gray), mScarlet (red), and nuclei (blue) of mature pancreatic endocrine cell reaggregates (stage 7 day 53) derived from INSEGFPGCGmScarlet reporter cell line transplanted into the mouse eye. Images were obtained on a Leica SP8 confocal microscope. Scale bars = 100 μm.

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To determine whether the INSEGFPGCGmScarlet cell line can be used for in vivo monitoring of insulin- and glucagon-expressing cells, we anesthetized the mice that received transplants and performed intravital imaging at 1 and 3 months following transplantation. We were able to image both EGFP- and mScarlet-expressing cells in vivo at high resolution (Fig. 6D). We observed the arrangement of EGFP- and mScarlet-expressing cells in the graft do not change dramatically pre- and post-transplant (compare Figs. 5,A and 6,D). We then validated these cells do indeed express their corresponding hormone in vivo using immunofluorescence (Fig. 6E and F), showcasing the INSEGFPGCGmScarlet cell line continues to reliably report insulin and glucagon expression post-transplantation.

Current pancreatic endocrine cell differentiation protocols generate heterogeneous cultures. Here we describe the generation and characterization of the INSEGFPGCGmScarlet reporter hESC line for the visualization and enrichment of hESC-derived β- and α-like cells. We validated this double knock-in reporter line faithfully reports insulin and glucagon expression with EGFP and mScarlet expression, respectively. Our phenotypic and functional studies of the cell line also show that the genetic modifications do not disrupt endogenous insulin and glucagon expression or secretion. We also demonstrated that this reporter line can enrich α- and β-like cells as well as track their differentiation. Lastly, we showed this cell line can easily be used to monitor hormone-expressing cells in vivo. Thus, the INSEGFPGCGmScarlet hESC reporter line can be used to study β- and α-cells during pancreatic endocrine cell differentiations and further improve the production of functional hESC-derived pancreatic endocrine cells for diabetes therapy.

Two insulin and glucagon dual reporter hESC cell lines have previously been reported (30,31). However, Labonne et al. (30) only validated the coexpression of hormones and their corresponding fluorescent reporters using their dual reporter cell line, so it is unclear whether the genetic modifications used interrupted the differentiation, expression profile, and/or function of pancreatic endocrine cells. More recently, Zanfrini et al. (31) performed a more rigorous characterization of the dual reporter cell line they generated; however, their focus in these analyses was on β-cells and not α-cells. Furthermore, it is unclear whether the α-like cells derived from their cell line were phenotypically or functionally different from those generated using the parental hESC line. Here we performed phenotypic and functional validation on both β- and α-like cells derived from our dual reporter cell line (Figs. 3 and 5,C and D). We also transplanted these cells into mice and found they respond to glucose challenge and fasting (Fig. 6A–C). Lastly, we observed increased levels of plasma human C-peptide over time in vivo, which has previously been reported by other groups using control hESC-derived β-like cells and suggests β-like cells derived from our dual reporter hESC line can further mature in vivo (Fig. 6B) (22,32–34).

Insulin- and glucagon-expressing bihormonal cells have previously been described in the developing human pancreas (35,36). These cells were shown to become less frequently observed over time in the human fetal pancreas, and their numbers become negligible in adulthood (35,36). On the basis of the transcription factor profile of these bihormonal cells, it has been suggested that these cells are α-cell precursors (35). Similar observations have been made in hESC-derived polyhormonal cells. Transcriptomic analyses have shown hESC-derived bihormonal cells possess a more α-like expression profile (10,22,26,37,38). The α-like cell differentiation protocols generated by other groups also found a similar reduction in the number of bihormonal cells accompanied by an increase in the number of glucagon-expressing monohormonal cells (38,39), suggesting these bihormonal cells are fated to become α-like cells. However, it was previously unclear whether this transition from bihormonal cells to α-like cells occurred in a cell-autonomous manner or whether death of bihormonal cells accompanied the differentiation of monohormonal α-cells from progenitors. Furthermore, it was not clear whether this transition was cell-intrinsic or driven by the differentiation reagents that enrich for α-like cells. To answer these questions, we used the INSEGFPGCGmScarlet reporter line to purify bihormonal cells in vitro and demonstrate that these cells are fated to become α-like cells even in a differentiation protocol that produces all pancreatic endocrine cell lineages, suggesting this transition from bihormonal cells to α-like cells is cell-autonomous. The dual reporter line can be used to further examine the fate transitions of these bihormonal cells in vitro and in vivo and to determine whether they can be directed toward the β-cell lineage.

Interestingly, α-like and β-like cells generated with our differentiation protocol clustered separately with very little cell-cell contact between the two cell types in vitro and in vivo (Supplementary Fig. 4B and Figs. 5A and 6D). Adult human islets commonly adopt a mixed cytoarchitecture where α- and β-cells intermingle; however, the cytoarchitecture of human islets is more dynamic during embryonic development. Human β- and α-cells form mantle-core islets 12 weeks postconception (wpc), then reorganize to form bigeminal islets 17 wpc, and, finally, reorganize again into islets with mixed cytoarchitecture 30 wpc (40). However, the cell organization processes involved in the assembly of mature islets remain largely unknown. The dual reporter line could be used to determine factors that drive the formation of adult human islet architecture in vitro and post-transplant to better mimic its in vivo counterparts.

In summary, we generated an INSEGFPGCGmScarlet reporter hESC line that reports insulin and glucagon without affecting the differentiation or function of hESC-derived β- and α-like cells. We also showed that insulin- and glucagon-expressing cells can be purified and tracked in vitro and in vivo using the INSEGFPGCGmScarlet hESC reporter line. This dual reporter line could therefore be used to further characterize and improve in vitro pancreatic endocrine differentiations for diabetes therapy.

This article contains supplementary material online at https://doi.org/10.2337/figshare.27737223.

Acknowledgments. The authors thank the members of the Lynn, Levings, Verchere, Wasserman, Johnson, and Kieffer Laboratories (Vancouver, British Columbia, Canada); the Bruin Laboratory (Carleton University); the P.E. MacDonald laboratory (University of Alberta); and the BC Children’s Hospital Research Institute Flow Cytometry and Imaging core facilities for technical support, discussion, and critical reading of the manuscript. The authors acknowledge that University of British Columbia and BC Children’s Hospital are situated on the traditional, ancestral, and unceded territories of the Coast Salish peoples, the Sḵwx̱wú7mesh (Squamish), səl̓ilwətaɁɬ (Tsleil-Waututh), and xwməθkwəy̓əm (Musqueam) Nations.

Funding. F.C.L. was supported by the Juvenile Diabetes Research Foundation (5-SRA-2020-1059-S-B, 3-COE-2022-1103-M-B) and Canadian Institutes of Health Research (ASD-173663). Salary (F.C.L.) was supported by the Michael Smith Foundation for Health Research (#5238 BIOM) and the BC Children’s Hospital Research Institute. Scholarship funding was provided by the Canadian Institutes of Health Research (S.M. and E.F.), The BC Children’s Hospital Research Institute (S.M. and E.F.), the University of British Columbia CELL Graduate Program One-Year Fellowship (S.M.), and the Canadian Islet Research and Training Network NSERC CREATE program (E.F.).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. S.M. researched data and wrote, reviewed, and edited the manuscript. E.F., S.S., M.M., D.Z., A.Y., and C.N. researched data and reviewed and edited the manuscript. F.C.L. acquired funding, contributed to discussion, and reviewed and edited the manuscript. F.C.L. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. A non–peer-reviewed version of this manuscript was submitted to the bioRxiv preprint server (https://doi.org/10.1101/2023.04.19.537542).

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