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.<...

<ol>
	<li>Atkinson, M. A., Eisenbarth, G. S., Michels, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+1+diabetes.">Type 1 diabetes.</a> Lancet. 383 (9911), 69-82 (2014).</li><li>Petersen, M. C., Shulman, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+insulin+action+and+insulin+resistance.">Mechanisms of insulin action and insulin resistance.</a> Physiological Reviews. 98 (4), 2133-2223 (2018).</li><li>Shapiro, A. 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=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> The New England Journal of Medicine. 343 (4), 230-238 (2000).</li><li>Gamble, A., Pepper, A. R., Bruni, A., Shapiro, A. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+journey+of+islet+cell+transplantation+and+future+development.">The journey of islet cell transplantation and future development.</a> Islets. 10 (2), 80-94 (2018).</li><li>Pagliuca, F. W., 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+functional+human+pancreatic+beta+cells+in+vitro.">Generation of functional human pancreatic beta cells in vitro.</a> Cell. 159 (2), 428-439 (2014).</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=Reversal+of+diabetes+with+insulin-producing+cells+derived+in+vitro+from+human+pluripotent+stem+cells.">Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.</a> Nature Biotechnology. 32 (11), 1121-1133 (2014).</li><li>Jennings, R. 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=Development+of+the+human+pancreas+from+foregut+to+endocrine+commitment.">Development of the human pancreas from foregut to endocrine commitment.</a> Diabetes. 62 (10), 3514-3522 (2013).</li><li>Jorgensen, M. C., 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=An+illustrated+review+of+early+pancreas+development+in+the+mouse.">An illustrated review of early pancreas development in the mouse.</a> Endocrine Reviews. 28 (6), 685-705 (2007).</li><li>Jensen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+factors+in+pancreatic+development.">Gene regulatory factors in pancreatic development.</a> Developmental Dynamics. 229 (1), 176-200 (2004).</li><li>Hald, 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=Generation+and+characterization+of+Ptf1a+antiserum+and+localization+of+Ptf1a+in+relation+to+Nkx6.1+and+Pdx1+during+the+earliest+stages+of+mouse+pancreas+development.">Generation and characterization of Ptf1a antiserum and localization of Ptf1a in relation to Nkx6.1 and Pdx1 during the earliest stages of mouse pancreas development.</a> Journal of Histochemistry and Cytochemistry. 56 (6), 587-595 (2008).</li><li>Villasenor, A., Chong, D. C., Henkemeyer, M., Cleaver, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+dynamics+of+pancreatic+branching+morphogenesis.">Epithelial dynamics of pancreatic branching morphogenesis.</a> Development. 137 (24), 4295-4305 (2010).</li><li>Rukstalis, J. M., Habener, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenin3:+a+master+regulator+of+pancreatic+islet+differentiation+and+regeneration.">Neurogenin3: a master regulator of pancreatic islet differentiation and regeneration.</a> Islets. 1 (3), 177-184 (2009).</li><li>Mastracci, T. L., Anderson, K. R., Papizan, J. B., Sussel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Neurod1+contributes+to+the+lineage+potential+of+Neurogenin3++endocrine+precursor+cells+in+the+pancreas.">Regulation of Neurod1 contributes to the lineage potential of Neurogenin3+ endocrine precursor cells in the pancreas.</a> PLoS Genetics. 9 (2), e1003278 (2013).</li><li>Balboa, D., 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=Functional,+metabolic+and+transcriptional+maturation+of+human+pancreatic+islets+derived+from+stem+cells.">Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells.</a> Nature Biotechnology. 40 (7), 1042-1055 (2022).</li><li>Du, 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=Human+pluripotent+stem-cell-derived+islets+ameliorate+diabetes+in+non-human+primates.">Human pluripotent stem-cell-derived islets ameliorate diabetes in non-human primates.</a> Nature Medicine. 28 (2), 272-282 (2022).</li><li>Hogrebe, N. J., Augsornworawat, P., Maxwell, K. G., Velazco-Cruz, L., Millman, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+cytoskeleton+to+direct+pancreatic+differentiation+of+human+pluripotent+stem+cells.">Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells.</a> Nature Biotechnology. 38 (4), 460-470 (2020).</li><li>Yoshihara, 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=Immune-evasive+human+islet-like+organoids+ameliorate+diabetes.">Immune-evasive human islet-like organoids ameliorate diabetes.</a> Nature. 586 (7830), 606-611 (2020).</li><li>Mahaddalkar, P. 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 ...

This protocol uses a static suspension culture strategy for differentiating pancreatic progenitors into islet clusters, reduces the stresses that cells experience during shaking culture, and greatly improves differentiation success. The hardware protocol is beginner-friendly, and save time for optimization of the first four stages. Personnel with basic stem cell culture techniques can reproduce this protocol with a good chance of generating functional islet-like clusters.To begin, precoat culture wells with hESC-qualified matrigel diluted in ice-cold DMEM/F12 and place the plate in a humidified 37-degrees-Celsius 5%carbon dioxide incubator for 30 minutes. 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 degrees Celsius for three to five minutes.After dissociation, rinse the cells by adding warm DMEM/F12 medium and transfer them into a 15-or 50-milliliter conical tube. Centrifuge the cell suspension at 300 G for five minutes and resuspend the pellet in a stem cell complete medium containing 10-micromolar Y-27632. Then, perform live cell counting using a hemocytometer.After counting aspirate diluted matrigel and immediately seed live cells at 1.6 to 1.8 times 10 to the five per square centimeter density on matrigel-coated wells. Incubate the cells in a humidified incubator for 24 hours. Next, on stage one, day one, prepare stage 1A medium.Replace the spent medium with stage 1A medium and incubate for 24 hours. The next day, replace the spent medium from the wells with freshly prepared stage 1B medium. On stage two, day one, prepare stage 2A medium and replace the medium from wells with stage 2A medium.Incubate for 24 hours before adding stage 2B medium into the wells;An stage three, day one, aspirate the spent medium from wells, then add freshly prepared stage 3 medium and incubate in a humidified incubator. On stage four, day one, replace the spent medium with freshly prepared stage 4 medium and incubate in a humidified incubator. On stage four, day five, pretreat the microwells with 500 microliters of anti-adherence rinsing solution per well.Centrifuge the microwell plate at 1, 300 G for five minutes and observe under a microscope to ensure no air bubbles are trapped in microwells. Next, aspirate the rinsing solution from the wells and rinse once with one milliliter of DPBS. Then, add one milliliter of aggregation medium to each well.After removing the spent medium from the pancreatic progenitor cultures, wash the culture once with DPBS before dissociating into single cells with dissociation reagent at 37 degrees Celsius for 10 to 12 minutes. Rinse the cells with warm DMEM/F12 and transfer them into a tube for centrifugation at 300 G for five minutes. Resuspend the pellet in the aggregation medium and perform live cell counting.Seed 2.4 to 3.6 million cells per well and add an aggregation medium to each well to achieve a final volume of two milliliters per well. Using a P-1000 pipette tip, gently pipette the cells up and down for even distribution. Centrifuge the cell suspension at 300 G for five minutes to capture the cells in the microwells and incubate in a humidified incubator for 24 hours to initiate aggregation.After aggregation, gently pipette the aggregates up and down several times to float any non-aggregated cells. Once the aggregates settle, carefully remove the spent medium containing floating non-aggregated cells without aspirating aggregates. Dispense fresh stage 5 medium onto the surface of the microwell plate to dislodge aggregates from the microwells, and transfer them into the ultra low-attachment six-well plate.After collecting all aggregates in the ultra low-attachment six wells, resuspend them in stage 5 medium to adjust the density to 20 clusters per 100 microliters. Then, using a multichannel pipette, dispense 50 microliters of stage 5 medium into each well of an ultra-low attachment ULA flat-bottom 96-well plate. Fill the corner and edge wells with 200 microliters of DPBS.Gently mix cluster resuspension every time before dispensing. Then, add 100 microliters of the cluster resuspension into each well of the ULA flat-bottom 96-well plate. Place the culture plates on a level surface in a humidified incubator for 24 hours.The next day or on stage five, day two, using a multi-channel pipette, gently remove 90 microliters of the spent medium from each well and refresh with 100 microliters of stage 5 medium. On stage six, day one, replace the 90 microliters of spent medium with 100 microliters of stage 6 medium per well. On stage seven, day one, remove the spent medium and add 100 microliters of stage 7 medium per well.In this study, using microwell plates, thousands of pancreatic progenitor clusters with uniform size and morphology were generated at a time, and the average aggregation efficiency was around 74%Importantly, expression of PDX1 and NKX6.1 was maintained at high levels in these clusters post-aggregation. Insulin expressing cells were gradually induced within endocrine clusters, as indicated by increased insulin GFP signals in living cultures over time. Cluster size increases from an average diameter of 150 microns to 220 microns between stages five and seven.Characterization of cell composition shows that stage seven hPSC islets generated by the hybrid protocol were primarily endocrine, and comprised of four major islet cell types. This hybrid protocol generated largely monohormonal islet cells and a minority of bihormonal cells. Differentiating beta cells express NKX6.1, MAFA, NEUROD1, PDX1, and GLUT1.Furthermore, 60%insulin and PDX1 double-positive cells and 50%C-peptide and NKX6.1 double-positive cells were induced in stage seven cultures. In vitro, static glucose-stimulated insulin secretion assays demonstrated that stage seven hPSC islets secrete high insulin levels in response to high glucose and direct depolarization. The total insulin content of hPSC islets is approximately half the amount of cadaveric islets.The multi-well-based static culture system for generating stem cell-derived islets facilitates various in situ assays, and can serve as a suitable platform for protocol development and small-scale screening studies. To minimize cluster fusion during a static culture in 96-well plate, do not tilt the plate when performing the medium change. Always keep the plate horizontal on a level surface.

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

Research

JoVE Journal

Developmental Biology

2.1K visualizações.  University of British Columbia. Este protocolo usa uma estratégia de cultura de suspensão estática para diferenciar progenitores pancreáticos em aglomerados de ilhotas, reduz os estresses que as células experimentam durante a cultura de agitação e melhora consideravelmente o sucesso da diferenciação. O protocolo de hardware é amigável para iniciantes e economiza tempo para otimização dos quatro primeiros estágios. Pessoal com técnicas básicas de cultura de células-tronco pode reproduzir esse protocolo com u...