This work was supported in part by funding from the Japan Society for the Promotion of Science (JSPS) through Scientific Research (C) (JSPS KAKENHI Grant Number15K09385 and 18K08510) to T.T., and Grant-in-Aid for JSPS Research Fellows (JSPS KAKENHI Grant Number 17J07622) to A.K., and the Japan Agency for Medical Research and Development (AMED) through its research grant &#8220;Core Center for iPS Cell Research, Research Center Network for Realization of Regenerative Medicine&#8221; to K.O. The authors thank Dr. Peter Karagiannis for reading the manuscript.

Experiments using hPSCs were approved by the ethics committee of the Department of Medicine and Graduate School of Medicine, Kyoto University.
1. Preparation of Materials
NOTE: Prepare all media and reagents for cell culture in a sterile environment. Warm up base culture media to room temperature (RT) before use. Medium for differentiation is used within 6 h at RT. Details of the reagents are listed in Table of Materials.
<ol>
	<li>Differentiation (Figure 1A)
	<ol>
		<li>Stage 1A medium: Transfer RPMI 1640 medium into a tube using a pipette. Add the serum-free supplement, activin A, CHIR99021, and Y-27632 to a concentration of 1x, 100 ng/mL, 3 &#956;M and 10 &#956;M, respectively.</li>
		<li>Stage 1B medium: Transfer RPMI 1640 medium into a tube using a pipette. Add the serum-free supplement, activin A and CHIR99021 to a concentration of 1x, 100 ng/mL, &#8804;1 &#956;M, respectively. 
		NOTE: The concentration of CHIR99021 should be lower than in Stage 1A medium, and addition is not necessary, but it increases the cell number.</li>
		<li>Stage 2 medium: Transfer Improved MEM (iMEM) medium into a tube using a pipette. Add the serum-free supplement and keratinocyte growth factor (KGF) to a concentration of 0.5x and 50 ng/mL, respectively.</li>
		<li>Stage 3 medium: Transfer iMEM medium into a tube using a pipette. Add the serum-free supplement, KGF, NOGGIN, 3-Keto-N-aminoethyl-N&#8217;-aminocaproyldihydrocinnamoyl cyclopamine (CYC) and 4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]-benzoic acid (TTNPB) by pipette to concentrations of 0.5x, 50 ng/mL, 100 ng/mL, 0.5 &#956;M and 10 nM, respectively.</li>
	</ol>
	</li>
	<li>Flow cytometry (FCM)
	<ol>
		<li>1x Permeabilization/Wash buffer: Transfer water into a tube by a pipette. Add Permeabilization/Wash buffer by a pipette to a concentration of 1x.</li>
		<li>FCM blocking solution: Transfer 1x Permeabilization/Wash buffer into a tube by a pipette. Add donkey serum by a pipette to a concentration of 2% (vol/vol).</li>
	</ol>
	</li>
	<li>Immunostaining
	<ol>
		<li>Blocking solution: Transfer Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) into a tube by a pipette. Add donkey serum and Triton X by pipette to concentrations of 5% (vol/vol) and 0.4% (vol/vol), respectively.</li>
	</ol>
	</li>
</ol>
2. hPSC Differentiation to Posterior Foregut Cells/Pancreatic Progenitors (PDX1+ Cells) 
NOTE: Conduct all procedures using sterile techniques. hPSCs are maintained on 6-well plates coated by a synthetic surface material for hPSCs with hPSC maintenance medium according to the manufacturer&#39;s instructions17,26. When cells reach 50-80% confluency (Stage 0) use them for differentiation.
<ol>
	<li>Prepare basement membrane matrix-coated plates.
	<ol>
		<li>Transfer 6 mL of RPMI 1640 (4 &#176;C) into a tube cooled to 4 &#176;C on ice. Add 2 mg of basement membrane matrix (4 &#176;C) with cooled 1 mL-pipette tips and mix well by gentle pipetting to make 0.33 mg/mL basement membrane matrix.</li>
		<li>Transfer 2 mL of diluted basement membrane matrix to each well of 6-well cell culture plates by a pipette. Place the plates at 37 &#176;C for 60-90 min in an incubator. Afterward, keep the plates at RT until use (within 3 h).</li>
	</ol>
	</li>
	<li>Seed the hPSCs and initiate differentiation to definitive endoderm (Stage 1A).
	<ol>
		<li>Aspirate the used medium and add 2 mL of 0.5 mM ethylenediaminetetraacetic acid (EDTA) (RT) per well by pipette to wash the hPSCs cultured in the 6-well plates.</li>
		<li>Aspirate 0.5 mM EDTA and add 2 mL of 0.5 mM EDTA per well by pipette. Incubate the plates at 37 &#176;C for 5 min.</li>
		<li>Aspirate 0.5 mM EDTA and add 1 mL of hPSC maintenance medium at RT&#160;supplemented with 10 &#956;M Y-27632 per well by pipette.&#160;Pipette gently but quickly to blow off attached cells on the plates and to dissociate clumped cells into single cells. Do not bubble the cell suspension during pipetting.</li>
		<li>Transfer the cell suspension in a 50 mL centrifuge tube containing 4 mL of hPSC maintenance medium at RT supplemented with 10 &#956;M Y-27632. Gently pipette the cell suspension.&#160;</li>
		<li>Count the cell number in the cell suspension using the Trypan Blue stain exclusion procedure.
		<ol>
			<li>Mix 15 &#956;L of cell suspension and 15 &#956;L of Trypan Blue in a tube.</li>
			<li>Transfer 10 &#956;L of the cell suspension diluted with Trypan Blue onto cell counting slides in duplicate by a 10 &#956;L pipette.</li>
			<li>Count the cell number with an automatic cell counter and calculate the cell density in the cell suspension. Viability is usually &#62;98%.</li>
		</ol>
		</li>
		<li>Aliquot the cell suspension in a 50 mL centrifuge tube at 1-1.5 x 106 cells per well of 6-well plates (1-1.5 x 105 cells/cm2).</li>
		<li>Centrifuge the 50 mL tube at 200 x g at RT for 5 min.</li>
		<li>Aspirate the supernatant using a pipette and resuspend the cells with 1 mL of Stage 1A medium (RT) per well. Gently pipette the cell suspension and add another 1 mL of Stage 1A medium (total, 2 mL per well).</li>
		<li>Aspirate the diluted basement membrane matrix in the wells of the cell culture plates prepared in step 2.1.2 by a 1,000 &#956;L pipette. Proceed to the next step immediately.</li>
		<li>Gently pipette the cell suspension in step 2.2.8 again. Immediately after mixing, transfer the cell suspension (2 mL) into each well of a 6-well plate. Cover the plate with an aluminum foil to protect the plate from light. Place the plate in the clean bench at RT for 10-15 min. 
		NOTE: Do not move the plate after seeding.</li>
		<li>Gently place the plate into a 37 &#176;C, 5% CO2 incubator (humidified atmosphere) and culture for 24 h. 
		NOTE: Do not shake the plate after seeding.</li>
	</ol>
	</li>
	<li>Induce the differentiation into definitive endoderm (Stage 1B).
	<ol>
		<li>Aspirate the used medium and add 2 mL of DPBS to the wells by pipette. Repeat the aspiration and addition of DPBS one time. 
		NOTE: To remove any dead cells from the monolayer, gently shake the plate before each aspiration.</li>
		<li>Aspirate the used DPBS by a pipette and add 4 mL of Stage 1B medium (37 &#176;C) per well. Gently place the plate into the 37 &#176;C incubator (humidified atmosphere of 5% CO2) and culture for 48 h.</li>
		<li>Aspirate the used medium and add 2 mL of DPBS to the well by pipette. Repeat the aspiration and addition of DPBS one time. 
		NOTE: Remove the dead cells from the monolayer by gentle shaking before each aspiration.</li>
		<li>Aspirate the used DPBS by pipette and add 4 mL of Stage 1B medium (37 &#176;C) per well. Gently place the plate into the incubator (37 &#176;C, humidified atmosphere of 5% CO2) and culture for 24 h.</li>
	</ol>
	</li>
	<li>Induce the differentiation into the primitive gut tube (Stage 2).
	<ol>
		<li>Aspirate the used medium and add 2 mL of DPBS to the well by pipette. 
		NOTE: Remove the dead cells from the monolayer by gentle shaking before each aspiration.</li>
		<li>Aspirate the used DPBS by pipette and add 4 mL of Stage 2 medium (37 &#176;C) per well. Place the plate into the incubator (37 &#176;C, humidified atmosphere of 5% CO2) and culture for 4 days.</li>
	</ol>
	</li>
	<li>Induce the differentiation into PDX1+ cells (Stage 3).
	<ol>
		<li>Aspirate the used medium and add 2 mL of DPBS to the well by pipette. Repeat the aspiration and addition of DPBS one time.</li>
		<li>Aspirate the used DPBS by pipette and add 4 mL of Stage 3 medium (37 &#176;C) per well. Place the plate into the incubator (37 &#176;C, humidified atmosphere of 5% CO2) and culture for 3 days.</li>
	</ol>
	</li>
	<li>Induce the differentiation into pancreatic endocrine cells.
	<ol>
		<li>Perform NKX6.1+ cell induction (Stage 4, for 8 days) followed by endocrine cell induction (Stage 5, for 12 days) with previously described protocols3,16,26. Characterize and validate the endocrine cell differentiation by immunostaining and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis. 
		NOTE: Refer to Toyoda et al.16 and Kimura et al.26 for detailed experimental procedures. Refer Table 1 for primer sequences used for qRT-PCR analysis.</li>
	</ol>
	</li>
</ol>
3. Flow Cytometry (FCM)
<ol>
	<li>Aspirate the used medium and add 2 mL of 0.5 mM EDTA to the well by pipette. Repeat the aspiration and addition of 0.5 mM EDTA one time. 
	NOTE: Shake the plate gently to remove any dead cells from the monolayer cells before each aspiration.</li>
	<li>To dissociate the cells (approximately 3-6 &#215; 106 cells per well), add 2 mL of 0.25% Trypsin containing 0.5 mM EDTA per well. Incubate the plates at 37 &#176;C for 5-10 min.</li>
	<li>Pipette gently but quickly to dissociate clumped cells into single cells. Do not bubble the cell suspension during pipetting.</li>
	<li>Add 8-10 mL of base medium (RPMI 1640 or iMEM, RT) containing 0.5x serum-free supplement and 10 &#956;M Y-27632 per well and mix the cell suspension. Transfer the cell suspension into a centrifuge tube.</li>
	<li>Centrifuge the tube at 300 x g at RT for 5 min.</li>
	<li>Aspirate the supernatant and resuspend the cells with 2 mL of 0.5 mM EDTA (RT). Gently pipette the cell suspension.</li>
	<li>Centrifuge the tube at 300 x g at RT for 5 min.</li>
	<li>Aspirate the supernatant and resuspend the cells with 100 &#956;L of fixation and permeabilization buffer per 106 cells under a hood. Gently pipette the cell suspension under a hood. Keep the tube at RT for 30 min.</li>
	<li>Centrifuge the tube at 400 x g at RT for 5 min.</li>
	<li>Aspirate the supernatant under a hood and resuspend the cells with 500 &#956;L FCM Blocking solution per 106 cells. Gently pipette the cell suspension. Keep the tube at RT for 30 min.</li>
	<li>Transfer 50 &#956;L of the cell suspension to a tube (approximately 1 x 105 cells). Centrifuge the tube at 400 x g at RT for 5 min. Remove the supernatant and resuspend the cells with 100 &#956;L of diluted primary antibody (see Table of Materials) in FCM blocking solution and incubate at 4 &#176;C for &#62;16 h.</li>
	<li>Centrifuge the tube at 400 x g at RT for 5 min. Remove the supernatant and resuspend the cells with 180 &#956;L of 1x Perm/Wash buffer.</li>
	<li>Centrifuge the tube at 400 x g at RT for 5 min. Remove the supernatant and resuspend the cells with 100 &#956;L of diluted secondary antibody (see Table of Materials) in FCM blocking solution. Incubate the cells at RT for 60 min or at 4 &#176;C for &#62;16 h with protection from light.</li>
	<li>Centrifuge the tube at 400 x g at RT for 5 min. Remove the supernatant and resuspend the cells with 180 &#956;L of 1x Perm/Wash buffer.</li>
	<li>Centrifuge the tube at 400 x g at RT for 5 min. Remove the supernatant and resuspend the cells with 180 &#956;L of 2% donkey serum in DPBS.</li>
	<li>Filter the cell suspension by transferring on a 5 mL round bottom polystyrene tube with a cell strainer (35 &#181;m nylon mesh) and keep at 4 &#176;C with protection from light until the analysis.</li>
	<li>Analyze a proportion of positively stained cells by a flow cytometer27.</li>
</ol>
4. Immunostaining 
<ol>
	<li>Aspirate the used medium and add 2 mL of DPBS to the well by pipette. Repeat the aspiration and addition of DPBS one time. 
	NOTE: Gently shake the plate to remove any dead cells from the monolayer cells before each aspiration.</li>
	<li>Add 2 mL of 4% paraformaldehyde (PFA) (4 &#176;C) per well by pipette under a hood. Keep the plate at 4 &#176;C for 20 min.</li>
	<li>Remove the PFA by pipette under a hood. Add 2 mL of DPBS at RT to the well by pipette under a hood. Repeat the aspiration and addition of DPBS one time.</li>
	<li>Add 2 mL of blocking solution per well and keep the plate at RT for 30 min.</li>
	<li>Aspirate the used solution and add 1 mL of primary antibody (see Table of Materials) in blocking solution per well. Incubate at RT for 60 min or at 4 &#176;C for &#62;16 h.</li>
	<li>Aspirate the used solution, add 2 mL of DPBS containing 0.4% Triton X-100 and incubate at RT for 10 min. Repeat the aspiration, addition of solution and incubation one time.</li>
	<li>Aspirate the used solution and add 1 mL of secondary antibody (see Table of Materials) in blocking solution per well. Incubate at RT for 60 min with protection&#160;from light.</li>
	<li>Aspirate the used solution, add 2 mL of DPBS containing 0.4% Triton X-100 (RT) and incubate at RT for 5 min with protection from light. Repeat the aspiration, addition of solution and incubation two times.</li>
	<li>Take micrographs to examine the differentiation efficiency under a fluorescence microscope28.</li>
</ol>

<ol>
	<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=Production+of+pancreatic+hormone-expressing+endocrine+cells+from+human+embryonic+stem+cells.">Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.</a> Nature Biotechnology. 24 (11), 1392-1401 (2006).</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>Kondo, Y., Toyoda, T., Inagaki, N., Osafune, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPSC+technology-based+regenerative+therapy+for+diabetes.">iPSC technology-based regenerative therapy for diabetes.</a> Journal of Diabetes Investigation. 9 (2), 234-243 (2017).</li><li>Millman, J. R., 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+stem+cell-derived+beta-cells+from+patients+with+type+1+diabetes.">Generation of stem cell-derived beta-cells from patients with type 1 diabetes.</a> Nature Communications. 7, 11463 (2016).</li><li>Zeng, H., 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+Isogenic+Human+ESC+Platform+for+Functional+Evaluation+of+Genome-wide-Association-Study-Identified+Diabetes+Genes+and+Drug+Discovery.">An Isogenic Human ESC Platform for Functional Evaluation of Genome-wide-Association-Study-Identified Diabetes Genes and Drug Discovery.</a> Cell Stem Cell. 19 (3), 326-340 (2016).</li><li>Hosokawa, 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=Insulin-producing+cells+derived+from+'induced+pluripotent+stem+cells'+of+patients+with+fulminant+type+1+diabetes:+Vulnerability+to+cytokine+insults+and+increased+expression+of+apoptosis-related+genes.">Insulin-producing cells derived from 'induced pluripotent stem cells' of patients with fulminant type 1 diabetes: Vulnerability to cytokine insults and increased expression of apoptosis-related genes.</a> Journal of Diabetes Investigation. , (2017).</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>Serup, P., 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=The+homeodomain+protein+IPF-1/STF-1+is+expressed+in+a+subset+of+islet+cells+and+promotes+rat+insulin+1+gene+expression+dependent+on+an+intact+E1+helix-loop-helix+factor+binding+site.">The homeodomain protein IPF-1/STF-1 is expressed in a subset of islet cells and promotes rat insulin 1 gene expression dependent on an intact E1 helix-loop-helix factor binding site.</a> Biochemical Journal. 310 (Pt 3), 997-1003 (1995).</li><li>Riedel, M. 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=Immunohistochemical+characterisation+of+cells+co-producing+insulin+and+glucagon+in+the+developing+human+pancreas.">Immunohistochemical characterisation of cells co-producing insulin and glucagon in the developing human pancreas.</a> Diabetologia. 55 (2), 372-381 (2012).</li><li>Mae, S. I., 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=Monitoring+and+robust+induction+of+nephrogenic+intermediate+mesoderm+from+human+pluripotent+stem+cells.">Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells.</a> Nature Communications. 4, 1367 (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>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=Rho-Associated+Kinases+and+Non-muscle+Myosin+IIs+Inhibit+the+Differentiation+of+Human+iPSCs+to+Pancreatic+Endoderm.">Rho-Associated Kinases and Non-muscle Myosin IIs Inhibit the Differentiation of Human iPSCs to Pancreatic Endoderm.</a> Stem Cell Reports. 9 (2), 419-428 (2017).</li><li>Chen, K. G., Mallon, B. S., McKay, R. D., Robey, P. G. <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+culture:+considerations+for+maintenance,+expansion,+and+therapeutics.">Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics.</a> Cell Stem Cell. 14 (1), 13-26 (2014).</li><li>Bauwens, C. L., 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=Control+of+human+embryonic+stem+cell+colony+and+aggregate+size+heterogeneity+influences+differentiation+trajectories.">Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories.</a> Stem Cells. 26 (9), 2300-2310 (2008).</li><li>Nguyen, Q. H., 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=Single-cell+RNA-seq+of+human+induced+pluripotent+stem+cells+reveals+cellular+heterogeneity+and+cell+state+transitions+between+subpopulations.">Single-cell RNA-seq of human induced pluripotent stem cells reveals cellular heterogeneity and cell state transitions between subpopulations.</a> Genome Research. 28 (7), 1053-1066 (2018).</li><li>Narsinh, K. H., 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=Single+cell+transcriptional+profiling+reveals+heterogeneity+of+human+induced+pluripotent+stem+cells.">Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells.</a> Journal of Clinical Investigation. 121 (3), 1217-1221 (2011).</li><li>Rosowski, K. A., Mertz, A. F., Norcross, S., Dufresne, E. R., Horsley, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Edges+of+human+embryonic+stem+cell+colonies+display+distinct+mechanical+properties+and+differentiation+potential.">Edges of human embryonic stem cell colonies display distinct mechanical properties and differentiation potential.</a> Scientific Reports. 5, 14218 (2015).</li><li>Torres-Padilla, M. E., Chambers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+heterogeneity+in+pluripotent+stem+cells:+a+stochastic+advantage.">Transcription factor heterogeneity in pluripotent stem cells: a stochastic advantage.</a> Development. 141 (11), 2173-2181 (2014).</li><li>Cahan, P., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+implications+of+pluripotent+stem+cell+variability+and+heterogeneity.">Origins and implications of pluripotent stem cell variability and heterogeneity.</a> Nature Reviews Molecular and Cell Biology. 14 (6), 357-368 (2013).</li><li>Chetty, 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=A+simple+tool+to+improve+pluripotent+stem+cell+differentiation.">A simple tool to improve pluripotent stem cell differentiation.</a> Nature Methods. 10 (6), 553-556 (2013).</li><li>Kimura, 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=Small+molecule+AT7867+proliferates+PDX1-expressing+pancreatic+progenitor+cells+derived+from+human+pluripotent+stem+cells.">Small molecule AT7867 proliferates PDX1-expressing pancreatic progenitor cells derived from human pluripotent stem cells.</a> Stem Cell Research. 24, 61-68 (2017).</li><li>Bhattacharya, 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=High+efficiency+differentiation+of+human+pluripotent+stem+cells+to+cardiomyocytes+and+characterization+by+flow+cytometry.">High efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry.</a> Journal of Visualized Experiments. (91), e52010 (2014).</li><li>Honvo-Houeto, E., Truchet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+Immunofluorescence+on+Frozen+Sections+of+Mouse+Mammary+Gland.">Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland.</a> Journal of Visualized Experiments. (106), (2015).</li><li>Okita, K., 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=A+more+efficient+method+to+generate+integration-free+human+iPS+cells.">A more efficient method to generate integration-free human iPS cells.</a> Nature Methods. 8 (5), 409-412 (2011).</li><li>Kajiwara, 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=Donor-dependent+variations+in+hepatic+differentiation+from+human-induced+pluripotent+stem+cells.">Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells.</a> Proceedings of the National Academy of Science U S A. 109 (31), 12538-12543 (2012).</li><li>Kikuchi, 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=Human+iPS+cell-derived+dopaminergic+neurons+function+in+a+primate+Parkinson's+disease+model.">Human iPS cell-derived dopaminergic neurons function in a primate Parkinson's disease model.</a> Nature. 548 (7669), 592-596 (2017).</li><li>Suemori, H., 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+establishment+of+human+embryonic+stem+cell+lines+and+long-term+maintenance+with+stable+karyotype+by+enzymatic+bulk+passage.">Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage.</a> Biochemical and Biophysical Research Communication. 345 (3), 926-932 (2006).</li><li>Gage, B. K., Webber, T. D., Kieffer, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+cell+seeding+density+influences+pancreatic+endocrine+development+during+in+vitro+differentiation+of+human+embryonic+stem+cells.">Initial cell seeding density influences pancreatic endocrine development during in vitro differentiation of human embryonic stem cells.</a> PLoS One. 8 (12), e82076 (2013).</li><li>Nostro, 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=Efficient+generation+of+NKX6-1++pancreatic+progenitors+from+multiple+human+pluripotent+stem+cell+lines.">Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines.</a> Stem Cell Reports. 4 (4), 591-604 (2015).</li><li>Jonsson, J., Carlsson, L., Edlund, T., Edlund, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-promoter-factor+1+is+required+for+pancreas+development+in+mice.">Insulin-promoter-factor 1 is required for pancreas development in mice.</a> Nature. 371 (6498), 606-609 (1994).</li><li>Kelly, O. G., 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-surface+markers+for+the+isolation+of+pancreatic+cell+types+derived+from+human+embryonic+stem+cells.">Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells.</a> Nature Biotechnology. 29 (8), 750-756 (2011).</li><li>Hohwieler, 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=Human+pluripotent+stem+cell-derived+acinar/ductal+organoids+generate+human+pancreas+upon+orthotopic+transplantation+and+allow+disease+modelling.">Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling.</a> Gut. 66 (3), 473-486 (2017).</li><li>Takeuchi, H., Nakatsuji, N., Suemori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endodermal+differentiation+of+human+pluripotent+stem+cells+to+insulin-producing+cells+in+3D+culture.">Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture.</a> Scientific Reports. 4, 4488 (2014).</li></ol>

The authors have nothing to disclose.

The generation of PDX1+ cells is comprised of multiple steps; therefore, it is critical to treat cells at the appropriate time. Among the steps, the induction efficiency of definitive endoderm largely affects the final induction efficiency, possibly by interference from other contaminating lineage cells (i.e., mesoderm and ectoderm), which may proliferate and/or secrete factors that disrupt specific differentiation. If the proportion of SOX17+ cells is lower than 80% on day 4 (Stage 1B), an efficien...

The pancreas mainly consists of exocrine and endocrine cells, and its dysfunction or overload causes several diseases, such as pancreatitis, diabetes and pancreatic cancer. To elucidate the pathogeny of pancreatopathy, it is necessary to analyze the developmental process and function of pancreatic cells. In addition, a stable cell supply with robust quality is required to establish cell/tissue supplementation therapy. Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for these purposes, and the differentiation protocol toward pancreatic cells has been intensively studied1,2,3,4. Recent advances in the in vitro generation of pancreatic &#946; cells mimic the generation of &#946; cells in adult human, and these cells show therapeutic efficacy upon implantation into diabetic model mice2,3. In addition, the analysis of &#946; cells generated from the induced pluripotent stem cells (iPSCs) of healthy and type 1 diabetes patient donors revealed no functional differences including when under stress5. Moreover, disease phenotypes have been partially reproduced in induced pancreatic cells with patient-derived iPSCs or hPSCs harboring genetic mutations in the same site as the patients6,7.&#160;
To generate pancreatic cells from hPSCs, stepwise directed differentiation that recapitulates developmental processes is used. The pancreas is derived from the endoderm layer of the early embryo, which expresses sex determining region Y-box 17 (SOX17) and forkhead box A2 (FOXA2)8. Based on the mouse studies, the endodermal layer forms the primitive gut tube, which is marked by the expression of hepatocyte nuclear factor 1-beta (Hnf1&#946;) and hepatocyte nuclear factor 4-alpha (Hnf4&#945;). The primitive gut tube elongates and develops into the respiratory apparatus, digestive tract, and organs. After elongation, the posterior foregut area becomes the presumptive pancreatic region, as marked by the expression of the transcriptional factor pancreas/duodenum homeobox protein 1 (PDX1)8,9,10. The dorsal and ventral parts of the PDX1+ gut tube thicken to form pancreatic buds, which are marked by the co-expression of pancreas transcription factor 1 subunit alpha (PTF1A) and NK6 homeobox 1 (NKX6.1)8,11. This expression marks the morphological start of pancreatic organogenesis. Pancreatic endoderm cells, which are components of the pancreatic buds, form a branched tubular network of epithelial structures12 and eventually differentiate into exocrine and endocrine cells, including insulin-secreting &#946;-cells and glucagon-secreting &#945;-cells. Expression of PDX1 is detected first at the presumptive pancreatic region, which is then observed throughout the entire pancreatic development, and shows localization to &#946;- and &#948;-cells9,13,14. Although the Pdx1+ cell area that does not express Ptf1a or Nkx6.1 differentiates into the gastric antrum, duodenum, extrahepatic bile duct and some intestinal cells at the middle to late stage of development in mice9, PDX1+ cells are considered the progenitors of the pancreas at the early developmental stage in humans.&#160;
Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells for the generation of pancreatic lineages. The protocol initiates differentiation by seeding dissociated single cells15,16,17. Generally, undifferentiated hPSCs are maintained as colonies or cell aggregates in suspension or in adhesion. As a result, most protocols initiate the differentiation shortly after passaging. However, in the state of colonies or aggregates, cells are prone to spatial and transcriptional heterogeneities18,19,20,21,22, which might hamper the first differentiation step toward definitive endoderm followed by inefficient differentiation to PDX1+ cells. The present protocol may offer easy handling to improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently to endodermal lineages and PDX1+ cells23,24,25.

<table border="0" cellpadding="0" cellspacing="0" style="width:617px;" width="616">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">3-Keto-N-aminoethyl-N&#8242;-aminocaproyldihydrocinnamoyl cyclopamine</td>
			<td style="width:100px;">Toronto Research Chemicals</td>
			<td style="width:123px;">K171000</td>
			<td style="width:172px;">CYC</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]-benzoic acid</td>
			<td style="width:100px;">Santa Cruz Biotechnology</td>
			<td style="width:123px;">SC-203303</td>
			<td style="width:172px;">TTNPB</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">50 mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">339652</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Anti-CDX2 antibody [EPR2764Y]</td>
			<td style="width:100px;">Abcam</td>
			<td style="width:123px;">Ab76541</td>
			<td style="width:172px;">Anti-CDX2, &#215; 1/1000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">B-27 Supplement (50 &#215;)</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">17504-044</td>
			<td style="width:172px;">Serum-free supplement</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">BD FACSAria II Cell Sorter</td>
			<td style="width:100px;">BD Biosciences</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">For flow cytometry</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Biomedical freezer</td>
			<td style="width:100px;">SANYO</td>
			<td style="width:123px;">MDF-U538</td>
			<td>For -30 &#176;C storing</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Cell Counting Slides for TC10/TC20 Cell Counter, Dual-Chamber</td>
			<td style="width:100px;">BIO-RAD</td>
			<td style="width:123px;">1450011</td>
			<td style="width:172px;">Counting slide glass</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">CELL CULTURE MULTIWELL PLATE, 6 WELL, PS, CLEAR</td>
			<td style="width:100px;">Greiner bio-one</td>
			<td style="width:123px;">657165</td>
			<td style="width:172px;">For differentiation culture/6-well plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Centrifuge</td>
			<td style="width:100px;">TOMY</td>
			<td style="width:123px;">AX-310</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Centrifuge</td>
			<td style="width:100px;">TOMY</td>
			<td style="width:123px;">MX-305</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">CHIR99021</td>
			<td style="width:100px;">Axon Medchem</td>
			<td style="width:123px;">Axon 1386</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">CLEAN BENCH</td>
			<td style="width:100px;">SHOWA KAGAKU&#160;</td>
			<td style="width:123px;">S-1601PRV</td>
			<td style="width:172px;">Clean bench</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Corning CellBIND 6-well plate</td>
			<td style="width:100px;">Corning</td>
			<td style="width:123px;">3335</td>
			<td style="width:172px;">For feeder-free culture of hPSCs/6-well plate</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Corning Matrigel Basement Membrane Matrix Growth Factor Reduced</td>
			<td style="width:100px;">Corning</td>
			<td style="width:123px;">354230</td>
			<td style="width:172px;">Basement membrane matrix</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Corning Synthemax II-SC Substrate</td>
			<td style="width:100px;">Corning</td>
			<td style="width:123px;">3535</td>
			<td style="width:172px;">For feeder-free culture of hPSCs/synthetic surface material for hPSCs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Cryostat</td>
			<td style="width:100px;">Leica</td>
			<td style="width:123px;">Leica CM1510 S</td>
			<td style="width:172px;">For immunostaining of aggregates.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:223px;">Cytofix/Cytoperm Kit</td>
			<td style="width:100px;">Becton Dickinson</td>
			<td style="width:123px;">554714</td>
			<td style="width:172px;">Perm/Wash buffer is&#160; Permeabilization/Wash buffer. Cytofix/Cytoperm buffer is fixation and permeabilization buffer.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Dako pen</td>
			<td style="width:100px;">Dako</td>
			<td style="width:123px;">S2002</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">dNTP mix (10 mM)</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td>18427-088</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">A11055</td>
			<td style="width:172px;">Secondary antibody, &#215; 1/500 dilution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">A10036</td>
			<td style="width:172px;">Secondary antibody, &#215; 1/500 dilution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">A10040</td>
			<td style="width:172px;">Secondary antibody, &#215; 1/500 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Donkey Serum</td>
			<td style="width:100px;">Merck Millipore</td>
			<td style="width:123px;">S30</td>
			<td style="width:172px;">Donkey serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">D-PBS(-) without Ca or Mg</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td style="width:123px;">14249-95</td>
			<td style="width:172px;">DPBS</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Essential 8 Medium</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">A1517001</td>
			<td style="width:172px;">For feeder-free culture of hPSCs/hPSC maintenance medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Falcon 5mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td style="width:100px;">Corning</td>
			<td style="width:123px;">352235</td>
			<td style="width:172px;">5 mL round bottom polystyrene tube with cell strainer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Filter Tip, 1000 &#181;L</td>
			<td style="width:100px;">Watoson</td>
			<td style="width:123px;">124-1000S</td>
			<td style="width:172px;">Use together with pipettes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Filter Tip, 20 &#181;L</td>
			<td style="width:100px;">Watoson</td>
			<td style="width:123px;">124-P20S</td>
			<td style="width:172px;">Use together with pipettes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Filter Tip, 200 &#181;L</td>
			<td style="width:100px;">Watoson</td>
			<td style="width:123px;">124-P200S</td>
			<td style="width:172px;">Use together with pipettes</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Fluorescence Microscope</td>
			<td style="width:100px;">Keyence</td>
			<td style="width:123px;">BZ-X700</td>
			<td style="width:172px;">For immunostaining</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Forma Steri-Cycle CO2 incubator</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">370A</td>
			<td style="width:172px;">Incubator</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">HNF-1&#946; Antibody (C-20)</td>
			<td style="width:100px;">Santa Cruz Biotechnology</td>
			<td style="width:123px;">sc-7411</td>
			<td style="width:172px;">Anti-HNF1&#946;, &#215; 1/200 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">HNF-4&#945; Antibody (H-171)</td>
			<td style="width:100px;">Santa Cruz Biotechnology</td>
			<td style="width:123px;">sc-8987</td>
			<td style="width:172px;">Anti-HNF4&#945;, &#215; 1/200 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Hoechst 33342</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">H3570</td>
			<td style="width:172px;">For nucleus staining, &#215; 1/200 dilution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Human Pancreas Total RNA</td>
			<td style="width:100px;">Ambion</td>
			<td style="width:123px;">AM7954</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Human PDX-1/IPF1 Antibody</td>
			<td style="width:100px;">R&#38;D Systems</td>
			<td style="width:123px;">AF2419</td>
			<td style="width:172px;">Anti-PDX1, goat IgG, &#215; 1/200 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Human SOX17 Antibody</td>
			<td style="width:100px;">R&#38;D Systems</td>
			<td style="width:123px;">AF1924</td>
			<td style="width:172px;">Anti-SOX17, &#215; 1/200 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Improved MEM Zinc Option medium</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">10373-017</td>
			<td style="width:172px;">iMEM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Incubation chamber</td>
			<td style="width:100px;">Cosmo Bio</td>
			<td style="width:123px;">10DO</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Latex Examination Gloves</td>
			<td style="width:100px;">Adachi</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">MAS coated slide glass</td>
			<td style="width:100px;">Matsunami Glass</td>
			<td>83-1881</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">MicroAmp Fast 96-well Reaction Plate</td>
			<td style="width:100px;">Applied Biosystems/Thermo Fisher Scientific</td>
			<td style="width:123px;">4346907</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Microscope</td>
			<td style="width:100px;">Olympus</td>
			<td style="width:123px;">CKX41N-31PHP</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Microtube</td>
			<td style="width:100px;">Watoson</td>
			<td style="width:123px;">131-515CS</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Monoclonal Anti-&#945;-Fetoprotein</td>
			<td style="width:100px;">SIGMA</td>
			<td style="width:123px;">A8452</td>
			<td style="width:172px;">Anti-AFP, &#215; 1/200 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Nanodeop 8000</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Oligo dT</td>
			<td style="width:100px;">FASMAC</td>
			<td style="width:123px;">Custom made Oligo&#160;</td>
			<td style="width:172px;">For RT-qPCR of sequence is &#34;TTTTTTTTTTTTTTTTTTTT&#34;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Paraformaldehyde, powder</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td style="width:123px;">26126-54</td>
			<td style="width:172px;">PFA, fixative, diluted in DPBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Pharmaceutical refrigerator</td>
			<td style="width:100px;">SANYO</td>
			<td style="width:123px;">MPR-514</td>
			<td>For 4 &#176;C storing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">PIPETMAN P&#160;</td>
			<td style="width:100px;">GILSON</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">Pipette</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Recombinant Human KGF/FGF-7</td>
			<td style="width:100px;">R&#38;D Systems</td>
			<td style="width:123px;">251-KG</td>
			<td style="width:172px;">KGF</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Recombinant Human Noggin</td>
			<td style="width:100px;">PeproTech</td>
			<td style="width:123px;">120-10C</td>
			<td style="width:172px;">NOGGIN</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Recombinant Human/Mouse/Rat Activin A</td>
			<td style="width:100px;">R&#38;D Systems</td>
			<td style="width:123px;">338-AC</td>
			<td style="width:172px;">Activin A</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">ReverTra Ace (100 U/&#956;L)</td>
			<td style="width:100px;">TOYOBO</td>
			<td style="width:123px;">TRT-101</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Rnase-Free Dnase Set (50)</td>
			<td style="width:100px;">QIAGEN</td>
			<td style="width:123px;">79254</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Rneasy Mini Kit</td>
			<td style="width:100px;">QIAGEN</td>
			<td style="width:123px;">74104</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">RPMI 1640 with L-Gln</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td style="width:123px;">30264-85</td>
			<td style="width:172px;">RPMI 1640</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sealing Film for Real Time</td>
			<td style="width:100px;">Takara</td>
			<td style="width:123px;">NJ500</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Serological pipettes 10 mL</td>
			<td style="width:100px;">Costar/Corning</td>
			<td style="width:123px;">4488</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Serological pipettes 25 mL</td>
			<td style="width:100px;">Costar/Corning</td>
			<td>4489</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Serological pipettes 5 mL</td>
			<td style="width:100px;">Costar/Corning</td>
			<td style="width:123px;">4487</td>
			<td style="width:172px;">For cell culturing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Sox2 (D6D9) XP Rabbit mAb</td>
			<td style="width:100px;">Cell signaling</td>
			<td style="width:123px;">3579S</td>
			<td style="width:172px;">Anti-SOX2, &#215; 1/200 dilution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">StepOnePlus</td>
			<td style="width:100px;">Applied Biosystems/Thermo Fisher Scientific</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Sucrose</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td>30406-25</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">TB Green 
			Premix Ex Taq II&#160;</td>
			<td style="width:100px;">Takara</td>
			<td>RR820B</td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">TC20 Automated Cell Counter</td>
			<td style="width:100px;">BIO-RAD</td>
			<td style="width:123px;">1450101J1</td>
			<td style="width:172px;">Automatic cell counter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Tissue-Tek OCT compound 4583&#160;</td>
			<td style="width:100px;">Sakura Finetechnical</td>
			<td>4583</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Tissue-Tek Cryomold Molds/Adapters</td>
			<td style="width:100px;">Sakura Finetechnical</td>
			<td>4566</td>
			<td style="width:172px;">For immunostaining of aggregates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Triton X-100</td>
			<td style="width:100px;">Nacalai tesque</td>
			<td style="width:123px;">35501-15</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Trypan Blue</td>
			<td style="width:100px;">BIO-RAD</td>
			<td style="width:123px;">1450021</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Ultracold freezer</td>
			<td style="width:100px;">SANYO</td>
			<td style="width:123px;">MDF-U33V</td>
			<td>For -80 &#176;C storing</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">UltraPure 0.5M EDTA, pH 8.0</td>
			<td style="width:100px;">Thermo Fisher Scientific</td>
			<td style="width:123px;">15575-038</td>
			<td style="width:172px;">Dilute with DPBS to prepare 0.5 mM EDTA</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:223px;">Veriti Thermal Cycler</td>
			<td style="width:100px;">Applied Biosystems/Thermo Fisher Scientific</td>
			<td style="width:123px;"></td>
			<td style="width:172px;">For RT-qPCR</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:223px;">Y-27632</td>
			<td style="width:100px;">Wako</td>
			<td style="width:123px;">251-00514</td>
			<td style="width:172px;"></td>
		</tr>
	</tbody>
</table>

efficient generation of pancreas/duodenum homeobox protein 1+ posterior foregut/pancreatic progenitors from hpscs in adhesion cultures

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

Propagating hiPSCs (585A129,30) are condensed and form a homogenous monolayer (Figure 1B) that is suitable for differentiation. Undifferentiated hiPSCs (Stage 0) are dissociated and re-seeded as single cells at low cell densities (1-1.5 x 105 cells/cm2). Within 1 h, the cells are attached to the plate and start to show protrusion. On day 1, the cells are proliferated and well distributed to cover 80-90% of the s...

Watch this Scientific Journal Video about Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures at JoVE.com

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

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

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

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

efficient-generation-pancreasduodenum-homeobox-protein-1-posterior

Research

JoVE Journal

Developmental Biology

6.0K Views.  Kyoto University. Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

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

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

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

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

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

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

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

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

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

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

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

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

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 ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

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

Summary

Explore More Videos

Chapters in this video

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

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

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

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

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

Generation of Organoids from Mouse Extrahepatic Bile Ducts