
==== Front
Mol Metab
Mol Metab
Molecular Metabolism
2212-8778
Elsevier

S2212-8778(24)00148-0
10.1016/j.molmet.2024.102017
102017
Brief Communication
An INSULIN and IAPP dual reporter enables tracking of functional maturation of stem cell-derived insulin producing cells
Bayly Carmen L. 12
Dai Xiao-Qing 45
Nian Cuilan 12
Orban Paul C. 12
Verchere C. Bruce 1237
MacDonald Patrick E. 45
Lynn Francis C. francis.lynn@ubc.ca
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126⁎
1 BC Children's Hospital Research Institute, Vancouver, BC, Canada
2 Department of Surgery, University of British Columbia, Vancouver, BC, Canada
3 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada
4 Department of Pharmacology, University of Alberta, Edmonton, AB, Canada
5 Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada
6 School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada
7 Centre of Molecular Medicine and Therapeutics, Vancouver BC, Canada
⁎ Corresponding author. francis.lynn@ubc.caTwitter iconTwitter icon
23 8 2024
11 2024
23 8 2024
89 10201717 4 2024
24 7 2024
20 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Objective

Human embryonic stem cell (hESC; SC)-derived pancreatic β cells can be used to study diabetes pathologies and develop cell replacement therapies. Although current differentiation protocols yield SCβ cells with varying degrees of maturation, these cells still differ from deceased donor human β cells in several respects. We sought to develop a reporter cell line that could be used to dynamically track SCβ cell functional maturation.

Methods

To monitor SCβ cell maturation in vitro, we created an IAPP-2A-mScar and INSULIN-2A-EGFP dual fluorescent reporter (INS2A-EGFP/+;IAPP2A-mScarlet/+) hESC line using CRISPR/Cas9. Pluripotent SC were then differentiated using a 7-stage protocol to islet-like cells. Immunohistochemistry, flow cytometry, qPCR, GSIS and electrophysiology were used to characterise resulting cell populations.

Results

We observed robust expression of EGFP and mScarlet fluorescent proteins in insulin- and IAPP-expressing cells without any compromise to their differentiation. We show that the proportion of insulin-producing cells expressing IAPP increases over a 4-week maturation period, and that a subset of insulin-expressing cells remain IAPP-free. Compared to this IAPP-free population, we show these insulin- and IAPP-expressing cells are less polyhormonal, more glucose-sensitive, and exhibit decreased action potential firing in low (2.8 mM) glucose.

Conclusions

The INS2A-EGFP/+;IAPP2A-mScarlet/+ hESC line provides a useful tool for tracking populations of maturing hESC-derived β cells in vitro. This tool has already been shared with 3 groups and is freely available to all.

Highlights

• Islet amyloid polypeptide (IAPP) is expressed during differentiation of stem cells to β cells.

• A double reporter insulin, IAPP stem cell line can be used to monitor IAPP expression.

• IAPP-expressing cells have an improved stimulation index and decreased basal electrical activity.

• IAPP expression marks a subset of stem cell-derived β cellsexhibiting traits of intermediate functional maturation.

Keywords

Islets
INS
Islet amyloid polypeptide
Beta cell maturation
Human embryonic stem cells
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pmc1 Introduction

Stem cell-derived islets and β cells (SC-islets and SCβ cells) serve both as model systems for studying diabetes and also as potential alternatives to deceased donor islets for type 1 diabetes (T1D) cell therapies [1,2]. Recent differentiation protocols have generated SC-islets with many similarities to their mature human counterparts, but they still carry structural, transcriptional, metabolic and functional differences which could diminish their utility [[3], [4], [5], [6]]. Some of these differences correspond to immature β cell traits, and are reduced or eliminated following transplantation [7,8]. This suggests the presence of important signals for β cell maturation in vivo that remain absent from in vitro culture conditions [3,9]. An improved understanding and recapitulation of β-cell maturation in vitro would enhance the utility of SC-islets and SCβ cells both for modelling disease and as diabetes cell therapies.

β cell maturation is a complex process marked by the acquisition of various traits, including those supporting glucose-stimulated insulin secretion (GSIS) [1,10]. Maturation occurs on a spectrum, with functions being acquired in part or in full; the extent to which different functions are displayed by different mature β cell subtypes remains an area of ongoing study [11,12]. One hallmark of β cell maturation is a reduction in β cell activity at fasting/low glucose levels, this occurs shortly after birth in mice and likely before birth in humans [13]. Fluorescent reporter cell lines of key β cell genes have played invaluable roles in elucidating successful differentiation conditions [[14], [15], [16], [17]]. While several reporter lines exist for studying earlier stages in differentiation, few have been generated for tracking SCβ cell maturation, potentially due to the complex and heterogeneous nature of the maturation process [1,18].

Here we explore the use of an islet amyloid polypeptide (IAPP) reporter for tracking maturation in SCβ cells. We chose IAPP as an intermediate maturation marker because of its specificity to β cells, its range and strength of expression during β and SCβ cell maturation [4,9,19,20] and its correlation with mature β cell function in human islets [21]. We created an IAPP and Insulin dual reporter (IIDR) hESC line using CRISPR/Cas9, by adding a 2A-mScarlet expression cassette downstream of the IAPP locus on our pre-existing INS-EGFP reporter line [15,17]. We show that EGFP and mScarlet expression report insulin and IAPP expression respectively, and that insertion of this expression cassette does not interfere with differentiation. Differentiation and maturation of this line gives rise to two fluorescing populations, one expressing EGFP only (EGFP+mScar−) and the other expressing both EGFP and mScarlet reporters (EGFP+mScar+). To demonstrate this cell line can be used to enrich for maturing SCβ cells, we sorted cells based on fluorescent reporter expression and analyzed each population. We found that the EGFP+mScar+ population expressed lower levels of non-β cell hormones and had lower basal insulin secretion, resulting in a more sensitive glucose response. Electrophysiological characterization of these two cell types shows that cells expressing both reporters possessed reduced basal exocytosis and minimal action potential firing at 2.8 mM glucose, suggesting IAPP expression is indicative of an elevated glucose threshold for glucose-sensitive insulin release and thereby a marker of the maturation process of SCβ cells.

2 Methods

2.1 Cell culture

Human embryonic stem cells (hESC) were used under approval from the BC Children's Hospital Research Ethics Board (H09-00676) and the CIHR Stem Cell Oversight Committee. Stem cells were maintained as previously described [17,22]. Undifferentiated H1-derived cell lines (WA01; XY) hESCs were maintained in mTeSR Plus (STEMCELL Technologies Inc.) at 5% CO2, 37 °C. Cells were split every 5 days using ReLeSR (STEMCELL Technologies Inc.) and plated 1 × 106 and 2 × 106 cells on Geltrex-coated (1:100, Thermo Fisher Scientific) 60 mm and 10 cm plates respectively, with 10 μM Y-27632 (STEMCELL Technologies Inc.).

2.2 CRISPR/Cas9 knock-in

The CRISPR/Cas9 system used to generate the INS2A-EGFP/+;IAPP2A-mScarlet/+ hESC line was as previously described [15,17,22,23]. The pCCC CRISPR/Cas9 plasmid [23] was modified to express gRNA (5′-GAGAGTTACATTGTCCTCTAA) for targeting the 3′ end of the endogenous islet amyloid polypeptide (IAPP) coding region. This plasmid, along with a plasmid carrying a 2A-mScarlet-loxP-PGK-Puro-loxP cassette targeting the IAPP locus with 800-bp homology arms (Fig. S2A), was transfected into the pre-existing reporter line INS2A-EGFP/+ [17]. Homologous recombination was verified using PCR across the homology arms of the selected CRISPR clones and confirmed by Sanger sequencing (Fig. S2B). For a list of genotyping primers used, see Table S1. The loxP-flanked PGK-puro sequence was removed by transfection of a Cre-expressing plasmid. Clones with the insertions were tested for common chromosomal abnormalities using the hPSC Genetic Analysis Kit (STEMCELL Technologies Inc.).

2.3 hESC differentiation

The protocol used to differentiate the hESC lines is based on previously published protocols [9,14,17,22,24,25]. Briefly, hESCs were dissociated using Accutase (STEMCELL Technologies Inc.) for 5 min at 37 °C, then diluted tenfold using DPBS with calcium and magnesium (Gibco). hESCs were centrifuged at 200×g for 5 min, their supernatant was removed, and then they were resuspended at 1 × 106 cells/mL in mTeSR Plus medium containing 10 μM Y-27632. 5.5 million cells were then seeded into each well of non-tissue culture treated 6-well plates (Greiner Bio-One). The plates were placed on a shaker (25 mm orbit; Celtron, Infors HT) set at 94 RPM and left at 37 °C, 5% CO2 overnight. The next day, media was aspirated off the stem cell clusters, which were then washed twice using 5.5 mL DPBS with calcium and magnesium. After removal of the wash buffer, 5.5 mL of differentiation media was added, and clusters were incubated again overnight, at 37 °C, 5% CO2 and shaking at 94 RPM. Clusters were then incubated continually in these conditions, with media being changed daily or every two days, as described in Table S2. For detailed formulations of differentiation media at each stage, see Table S2.

2.4 Flow cytometry and cell sorting

For live cell flow cytometry analysis, ∼100 SC-islets were transferred to 1.5 mL microcentrifuge tubes containing 1 mL PBS. After allowing the SC-islets to settle, the PBS was removed and SC-islets were dissociated using 150 μL Accutase per tube (STEMCELL Technologies Inc.) for 8 min at 37 °C, with firm tapping every 2 min. Following addition of 1.25 mL FACS buffer (2% FBS in PBS with 10 μM Y-27632), dissociated cells were centrifuged for 5 min at 200×g and resuspended in 100 μL Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific, 1:1000 in FACS buffer) for 30 min at 4 °C. Cells were subsequently centrifuged for 5 min at 200×g and resuspended in 350 μL FACS buffer. These cell suspensions were then filtered into a 5 mL tube with a 35 μm strainer cap (Falcon) and kept on ice until analysis. Analysis was carried out on a BD Biosciences FORTESSA X-20 using unstained and single stain controls, and the data were processed with FlowJo using the gating strategy shown in Fig. S3.

For fixed cell flow cytometry analysis (Fig. S2), following staining with the fixable viability dye, cells were washed with PBS, resuspended in 4% paraformaldehyde (Fisher Scientific) and incubated on a rotator for 30 min at 22 °C. Cells were then centrifuged for 5 min at 200×g and resuspended in 500 μL PBS for long term storage. On the day of analysis, cells were filtered and analyzed on a BD LSR II using unstained and single stain controls, and the data were processed using FlowJo.

To sort EGFP-positive, mScarlet-positive (EGFP+mScar+) and EGFP-positive, mScarlet negative (EGFP+mScar−) populations, SC-islets were washed with PBS and dissociated using Accumax (Thermo Fisher) for 8 min at 37 °C [14], tapping firmly each 2 min. Dissociated cells were resuspended in FACS buffer and kept on ice until sorting, and were sorted into cold stage 7 (S7) media with 10 μM Y-27632 using a BD FACS Aria (BC Children's Hospital Research Institute Flow Cytometry Core Facility). Following sorting, cells were centrifuged at 200×g for 5 min, then resuspended in prewarmed S7 media with 10 μM Y-27632. Cells were reaggregated by seeding in AggreWell 800 plates (StemCell Technologies), at 200,000–250,000 cells per well, followed by incubation at 37 °C and 5% CO2 for 48 h. After this they were transferred into 6-well plate wells in 4.5 mL of S7 media, and maintained at 37 °C, 5% CO2 and shaking at 94 RPM until used for assays.

2.5 Gene expression analyses

RNA extraction and RT-qPCR using sorted cells was performed as previously described on an ABI ViiA7 qPCR system [17,26,27]. Gene expression was normalized to the housekeeping gene TBP. For the list of primers and FAM/ZEN/IBHQ probes used, see Table S3. For adult human islet samples, sample preparation and NanoString gene expression analysis is described in detail at humanIslets.com [21]. For comparison with SC-islet cells, only non-diabetic adult human islets assayed with the same codeset as SC-islets were used. For SC-islets, NanoString gene expression analysis was performed as previously described [22]. Briefly, 50,000 SC-islet cells were resuspended in 100 μL RLT Buffer (Qiagen) with 1% β-mercaptoethanol (Sigma–Aldrich). These samples were read on a NanoString nCounter SPRINT profiler and analyzed for gene expression using nSolver 4.0. NanoString data were normalized to six housekeeping genes: B2M, GAPDH, GUSB, HPRT1, POLR2A, and TBP. Resulting counts were log2-transformed prior to plotting and statistics. Target and housekeeping gene sequences are listed in Table S4.

2.6 Immunohistochemical analyses

Immunohistochemical analyses were performed as previously described [17]. 100–200 SC-islets were fixed in 4% PFA for 30 min at 22 °C before being embedded in 2% agarose. The agarose containing the SC-islets was then dehydrated and embedded into paraffin blocks. The paraffin blocks were sliced into 5 μm sections, which were then rehydrated, heated in antigen retrieval buffer (0.0433% v/v citraconic anhydride, 98%, from Alfa Aesar, in dH2O), and blocked for 30 min in 5% v/v horse serum in PBS. For IAPP and mScarlet, the sections were stained with the following primary antibodies, diluted in 5% v/v horse serum overnight at 4 °C: rabbit anti-IAPP antibody (1:200, BMA biomedicals), and anti-RFP (1:1000, ChromoTek). These were then washed and stained with the following secondary antibodies for 1 h at 22 °C in the dark: anti-rat rhodamine red (1:250, Jackson ImmunoResearch) and anti-rabbit FITC (1:450, Jackson ImmunoResearch) along with DAPI nuclear stain (1 ug/mL) to visualize the nuclei. For C-peptide and EGFP, sections were stained with the following conjugated antibodies, diluted in 5% v/v horse serum for 1 h at 22 °C in the dark: Alexa Fluor 647 Mouse Anti-C-Peptide (1:350, BD Biosciences) and Alexa Fluor 488 GFP Polyclonal Antibody (1:350, Invitrogen), along with DAPI nuclear stain (1 ug/mL) to visualize the nuclei. All sections were mounted with Slowfade Diamond Antifade Mounting Medium (Thermo Fisher Scientific) prior to imaging. Images were taken using a 20× oil immersion objective on a Leica TCS SP8 confocal microscope.

2.7 Glucose-stimulated insulin secretion assay

Fifty size-matched aggregates were used per treatment and pre-incubated in 500 μl Krebs-ringer buffered with HEPES (KRBH buffer, 114 mM NaCl, 20 mM HEPES, 4.7 mM KCl, 1.2 mM KH2PO4, 2.5 mM CaCl2, 1.17 mM MgSO4, 0.2% BSA, pH 7.4) supplemented with 2.8 mM d-glucose (Sigma–Aldrich) for at 37 °C for 1 h. After preincubation, the buffer was removed and 250 μl fresh KRBH with 2.8 mM d-glucose was added. Aggregates were incubated for 1 h at 37 °C in 5% CO2, then this buffer was removed from the aggregates and transferred to a fresh tube, for further processing. 250 μL of 16.7 mM d-glucose in KRBH was then added to the aggregates, which were then incubated at 37 °C for 1 h. The 2.8 mM d-glucose in KRBH that was removed from the aggregates was centrifuged at 5000×g for 10 min, then the supernatant was transferred again to a new tube and stored at −20 °C. Once the aggregates completed their 1-h incubation at 16 mM glucose-KRBH, this buffer was transferred to a fresh tube. 250 μL of KRBH with 16.7 mM glucose and 30 mM KCl was added to the aggregates, which were then left to incubate for 1 h at 37 °C. The 16.7 mM d-glucose in KRBH that was removed from the aggregates was centrifuged at 5000×g for 10 min, then the supernatant was transferred again to a new tube and stored at −20 °C. Once the aggregates finished their hour-long incubation, the 16.7 mM glucose and 30 mM KCl in KRBH was transferred from the aggregates to fresh tube. Then 500 μL of acid ethanol (1% HCl in 70% ethanol) was added to the tubes carrying the aggregates, which were each vortexed for 5 s and then incubated overnight at −20 °C. The 16.7 mM d-glucose with 30 mM KCl in KRBH that was removed from the aggregates was centrifuged at 5000×g for 10 min, then the supernatant was transferred again to a new tube and stored at −20 °C. The next day, the aggregates were vortexed again for 5 s, allowed to settle, then the acid-ethanol was transferred to a fresh tube. This was centrifuged at 21,000×g for 10 min, then the supernatant was transferred into a fresh tube, and the stored at −20 °C. Samples were then assayed using a human C-peptide ELISA (Alpco Stellux).

2.8 Patch-clamp electrophysiology

Electrophysiological characterization was carried out as previously described [28]. SC-islets, shipped from Vancouver to Edmonton, were dissociated into single cells using Accutase (Thermo Fisher) on the same day they were received. Dispersed cells were cultured in 5.5 mM DMEM with 10 μM Y-27632 for up to 2 days.

Membrane potential recordings were collected as described [28], using a HEKA EPC10 amplifier and PatchMaster Software (v2 × 91, HEKA Instruments Inc, Lambrecht/Pfalz, Germany) in the current-clamp mode in the perforated patch-clamp configuration. All the measures were done in a heated chamber (32–35 °C), performed with patch pipettes pulled from thick-walled borosilicate glass tubes (Sutter Instrument), that had resistances of 8–10 MΩ when filled with 76 mM K2SO4, 10 mM KCl, 10 mM NaCl, 1 mM MgCl2 and 5 mM Hepes (pH 7.25 with KOH), and back-filled with 0.24 mg/mL amphotericin B (Sigma Aldrich, cat# a9528). The extracellular solution consisted of 140 mM NaCl, 3.6 mM KCl, 1.5 mM CaCl2, 0.5 mM MgSO4, 10 mM Hepes, 0.5 mM NaH2PO4, 5 mM NaHCO3, and either 5, 2.8 or 16.7 mM glucose as indicated (pH 7.3 with NaOH).

Measurement of exocytosis was performed as described [28], in the whole-cell voltage-clamp configuration using the same hardware and software as above. Fire polished thin-walled borosilicate pipettes coated with Sylgard (3–5 MΩ), with an intracellular solution of (in mM): 125 Cs-glutamate, 10 CsCl, 10 NaCl, 1 MgCl2, 0.05 EGTA, 5 HEPES, 0.1 cAMP, and 3 MgATP (pH 7.15 with CsOH). The bath solution contained (in mM): 118 NaCl, 20 Tetraethylammonium-Cl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 5 HEPES, and either 2.8 mM or 16.7 mM glucose (pH 7.4 with NaOH). Quality control was assessed by the stability of seal (>10 GΩ) and access resistance (<15 MΩ) over the course of the experiment. Exocytosis was elicited by a series of ten 500 ms depolarizing pulses from −70 to 0 mV and recorded as increases in cell capacitance (surface area) normalized to initial cell size (fF/pF). Voltage-dependent Ca2+ and Na + currents were measured during a 500-ms depolarization to −10 mV, and also normalized to initial cell size (pA/pF). Data were analysed using FitMaster (HEKA Instruments Inc) and Prism (GraphPad Software Inc., version 9, San Diego, CA).

2.9 Online data sourcing

Data in Fig. S1A were downloaded from isletgenomics.org [29] on July 9th 2024, using the Bubbleplot/Heatmap function of the Shinycell application for plotting scRNAseq data of purified islets of 65 donors. The ‘Group by’ option for the x-axis used was ‘Cell Type Grouped’, and the following list of genes added for the Y axis: IAPP, UCN3, MAFA, SIX3, CHGB, HOPX, G6PC2, MNX1, ENTPD3, SIX2, CPE, FXYD2, and RBP4. To plot data only from non-diabetic subjects, in the ‘Diabetes Status’ option of the ‘Toggle to subset cells’ section was selected, with only the ND box checked. For Fig. S1B, GSIS and processed Nanostring expression data for human donors aged 18–80 was downloaded from humanislets.com [21] on July 4th 2024. For Figs. S1C–D, stimulation and secretion coefficients with associated adjusted p-values were obtained using the ‘Feature view’ application of humanislets.com [21], which returns gene lists with coefficients and adjusted p-values when queried with functional datatypes. For Fig. S1E, unprocessed Nanostring data for non-diabetic adult human islets was downloaded from humanislets.com [21]. Fig. S1F, shows IAPP protein abundance and %CV data reported by Kolic et al. [6]. For Fig. S6, total exocytosis data for non-diabetic human adults was also downloaded from humanislets.com [21] on July 12th 2024.

2.10 Statistical analyses

Statistical analyses were performed using Prism 9 (GraphPad Software). Data are presented as mean ± SEM, except for Fig. S1F, which shows %CV. Comparisons between two groups were analyzed using unpaired, two-tailed t-tests, with Welch's t test applied where variances were significantly different, and Mann–Whitney tests applied for groups with skewed distributions. Comparisons between more than two groups were evaluated using one-way ANOVA followed by a Tukey post-hoc test for multiple comparisons. Comparison of a null hypothesis value to a single group (Figure 4F) was done using a one-sample t-test. Comparisons between two groups over multiple timepoints were evaluated using two-way ANOVA followed by a Tukey post-hoc test for multiple comparisons. Electrophysiological data for SCβ cells were evaluated using multiple Mann–Whitney tests (one comparison between groups for each glucose condition), using the Holm-Šídák post hoc test for multiple comparisons. A one-way ANOVA with a Kruskall-Wallis post-hoc test for multiple comparisons was used to compare SCβ cell exocytosis data to that of adult human β cells. Adjusted p-values for stimulation index and secretion coefficients were calculated as described at humanislets.com [21]. Calculation of Benjamini-Hochberg-adjusted p-values in Fig. S1F are described by Kolic et al. [6].

3 Results

3.1 Choice of IAPP as an intermediate maturation marker

We chose the β cell hormone IAPP for its high expression level relative to other β cell-specific maturation markers (Fig. S1A) [29], its broad range of expression over the course of β cell and SCβ cell maturation [4,9,19,20,30], and its correlation with β cell function in human islets (Figs. S1B,C,D) [21]. IAPP is secreted by β cells at an approximate ratio of 1:100 with insulin, released from the same granules as insulin, and modified post-translationally by the same enzymes [31]. Knockout studies suggest that IAPP does not drive β cell maturation [32,33], rather it contributes to glucose homeostasis by regulating satiety and inhibiting gastric emptying [34,35]. In human fetal β cells, low levels of IAPP protein have been observed as early as 13–14 weeks of development (WD) [36,37], and by 17 WD, IAPP is found at low levels in 1–3% of β cells [19]. In non-diabetic human adult islets, IAPP has been detected at variable levels in 40–60% of β cells, and at much higher levels than their fetal counterparts [35,[38], [39], [40]]. However, as IAPP translation is affected by blood glucose levels [41], not typically controlled for in these reports, these percentages may underestimate the true proportion of IAPP-producing β cells.

In SCβ cells, the timing and extent of IAPP upregulation varies between differentiation protocols [30]. Transcripts are present at low levels after the start of stage 6, and increase during in vitro and in vivo maturation, eventually reaching levels comparable to those of adult human β cells [9,20] (Fig. S1E). Similarly, the IAPP protein content and secretion in human and transplanted SCβ cells exceeds that detected in stage 6 SCβ cells grown in vitro [4,42] (Fig. S1F). Based on this previous work, we hypothesize that a fluorescent reporter of IAPP would be detectable at an intermediate stage during in vitro maturation, identifying SCβ cells with enhanced maturation traits and enabling improvements to SCβ cell maturation protocols.

3.2 Generation of the INS2A-EGFP/+;IAPP2A-mScarlet/+ hESC reporter line using CRISPR/Cas9

To create the INS2A-EGFP/+;IAPP2A-mScarlet/+ (Insulin IAPP Dual Reporter; IIDR) hESC line, we used CRISPR/Cas9 to add a 2A-mScarlet cassette into exon 3 of the IAPP gene of an established INS2A-EGFP/+ reporter line [15] derived from the H1 hESCs (Figure 1A, S2A) [43]. For both insulin and IAPP, the stop codon was removed and a 2A site added immediately downstream, preceding the fluorescent protein sequences. The 2A site induces ribosomal skipping, resulting in production of approximately equimolar amounts of reporter and target genes [44,45]. A nuclear localization signal (NLS) downstream of the mScarlet reporter was also added to the cassette, to facilitate reporter detection through its concentration in the nucleus.Figure 1 Engineered EGFP and mScarlet (mScar) reporters reflect insulin and IAPP expression in stem cell-derived islets. (A) Schematic of fluorescent coding sequences inserted downstream of insulin and IAPP to create the INS2A−EGFP/+;IAPP2A-mScarlet/+ (Insulin IAPP Dual Reporter; IIDR) hESC line. Fluorescent proteins are preceded by 2A self-cleaving peptides (2A), and a nuclear localization signal (NLS) downstream of mScarlet targets it to the nucleus. (B) Schematic of differentiation procedure used to generate INS and IAPP-producing β-like cells, along with other endocrine-like cells as part of a maturing SC-islet. Green and red show intended timeframe for appearance of both reporters. (C) Confocal images of immunostained sections of SC-islets showing overlap of fluorescent protein and reported gene, at stage 7 (scale bars = 50 μm). (D) Quantification of stained cells for C-peptide and EGFP. (E) Quantification of stained cells for IAPP and mScarlet. Data in C and D show quantification of 3 images per differentiation, for 3 differentiations. ✱✱✱✱ = p ≤ 0.0001 by one-way ANOVA, followed by a Tukey post-hoc test for multiple comparisons. Error bars indicate ± SEM.

Figure 1

3.3 The IIDR line reports insulin and IAPP in hESC-derived pancreatic endocrine cells without affecting their differentiation capacity

To ensure that insertion of the mScarlet reporter cassette does not affect the differentiation of hESC-derived pancreatic endocrine cells, we compared the differentiations of pancreatic endocrine cells derived from the double knock-in hESC line clones to its single knock-in parent up to stage 7 (S7) week 1 (Fig. S2E). Following a suspension-based differentiation protocol (Figure 1B, Table S2), we observed that the morphology and differentiation efficiency (the proportion of cells expressing the insulin reporter EGFP) of the SC-islets derived from the IIDR clones remained comparable to those generated using INS2A−EGFP/+ hESCs (Figs. S2C–E). The proportion of EGFP-positive (EGFP+) cells generated by clone C4 differentiations, which was comparable to the other two clones, was further monitored alongside its INS2A-EGFP/+ parent line over a 4-week maturation period (Fig. S2F). Starting at stage 6.2, the differentiation efficiencies of the single and double knock-ins were 47.4 ± 4.2% and 47.2 ± 8.6% respectively, this decreased to 21.6 ± 4.9 % and 19.5 ± 7.8 % by stage 7 week 1. The proportion of EGFP+ cells further dropped to 15.2 ± 1.2% and 13.8 ± 5.3%, showing that the cell lines remain comparable through a 4-week maturation period. Taken together, these data show the insertion of the mScarlet expression cassette does not affect the morphology or differentiation efficiency of the hESC-derived pancreatic endocrine cell clusters during differentiation or maturation.

To verify that EGFP and mScarlet report insulin/C-peptide and IAPP expression respectively, maturing endocrine cells differentiated from the IIDR hESC line were immunostained for C-peptide, IAPP and their corresponding fluorescent reporters (Figure 1C). Using immunofluorescence microscopy, we found that 96.2 ± 0.7% of all C-peptide-producing cells are also EGFP-positive (Figure 1D) while 94.0 ± 5.2% of IAPP-producing cells also produce mScarlet (Figure 1E). Together, these data suggest that EGFP and mScarlet report expression of insulin/C-peptide and IAPP respectively in the IIDR cell line.

3.4 IAPPmScarlet expression increases in SCβ cells through a 4-week maturation period

To determine the extent to which mScarlet expression becomes visible in EGFP+ cells over the course of a 4-week in vitro maturation period, we took weekly measurements of SC-islets by flow analysis, and by live-cell confocal fluorescence microscopy (Figure 2). These show mScarlet fluorescence is initially present in a very low proportion of EGFP+ cells at stage 6.2, but increases over time. This is consistent with the significantly lower levels of IAPP transcripts we detect in SCβ cells generated with our protocol compared to those of non-diabetic adult human islets (Fig. S1E). Flow analysis shows that the percentage of cells expressing only EGFP (EGFP+mScar− cells) drops from 55.1 ± 3.7 % to 6.3 ± 0.1 %, while the population expressing both reporters (EGFP+mScar+ cells) increases from 1.0 ± 0.5 % to 16.1 ± 2.1 % (Figure 2B, S3). Therefore, while the overall EGFP+ population decreases to 22.5 ± 2.3 % by the end of the 4-week maturation period, the proportion of these cells with mScarlet fluorescence reaches 71.7 ± 6.1 % (Figure 2C). Furthermore, the EGFP+mScar+ population shows significant increases in the mean fluorescence intensity (MFI) of both reporters, while in the EGFP+mScar− population, the MFI does not change significantly for either (Fig. S4). These data suggest that IAPP expression in insulin-expressing cells increases significantly over the 4-week maturation period.Figure 2 The proportion of EGFP + cells expressing mScarlet increases throughout a 4-week maturation period. (A) Representative flow cytometry plots showing changes in fluorescent cell populations throughout a 4-week maturation period, from stage 6.2 (S6.2) up to stage 7 (S7) week 4. (B) Quantification of fluorescing populations within each quadrant over the 4-week maturation period. (D) Proportion of EGFP + cells expressing mScar over time. (D) Live-cell confocal images of SC-islets differentiated from the IIDR cell line, obtained with a Leica SP8 confocal microscope. Scale bars indicate 60 μm. For C and D, n = 3–7, ns = p > 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001. ∗∗∗∗ = p ≤ 0.0001 by one-way ANOVA followed by a Tukey post-hoc test for multiple comparisons. Error bars indicate ± SEM.

Figure 2

3.5 The IIDR line enriches for less polyhormonal SCβ cells

To determine whether fluorescence of both reporters selects for SCβ cells with a more mature gene expression profile than those with EGFP fluorescence alone, we used FACS to sort for both EGFP+mScar+ and EGFP+mScar− cell populations (Figure 3A). Our gating was informed by the flow analysis data that tracked fluorescing populations during maturation (Figure 2A). These analyses show that with time, the population of mScar-expressing cells bifurcates into clearly positive and negative populations, and that EGFP+mScar+ cells possess high EGFP fluorescence intensity (Fig. S4). In contrast, EGFP+mScar− cells possess a range of EGFP intensities throughout the maturation period. Therefore, the gating of the EGFP+mScar− population for comparison to the EGFP+mScar+ population was set to include cells of comparable EGFP fluorescence intensity. We also defined the gating of our populations stringently to maintain cell population purity.Figure 3 EGFP+mScar+ cells are less polyhormonal than EGFP+ cells with no mScarlet fluorescence. (A) Gates for sorting SC-islet cells into EGFP+mScar+ and EGFP+mScar− populations. (B) RT-qPCR analysis of these two populations for endocrine hormones. Populations were assessed midway through stage 7 (S7) week 1 (top) and at the beginning of S7 week 3 (bottom). For all genes tested, expression was normalized to TBP. n = 4–6, bold bars indicate mean ± SEM. ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗p ≤ 0.001 by unpaired t-tests or by Welch's t-tests where variances were significantly different.

Figure 3

Consistent with the presence of fluorescent reporters, our RT-qPCR analysis shows that EGFP+mScar+ cells possess higher levels of INS and IAPP transcripts compared to EGFP+mScar− cells (Figure 3B). While the two populations do not differ in their levels of late-stage maturation markers (Fig. S5), they showed significantly lower levels of non-β cell hormones, namely glucagon and somatostatin. At stage 7 week 1, populations differed significantly in both glucagon and somatostatin transcript levels, but by stage 7 week 3, populations only differed significantly in somatostatin transcript levels. These data suggest that throughout the 4-week maturation period, dual fluorescence marks SCβ cells that are less polyhormonal.

3.6 EGFP+mScar+ cells show improved glucose sensitivity

To determine whether EGFP+mScar+ cells possessed higher sensitivity to glucose, we reaggregated FACS-sorted populations and evaluated them using glucose-stimulated insulin secretion tests. Maturing SC-islet cells were sorted at the beginning of stage 7 week 3, and reaggregated into pure EGFP+mScar+ and EGFP+mScar− populations. These were cultured for an additional 6 days to permit recovery, and confocal imaging was used to verify that their identities did not change over time (Figure 4A). We found that C-peptide secretion was stimulated at high glucose concentrations for both populations, suggesting that the EGFP+mScar− cells are also β-like in function (Figure 4B). The KCl response from EGFP+mScar− cells is significantly higher, however, immature SC-islets differentiated with the protocol used in this study are shown to have an exaggerated response to KCl compared to human islets [6]. The less exaggerated KCl response from the EGFP+mScar+ cells, which is relatively more similar to human islets, could also be an indicator of maturity. While both populations secreted comparable levels of C-peptide at high glucose, basal secretion levels trended lower (Figure 4B) for the EGFP+mScar+ aggregates (1.9 ± 0.2 ng/mL vs. 2.40 ± 0.3 ng/mL for EGFP+mScar− aggregates), leading to a significantly higher stimulation index (Figure 4C; 1.5 ± 0.1 vs. 1.2 ± 0.1-fold change over basal insulin). Since C-peptide levels at high glucose are comparable between the two fluorescent populations (Figure 4B), this significance derives from differences in basal secretion. Notably, if data are segregated by differentiation, the EGFP+mScar+ aggregates secrete less C-peptide at 2.8 mM than their EGFP+mScar− counterparts (Figure 4E). If the ratio of basal secretion for each population within each differentiation is compared to a hypothetical ratio of 1 (the null hypothesis being they are not different), then there is a significant reduction (by 21%) in basal secretion in the EGFP+mScar+ aggregates using a 1-sample t-test (Figure 4F).Figure 4 Dual fluorescing cells show elevated glucose sensitivity. (A) EGFP+mScar+ (orange) and EGFP+mScar− (green) cell populations were sorted at the start of week 3, reaggregated and imaged by confocal microscopy 6 days post-reaggregation. Scale bars indicate 50 μm. (B) C-peptide secreted by 50 aggregates in 250 μL buffer during a static glucose-stimulated insulin secretion (GSIS) assay. (C) Stimulation index (fold change of the high-glucose condition over the low) for both aggregate types. (D) Total C-peptide released during a static glucose-stimulated insulin secretion (GSIS) assay. (E) Basal secretion (2.8 mM glucose) is lower in dual fluorescing β cells from the same differentiation. (F) Basal secretion ratio of EGFP+mScar+ aggregates to EGFP + mScar- aggregates from the same differentiation. C-E were analyzed with student t-tests, F was analyzed by comparison to a hypothetical value of 1.0 (a ratio of identical basal secretions), using a 1-sample t-test. n = 7, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01. Bold bars indicate mean ± SEM.

Figure 4

3.7 EGFP+mScar+ cells show reduced exocytosis, membrane potential and action potential firing in low glucose

To evaluate potential causes of reduced basal insulin secretion, we measured exocytosis, calcium currents and sodium currents within matured (post-week 4) cells from both fluorescing populations, under low (2.8 mM) and high (16.7 mM) glucose conditions. Depolarization-activated Na+ and Ca2+ currents did not differ significantly between populations in either glucose condition (not shown). Exocytosis was measured as change in membrane capacitance over a series of ten membrane depolarizations. Under basal conditions, exocytosis was significantly lower in the EGFP+mScar+ cells compared to EGFP+mScar− cells (16.9 ± 2.2 fF/pF and 39.2 ± 6.39 fF/pF, respectively) (Figure 5A), and did not differ significantly from that of human islets evaluated under similar conditions (15.3 ± 0.7 fF/pF, Fig. S6) [21]. In contrast, basal exocytosis in EGFP+mScar− cells was significantly higher. We also assessed the membrane potential and action potential firing of both cell populations at low and high glucose. While the membrane potential in the EGFP+mScar− cells did not change, remaining at −57.4 ± 1.0 mV for 2.8 mM and −57.1 ± 1.3 mV in 16.7 mM glucose, the membrane potential of EGFP+mScar+ cells was significantly lower (−65.4 ± 0.9 mV) at 2.8 mM glucose, rising to −56.1 ± 0.8 mV at 16.7 mM glucose (Figure 5B). This basal membrane potential in EGFP+mScar+ cells is nearer to the typical −70 mV value for human β cells, which initiate action potential firing at voltages above −60 mV [46]. Consistent with this, 92% of EGFP+mScar− cells continued to fire action potentials at low glucose (Figure 5C,D), while 93.4% of EGFP+mScar+ cells, like human β cells, remained electrically silent [46,47]. Taken together, this suggests that dual fluorescence from the IIDR line may indicate SCβ cells possessing more mature glucose responsiveness.Figure 5 Electrophysiological characteristics of EGFP+mScar− (green) and EGFP+mScar+ (orange) cell populations. (A) Exocytosis for each fluorescing cell type, at low (2.8 mM) and high (16.7 mM) glucose concentrations (B) Membrane potentials at low and high glucose concentrations. (C) Action potential firing frequency at low and high glucose concentrations. (D) Representative action potential traces for each cell type. A-C represent individual cells from 3 differentiations; ∗ = p ≤ 0.05 by Mann–Whitney test. Bold bars indicate mean ± SEM.

Figure 5

4 Discussion

β cell maturation is marked by changes at the transcriptomic, functional and metabolic levels [1]. Recent differentiation protocols can now produce SCβ cells capable of glucose-stimulated insulin secretion (GSIS), but their responses are not equivalent to those observed in primary islets [6]. In GSIS and other β cell functions, SCβ cells continue to exhibit traits of immaturity [3]. To facilitate further improvements to in vitro maturation protocols, we modified a pre-existing INSEGFP reporter hESC line with an IAPPmScarlet cassette for discerning and tracking intermediate maturation in SCβ cells in vitro. We selected IAPP as an intermediate maturation marker due to its steady increase in expression over the course of β cell and SCβ cell maturation [4,9,19,20], and for its correlation to insulin expression and function in mature β cells [35,48,49].

Our phenotypic and functional studies of this Insulin IAPP Dual Reporter (IIDR) hESC line show that mScarlet expression within EGFP-positive (EGFP+) cells increases throughout a 4-week maturation period, culminating in ∼71% of EGFP+ cells expressing the IAPP reporter. It also shows an overall decrease in the proportion of EGFP+ cells, consistent with these cells progressing toward their terminal identity and state [17]. Compared to cells expressing EGFP alone (EGFP+mScar− cells), cells expressing both reporters (EGFP+mScar+ cells) are less polyhormonal and secrete lower amounts of C-peptide at 2.8 mM glucose, consistent with similar results preprinted by Davies et al. [42]. EGFP+mScar+ cells also exhibit lower exocytosis and membrane potentials at 2.8 mM glucose, as well as complete suppression of action potentials for 93.3% of cells tested (in contrast to the 8% observed for EGFP+mScar− cells). These results suggest that IAPPmScarlet expression in SCβ cells may be used to indicate a more mature β cell state.

Several groups have used β cell markers for live-cell monitoring and enrichment for β cell identity and maturation state [2,22,42]. INS-GFP reporters have been used to study both primary and stem cell-derived β cells, and have enabled improved SCβ cell maturation through a sorting and reaggregation step [14,50]. While successful at enriching for SCβ cells, the INS-GFP reporter alone is less discriminating between mature β cells, immature β cells and ɑ-cell-fated polyhormonal cells [17,20]. Simultaneous use of multiple markers enables further specification of cell identities and states: dual reporters for insulin and glucagon enable discernment of SCβ cells from polyhormonal (glucagon-expressing) cells [17,51], and INS-PDX1 reporters have been used to classify β cell subtypes according to functional maturation state [52], albeit in mice. However, stem cell lines with reporters specifically for maturation markers have not yet been documented in peer-reviewed journals. This work provides an account of IAPPmScarlet expression dynamics during SCβ cell maturation in vitro, and physiological and electrophysical characterization of resulting fluorescent populations.

EGFP+mScar+ cells begin to appear during Stage 6.2, and by S7 week 1 they mark SC- β cells expressing lower levels of non-β cell hormones. Thus, using the IIDR line to enrich for maturing β cells at this stage may also facilitate exclusion of polyhormonal cells reported by other groups [2,20]. In static GSIS, the EGFP+mScar+ cells showed reduced basal C-peptide secretion compared to EGFP+mScar− cells, potentially corresponding to an increased threshold for glucose secretion. β cell maturation is marked by an increase in glucose threshold for insulin secretion, a trait mediated in part through β cell electrophysiological activity [13]. Balboa et al. showed that exocytosis in immature SC-islets is unaffected by low and high glucose concentrations, while mature SC-islets released half as much insulin at low glucose [9]. These mature and immature exocytosis patterns match the exocytosis patterns observed in EGFP+mScar+ cells and EGFP+mScar− cells respectively. Consistent with this, EGFP+mScar+ cells possess a lower membrane potential at low glucose, below the threshold for electrical activity in human β cells [46,47]. This likely contributes to the complete absence of action potentials in >90% EGFP+mScar+ cells tested at low glucose, similar to the behavior of mature SC-islets, and adult human islets (Fig. S6) [9,46,47]. Taken together, these features point to EGFP+mScar+ cells possessing an elevated glucose threshold for insulin secretion.

In summary, we developed an INS2A-EGFP/+;IAPP2A-mScarlet/+ dual reporter hESC line that reports insulin and IAPP without affecting the differentiation of SCβ cells. We showed that IAPP can be tracked in insulin-producing cells using the bright red fluorescent mScarlet reporter, and that the proportion of insulin-producing cells expressing IAPP increases over a 4-week maturation period. We show that IAPPmScarlet expression in insulin-producing cells is indicative of SCβ cells with a more mature hormone profile and elevated glucose sensitivity. We further show tight correlation between co-expression of IAPP and insulin and more mature electrophysiological properties at low glucose. This dual reporter hESC line could therefore be used to track aspects of maturation in SCβ cells and allow visualization and isolation of less polyhormonal SCβ cells with improved glucose responsiveness in a heterogeneous culture.

CRediT authorship contribution statement

Carmen L. Bayly: Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. Xiao-Qing Dai: Writing – review & editing, Writing – original draft, Investigation, Formal analysis. Cuilan Nian: Writing – review & editing, Investigation. Paul C. Orban: Writing – review & editing, Investigation. C. Bruce Verchere: Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization. Patrick E. MacDonald: Writing – review & editing, Supervision, Resources, Funding acquisition. Francis C. Lynn: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following are the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Multimedia component 2

Multimedia component 2

Data availability

Data will be made available on request.

Acknowledgements

The authors thank the members of the Lynn and Verchere Laboratories (Vancouver, British Columbia, Canada); the PE MacDonald Laboratory (University of Alberta); and the BCCHR Flow Cytometry and Imaging core facilities for technical support, discussion, and critical reading of the manuscript. FCL, BV and PEM were supported by operating grants from the JDRF (5-SRA-2020-1059-S-B, FCL, BV, PEM; and 3-COE-2022-1103- M-B, FCL, BV) and Canadian Institutes of Health Research (ASD-173663, FCL, BV, and PEM). Salary (FCL) was supported by the Michael Smith Foundation for Health Research (#5238 BIOM) and the BC Children's Hospital Research Institute (IGAP awards). Salary (CLB) was provided by BC Children's Hospital Research Institute Canucks for Kids Childhood Diabetes Laboratories Postdoctoral Fellowship. PEM holds a Canada Research Chair in Islet Biology.

This work includes data and/or analyses from HumanIslets.com funded by the Canadian Institutes of Health Research, JDRF Canada, and Diabetes Canada (5-SRA-2021-1149-S-B/TG 179092) with data from islets isolated by the Alberta Diabetes Institute IsletCore with the support of the Human Organ Procurement and Exchange (HOPE) program, Trillium Gift of Life Network (TGLN), and other Canadian organ procurement organizations with written informed donor consent as approved by the Human Research Ethics Board at the University of Alberta (Pro00013094).

The authors acknowledge that UBC and BC Children's Hospital are situated on the traditional, ancestral, and unceded territories of the Coast Salish peoples, the Sḵw x_ wú7mesh (Squamish), səĺ ilwətaɁɬ (Tsleil-Waututh), and xʷməθkʷəýəm (Musqueam) Nations; and the University of Alberta is located on Treaty 6 territory, a traditional gathering place for diverse Indigenous peoples including the Cree, Blackfoot, Métis, Nakota Sioux, Iroquois, Dene, Ojibway/Saulteaux/Anishinaabe, Inuit, and many others

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.molmet.2024.102017.
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