Modeling disease progression in an accurate and reproducible manner is a significant challenge for researchers studying diabetes and other metabolic disorders. The gold standard in diabetes modeling and drug discovery involves the use of primary islets—small communities of hormone-producing cells, including insulin-producing beta cells—found in the islets of Langerhans in the pancreas. Aside from being difficult and costly to obtain, primary islets come from different genetic backgrounds, which confounds the study of insulin production and secretion by adding noise and variability to models and assays. Raising the bar, a renewable source of single-origin beta cells derived from a human induced pluripotent stem cell (hiPSC) line would be a consistent screening tool for drug discovery and a physiologically relevant model of insulin production and release.
To address the need for a consistent and reliable source of beta cells, we have developed a standardized differentiation protocol that mimics the typical stages of pancreatic development to create industrial-scale quantities of hiPSC-derived beta cells. Derived from a healthy hiPSC donor line, these beta cells: express insulin, C-peptide, MAFA, NKX6.1, PDX1, and UCN3; respond to incretin stimulation with insulin and C-peptide secretion; and, have functional potassium and calcium channels. These reproducibly derived, mature, and genetically identical beta cells respond consistently in experimental assays and are a major improvement over primary islets.
Creating hiPSC-derived beta cells
Our differentiation strategy is based on a four-step protocol that resembles normal embryonic development of beta cells (Figure 1). hiPSCs are first differentiated into SOX17+ definitive endoderm. Following endodermal commitment, the cells are further differentiated into PDX1/NKX6.1 double-positive pancreatic endoderm. Next, differentiation continues into endocrine progenitor cells, which are NGN3/NKX2.2 double-positive. Finally, the progenitors mature into terminally differentiated beta cells.
hiPSC-derived beta cells express relevant mRNAs
We first assayed the hiPSC-derived beta cells for the expression of mRNA transcripts known to be present in mature beta cells: PDX1, GCK, GLUT1, GLUT3, MAFB, NKX2.2, NKX6.1, and NeuroD1. RT-qPCR was performed on three different batches of hiPSC-derived beta cells, along with a batch of dissociated primary islets. hiPSC-derived beta cell expression levels were normalized to the single batch of primary islets for comparison. All transcripts were present in the hiPSC-derived beta cells (Figure 2), with some being expressed at comparable levels and others at slightly lower levels. Critically, the three different batches of hiPSC-derived beta cells all expressed mRNA at consistent levels, demonstrating reproducibility in gene expression.
We next assayed the hiPSC-derived beta cells for the expression of key proteins, including insulin and C-peptide; MAFA, and NKX6.1 (both transcription factors that regulate the insulin gene); PDX1 (a transcription factor important for pancreatic development); and UCN3 (a paracrine factor and regulator of insulin secretion present in mature beta cells). All proteins were present in the hiPSC-derived beta cells (Figures 3 and 4).
hiPSC-derived beta cells secrete insulin in response to KCl
KCl causes a depolarization of the membrane and activates calcium influx, inducing insulin release. To test the functionality of the hiPSC-derived beta cells in response to KCl stimulation, hiPSC-derived beta cells were incubated in KREBS buffer supplemented with 2.8 mM glucose. Next, the cells were incubated in the same KREBS buffer spiked with KCl. Lastly, the KREBS buffer was spiked with KCl and isradipine (a calcium channel blocker). The cells responded appropriately to KCl stimulation, as well as KCl plus isradipine administration, indicating that the hiPSC-derived beta cells have functional calcium channels (Figure 5).
hiPSC-derived beta cells secrete insulin in response to incretins
Next, we tested the functionality of the hiPSC-derived beta cells in response to incretin stimulation. hiPSC-derived beta cells were subjected to a C-peptide secretion assay in which the cells were incubated in 5.5 mM glucose, then spiked with either GLP-1 or exenatide (incretins known to stimulate insulin secretion). The cells responded to both incretin stimulations with an increase in insulin/C-peptide secretion (Figure 6).
G-protein-coupled receptors (GPR) 40, 41, and 43 are free fatty acid receptors (FFARs) that are known drug targets for diabetes and other metabolic disorders. To examine the gene expression of these receptors, we analyzed four different batches of hiPSC-derived beta cells and found that all batches expressed the FFARs (Figure 7). FFAR expression was highly consistent across batches, demonstrating the hiPSC-derived beta cells' suitability as a reliable cellular model for diabetes and metabolism studies.
GPR40 (FFAR1) mediates the effect of free fatty acids on insulin secretion. GPR40 agonists have been shown to increase insulin secretion and are promising therapeutics for diabetes. To examine the functionality of FFARs in the hiPSC-derived beta cells, we used sodium palmitate treatment to stimulate GPR40, which should cause an increase in insulin secretion. Indeed, Figure 8 shows that insulin secretion increased in response to palmitate treatment, indicating that the cells express functional GPR40.
To date, human primary islets have been considered the gold standard for diabetes research as they provide a model to assess key criteria such as insulin production, insulin secretion, and in vitro toxicity. However, availability of these cells can be limiting. Moreover, primary islets can be unreliable: they show large donor variation, preventing the generation of consistent results.
Beta cells derived from human induced pluripotent stem cells are a powerful alternative to primary islets, as they offer a virtually unlimited source of cells, are easy to culture, and provide reliable and reproducible models for drug discovery and toxicity testing.
Cellartis hiPSC-derived beta cells provide a reliable, off-the-shelf source of beta cells. These cells consistently demonstrate high expression of known beta cell mRNA transcripts and proteins, and they display appropriate responsiveness to incretin and KCl stimulation. Our hiPSC-derived beta cells are an excellent tool for studying the differences between diabetic and healthy individuals, modeling other metabolic disorders, and supporting screening efforts for drug discovery. In the future, we aim to develop a universal differentiation protocol that can be applied to any pluripotent stem cell, from both healthy and affected individuals, to enable the generation of disease-relevant beta cells from a wide variety of genetic backgrounds. This will further broaden the ability to screen and develop more effective and specific therapies.
Beta cell culture
Cellartis hiPSC-derived beta cells were cultured for 14 days post-thaw according to the Cellartis hiPS Beta Cells (from ChiPSC12) Kit User Manual. Specific assays were performed as described below.
mRNA expression (Figures 2 and 7)
RNA was prepared using the MagMax Total RNA Isolation Kit (Thermo Fisher Scientific), and cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). TaqMan analysis of mRNA was performed from three (Figure 2) or four (Figure 7) separate batches of Cellartis hiPSC-derived beta cells. All TaqMan designs were acquired from Applied Biosystems. Data is represented as mean ± SEM.
Immunocytochemistry (Figures 3 and 4)
Cells were fixed in 4% paraformaldehyde, permeabilized in 0.3% Triton X-100, blocked in TNB blocking buffer (PerkinElmer), and immunolabeled with primary antibodies specific to the marker of interest: insulin ab (Abcam), C-peptide ab (Cedarlane Laboratories), MAFA ab (Abcam), UCN3 ab (Abcam), NKX6.1 ab (DSHB), and/or PDX1 ab (Abcam). All primary antibodies were diluted 1:500 in TNB blocking buffer except for PDX1, which used a 1:8,000 dilution. Fluorescent-conjugated secondary antibodies (Alexa Fluor 488 or Alexa Fluor 594, Thermo Fisher Scientific) specific to the primary antibody host species were added to visualize the marker of interest. Secondary antibody was diluted 1:1,000 in TNB blocking buffer.
KCl/isradipine response (Figure 5)
Cells were incubated in KREBS buffer with 2.8 mM glucose for 45 minutes (control) followed by a second step in which the same buffer was supplemented with 30 mM KCl and incubated for 45 minutes. In the third step, both KCl and the calcium channel inhibitor isradipine (10 µM) were added, and the cells were incubated for 45 minutes. Samples were collected, and C-peptide secretion was measured by Mercodia C-peptide ELISA (the kit recognizes human C-peptide; Mercodia). The experiment was performed on two batches of Cellartis hiPSC-derived beta cells and three biological samples per batch (n = 6). The error bars are shown as SEM, and the statistical significance was calculated using Student's t-test; ***indicates p<0.001.
Incretin stimulation (Figure 6)
Cells were incubated in KREBS buffer with 5.5 mM glucose for 45 minutes (control) followed by a second step in which the same buffer was supplemented with incretins (100 nM GLP-1 or 100 nM exenatide), then incubated for 45 minutes. C-peptide secretion was measured by Mercodia C-peptide ELISA. Mean ± SEM (n = 5, GLP-1; n = 4, exenatide); *indicates p<0.05.
Sodium palmitate stimulation (Figure 8)
Cells were incubated in KREBS buffer with 2.8 mM glucose for 45 minutes (control) followed by a second step in which the same buffer was supplemented with sodium palmitate (0.5 mM) and incubated for 45 minutes. C-peptide secretion was measured by Mercodia C-peptide ELISA. Mean ± SEM (n = 5); **indicates p<0.01.