Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

This protocol is based on work with hPSC lines, including H1, HUES4 PDXeG, and Mel1 INS^GFP/W, in feeder-free conditions. A step-by-step procedure is detailed in this section, with supporting data from the differentiation of Mel1 INS^GFP/W in the representative results section. We recommend that further optimization is needed when working with other hPSC lines that are not stated here. See the Table of Materials for details related to all reagents and solutions used in this protocol.

1. Preparation of differentiation media and solutions

NOTE: See Table 1 for all solutions to be prepared and Table 2 for the differentiation media.

1. Prepare all media and reagents for cell culture in a sterile biosafety cabinet. Briefly warm up cell culture-related solutions and basal media to 37 °C in a bath before use.
   ​NOTE: As described below, make fresh differentiation media daily by adding supplements and use them on the same day. Alternatively, the pancreatic progenitor kit provides the option to prepare a larger volume in advance that can be used over several days following the manufacturer's protocol. Both methods for media preparation prove to work well. Note that it is not recommended to leave media for an extended time in the water bath. When preparing media fresh each day, it is recommended to aliquot supplements to avoid repeated freeze-thaw cycles.

2. Differentiation of hPSCs into pancreatic progenitor cells

1. Maintain undifferentiated hPSCs on Matrigel-coated vessels in mTeSR1 complete medium and perform medium changes daily. During maintenance culture, passage hPSCs every 3-4 days when cells reach ~70% confluency using the cell dissociation reagent following the manufacturer's protocol. To initiate differentiation, dissociate the cells into single cells and seed them onto 6-well or 12-well tissue culture-treated plates (with media volumes of 2 mL or 1 mL per well, respectively).
   NOTE: See Table 1 for instructions related to the storage of endoderm basal medium and its aliquots.
2. Precoat culture wells with hESC-qualified Matrigel (diluted in ice-cold DMEM/F12 as recommended by the manufacturer's protocol) and place the plate in a humidified 37 °C, 5% CO₂ incubator for 30 min.
3. Move the Matrigel-coated plate to room temperature (use within 3 h).
4. Aspirate the spent medium from the hPSC culture and rinse once with DPBS. Dissociate hPSC culture into single cells with cell dissociation enzyme at 37 °C for 3-5 min. Rinse the cells off by adding warm DMEM/F12 medium, transfer into a 15 mL or 50 mL conical tube, and spin at 300 × g for 5 min.
5. Resuspend the cell pellet with stem cell complete medium plus 10 µM Y-27632 and perform live cell (trypan blue negative) counting with a hemocytometer or automated cell counter. Aspirate diluted Matrigel and immediately seed live cells at a density of 1.60-1.80 × 10⁵/cm² on Matrigel-coated wells and incubate in a humidified 37 °C, 5% CO₂ incubator for 24 h. Proceed to Stage 1.
   NOTE: This will result in ~95% starting confluency for initiating differentiation. According to the manufacturer's protocol, a seeding density of 2.6 × 10⁵/cm² is recommended. It is recommended to test various seeding densities to determine the optimal value for a cell line and to check cell viability. If the viability is below 80%, do not proceed with differentiation. Low cell viability can be a result of overdigestion or overtrituration during harvest or leaving cells for an extended time in DMEM/F12 medium prior to seeding.
6. On Stage 1 day 1, briefly warm endoderm basal medium to 37 °C in a bath and thaw Supplement MR and CJ. Prepare Stage 1A medium by adding Supplement MR and CJ to the endoderm basal medium. Mix well and use immediately.
7. Aspirate the spent medium from wells, add Stage 1A medium and incubate in a humidified 37 °C, 5% CO₂ incubator for 24 h.
   ​NOTE: Washing steps are unnecessary. Remove any unattached cells from the culture by gently shaking the culture plate before the medium exchange. Avoid disrupting the monolayer by not touching the pipette tip to the well bottom during medium removal and through gentle medium addition.
8. On Stage 1 day 2, briefly warm the endoderm basal medium in a 37 °C bath and thaw Supplement CJ. Prepare Stage 1B medium by adding Supplement CJ to the endoderm basal medium. Mix well and use immediately.
9. Aspirate the spent medium from the wells, add Stage 1B medium, and incubate in a humidified 37 °C, 5% CO₂ incubator for 24 h.
10. On Stage 1 day 3, repeat steps 2.8-2.9. Proceed to Stage 2.
11. On Stage 2 day 1, warm pancreatic Stage 2-4 Basal Medium in a 37 °C bath and thaw Supplement 2A and 2B. Prepare Stage 2A medium by adding Supplement 2A and 2B to the pancreatic Stage 2-4 Basal Medium. Mix well and use immediately.
12. Aspirate the spent medium from wells, add Stage 2A medium, and incubate in a humidified 37 °C, 5% CO₂ incubator for 24 h.
13. On Stage 2 day 2, warm pancreatic Stage 2-4 Basal Medium in a 37 °C bath and thaw Supplement 2B. Prepare Stage 2B medium by adding Supplement 2B to the pancreatic Stage 2-4 Basal Medium. Mix well and use immediately.
14. Aspirate the spent medium from wells, add Stage 2B medium, and incubate in a humidified 37 °C, 5% CO₂ incubator for 24 h.
15. On Stage 2 day 3, repeat steps 2.13-2.14. Proceed to Stage 3.
16. On Stage 3 day 1, warm pancreatic Stage 2-4 Basal Medium in a 37 °C bath and thaw Supplement 3. Prepare Stage 3 medium by adding Supplement 3 to the pancreatic Stage 2-4 Basal Medium. Mix well and use immediately.
17. Aspirate the spent medium from wells, add Stage 3 medium, and incubate in a humidified 37 °C, 5% CO₂ incubator for 24 h.
18. On Stage 3 day 2 and day 3, repeat steps 2.16-2.17. Proceed to Stage 4.
19. On Stage 4 day 1, warm pancreatic Stage 2-4 Basal Medium in a 37 °C bath and thaw Supplement 4. Prepare Stage 4 medium by adding Supplement 4 to the pancreatic Stage 2-4 Basal Medium. Mix well and use immediately.
20. Aspirate the spent medium from wells, add Stage 4 medium, and incubate in a humidified 37 °C, 5% CO₂ incubator for 24 h.
21. On Stage 4 day 2 to day 4, repeat steps 2.19-2.20. Proceed to aggregation steps.

3. Aggregation into pancreatic progenitor clusters using 400 µm diameter microwell plates

1. On Stage 4 day 5, pretreat the microwells (e.g., 24-well plate format) with 500 µL of Anti-Adherence Rinsing Solution per well.
2. Prepare a balance plate and centrifuge the microwell plate at 1,300 × g for 5 min.
   NOTE: Check the plate under a microscope to ensure no bubbles are left in the microwells. Centrifuge again for another 5 min if bubbles remain trapped in any microwells.
3. Aspirate the Anti-Adherence Rinsing Solution from the wells and rinse once with 1 mL of DPBS. Aspirate the DPBS from the wells and add 1 mL of Aggregation medium to each well.
4. Remove the spent medium from the pancreatic progenitor cultures and wash once with DPBS. Dissociate the cultures into single cells with dissociation reagent at 37 °C for 10-12 min. Rinse the cells off with warm DMEM/F12, transfer them into a tube, and spin at 300 × g for 5 min.
5. Resuspend the cell pellet in the Aggregation medium and perform live cell counting. Seed 2.4-3.6 million total cells per well (i.e., 2,000-3,000 cells per microwell) and add Aggregation medium to each well to achieve a final volume of 2 mL per well.
6. Gently pipette the cells up and down several times with a P1000 pipette tip to ensure an even distribution of cells throughout the well. Do not introduce bubbles into microwells. Centrifuge the microwell plate at 300 × g for 5 min to capture the cells in the microwells. Incubate the microwell plate in a humidified 37 °C, 5% CO₂ incubator for 24 h aggregation and proceed to Stages 5-7 differentiation.
   ​NOTE: Be careful not to disturb the plate during the time of aggregation. Up to 48 h of incubation time may be needed for optimal aggregate formation.

4. Differentiation of kit-derived pancreatic progenitor clusters into hPSC-islets

1. On Stage 5 day 1, warm Stage 5-7 basal medium in a 37 °C bath and thaw supplements (see Table 1 and Table 2). Prepare Stage 5 medium by adding supplements (to final working concentrations) to the Stage 5-7 basal medium. Mix well and use immediately.
2. After aggregate formation, gently pipette the aggregates up and down several times with a P1000 pipette tip to float any non-aggregated cells. Allow the aggregates to settle down by gravity (wait for ~1 min). Gently remove the spent medium (containing the floating non-aggregated cells) from the well as much as possible while avoiding aspirating aggregates.
3. Dispense fresh Stage 5 medium vigorously onto the surface of the microwell plate to dislodge aggregates from the microwells. Transfer aggregates into ultralow attachment (ULA) 6-wells. Rinse once with Stage 5 medium to completely retrieve aggregates from the microwells.
4. Collect all aggregates in the ULA 6-wells, resuspend aggregates in Stage 5 medium, and adjust the density to 20 clusters per 100 µL (e.g., ~1,200 clusters are expected to be retrieved from each well. Resuspend 1,200 clusters in 6 mL of Stage 5 medium). Use a multichannel pipette to dispense 50 µL of Stage 5 medium into each well of a ULA flat bottom 96-well plate. Fill the corner and edge wells with 200 µL of DPBS.
   NOTE: Avoid using the edge wells for culturing aggregates since they are prone to more evaporation; otherwise, use a 96-well plate that is designed to minimize evaporation along the edges (e.g., 96-well plates with built-in moats) or place the culture plate in a high-humidity microclimate cell culture incubator.
5. Add 100 µL of the cluster resuspension into each well of the ULA flat bottom 96-well plate, resulting in a total of 150 µL culture medium containing an average of 20 clusters per well.
   NOTE: Gently mix the cluster resuspension every time before dispensing into 96-wells.
6. Place culture plates on a level surface in a humidified 37 °C, 5% CO₂ incubator for 24 h.
7. On Stage 5 day 2, prepare Stage 5 medium following step 4.1.
8. Use a multichannel pipette to remove ~90 µL of the spent medium from each well, and refresh with 100 µL of Stage 5 medium.
9. On Stage 5 day 3, repeat steps 4.7-4.8. Proceed to Stage 6.
10. On Stage 6 day 1, warm Stage 5-7 basal medium in a 37 °C bath and thaw supplements (see Table 1 and Table 2). Prepare Stage 6 medium by adding supplements (to final working concentrations) to the Stage 5-7 basal medium. Mix well and use immediately.
11. Use a multichannel pipette to remove ~90 µL of the spent medium and refresh with 100 µL of Stage 6 medium per well.
12. On Stage 6 day 2 to day 8, repeat steps 4.10-4.11. Proceed to Stage 7.
13. On Stage 7 day 1, warm Stage 5-7 basal medium in a 37 °C bath and thaw supplements (see Table 1 and Table 2). Prepare Stage 7 medium by adding supplements (to final working concentrations) to the Stage 5-7 basal medium. Mix well and use immediately.
14. Use a multichannel pipette to remove ~90 µL of the spent medium and refresh with 100 µL of Stage 7 medium per well.
15. On Stage 7 day 2 to day 12, repeat steps 4.13-4.14. Proceed to in vitro characterization.

5. Flow cytometric analysis

1. Remove culture media and wash once with DPBS. Dissociate the cultures (planar progenitor cells or differentiating clusters) into single cells with dissociation reagent by incubating at 37 °C for 10-12 min. Rinse with FACS buffer and spin at 300 × g for 5 min.
2. Resuspend the cell pellet in FACS buffer and perform cell counting. Spin cells at 300 × g for 5 min. Resuspend single cells in 500 µL of Fix/Perm buffer for 10 min at room temperature.
   NOTE: Bring the Fix/Perm buffer to room temperature before use. The Fix/Perm buffer contains paraformaldehyde (PFA). Carefully handle PFA in a fume hood and dispose according to institutional guidelines.
3. Spin at 500 × g for 3 min, wash twice with 500 µL of 1x Perm/Wash buffer, and resuspend in 300 µL of 1x Perm/Wash buffer. Transfer 100 µL of single-cell resuspension (each containing 2.5-3 × 10⁵ cells) to three microfuge tubes for unstained control, isotype control, and antibody staining (see Table of Materials for sample antibody and dilution information). Incubate for 45 min at room temperature and cover with foil.
4. Spin at 500 × g for 3 min. Wash twice with 500 µL of 1x Perm/Wash buffer. Resuspend the samples in 100 µL of FACS buffer and analyze the stained cells (see Table of Materials for intracellular staining of markers at specific stages) with a flow cytometer and software (e.g., FlowJo).
   ​NOTE: If the samples are not assayed immediately, store them at 4°C protected from light. For the gating strategy, the flow data are first gated by scatter properties (FSC-A and SSC-A) to remove small cellular debris followed by FSC-A and FSC-width properties to identify singlets. Positive and negative gating is determined by stem cell controls and/or unstained, isotype controls.

6. Whole mount immunostaining

1. Collect clusters in a microfuge tube. Settle clusters by gravity. Aspirate the spent medium and rinse once with 1 mL of PBS (with calcium and magnesium). Fix the clusters with 1 mL of 4% PFA at 4°C overnight.
2. Rinse once with PBST and permeabilize 20-30 clusters with 500 µL of 0.3% TritonX-100 in a microfuge tube at ambient temperature for at least 6 h on a tilt shaker.
   NOTE: A longer permeabilization time (up to 24 h) is needed for large clusters.
3. Incubate the clusters in 100 µL of blocking buffer at ambient temperature for 1 h on a tilt shaker. Remove the blocking buffer and incubate the clusters with 100 µL of primary antibodies diluted in antibody dilution buffer at ambient temperature overnight on a tilt shaker.
   NOTE: If the background is strong during imaging, incubate with primary antibodies at 4 °C.
4. Wash 3 x 30 min with 500 µL of PBST thoroughly on a tilt shaker (tilt angle set at 8, speed set at 20).
5. Incubate the clusters with 100 µL of secondary antibodies in antibody dilution buffer at ambient temperature overnight on a tilt shaker. Cover the tubes with foil.
   NOTE: If the background is strong during imaging, incubate with secondary antibodies at 4 °C.
6. Rinse once with 500 µL of PBST. Add 100 µL of 4',6-diamidino-2-phenylindole (DAPI) solution (10 µg/mL in PBST) and stain at ambient temperature for 10-15 min. Cover with foil and protect from light.
7. Wash 3 x 30 min with 500 µL of PBST thoroughly on a tilt shaker. Cover tubes with foil during the washing step.
8. Attach the clusters onto imaging chamber slides (see the Table of Materials) precoated with tissue adhesive according to the manufacturer's protocol and published protocols^20,21.
9. Mount with tissue clearing medium (80% glycerol in PBS solution) and cover with glass coverslips. Protect from light until confocal imaging.
   ​NOTE: If samples are not imaged immediately, store them at 4 °C protected from light.

7. Static GSIS assay

1. Warm low-glucose Kreb's Ringer bicarbonate (3.3G KRB) buffer, high-glucose (16.7G) KRB buffer, and 30 mM KCl KRB buffer in a 37 °C bath.
2. Hand-pick size-matched clusters and rinse with 2 mL of warm 3.3G KRB buffer. Transfer the clusters into non-tissue culture-treated 6-well plate and equilibrate in 2 mL o warm 3.3G KRB buffer for 1 h in a humidified 37 °C, 5% CO₂ incubator.
3. After equilibration, fill the assay chamber (see the Table of Materials) with 100 µL of warm 3.3G KRB buffer. Hand-pick five clusters and transfer them to the center of the assay chamber. Incubate for 30 min in a humidified 37 °C, 5% CO₂ incubator.
   NOTE: Use 10 µL pipette tips for transferring clusters with minimal medium volume.
4. Carefully collect ~100 µL of the supernatant and store it at -30 °C until measurement of the basal insulin secretion (see step 7.9). Do not dry or lose any clusters in this step. Immediately add 100 µL of warm 16.7G KRB buffer into the assay chamber and incubate the same clusters for 30 min in a humidified 37 °C, 5% CO₂ incubator.
5. Carefully collect ~100 µL of the supernatant and store it at -30 °C until measurement of the glucose-stimulated insulin secretion (see step 7.9). Immediately add 100 µL of warm 30 mM KCl KRB buffer into the assay chamber and incubate the same clusters for 30 min in a humidified 37 °C, 5% CO₂ incubator.
6. Carefully collect ~100 µL of the supernatant and store at -30 °C until measurement of the KCl-stimulated insulin secretion (see step 7.9). Immediately add 100 µL of RIPA lysis buffer into the assay chamber and transfer the lysis buffer containing all five clusters into a microfuge tube.
7. Optional) Break the clusters manually by vigorous trituration with a P200 pipette tip. Vortex for 30 s and incubate on ice for 30 min followed by another 30 s of vortexing.
   NOTE: Ensure complete lysis by vortexing and sufficient time of incubation on ice.
8. Spin at 8,000 × g for 1 min. Collect the supernatant and store at -30 °C until measurement of total insulin content (see step 7.9).
9. Measure human insulin in the supernatant samples using a Human Insulin ELISA Kit according to the manufacturer's protocol and published protocols^20,22.
   NOTE: Avoid repeated freeze and thaw of supernatant samples. Samples with more than three freeze-and-thaw cycles should not be assayed.