We gratefully acknowledge the support from STEMCELL Technologies, Michael Smith Health Research BC, Stem Cell Network, JDRF, and the Canadian Institutes of Health Research. Jia Zhao and Shenghui Liang are recipients of the Michael Smith Health Research BC Trainee Award. Mitchell J.S. Braam is a recipient of the Mitacs Accelerate Fellowship. Diepiriye G. Iworima is a recipient of the Alexander Graham Bell Canada Graduate Scholarship and the CFUW 1989 Ecole Polytechnique Commemorative Award. We sincerely thank Dr. Edouard G. Stanley from MCRI and Monash University for sharing the Mel1 INS GFP/W line and the Alberta Diabetes Institute Islet Core for isolating and distributing human islets. We also acknowledge the support from the Life Sciences Institute Imaging and Flow Cytometry facilities at the University of British Columbia. Figure 1 was created with BioRender.com.

This protocol is based on work with hPSC lines, including H1, HUES4 PDXeG, and Mel1 INSGFP/W, in feeder-free conditions. A step-by-step procedure is detailed in this section, with supporting data from the differentiation of Mel1 INSGFP/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.
<ol>
	<li>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 &#176;C in a bath before use. 
	&#8203;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&#39;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.</li>
</ol>
2. Differentiation of hPSCs into pancreatic progenitor cells
<ol>
	<li>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&#39;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.</li>
	<li>Precoat culture wells with hESC-qualified Matrigel (diluted in ice-cold DMEM/F12 as recommended by the manufacturer&#39;s protocol) and place the plate in a humidified 37 &#176;C, 5% CO2 incubator for 30 min.</li>
	<li>Move the Matrigel-coated plate to room temperature (use within 3 h).</li>
	<li>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 &#176;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 &#215; g for 5 min.</li>
	<li>Resuspend the cell pellet with stem cell complete medium plus 10 &#181;M 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 &#215; 105/cm2 on Matrigel-coated wells and incubate in a humidified 37 &#176;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&#39;s protocol, a seeding density of 2.6 &#215; 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.</li>
	<li>On Stage 1 day 1, briefly warm endoderm basal medium to 37 &#176;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.</li>
	<li>Aspirate the spent medium from wells, add Stage 1A medium and incubate in a humidified 37 &#176;C, 5% CO2 incubator for 24 h. 
	&#8203;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.</li>
	<li>On Stage 1 day 2, briefly warm the endoderm basal medium in a 37 &#176;C bath and thaw Supplement CJ. Prepare Stage 1B medium by adding Supplement CJ to the endoderm basal medium. Mix well and use immediately.</li>
	<li>Aspirate the spent medium from the wells, add Stage 1B medium, and incubate in a humidified 37 &#176;C, 5% CO2 incubator for 24 h.</li>
	<li>On Stage 1 day 3, repeat steps 2.8-2.9. Proceed to Stage 2.</li>
	<li>On Stage 2 day 1, warm pancreatic Stage 2-4 Basal Medium in a 37 &#176;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.</li>
	<li>Aspirate the spent medium from wells, add Stage 2A medium, and incubate in a humidified 37 &#176;C, 5% CO2 incubator for 24 h.</li>
	<li>On Stage 2 day 2, warm pancreatic Stage 2-4 Basal Medium in a 37 &#176;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.</li>
	<li>Aspirate the spent medium from wells, add Stage 2B medium, and incubate in a humidified 37 &#176;C, 5% CO2 incubator for 24 h.</li>
	<li>On Stage 2 day 3, repeat steps 2.13-2.14. Proceed to Stage 3.</li>
	<li>On Stage 3 day 1, warm pancreatic Stage 2-4 Basal Medium in a 37 &#176;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.</li>
	<li>Aspirate the spent medium from wells, add Stage 3 medium, and incubate in a humidified 37 &#176;C, 5% CO2 incubator for 24 h.</li>
	<li>On Stage 3 day 2 and day 3, repeat steps 2.16-2.17. Proceed to Stage 4.</li>
	<li>On Stage 4 day 1, warm pancreatic Stage 2-4 Basal Medium in a 37 &#176;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.</li>
	<li>Aspirate the spent medium from wells, add Stage 4 medium, and incubate in a humidified 37 &#176;C, 5% CO2 incubator for 24 h.</li>
	<li>On Stage 4 day 2 to day 4, repeat steps 2.19-2.20. Proceed to aggregation steps.</li>
</ol>
3. Aggregation into pancreatic progenitor clusters using 400 &#181;m diameter microwell plates
<ol>
	<li>On Stage 4 day 5, pretreat the microwells (e.g., 24-well plate format) with 500 &#181;L of Anti-Adherence Rinsing Solution per well.</li>
	<li>Prepare a balance plate and centrifuge the microwell plate at 1,300 &#215; 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.</li>
	<li>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.</li>
	<li>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 &#176;C for 10-12 min. Rinse the cells off with warm DMEM/F12, transfer them into a tube, and spin at 300 &#215; g for 5 min.</li>
	<li>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.</li>
	<li>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 &#215; g for 5 min to capture the cells in the microwells. Incubate the microwell plate in a humidified 37 &#176;C, 5% CO2 incubator for 24 h aggregation and proceed to Stages 5-7 differentiation. 
	&#8203;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.</li>
</ol>
4. Differentiation of kit-derived pancreatic progenitor clusters into hPSC-islets
<ol>
	<li>On Stage 5 day 1, warm Stage 5-7 basal medium in a 37 &#176;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Collect all aggregates in the ULA 6-wells, resuspend aggregates in Stage 5 medium, and adjust the density to 20 clusters per 100 &#181;L (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 &#181;L of Stage 5 medium into each well of a ULA flat bottom 96-well plate. Fill the corner and edge wells with 200 &#181;L 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.</li>
	<li>Add 100 &#181;L of the cluster resuspension into each well of the ULA flat bottom 96-well plate, resulting in a total of 150 &#181;L culture medium containing an average of 20 clusters per well. 
	NOTE: Gently mix the cluster resuspension every time before dispensing into 96-wells.</li>
	<li>Place culture plates on a level surface in a humidified 37 &#176;C, 5% CO2 incubator for 24 h.</li>
	<li>On Stage 5 day 2, prepare Stage 5 medium following step 4.1.</li>
	<li>Use a multichannel pipette to remove ~90 &#181;L of the spent medium from each well, and refresh with 100 &#181;L of Stage 5 medium.</li>
	<li>On Stage 5 day 3, repeat steps 4.7-4.8. Proceed to Stage 6.</li>
	<li>On Stage 6 day 1, warm Stage 5-7 basal medium in a 37 &#176;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.</li>
	<li>Use a multichannel pipette to remove ~90 &#181;L of the spent medium and refresh with 100 &#181;L of Stage 6 medium per well.</li>
	<li>On Stage 6 day 2 to day 8, repeat steps 4.10-4.11. Proceed to Stage 7.</li>
	<li>On Stage 7 day 1, warm Stage 5-7 basal medium in a 37 &#176;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.</li>
	<li>Use a multichannel pipette to remove ~90 &#181;L of the spent medium and refresh with 100 &#181;L of Stage 7 medium per well.</li>
	<li>On Stage 7 day 2 to day 12, repeat steps 4.13-4.14. Proceed to in vitro characterization.</li>
</ol>
5. Flow cytometric analysis
<ol>
	<li>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 &#176;C for 10-12 min. Rinse with FACS buffer and spin at 300 &#215; g for 5 min.</li>
	<li>Resuspend the cell pellet in FACS buffer and perform cell counting. Spin cells at 300 &#215; g for 5 min. Resuspend single cells in 500 &#181;L 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.</li>
	<li>Spin at 500 &#215; g for 3 min, wash twice with 500 &#181;L of 1x Perm/Wash buffer, and resuspend in 300 &#181;L of 1x Perm/Wash buffer. Transfer 100 &#181;L of single-cell resuspension (each containing 2.5-3 &#215; 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.</li>
	<li>Spin at 500 &#215; g for 3 min. Wash twice with 500 &#181;L of 1x Perm/Wash buffer. Resuspend the samples in 100 &#181;L 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). 
	&#8203;NOTE: If the samples are not assayed immediately, store them at 4&#176;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.</li>
</ol>
6. Whole mount immunostaining
<ol>
	<li>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&#176;C overnight.</li>
	<li>Rinse once with PBST and permeabilize 20-30 clusters with 500 &#181;L 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.</li>
	<li>Incubate the clusters in 100 &#181;L of blocking buffer at ambient temperature for 1 h on a tilt shaker. Remove the blocking buffer and incubate the clusters with 100 &#181;L 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 &#176;C.</li>
	<li>Wash 3 x 30 min with 500 &#181;L of PBST thoroughly on a tilt shaker (tilt angle set at 8, speed set at 20).</li>
	<li>Incubate the clusters with 100 &#181;L 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 &#176;C.</li>
	<li>Rinse once with 500 &#181;L of PBST. Add 100 &#181;L of 4&#39;,6-diamidino-2-phenylindole (DAPI) solution (10 &#181;g/mL in PBST) and stain at ambient temperature for 10-15 min. Cover with foil and protect from light.</li>
	<li>Wash 3 x 30 min with 500 &#181;L of PBST thoroughly on a tilt shaker. Cover tubes with foil during the washing step.</li>
	<li>Attach the clusters onto imaging chamber slides (see the Table of Materials) precoated with tissue adhesive according to the manufacturer&#39;s protocol and published protocols20,21.</li>
	<li>Mount with tissue clearing medium (80% glycerol in PBS solution) and cover with glass coverslips. Protect from light until confocal imaging. 
	&#8203;NOTE: If samples are not imaged immediately, store them at 4 &#176;C protected from light.</li>
</ol>
7. Static GSIS assay
<ol>
	<li>Warm low-glucose Kreb&#39;s Ringer bicarbonate (3.3G KRB) buffer, high-glucose (16.7G) KRB buffer, and 30 mM KCl KRB buffer in a 37 &#176;C bath.</li>
	<li>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 &#176;C, 5% CO2 incubator.</li>
	<li>After equilibration, fill the assay chamber (see the Table of Materials) with 100 &#181;L 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 &#176;C, 5% CO2 incubator. 
	NOTE: Use 10 &#181;L pipette tips for transferring clusters with minimal medium volume.</li>
	<li>Carefully collect ~100 &#181;L of the supernatant and store it at -30 &#176;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 &#181;L of warm 16.7G KRB buffer into the assay chamber and incubate the same clusters for 30 min in a humidified 37 &#176;C, 5% CO2 incubator.</li>
	<li>Carefully collect ~100 &#181;L of the supernatant and store it at -30 &#176;C until measurement of the glucose-stimulated insulin secretion (see step 7.9). Immediately add 100 &#181;L of warm 30 mM KCl KRB buffer into the assay chamber and incubate the same clusters for 30 min in a humidified 37 &#176;C, 5% CO2 incubator.</li>
	<li>Carefully collect ~100 &#181;L of the supernatant and store at -30 &#176;C until measurement of the KCl-stimulated insulin secretion (see step 7.9). Immediately add 100 &#181;L of RIPA lysis buffer into the assay chamber and transfer the lysis buffer containing all five clusters into a microfuge tube.</li>
	<li>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.</li>
	<li>Spin at 8,000 &#215; g for 1 min. Collect the supernatant and store at -30 &#176;C until measurement of total insulin content (see step 7.9).</li>
	<li>Measure human insulin in the supernatant samples using a Human Insulin ELISA Kit according to the manufacturer&#39;s protocol and published protocols20,22. 
	NOTE: Avoid repeated freeze and thaw of supernatant samples. Samples with more than three freeze-and-thaw cycles should not be assayed.</li>
</ol>

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U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+pancreatic+beta+cells+from+CD177(+)+anterior+definitive+endoderm.">Generation of pancreatic beta cells from CD177(+) anterior definitive endoderm.</a> Nature Biotechnology. 38 (9), 1061-1072 (2020).</li><li>Liang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+stem+cell-derived+pancreatic+progenitors+into+insulin-secreting+islet+clusters+in+a+multiwell-based+static+3D+culture+system.">Differentiation of stem cell-derived pancreatic progenitors into insulin-secreting islet clusters in a multiwell-based static 3D culture system.</a> Cell Reports Methods. 3, 10046 (2023).</li><li>Zhao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+beta-cell+function+reveals+glucose-mediated+heterogeneity+of+beta-cell+functional+development.">In vivo imaging of beta-cell function reveals glucose-mediated heterogeneity of beta-cell functional development.</a> Elife. 8, e41540 (2019).</li><li>Zhao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+calcium+activities+from+pancreatic+beta-cells+in+zebrafish+embryos+using+spinning-disc+confocal+and+two-photon+light-sheet+microscopy.">In vivo imaging of calcium activities from pancreatic beta-cells in zebrafish embryos using spinning-disc confocal and two-photon light-sheet microscopy.</a> Bio-protocol. 11 (23), e4245 (2021).</li><li>Liang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+monoxide+enhances+calcium+transients+and+glucose-stimulated+insulin+secretion+from+pancreatic+beta-cells+by+activating+phospholipase+C+signal+pathway+in+diabetic+mice.">Carbon monoxide enhances calcium transients and glucose-stimulated insulin secretion from pancreatic beta-cells by activating phospholipase C signal pathway in diabetic mice.</a> Biochemical and Biophysical Research Communications. 582, 1-7 (2021).</li><li>Bruin, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+and+function+of+human+embryonic+stem+cell-derived+pancreatic+progenitors+in+macroencapsulation+devices+following+transplant+into+mice.">Maturation and function of human embryonic stem cell-derived pancreatic progenitors in macroencapsulation devices following transplant into mice.</a> Diabetologia. 56 (9), 1987-1998 (2013).</li><li>Toyoda, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+aggregation+optimizes+the+differentiation+of+human+ESCs+and+iPSCs+into+pancreatic+bud-like+progenitor+cells.">Cell aggregation optimizes the differentiation of human ESCs and iPSCs into pancreatic bud-like progenitor cells.</a> Stem Cell Research. 14 (2), 185-197 (2015).</li><li>Russ, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+induction+of+human+pancreatic+progenitors+produces+functional+beta-like+cells+in+vitro.">Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro.</a> EMBO Journal. 34 (13), 1759-1772 (2015).</li><li>Veres, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+cellular+identity+during+human+in+vitro+beta-cell+differentiation.">Charting cellular identity during human in vitro beta-cell differentiation.</a> Nature. 569 (7756), 368-373 (2019).</li><li>D'Amour, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+differentiation+of+human+embryonic+stem+cells+to+definitive+endoderm.">Efficient differentiation of human embryonic stem cells to definitive endoderm.</a> Nature Biotechnology. 23 (12), 1534-1541 (2005).</li><li>Jiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+pancreatic+progenitors+from+human+pluripotent+stem+cells+by+small+molecules.">Generation of pancreatic progenitors from human pluripotent stem cells by small molecules.</a> Stem Cell Reports. 16 (9), 2395-2409 (2021).</li><li>Tran, R., Moraes, C., Hoesli, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+clustering+enhances+PDX1+and+NKX6.1+expression+in+pancreatic+endoderm+cells+derived+from+pluripotent+stem+cells.">Controlled clustering enhances PDX1 and NKX6.1 expression in pancreatic endoderm cells derived from pluripotent stem cells.</a> Scientific Reports. 10 (1), 1190 (2020).</li><li>Mamidi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosignalling+via+integrins+directs+fate+decisions+of+pancreatic+progenitors.">Mechanosignalling via integrins directs fate decisions of pancreatic progenitors.</a> Nature. 564 (7734), 114-118 (2018).</li><li>Rezania, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+human+embryonic+stem+cell-derived+NKX6.1-expressing+pancreatic+progenitor+cells+accelerates+the+maturation+of+insulin-secreting+cells+in+vivo.">Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo.</a> Stem Cells. 31 (11), 2432-2442 (2013).</li><li>Sander, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeobox+gene+Nkx6.1+lies+downstream+of+Nkx2.2+in+the+major+pathway+of+beta-cell+formation+in+the+pancreas.">Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas.</a> Development. 127 (24), 5533-5540 (2000).</li></ol>

Timothy J. Kieffer was an employee of ViaCyte during the preparation of this manuscript. The other authors declare no competing interests.

This paper describes a seven-stage hybrid protocol that allows for the generation of hPSC islets capable of secreting insulin upon glucose challenge within 40 days of culture in vitro. Among these multiple steps, efficient induction of definitive endoderm is believed to set an important starting point for the final differentiation outcomes18,27,28. In the manufacturer&#39;s protocol, a seeding density at 2.6 &#215; 10<s...

Pancreatic beta cells secrete insulin responding to rises in blood glucose levels. Patients lacking sufficient insulin production due to the autoimmune destruction of beta cells in type 1 diabetes (T1D)1, or due to beta cell dysfunction in type 2 diabetes (T2D)2, are typically treated with the administration of exogenous insulin. Despite this life-saving therapy, it cannot precisely match the exquisite control of blood glucose as achieved by dynamic insulin secretion from bona fide beta cells. As such, patients often suffer the consequences of life-threatening hypoglycemic episodes and other complications resulting from chronic hyperglycemic excursions. Transplantation of human cadaveric islets successfully restores tight glycemic control in T1D patients but is limited by the availability of islet donors and difficulties in purifying healthy islets for transplantation3,4. This challenge can, in principle, be solved by using hPSCs as an alternative starting material.
Current strategies for generating insulin-secreting islets from hPSCs in vitro often aim to mimic the process of embryonic pancreas development in vivo5,6. This requires knowledge of the responsible signaling pathways and timed addition of corresponding soluble factors to mimic critical stages of the developing embryonic pancreas. The pancreatic program initiates with the commitment into definitive endoderm, which is marked by transcription factors forkhead box A2 (FOXA2) and sex-determining region Y-box 17 (SOX17)7. Successive differentiation of definitive endoderm involves the formation of a primitive gut tube, patterning into a posterior foregut that expresses the pancreatic and duodenal homeobox 1 (PDX1)7,8,9, and epithelial expansion into pancreatic progenitors that co-express PDX1 and NK6 homeobox 1 (NKX6.1)10,11.
Further commitment to endocrine islet cells is accompanied by the transient expression of pro-endocrine master regulator neurogenin-3 (NGN3)12 and stable induction of key transcription factors neuronal differentiation 1 (NEUROD1) and NK2 homeobox 2 (NKX2.2)13. The major hormone-expressing cells, such as insulin-producing beta cells, glucagon-producing alpha cells, somatostatin-producing delta cells, and pancreatic polypeptide-producing PPY cells, are subsequently programmed. With this knowledge, as well as discoveries from extensive, high-throughput drug screening studies, recent advancements have enabled the generation of hPSC-islets with cells resembling beta cells capable of insulin secretion14,15,16,17,18,19.
Step-wise protocols have been reported for generating glucose-responsive beta cells6,14,18,19. Built upon these studies, the present protocol involves the use of a pancreatic progenitor kit for generating PDX1+/NKX6.1+ pancreatic progenitor cells in a planar culture, followed by microwell plate aggregation into uniform-sized clusters and further differentiation toward insulin-secreting hPSC-islets with the R-protocol in a static 3D suspension culture. Quality control analyses, including flow cytometry, immunostaining, and functional assessment, are performed for rigorous characterization of the differentiating cells. This paper provides a detailed description of each step of the directed differentiation and outlines the in vitro characterization approaches.

<table><tbody><tr><td>3,3&amp;rsquo;,5-Triiodo-L-thyronine (T3)</td><td>Sigma</td><td>T6397</td><td>Thyroid hormone</td></tr><tr><td>4% PFA solution</td><td>Santa Cruz Biotechnology</td><td>sc-281692</td><td>Should be handled in fume hood</td></tr><tr><td>96-Well, Ultralow Attachment, flat bottom</td><td>Corning Costar (VWR)</td><td>CLS3474</td><td>Flat bottom; for static suspension culture in the last three stages</td></tr><tr><td>Accutase</td><td>STEMCELL Technologies</td><td>07920</td><td>Dissociation reagent for Stage 4 cells</td></tr><tr><td>Aggrewell400 plates</td><td>STEMCELL Technologies</td><td>34415</td><td>400 &amp;micro;m diameter microwell plates</td></tr><tr><td>Aggrewell800 plates</td><td>STEMCELL Technologies</td><td>34815</td><td>800 &amp;micro;m diameter microwell plates</td></tr><tr><td>Alexa Fluor 488 Goat anti-Human FOXA2 (goat IgG)</td><td>R&amp;amp;D Systems</td><td>IC2400G</td><td>1:100 in flow cytometry; used for assaying Stage 1 cells</td></tr><tr><td>Alexa Fluor 488 Goat IgG Isotype Control</td><td>R&amp;amp;D Systems</td><td>IC108G</td><td>1:100 in flow cytometry</td></tr><tr><td>Alexa Fluor 488 Mouse anti-Human SST (mouse IgG2B)</td><td>BD Sciences</td><td>566032</td><td>1:250 in flow cytometry; used for assaying Stage 7 cells</td></tr><tr><td>Alexa Fluor 488 Mouse IgG2B Isotype Control</td><td>R&amp;amp;D Systems</td><td>IC0041G</td><td>1:500 in flow cytometry</td></tr><tr><td>Alexa Fluor 647 Mouse anti-Human C-peptide (mouse IgG1&amp;kappa;)</td><td>BD Pharmingen</td><td>565831</td><td>1:2,000 in flow cytometry; used for assaying Stage 7 cells</td></tr><tr><td>Alexa Fluor 647 Mouse anti-Human INS (mouse IgG1&amp;kappa;)</td><td>BD Sciences</td><td>565689</td><td>1:2,000 in flow cytometry</td></tr><tr><td>Alexa Fluor 647 Mouse anti-Human NKX6.1 (mouse IgG1&amp;kappa;)</td><td>BD Sciences</td><td>563338</td><td>1:33 in flow cytometry; used for assaying Stage 4 cells</td></tr><tr><td>Alexa Fluor 647 Mouse anti-Human SOX17 (mouse IgG1&amp;kappa;)</td><td>BD Sciences</td><td>562594</td><td>1:50 in flow cytometry; used for assaying Stage 1 cells</td></tr><tr><td>Alexa Fluor 647 Mouse IgG1&amp;kappa; Isotype Control</td><td>BD Sciences</td><td>557714</td><td>1:50 in flow cytometry</td></tr><tr><td>ALK5i II</td><td>Cayman Chemicals</td><td>14794</td><td>TGF-beta signaling inhibitor</td></tr><tr><td>Anti-Adherence Rinsing Solution&amp;nbsp;</td><td>STEMCELL Technologies</td><td>7010</td><td>Microwell Rinsing Solution</td></tr><tr><td>Assay chamber</td><td>Cellvis</td><td>D35-10-1-N</td><td>For static GSIS and confocal imaging purposes</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Thermo Fisher Scientific</td><td>BP1600-100</td><td>For immunostaining procedure</td></tr><tr><td>CK19 antibody</td><td>DAKO</td><td>M0888</td><td>1:50 in whole mount immunofluorescence</td></tr><tr><td>D-glucose</td><td>Sigma</td><td>G8769</td><td>Medium supplement</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td>For nuclear counterstaining</td></tr><tr><td>DMEM/F12, HEPES</td><td>Thermo Fisher Scientific</td><td>11330032</td><td>Matrix diluting solution</td></tr><tr><td>Donkey anti-goat Alexa Fluor 555</td><td>Life technologies</td><td>A21432</td><td>1:500 in whole mount immunofluorescence</td></tr><tr><td>Donkey anti-goat Alexa Fluor 647</td><td>Life technologies</td><td>A21447</td><td>1:500 in whole mount immunofluorescence</td></tr><tr><td>Donkey anti-mouse Alexa Fluor 555</td><td>Life technologies</td><td>A31570</td><td>1:500 in whole mount immunofluorescence</td></tr><tr><td>Donkey anti-mouse Alexa Fluor 647</td><td>Life technologies</td><td>A31571</td><td>1:500 in whole mount immunofluorescence</td></tr><tr><td>Donkey anti-rabbit Alexa Fluor 555</td><td>Life technologies</td><td>A31572</td><td>1:500 in whole mount immunofluorescence</td></tr><tr><td>Donkey anti-rabbit Alexa Fluor 647</td><td>Life technologies</td><td>A31573</td><td>1:500 in whole mount immunofluorescence</td></tr><tr><td>Donkey anti-sheep Alexa Fluor 647</td><td>Life technologies</td><td>A21448</td><td>1:500 in whole mount immunofluorescence</td></tr><tr><td>DPBS</td><td>Sigma</td><td>D8537</td><td>Without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>ELISA, insulin, human</td><td>Alpco</td><td>80-INSHU-E01.1</td><td>For human insulin measurement</td></tr><tr><td>Fatty acid-free BSA</td><td>Proliant</td><td>68700</td><td>Medium supplement</td></tr><tr><td>Fixation and Permeabilization Solution Kit</td><td>BD Sciences</td><td>554714</td><td>Fix/Perm and 10x Perm/Wash solutions included</td></tr><tr><td>Gentle Cell Dissociation Reagent</td><td>STEMCELL Technologies</td><td>7174</td><td>For clump passaging hPSCs during maintenance culture</td></tr><tr><td>Glucagon antibody</td><td>Sigma</td><td>G2654</td><td>1:400 in whole mount immunofluorescence</td></tr><tr><td>GLUT1 antibody</td><td>Thermo Fisher Scientific</td><td>PA1-37782</td><td>1:200 in whole mount immunofluorescence</td></tr><tr><td>GlutaMAX-I (100x)</td><td>Gibco</td><td>35050061</td><td>L-glutamine supplement</td></tr><tr><td>Glycerol</td><td>Thermo Fisher Scientific</td><td>G33-4</td><td>For tissue clearing and mounting</td></tr><tr><td>GSi XX</td><td>Sigma Millipore</td><td>565789</td><td>Notch inhibitor</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3149</td><td>Medium supplement</td></tr><tr><td>ITS-X (100x)</td><td>Thermo Fisher Scientific</td><td>51500056</td><td>Insulin-Transferrin-Selenium-Ethanolamine; medium supplement</td></tr><tr><td>LDN193189&amp;nbsp;</td><td>STEMCELL Technologies</td><td>72147</td><td>BMP antagonist</td></tr><tr><td>MAFA antibody</td><td>Abcam</td><td>ab26405</td><td>1:200 in whole mount immunofluorescence</td></tr><tr><td>Matrigel, hESC-qualified</td><td>Thermo Fisher Scientific</td><td>08-774-552</td><td>Extracellular matrix for vessel surface coating</td></tr><tr><td>MCDB131 medium</td><td>Life technologies</td><td>10372019</td><td>Base medium</td></tr><tr><td>mTeSR1 Complete Kit</td><td>STEMCELL Technologies</td><td>85850</td><td>stem cell medium and 5x supplement included</td></tr><tr><td>N-Cys (N-acetyl cysteine)</td><td>Sigma</td><td>A9165</td><td>Antioxidant</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6297</td><td>Medium supplement</td></tr><tr><td>NEUROD1 antibody</td><td>R&amp;amp;D Systems</td><td>AF2746</td><td>1:20 in whole mount immunofluorescence</td></tr><tr><td>NKX6.1 antibody</td><td>DSHB</td><td>F55A12-c</td><td>1:50 in whole mount immunofluorescence</td></tr><tr><td>Pancreatic polypeptide antibody</td><td>R&amp;amp;D Systems</td><td>AF6297</td><td>1:200 in whole mount immunofluorescence</td></tr><tr><td>PBS</td><td>Sigma</td><td>D8662</td><td>With Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>PDX1 antibody</td><td>Abcam</td><td>ab47267</td><td>1:200 in whole mount immunofluorescence</td></tr><tr><td>PE Mouse anti-Human GCG (mouse IgG1&amp;kappa;)</td><td>BD Sciences</td><td>565860</td><td>1:2,000 in flow cytometry; used for assaying Stage 7 cells</td></tr><tr><td>PE Mouse anti-Human NKX6.1 (mouse IgG1k)</td><td>BD Sciences</td><td>563023</td><td>1:250 in flow cytometry</td></tr><tr><td>PE Mouse anti-Human PDX1 (mouse IgG1k)</td><td>BD Sciences</td><td>562161</td><td>1:200 in flow cytometry; used for assaying Stage 4 cells</td></tr><tr><td>PE Mouse IgG1&amp;kappa; Isotype Control</td><td>BD Sciences</td><td>554680</td><td>1:2,000 in flow cytometry</td></tr><tr><td>PE Mouse-Human Chromogranin A (CHGA, mouse IgG1k)</td><td>BD Sciences</td><td>564563</td><td>1:200 in flow cytometry</td></tr><tr><td>R428&amp;nbsp;</td><td>Cayman Chemicals</td><td>21523</td><td>AXL tyrosine kinase inhibitor</td></tr><tr><td>Retinoid acid, all-trans</td><td>Sigma</td><td>R2625</td><td>Light-sensitive</td></tr><tr><td>RIPA lysis buffer, 10x</td><td>Sigma</td><td>20-188</td><td>For hormone extraction</td></tr><tr><td>SANT-1</td><td>Sigma</td><td>S4572</td><td>SHH inhibitor</td></tr><tr><td>SLC18A1 antibody</td><td>Sigma</td><td>HPA063797</td><td>1:200 in whole mount immunofluorescence</td></tr><tr><td>Somatostatin antibody</td><td>Sigma</td><td>HPA019472</td><td>1:100 in whole mount immunofluorescence</td></tr><tr><td>STEMdiff Pancreatic Progenitor Kit</td><td>STEMCELL Technologies</td><td>05120</td><td>Basal media and supplements included</td></tr><tr><td>Synaptophysin antibody</td><td>Novus</td><td>NB120-16659</td><td>1:25 in whole mount immunofluorescence</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td>For permeabilization</td></tr><tr><td>Trolox&amp;nbsp;</td><td>Sigma Millipore</td><td>648471</td><td>Vitamin E analog</td></tr><tr><td>TrypLE Enzyme Express</td><td>Life technologies</td><td>12604-021</td><td>cell dissociation enzyme reagent for single cell passaging hPSCs</td></tr><tr><td>Trypsin1/2/3 antibody</td><td>R&amp;amp;D Systems</td><td>AF3586</td><td>1:25 in whole mount immunofluorescence</td></tr><tr><td>Y-27632</td><td>STEMCELL Technologies</td><td>72304</td><td>ROCK inhibitor</td></tr><tr><td>Zinc sulfate</td><td>Sigma</td><td>Z0251</td><td>Medium supplement</td></tr></tbody></table>

differentiation of human pluripotent stem cells into insulin-producing islet clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

We developed a hybrid strategy to differentiate stem cells into insulin-secreting hPSC-islets in seven steps, which utilizes a pancreatic progenitor kit for the first four stages in planar culture, followed by a modified protocol built upon a previously reported method6 in a static suspension culture for the last three stages (Figure 1). With this protocol, ensuring a near confluency (90%-100%) culture at 24 h after cell seeding (Stage 0) is critical for initiating an...

Watch this Scientific Journal Video about Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters at JoVE.com

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

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

differentiation-human-pluripotent-stem-cells-into-insulin-producing

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JoVE Journal

Developmental Biology

2.8K Views.  University of British Columbia. Dit protocol maakt gebruik van een statische suspensiecultuurstrategie voor het differentiëren van voorlopercellen van de alvleesklier in eilandjesclusters, vermindert de spanningen die cellen ervaren tijdens het schudden van de cultuur en verbetert het differentiatiesucces aanzienlijk. Het hardwareprotocol is beginnersvriendelijk en bespaart tijd voor optimalisatie van de eerste vier fasen. Personeel met elementaire stamcelkweektechnieken kan dit protocol reproduceren met een goede kans op ...

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

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Medicine

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Biology

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

Differentiating Human Pluripotent Stem Cells into Pancreatic β-Cells

Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic &#946;-cells. hPSCs can either be embryonic stem cells (hESCs) derived from the blastocyst or induced pluripotent cells (hiPSCs) generated directly from somatic cells using a reprogramming process. Here a video-based protocol is presented to outline the optimal culture and passage conditions for hPSCs, prior to their differentiation and subsequent generation of insulin-producing pancreatic cells. This methodology follows the six-stage process for &#946;-cell directed differentiation, wherein hPSCs differentiate into definitive endoderm (DE), primitive gut tube, posterior foregut fate, pancreatic progenitors, pancreatic endocrine progenitors, and ultimately pancreatic &#946;-cells. It is noteworthy that this differentiation methodology takes a period of 27 days to generate human pancreatic &#946;-cells. The potential of insulin secretion was evaluated through two experiments, which included immunostaining and glucose-stimulated insulin secretion.

Author Spotlight: Advancements and Challenges in &#946;-Cells Differentiation from Pluripotent Stem Cells

This article presents a protocol for directed differentiation and functional analysis of &#946;-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional &#946;-cell like cells secreting insulin in response to glucose.

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

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