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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3,3&#8217;,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 &#181;m diameter microwell plates</td>
		</tr>
		<tr>
			<td>Aggrewell800 plates</td>
			<td>STEMCELL Technologies</td>
			<td>34815</td>
			<td>800 &#181;m diameter microwell plates</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat anti-Human FOXA2 (goat IgG)</td>
			<td>R&#38;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&#38;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&#38;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&#954;)</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&#954;)</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&#954;)</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&#954;)</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&#954; 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&#160;</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 Ca2+ and Mg2+</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&#160;</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>NaHCO3</td>
			<td>Sigma</td>
			<td>S6297</td>
			<td>Medium supplement</td>
		</tr>
		<tr>
			<td>NEUROD1 antibody</td>
			<td>R&#38;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&#38;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 Ca2+ and Mg2+</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&#954;)</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&#954; 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&#160;</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&#160;</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&#38;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 ...

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JoVE Journal

Developmental Biology

2.4K Views.  University of British Columbia. 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.

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

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 ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...