Recent advances in human pluripotent stem cell (hPSC) differentiation protocols have generated insulin-producing cells resembling pancreatic β cells. While these stem cell-derived β (SC-β) cells are capable of undergoing glucose-stimulated insulin secretion (GSIS), insulin secretion per cell remains low compared with islets and cells lack dynamic insulin release. Herein, we report a differentiation strategy focused on modulating transforming growth factor β (TGF-β) signaling, controlling cellular cluster size, and using an enriched serum-free media to generate SC-β cells that express β cell markers and undergo GSIS with first- and second-phase dynamic insulin secretion. Transplantation of these cells into mice greatly improves glucose tolerance. These results reveal that specific time frames for inhibiting and permitting TGF-β signaling are required during SC-β cell differentiation to achieve dynamic function. The capacity of these cells to undergo GSIS with dynamic insulin release makes them a promising cell source for diabetes cellular therapy.

Here we report a six-stage differentiation strategy that generates almost pure populations of endocrine cells containing β-like cells that secrete high levels of insulin and express β cell markers. This is achieved by modulating Alk5i exposure to inhibit and permit TGF-β signaling during key stages in combination with cellular cluster resizing and enriched serum-free media (ESFM) culture. These cells are glucose responsive, exhibiting first- and second-phase insulin release, and respond to multiple secretagogues. Transplanted cells greatly improve glucose tolerance in mice. We identify that inhibiting TGF-β signaling during stage 6 greatly reduces the function of these differentiated cells while treatment with Alk5i during stage 5 is necessary for a robust β-like cell phenotype.

We () and others () published similar approaches for generating SC-β cells in vitro that in part use the compound Alk5 inhibitor type II (Alk5i) to inhibit transforming growth factor β (TGF-β) signaling during the last stages of differentiation. These approaches produced SC-β cells capable of undergoing glucose-stimulated insulin secretion (GSIS) in static incubations, expressing β cell markers, and controling blood sugar in diabetic mice after several weeks. However, even with this significant breakthrough, these cells had inferior function compared with human islets, including lower insulin secretion and little to no first- and second-phase insulin release in response to a high glucose challenge, demonstrating that these SC-β cells were less mature than β cells from islets. Several follow-up studies have been performed introducing additional differentiation factors or optimizing the process but have failed to bring SC-β cell function equivalent to human islets ().

Differentiation of human pluripotent stem cells (hPSCs) into stem cell-derived β cells (SC-β cells) is a promising alternative cell source for diabetes cell replacement therapy as well as other applications, such as modeling disease and studying pancreatic development (). Through modulation of pathways identified from embryonic development, studies with hPSCs have detailed protocols for generating cells that resemble early endoderm and pancreatic progenitors (), the latter of which can be transplanted into rodents and spontaneously differentiated into β-like cells after several months ().

Diabetes mellitus is a global health problem affecting over 400 million people worldwide and is increasing in prevalence (). Diabetes is principally caused by the death or dysfunction of insulin-producing β cells found within the islets of Langerhans in the pancreas, resulting in improper insulin secretion and failure of patients to maintain normal glycemia, which in severe cases can cause ketoacidosis and death. Patients are often reliant on insulin injections but can still suffer from long-term complications, including retinopathy, neuropathy, nephropathy, and cardiovascular disease (). An alternative treatment is replacement of the endogenous β cells by transplantation of pancreatic islets (). While this therapy has had clinical success, limited availability of cadaveric donor islets largely hampers its widespread application ().

Method for the isolation of intact islets of Langerhans from the rat pancreas.

Deaths attributable to diabetes in the United States: comparison of data sources and estimation approaches.

Projections of global mortality and burden of disease from 2002 to 2030.

Finally, we studied the role of Alk5i during stage 5 of differentiation to evaluate its effects on differentiation toward pancreatic endocrine cells, as it had been used previously for endocrine induction (). These experiments were performed as outlined in Figure 1 A in the presence or absence of Alk5i. We observed that the fraction of cells differentiated to endocrine cells (CHGA+) was unchanged but the fraction of cells differentiated to a C-peptide+ phenotype was decreased by omitting Alk5i ( Figures 7 A–7C). Similarly, the fraction of cells co-expressing C-peptide and NKX6-1, an important transcription factor for specifying β cells (), was decreased by omitting Alk5i. INS and GCG gene expression decreased with Alk5i omission, but surprisingly SST expression was slightly increased ( Figure 7 D). Expression of NKX6-1 and PDX1 were reduced without Alk5i ( Figure 7 E), while expression of several pancreatic endocrine markers were either unchanged or only slightly changed ( Figure 7 F). To further test the importance of Alk5i during stage 5, cells treated with or without Alk5i during stage 5 were further cultured for 7 days in stage 6 without Alk5i nor cluster resizing, and insulin secretion was substantially higher in cells treated with Alk5i during stage 5 ( Figure 7 G). Taken together, these data show that Alk5i treatment during stage 5 positively influences specification to β-like cell fate, not necessary to specify endocrine cells, and is necessary for high insulin secretion of resulting SC-β cells. In addition, these observations illustrate the importance of stage-specific treatment of the TGF-β signaling-inhibitor Alk5i to both generate and functionally mature SC-β cells.

(G) Human insulin secretion at 20 mM glucose of cells cultured in stage 5 in either DMSO or Alk5i plus an additional 7days in stage 6 without Alk5i and without cluster resizing (n = 3). ∗∗ p < 0.01 by unpaired two-way t test. All data shown are from HUES8.

(D–F) Real-time PCR measuring relative gene expression of stage 5 cells cultured with DMSO or Alk5i for pancreatic hormones (D), β cell markers (E), or endocrine markers (F) (n = 6). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, or n.s., not significant by unpaired two-way t test.

(C) Fraction of cells expressing the indicated markers (n = 4 except CHGA, which was n = 3). ∗ p < 0.05, ∗∗ p < 0.01 or n.s., not significant by unpaired two-way t test.

(A and B) Representative flow cytometric dot plots of dispersed stage 5 clusters immunostained for CHGA and NKX6-1 (A) or C-peptide and NKX6-1 (B).

Our studies with Alk5i during stage 6 suggested that permitting TGF-β signaling was necessary for robust functional maturation of SC-β cells, as inhibition of TGFBR1 is the canonical function of Alk5i. To test this hypothesis, we first used western blot analysis to validate that TGF-β signaling was occurring in our stage 6 cells via SMAD phosphorylation ( Figure 6 A). Alk5i treatment diminished phosphorylated SMAD, confirming that TGF-β signaling was indeed occurring and inhibited by Alk5i. SMAD phosphorylation was observed in stage 6 clusters regardless of whether they were resized, consistent with observations that Alk5i treatment reduced GSIS regardless of resizing ( Figure S7 ). Next, we generated two lentiviruses carrying short hairpin RNA (shRNA) designed to knock down TGFBR1 (TGFBR1 no. 1 and no. 2). These viruses were capable of reducing TGFBR1 transcript compared with control virus targeting GFP in stage 6 cells ( Figure 6 B) and reduced SMAD phosphorylation ( Figures 6 C and S7 C), albeit to much lesser extent than Alk5i treatment ( Figure 6 A). Similar to Alk5i treatment ( Figures 5 A and 5F), stage 6 cells transduced with shRNA against TGFBR1 had reduced insulin secretion and reduced positive glucose responsiveness in the static GSIS assay ( Figure 6 C) and blunted glucose response in the dynamic GSIS assay ( Figure 6 D). These data show that permitting TGF-β signaling during stage 6 is important for SC-β cell functional maturation, which is inhibited by treatment with Alk5i.

(E) Dynamic human insulin secretion of stage 6 cells transduced with lentiviruses containing GFP or TGFBR1 no. 1 shRNA in a perfusion GSIS assay. Cells are perfused with low glucose (2 mM) except where high glucose (20 mM) is indicated (n = 4). Data shown are from HUES8.

(D) Human insulin secretion of stage 6 cells in static GSIS assay transduced with lentiviruses containing GFP, TGFBR1 no. 1 or no. 2 shRNA (n = 3). ∗∗ p < 0.01 by paired two-way t test. ##p < 0.01 by one-way ANOVA Dunnett multiple comparison test compared with GFP. Data shown are from HUES8.

(C) Western blot of stage 6 cells transduced with lentiviruses containing GFP or TGFBR1 no. 1 shRNA. Data shown are from 1013-4FA.

(B) Real-time PCR of stage 6 cells transduced with lentiviruses containing GFP (control) or one of two sequences against TGFBR1 (TGFBR1 no. 1 and no. 2) (n = 3) shRNA. ∗∗∗∗ p < 0.0001 by one-way ANOVA Dunnett multiple comparison test compared with GFP.

(A) Western blot of stage 6 cells cultured with DMSO or Alk5i stained for phosphorylated SMAD 2/3 (pSMAD2/3), total SMAD 2/3 (tSMAD2/3), and actin. Data shown are from HUES8.

After having evaluated SC-β cells generated with our protocol, we investigated the protocol changes that were made to gain insights into SC-β cell differentiation and maturation. We found that, while inclusion of Alk5i during stage 6 resulted in relatively weak but statistically significant GSIS in a static assay, similar to data from the Pagliuca protocol ( Figure 1 D), omission of Alk5i drastically increased insulin secretion and glucose stimulation ( Figures 5 A and S6 A). Insulin content also increased with removal of Alk5i during stage 6 ( Figure 5 B), but the proinsulin/insulin ratio remained similar ( Figure 5 C), suggesting that the increased insulin content is not due to hormone processing. Furthermore, the fraction of cells expressing pancreatic endocrine markers, including C-peptide, remained similar between DMSO- and Alk5i-treated cells ( Figures 5 D, 5E, and S6 B). Gene expression was similar overall with and without Alk5i treatment, with cluster resizing typically having a larger effect ( Figure S6 C). Cells treated with Alk5i during stage 6 also had dramatically reduced insulin secretion with the dynamic GSIS assay, displaying weak to no first- and second-phase response ( Figure 5 F), similar to cells generated with the Pagliuca protocol ( Figure 1 F). These data show that Alk5i treatment during stage 6 inhibits functional maturation of SC-β cells.

(F) Dynamic human insulin secretion of stage 6 cells treated with DMSO or Alk5i in a perfusion GSIS assay. Cells are perfused with low glucose (2 mM) except where high glucose (20 mM) is indicated (n = 12). All data shown are with HUES8.

(D and E) Representative flow cytometric dot plots of dispersed stage 6 clusters immunostained for CHGA and PDX1 (D) or C-peptide and NKX6-1 (E).

(C) Cellular proinsulin/insulin content ratio of stage 6 cells treated with DMSO or Alk5i (n = 17). n.s., not significant by two-way unpaired t test.

(A) Box-and-whiskers plot of human insulin secretion of stage 6 cells in static GSIS assay treated with DMSO or Alk5i (n = 9). ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by two-way paired t test; ####p < 0.0001 by two-way unpaired t test.

To further characterize dynamic insulin secretion, we performed perifusion experiments to assay whether SC-β cells could respond to sequential challenges with several known secretagogues ( Figure 4 B). After an initial high glucose challenge, SC-β cells were able to respond to a second high glucose-only challenge, albeit less strongly than the first challenge, and extending the first glucose challenge to 1 hr in a separate experiment did not reduce insulin secretion ( Figure 4 C). Addition of other secretagogues during the second challenge further increased insulin secretion ( Figure 4 B). Membrane depolarizers KCl and L-arginine had the largest increases. Tolbutamide (blocks potassium channel), 3-isobutyl-1-methylxanthine (IBMX) (raises cytosolic cAMP), and exendin-4 (agonist of GLP-1 receptor) also increased insulin secretion over high glucose alone. Not only was insulin secretion increased but it rose faster than with high glucose alone. However, we noted that the response of stage 6 cells to KCl challenge was stronger than in human islets ( Figure S5 B), an observation made by others comparing β-like cells to human islets (), possibly indicative of continued immature or juvenile β cell phenotype. Taken together, these data show that SC-β cells can respond to several secretagogues that have diverse modes of action and have potential application in drug screening.

Since the differentiation protocol produces cells that are capable of dynamic insulin secretion, we studied this phenotype in more detail. We performed dynamic GSIS on cells as they progressed through stage 6 ( Figure 4 A). We observed that robust dynamic function was transient, with cells at 5 days secreting low amounts of insulin and exhibiting weak first- and second-phase response, with later time points (9–26 days) secreting higher amounts of insulin with a clear first- and second-phase response. During this time, the fraction of C-peptide+ cells decreased slightly ( Figure S5 A). By 35 days, insulin secretion at low glucose had risen such that first and second phases were difficult to clearly identify. These data show that SC-β cells require 9 days in stage 6 to acquire dynamic function, this function persists for weeks, but after extended in vitro culture glucose responsiveness is lost. Similarly, cadaveric human islets are known to have a limited functional lifetime in vitro, but the cause of this is not clear. These data further suggest an optimal time frame for these cells to be used in transplantation and drug-screening studies.

(C) Dynamic human insulin secretion of stage 6 cells in a perfusion GSIS assay with an extended high glucose treatment. Cells are perfused with low glucose (2 mM) except where high glucose (20 mM) is indicated (n = 3). All data shown are with HUES8.

(B) Dynamic human insulin secretion of stage 6 cells in a perfusion GSIS assay treated with multiple secretagogues. Cells are perfused with low glucose (2 mM) except where high (20 mM) glucose is indicated (Glu), then perfused with a second challenge of high glucose alone or with additional compounds (tolbutamide, IBMX, and Extendin-4 on the left; KCL and L-arginine on the right) where indicated (Glu + Factor). Note that the high glucose-only challenge is shown in both left and right graphs and the scale change (n = 3 except glucose, which is n = 2).

(A) Dynamic human insulin secretion cells in stage 6 for 5, 9, 15, 22, 26, and 35 days in a perfusion GSIS assay. Data for each individual time point is shown as mean ± SEM and the final graph shows only the means of each graph. Cells are perfused with low glucose (2 mM) except where high glucose (20 mM) is indicated (n = 3 for each stage 6 time point).

SC-β Cells have Transient Dynamic Function In Vitro, Respond to Multiple Stimuli, and Sustain Second-Phase Insulin Secretion at High Glucose

Figure 4 SC-β Cells have Transient Dynamic Function In Vitro, Respond to Multiple Stimuli, and Sustain Second-Phase Insulin Secretion at High Glucose

To evaluate the functional potential of stage 6 cells in vivo, we first transplanted cells under the renal capsule of non-diabetic mice and evaluated the ability of the graft to respond to a glucose challenge ( Figure 3 A). We observed that, even after extended time post-transplantation (6 months), the grafts responded to a glucose injection by increasing human insulin by a factor of 1.9 ± 0.5. Excision and immunostaining of the transplanted kidneys revealed C-peptide+ cells that tended to be clustered together in addition to other pancreatic endocrine and exocrine markers ( Figures 3 B and S4 A). To more rigorously evaluate stage 6 cells in vivo, we transplanted a separate mouse cohort that had been chemically induced to be diabetic with streptozotocin (STZ) and evaluated function at early (10 and 16 day) and late (10 week) time points. After only 10 days post-transplantation, STZ-treated mice receiving stage 6 cells had greatly improved glucose tolerance compared with STZ-treated sham mice and had similar glucose clearance as the non-STZ-treated mice ( Figures 3 C and 3D). Measurements of human insulin 16 days after transplantation revealed high insulin concentrations that increased by a factor of 2.3 ± 0.6 with a glucose injection to 16.6 ± 3.1 μIU/mL ( Figure 3 E). These values are greater than what was reported inunder similar conditions, which had an insulin increase of 1.4 ± 0.3 and concentration of 3.8 ± 0.8 μIU/mL. Observing our cohort 10 weeks after transplantation revealed similar results as the 10- and 16-day data, with transplanted mice having greatly improved glucose tolerance ( Figures 3 F and 3G) and glucose-responsive insulin secretion ( Figure 3 H). Mice not receiving STZ had similar glucose tolerance as mice receiving a therapeutic dose of human islets (). Mice that did not receive stage 6 cells had undetectable human insulin and mice that received STZ had drastically reduced mouse C-peptide compared with non-STZ-treated mice ( Figures S4 B and S4C). Grafts from these STZ-treated mice contained cells that expressed β cell markers in addition to other endocrine and exocrine markers ( Figure S4 D). Overall these data demonstrate that stage 6 cells generated with our protocol are functional both at early and late time points in vivo, greatly improving glucose tolerance to equal that of non-STZ-treated mice.

(H) Serum human insulin of STZ, Txp mice (n = 5) fasted overnight 0 and 60 min after an injection of 2 g/kg glucose. ∗∗ p < 0.01 by one-sided paired t test. All data shown are with HUES8. (A and B) are severe combined immunodeficiency (SCID)/Beige and (C–H) are non-obese diabetic/SCID mice.

(F) GTT 10 weeks after surgery for STZ, No Txp mice (n = 6), No STZ, No Txp mice (n = 4), and STZ, Txp mice (n = 5). ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by two-way ANOVA Tukey multiple comparison test.

(E) Serum human insulin of STZ, Txp mice (n = 5) fasted overnight 0 and 60 min after an injection of 2 g/kg glucose. ∗∗ p < 0.01 by one-sided paired t test.

(D) Area under the curve (AUC) calculations for data shown in (C). ∗∗ p < 0.01 by one-way ANOVA Tukey multiple comparison test.

(C) Glucose tolerance test (GTT) 10days after surgery for STZ-treated mice cohort without a transplant (STZ, No Txp; n = 6), untreated mice without a transplant (No STZ, No Txp; n = 5), and STZ-treated mice with a transplant (STZ, Txp; n = 6). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by two-way ANOVA Tukey multiple comparison.

(B) Immunostaining of sectioned explanted kidneys of non-STZ-treated mice 6 months after transplantation for C-peptide, PDX1, or with DAPI. The white dashed line is manually drawn to show the border between kidney and graft ( ∗ ). Scale bars, 50 μm.

(A) Serum human insulin of a non-STZ-treated mouse cohort (n = 3) 6 months after transplantation fasted overnight 0 and 60 min after an injection of 2 g/kg glucose. ∗∗ p < 0.01 by one-sided paired t test.

We measured expression of several genes compared with stage 6 cells generated with the Pagliuca protocol and human islets ( Figures 2 D and S3 ). Many islet and β cell genes were increased compared with the Pagliuca protocol, including INS, CHGA, NKX2-2, PDX1, NKX6-1, MAFB, GCK, and GLUT1. Interestingly, LDHA and SLC16A1, disallowed β cell genes, had reduced expression in our stage 6 cells compared with both the Pagliuca protocol and human islets (LDHA) and the Pagliuca protocol (SLC16A1). Our stage 6 cells had increased expression of CHGA, NKX6-1, MAFB, GCK, and GLUT1 compared with human islets. However, INS, GCG, SST, and particularly MAFA and UCN3 had reduced expression compared with stage 6 cells. However, several recent reports have provided evidence that question the utility of MAFA and UCN3 in evaluating human SC-β cell maturation. MAFA expression is low in juvenile human β cells (). MAFB is expressed in human but not mouse β cells (). UCN3 expression is much higher in mouse than human β cells () and is also expressed by human α cells (). These data show that our stage 6 cells have improved gene expression for many markers compared with the Pagliuca protocol and, while the expression of several β cell markers are equal to or great than human islets, other markers remain low.

Systematic single-cell analysis provides new insights into heterogeneity and plasticity of the pancreas.

Systematic single-cell analysis provides new insights into heterogeneity and plasticity of the pancreas.

To further characterize stage 6 cells generated with our differentiation protocol, we immunostained cells with a panel of pancreatic islet markers ( Figures 2 A–2C and S2 ). The vast majority of cells expressed CHGA (96% ± 1%), a pan-endocrine marker, and most cells expressed C-peptide (73% ± 3%) ( Figure 2 ). These fractions are higher than in stage 6 cells generated with the Pagliuca protocol ( Figure S2 ) and reported in. Many C-peptide+ cells from both protocols expressed other markers found in β cells and expression of the other pancreatic hormones was observed ( Figures 2 and S2 ). The majority of C-peptide+ cells expressed NKX6-1 ( Figure 2 ) and were monohormonal, which we presumed to be the SC-β cell population as done previously (). The fraction of C-peptide+ cells not expressing another hormone was increased compared with stage 6 cells generated with the Pagliuca protocol and reported in, while the fraction of these cells expressing another hormone was comparable ( Figures 2 and S2 ). These data show that stage 6 cells generated with our differentiation strategy are predominantly pancreatic endocrine with the majority expressing C-peptide.

(D) Real-time PCR analysis of stage 6 cells generated with the protocol from this study (n = 8), stage 6 cells generated with the Pagliuca protocol (n = 5), and cadaveric human islets (n = 7). n.s., not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by one-way ANOVA Dunnett multiple comparison test compared with this study. All stage 6 data shown are with HUES8.

(C) Box-and-whiskers plots quantifying fraction of cells expressing the indicated markers. Each point is an independent experiment.

(A) Immunostaining of dispersed stage 6 clusters plated overnight and stained for chromogranin A (CHGA), GCG, somatostatin (SST), NEUROD1, NKX6-1, PDX1, PAX6, C-peptide (CP), or with DAPI. Scale bar, 100 μm.

With dynamic GSIS, stage 6 cells displayed a rapid first-phase insulin release within 3–5 min of high glucose exposure, increasing insulin secretion by a factor of 7.6 ± 1.3 to 159 ± 21 μIU/μg DNA, higher than stage 6 cells generated from the Pagliuca protocol (1.7 ± 0.2× increase to 11 ± 1 μIU/μg DNA) but lower than human islets (15.0 ± 2.4× increase to 245 ± 26 μIU/μg DNA) ( Figure 1 F). Second-phase insulin secretion was observed with continued high glucose exposure, with cells maintaining 2.1 ± 0.3 higher insulin secretion than the initial low glucose, a higher increase than with the Pagliuca protocol (0.9 ± 0.1) but lower than human islets (6.7 ± 0.8) ( Figure 1 F). When the cells were returned to low glucose, insulin secretion from stage 6 cells returned to a reduced rate. Elevating insulin secretion and displaying first- and second-phase insulin release to a high glucose challenge are key features of β cell behavior. Overall, stage 6 cells generated with this differentiation strategy produces cells with clear first- and second-phase insulin secretion, which was not demonstrated byandand not seen with stage 6 cells produced with the Pagliuca protocol. However, when compared with human islets containing β cells, these stage 6 cells still have lower insulin secretion per cell at high glucose, lower glucose stimulation on average, and slightly slower first-phase insulin release.

We tested function of stage 6 cells generated with our differentiation protocol using both static ( Figures 1 D, 1E, and S1 C) and dynamic GSIS assays ( Figures 1 F and S1 D) and found that not only do the cells secrete insulin but also increase insulin release when moved from low to high glucose. With static GSIS, while there was some variability, stage 6 cells increased insulin secretion on average by a factor of 3.0 ± 0.1 when moved from 2 to 20 mM glucose. This is an improvement compared with cells generated with the protocol described in(1.4 ± 0.1), referred to here as the Pagliuca protocol, but less than human islets (3.2 ± 0.1) on average ( Figure 1 D). Stage 6 cells from this study did not increase insulin secretion in response to 5.6 mM glucose but did increase secretion in response to higher concentrations (11.1 and 20 mM), indicating that the cells are only stimulated at higher glucose threshold ( Figure 1 E). In terms of insulin secretion per cell, stage 6 cells secreted on average 5.3 ± 0.5 μIU/10cells at 20 mM glucose, 9.2 ± 1.1 times more than cells generated with the Pagliuca protocol and 2.3 ± 0.3 times less than human islets, on average ( Figure 1 D). It is important to note that our insulin values with the Pagliuca protocol are within range of the 2014 report but were lower on average, with differentiated HUES8 reported to secrete 0.2–2.6 μIU/10cells (average 1.4) and increase secretion by 0.4–4.1 (average 1.7) to high glucose.

Combining these modifications resulted in our six-stage differentiation protocol outlined in Figure 1 A. Stage 6 cells are grown as clusters in suspension culture ( Figure 1 B) that averaged 172 ± 34 μm (mean ± SD; n = 353 individual clusters) in diameter, less than half the diameter of the clusters before resizing, which was 364 ± 55 μm (n = 155 individual clusters). Stage 6 clusters stained red for the zinc-chelating dye dithizone, which stains β cells. Immunostaining of sectioned clusters revealed most cells to be C-peptide+, a protein also produced by the INS gene, in addition to PDX1+ and NKX6-1+, β cell markers ( Figure 1 C). A subset of cells stained positive for GCG or were polyhormonal, staining positive for both C-peptide and GCG. These polyhormonal cells are known to not to resemble adult β cells and are not functional ().

(F) Dynamic human insulin secretion of stage 6 cells generated with the protocol from this study (n = 12), stage 6 cells generated with the Pagliuca protocol (n = 4), and cadaveric human islets (n = 12) in a perfusion GSIS assay. Cells are perfused with low glucose (2 mM) except where high glucose (20 mM) is indicated. Act A, activin A; CHIR, CHIR9901; KGF, keratinocyte growth factor; RA, retinoic acid; Y, Y27632; LDN, LDN193189; PdbU, phorbol 12,13-dibutyrate; T3, triiodothyronine; Alk5i, Alk5 inhibitor type II; ESFM, enriched serum-free medium. All stage 6 data shown are with HUES8.

(E) Static GSIS assay of stage 6 cells from this study subjected to either 2, 5.6, 11.1, or 20 mM glucose (n = 4). ∗ p < 0.05, ∗∗∗ p < 0.001, n.s., not significant by one-way ANOVA Dunnett multiple comparison test compared with 2 mM glucose.

(D) Human insulin secretion of stage 6 cells generated with the protocol from this study (n = 16), stage 6 cells generated with the Pagliuca protocol (n = 12), and cadaveric human islets (n = 12) in a static GSIS assay. ∗∗ p < 0.01, ∗∗∗∗ p < 0.001 by one-sided paired t test. ###p < 0.001, ####p < 0.0001 by one-way ANOVA Dunnett multiple comparison test compared with this study.

(C) Immunostaining of sectioned stage 6 clusters stained for glucagon (GCG), NKX6-1, PDX1, C-peptide (CP), or with the nuclei marker DAPI. Scale bar, 100 μm.

(B) Images of unstained whole stage 6 clusters under phase contrast (top) or stained with dithizone (DTZ) (bottom) imaged under bright field. Scale bars, 400 μm.

We set out to develop an improved differentiation protocol starting from the approach we described inusing the HUES8 cell line. We included Y27632 during stages 3 to 4 and activin A during stage 4 as we reported previously () to help maintain cluster integrity and shortened stage 3 from 2 days to only 1 day to enhance progenitors (). We also developed an ESFM for stage 6 to replace the serum-containing media used previously to have a serum-free protocol. During our protocol pilot studies, we observed that both resizing clusters and removal of Alk5i and T3 increased insulin secretion while maintaining the C-peptide+ population ( Figures S1 A and S1B).

Discussion

Here we demonstrate that enhanced functional maturation of SC-β cells is achieved with our six-stage differentiation strategy. These cells secrete a large amount of insulin and are glucose responsive, displaying both first- and second-phase insulin release. This differentiation procedure generates almost pure endocrine cell populations without selection or sorting, and most cells express C-peptide and other β cell markers. Upon transplantation into STZ-treated mice, glucose tolerance is rapidly restored and function persists for months. These SC-β cells respond to multiple secretagogues in a perifusion assay. We found modulating TGF-β signaling to be crucial for success, with inhibition during stage 5 increasing SC-β cell differentiation but inhibition during stage 6 reducing function and insulin content. Permitting TGF-β signaling during stage 6 was necessary for robust dynamic function.

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et al. Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes. Our strategy enhances the utility of in-vitro-differentiated SC-β cells for drug screening due to their improved kinetics. Proper dynamic insulin release is an important feature of β cell metabolism that is commonly lost in diabetes (). We have established a renewable resource of SC-β cells with dynamic insulin release that can be used to better study the mechanism of β cell failure in diabetes and demonstrated their response to several secretagogues.

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Honore C. Single-cell gene expression analysis of a human ESC model of pancreatic endocrine development reveals different paths to beta-cell differentiation. The culmination of numerous modifications to the protocol produced SC-β cells exhibiting dynamic glucose response. In addition to modulating TGF-β signaling, other notable changes included the removal of serum, reducing cluster size, and the lack of several additional factors (T3, N-acetyl cysteine, Trolox, H1152, and R428) used in other reports during the last stage. We hope that these insights provide the basis for further innovations for differentiating SC-β cells and improving function, especially as a recent report indicates there may be multiple pathways to β cells (). While we demonstrate reproducibility of the protocol across multiple cell lines, marker expression and function were greatest in the HUES8 cell line. As this protocol was initially developed for this line, we suspect additional optimization to be beneficial when applying this protocol to additional lines.