This work was funded by a grant from Qatar National Research Fund (QNRF) (Grant No. NPRP10-1221-160041).

The study has been approved by the appropriate institutional research ethics committee and performed following the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The protocol was approved by the Institutional Review Board (IRB) of HMC (no. 16260/16) and Qatar Biomedical Research Institute (QBRI) (no. 2016-003). This work is optimized for hESCs such as H1, H9, and HUES8. Blood samples were obtained from healthy individuals from Hamad Medical Corporation (HMC) hospital with full informed consent. The iPSCs are generated from peripheral blood mononuclear cells (PBMCs) of control, healthy individual23.
1. Preparation of the culture media
<ol>
	<li>Prepare human pluripotent stem cells (hPSC) culture media
	<ol>
		<li>Prepare hPSC culture media from the commercially available medium for maintaining and expanding human embryonic stem cells by supplementing 100 units/mL of Penicillin and 100 ug/mL of Streptomycin (see Table of Materials). Aliquot and store the complete media at -20 &#176;C for the long term or 4 &#176;C for immediate use.</li>
	</ol>
	</li>
	<li>Prepare Stage 1 differentiation media (for Definitive Endoderm (DE)).
	<ol>
		<li>Prepare Basal media from MCDB 131 media by supplementing with 0.5% of fatty acid-free Bovine serum albumin (FFA-BSA), 1.5 g/L of Sodium bicarbonate (NaHCO3), 10 mM of glucose, 2 mM of Glutamax, 100 units/mL of Penicillin, and 100 &#181;g/mL of Streptomycin (see Table of Materials). Filter the prepared media using a 0.2 &#181;m filter and store at 4 &#176;C.</li>
		<li>Prepare Stage 1 media containing CHIR99021 from the Basal media (prepared in step 1.2.1) by warming the basal media at 37 &#176;C and then supplementing with 2 &#181;M of CHIR99021, 100 ng/mL of Activin A, 10 &#181;M of Rock inhibitor (Y-27632), and 0.25 mM of Vitamin C (see Table of Materials). Mix the supplemented media well and cover it with aluminum foil to protect it from light. 
		NOTE: This media will be used only on the first day of differentiation.</li>
		<li>Prepare Stage 1 media without CHIR99021 from the Basal media by warming it at 37 &#176;C and supplementing it with 100 ng/mL of Activin A and 0.25 mM of Vitamin C and mix well. 
		NOTE: 5 ng/mL of basic FGF or FGF2 can be optionally added to this media. This media will be used for the remaining days of Stage 1.</li>
	</ol>
	</li>
	<li>Prepare Stage 2 differentiation media (for Primitive Gut Tube; PGT).
	<ol>
		<li>Prepare Stage 2 differentiation media containing Rock inhibitor from the Basal media by warming it at 37 &#176;C and supplementing with 50 ng/mL of FGF10, 50 ng/mL of NOGGIN, 0.25 &#181;M of CHIR99021, 10 &#181;M of Y-27632 (Rock inhibitor), and 0.25 mM of Vitamin C. 
		NOTE: This media is used on the day of dissociation of endodermal cells.</li>
		<li>Prepare Stage 2 differentiation media without Rock inhibitor following the same procedure for the Media prepared in step 1.3.1, excluding the Rock inhibitor. 
		NOTE: This media is used on the second day of Stage 2.</li>
	</ol>
	</li>
	<li>Prepare Stage 3 differentiation media (for Posterior Foregut).
	<ol>
		<li>Prepare DMEM media containing 4.5 g/L of Glucose, then supplement with 1% of Penicillin-streptomycin and 2 mM of Glutamax and use as basal media for Stages 3 and 4.</li>
		<li>Warm the DMEM media (prepared in step 1.4.1) and supplement with 2 &#181;M of Retinoic acid, 0.25 &#181;M of SANT-1, 50 ng/mL of FGF10, 50 ng/mL of NOGGIN, 0.25 mM of Vitamin C, and 1% B27 supplement without vitamin A (see Table of Materials). 
		NOTE: This media is used for 4 days of Stage 3 for P2-D (Protocol 2, optimized, dissociated) and 2 days of Stage 3 for P1-ND (Protocol 1, non-optimized, non-dissociated).</li>
	</ol>
	</li>
	<li>Prepare Stage 4 differentiation media (for Pancreatic Progenitors).
	<ol>
		<li>Add DMEM containing 4.5 g/L of Glucose with 100 ng/mL of EGF, 10 mM of Nicotinamide, 50 ng/mL of NOGGIN, 0.25 mM of Vitamin C, and 1% of B27 supplement without Vitamin A (see Table of Materials). Use this media for all days of Stage 4. 
		NOTE: Maintain all reagents at appropriate temperatures, and all cytokine and sensitive reagents need to be aliquoted. Thaw the aliquoted reagents and add to the basal media at the time of changing the differentiation media. The compositions of the different differentiation media are provided in Table 1.</li>
	</ol>
	</li>
</ol>
2. Preparation of basement membrane matrix coated dishes
<ol>
	<li>Thaw commercially available basement matrix (see Table of Materials) and aliquot on ice; freeze the aliquots at -20 &#176;C.</li>
	<li>Before coating the tissue culture dishes, thaw the frozen aliquots on ice. Add appropriate volume of the membrane matrix solution to the chilled KO-DMEM/F-12 media (KnockOut DMEM/F-12) (see Table of Materials) to achieve the desired diluted concentration and mix well. Store the diluted matrix solution at 4 &#176;C for immediate use.</li>
	<li>Cover the surface of the tissue culture-treated plates (see Table of Materials) with diluted membrane matrix solution and place the plates at 37 &#176;C for at least 60 min before plating the cells. Use a dilution of 1:50 of the membrane matrix solution in KO-DMEM/F-12 for plating cells for pancreatic differentiation experiment and 1:80 for expansion of undifferentiated hPSCs.</li>
</ol>
3. Culture of undifferentiated hPSCs
<ol>
	<li>Passage the hPSCs when the colonies reach a 70%-80% confluency.</li>
	<li>To passage, wash the hPSCs once with warm PBS, aspirate using a portable vacuum aspirator inside the tissue culture hood, and add 0.5 mM of EDTA solution in PBS to cover the colony surface. Incubate at 37 &#176;C, 5% CO2 for 1 min or until the colonies&#39; borders detach from the plate surface.</li>
	<li>Remove EDTA solution and collect the detaching colonies with hPSC culture medium using a P1000 micropipette. Centrifuge the collected cells at 128 x g for 4 min at room temperature. Discard the supernatant and supplement the cells with hPSC culture media containing 10 &#181;M of Y-27632 (Rock inhibitor)23,24. 
	&#8203;NOTE: Passage the hPSCs in at least a 1:3 ratio on 1:80 membrane matrix-coated dishes. However, for differentiation experiments, plate the hPSCs on 1:50 coated dishes.</li>
</ol>
4. Induction of Definitive Endoderm (DE) differentiation in hPSCs (Stage 1)
<ol>
	<li>When the hPSC colonies reach a confluency of 70%-80%, wash them twice with warm PBS and aspirate using a portable vacuum aspirator inside the tissue culture hood to begin Stage 1 differentiation.</li>
	<li>Add Stage 1 differentiation medium containing CHIR99021 (from step 1.2.2) to the colonies, 2 mL per 6-well plate, and incubate at 37 &#176;C for 24 h.</li>
	<li>The next day, replace the spent media with Stage 1 differentiation medium without CHIR99021 (step 1.2.3).</li>
	<li>Every 24 h, aspirate the spent media using a portable vacuum aspirator inside the tissue culture hood and replace it with a fresh Stage 1 differentiation medium without CHIR99021. 
	&#8203;NOTE: Stage 1 can be extended up to 4 days. The length of Stage 1 is dependent on the hPSC line being used and should be optimized accordingly.</li>
</ol>
5. Immunofluorescence analysis of hPSC-derived DE (Stage 1)
<ol>
	<li>For immunofluorescence, aspirate the spent media using a portable vacuum aspirator inside the tissue culture hood from the wells and wash twice with warm PBS. Swirl the plate to get rid of any cell debris.</li>
	<li>Cover the surface of the wells with 4% of paraformaldehyde (PFA) to fix the DE cells; for example, add 250 uL of PFA per well of a 24-well plate. Place the plate on a 2D shaker at 20 x g for 20 min.</li>
	<li>After fixation, wash the DE cells with tris-buffered saline with 0.5% of Tween (TBST) (see Table of Materials) and place the plate on the shaker at 20 x g for 10 min. Repeat this step once more.</li>
	<li>Permeabilize the fixed cells by adding a generous volume of phosphate-buffered saline with 0.5% of Triton X-100 (PBST); for example, add 1 mL of PBST per well of a 24-well plate and place the plate back on the shaker at 20 x g for 20 min.</li>
	<li>Freshly prepare 5%-6% of BSA in PBST as blocking buffer and add it to the permeabilized cells. Incubate the plate for at least 1 h in the blocking solution on the shaker.</li>
	<li>Dilute the primary antibodies against SOX17 and FOXA2 together (see Table of Materials) in 2%-3% BSA in PBST solution. Add the combined antibodies to blocked cells and place the plate on the shaker at 4 &#176;C overnight at a low speed with gentle shaking. 
	NOTE: SOX17 and FOXA2 are well-established DE markers25,26.</li>
	<li>The next day, aspirate the primary antibodies using a portable vacuum filter and wash the wells with TBST three times, each wash for 10 mins on the shaker.</li>
	<li>Prepare 1:500 dilution of Alexa fluor 488- and 568- conjugated secondary antibodies (see Table of Materials) against the species the primary antibodies were raised in.</li>
	<li>Add the secondary antibody combination to the stained well and cover the plate with aluminum foil to protect from light. Place the plate on the shaker for 1 h at room temperature.</li>
	<li>Aspirate the secondary antibody solution using a portable vacuum filter and wash the stained wells with TBST on the shaker for 10 min, covering the plate with foil. Repeat the wash step a total of three times.</li>
	<li>Prepare a 1 &#181;g/mL of Hoechst 33342 dilution in PBS to stain the nuclei. Add the Hoechst solution to the wells and place the plate on the shaker for 2-3 min.</li>
	<li>Aspirate the Hoechst solution using a portable vacuum filter and rinse the wells with PBS twice.</li>
	<li>Finally, add PBS to the stained cells and image them using an inverted fluorescence microscope (see Table of Materials) in the dark. Keep the plate covered with foil when not imaging to minimize the fluorophore bleaching. 
	&#8203;NOTE: Alternatively, flow-cytometry can be used to assess DE efficiency as described in step 9.2.</li>
</ol>
6. Generation of the primitive gut tube (PGT) from hPSCs (Stage 2)
NOTE: If the immunofluorescence analysis in step 5.13 is determined to be a SOX17-FOXA2 co-expression of 80% and above, the experiment proceeds to Stage 2. If the efficiency is &#60;80%, extend the duration of Stage 1 to 4 days.
<ol>
	<li>On day 1 of Stage 2, dissociate the hPSC-derived endodermal cells using TrypLE or Accutase (see Table of Materials) for the optimized P2-D protocol. Wash the adherent cells with warm PBS and add warm 1 mL of TrypLE or Accutase solution per well of a 6-well plate for 3-5 min at 37 &#176;C, 5% CO2, or until the cells begin to detach from one another.</li>
	<li>Dissociate the detached sheets or monolayer of cells in the wells and then collect them together in a 15 mL polypropylene tube using basal Stage 1/2 media without cytokines containing at least 0.5% of either fetal bovine serum (FBS) or KnockOut serum (KOSR) (see Table of Materials).</li>
	<li>Spin down the cells at 800 x g for 5 min at 4 &#176;C and discard the supernatant. Add 1 mL of sterile PBS and resuspend the pellet into single cells.</li>
	<li>Count the cells using an automated counter (see Table of Materials) by loading the recommended volume of cells in the chamber slide. Spin the resuspended cells at 800 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the pellet in the appropriate volume of Stage 2 differentiation medium containing Rock inhibitor at a density of 2.5-3.5 x 105 cells/cm2. 
	NOTE: This count may come down to a 1:2 splitting ratio for most cell lines, dependent on the proliferation rate. The total volume will be 2 mL of media with resuspended cells in 1 a well of 6-well plate.</li>
	<li>Plate the resuspended cells on 1:50 membrane matrix-coated plates (prepared in step 2) and incubate them at 37 &#176;C, 5% CO2 in the incubator.</li>
	<li>24 h later, replace the media with Stage 2 differentiation medium without Rock inhibitor (prepared in step 1.3.2).</li>
</ol>
7. Generation of posterior foregut from hPSCs (Stage 3)
<ol>
	<li>Aspirate the spent Stage 2 media using a portable vacuum aspirator inside the tissue culture hood and wash the cells with warm PBS.</li>
	<li>Add Stage 3 differentiation media from step 1.4 to the cells and incubate at 37 &#176;C, 5% CO2.</li>
	<li>After 24 h, replace the spent media with freshly prepared Stage 3 differentiation media. Repeat this for a total of 4 days for P2-D protocol (optimized) and only 2 days for the non-dissociated P1-ND.</li>
</ol>
8. Generation of Pancreatic Progenitors from hPSCs (Stage 4)
<ol>
	<li>After 4 days of Stage 3 treatment, wash the cells with warm PBS, gently swirl the plate, and aspirate using a portable vacuum aspirator. Then, add Stage 4 differentiation media from step 1.5 to the cells.</li>
	<li>After 24 h, replace the spent media with freshly prepared Stage 4 media. Repeat this for a total of 4 days.</li>
</ol>
9. Assessment of differentiation efficiency of generating pancreatic progenitors from hPSCs
<ol>
	<li>Perform the immunofluorescence analysis of the hPSC-derived pancreatic progenitors (Stage 4) for expression of PDX1 and NKX6.1. 
	NOTE: PDX1 and NKX6.1 are well-established pancreatic progenitor markers17,18.
	<ol>
		<li>Perform fixation, permeabilization, blocking, and antibody incubation and washes according to step 5.</li>
		<li>Stain the hPSC-derived pancreatic progenitors with a combination of PDX1 and NKX6.1 antibodies (see Table of Materials) diluted in 2%-3% BSA in PBST.</li>
		<li>Use 1:500 dilutions of appropriate Alexa fluor 488- and 568- conjugated secondary antibodies (see Table of Materials).</li>
	</ol>
	</li>
	<li>Perform flow-cytometry analysis of hPSC-derived pancreatic progenitors for expression of PDX1 and NKX6.1. 
	NOTE: Flow-cytometry analysis of pancreatic markers in the generated hPSC-derived pancreatic progenitors provides a way to quantify the PDX1 and NKX6.1 co-expressing cells.
	<ol>
		<li>At the end of Stage 4, wash the cells twice with warm PBS and add enough TrypLE or Accutase to cover the surface of the wells, for example, 1 mL of TrypLE or Accutase per well of 6-well plate. Place the plate in the incubator for 5-7 min or until the cells detach from the surface.</li>
		<li>Dissociate the adherent sheets of cells within the well using a P1000 micropipette before collecting them in a 15 mL polypropylene tube.</li>
		<li>Spin down the cells in hPSC-derived pancreatic progenitors at 800 x g for 5 min at 4 &#176;C, then discard the supernatant. Wash the cells with PBS by dissociating them into single cells. Count the cells using an automated counter (see Table of Materials) and note the concentration in the number of cells per mL.</li>
		<li>Spin at 800 x g for 5 min at 4 &#176;C, and discard the supernatant. Add 200 &#181;L of chilled PBS to the pellet and dissociate.</li>
		<li>Add 2 mL of chilled 80% ethanol dropwise, with the tube on a vortex at low-medium speed (400 x g at room temperature). Close the caps tightly and place the tubes slightly tilted on the shaker at 4 &#176;C overnight.</li>
		<li>Spin down the cells at 800 x g for 5 min at 4 &#176;C, and wash with PBS to dissociate any clumps of fixed cells.</li>
		<li>Block the fixed cells with 5%-6% BSA solution in PBST for at least 1 h at room temperature or 4 &#176;C overnight on the shaker.</li>
		<li>Stain for the pancreatic progenitor markers following the steps below.
		<ol>
			<li>Distribute 2,00,000 cells per condition, including appropriate isotype controls dependent on the IgG subclass of the host species of the primary antibody, unstained and secondary antibody controls in a 96-well V bottom plate or 1.5 mL centrifuge tubes.</li>
			<li>Spin down the plate at 800 x g for 5 min at 4 &#176;C and flip the plate with a swift motion to discard the supernatant without losing the pellets. Prepare primary antibody dilutions in 3% BSA solution (see Table of Materials). 
			NOTE: The concentration of primary antibodies can be between 1:50 to 1:200.</li>
			<li>Incubate the stained cells for at least 2 h at room temperature or overnight at 4 &#176;C on a shaker with gentle shaking at low speed.</li>
			<li>Wash the stained cells with TBST thrice by pipetting the cells up and down in the wells. Spin and discard the supernatant as in step 9.2.8.2.</li>
			<li>Add 1:500 dilution of secondary antibodies (Alexa fluor 488- and 647-conjugated antibodies) prepared in PBS (see Table of Materials). Incubate for 30 min at room temperature.</li>
			<li>Wash the stained cells with TBST at least twice by pipetting the cells up and down. Spin the plate and discard the supernatant as in step 9.2.8.2.</li>
			<li>Collect the stained cells in at least 100 &#181;L of PBS and transfer them to light-protected FACS tubes (see Table of Materials). Run the samples on a flow-cytometry machine.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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M. <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+human+pluripotent+stem+cells+into+two+distinct+NKX6.1+populations+of+pancreatic+progenitors.">Differentiation of human pluripotent stem cells into two distinct NKX6.1 populations of pancreatic progenitors.</a> Stem Cell Research &amp; Therapy. 9 (1), 83 (2018).</li><li>Ali, 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=Keratinocytes+derived+from+patient-specific+induced+pluripotent+stem+cells+recapitulate+the+genetic+signature+of+psoriasis+disease.">Keratinocytes derived from patient-specific induced pluripotent stem cells recapitulate the genetic signature of psoriasis disease.</a> Stem Cells and Development. 29 (7), 383-400 (2020).</li><li>Elsayed, A. 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=Generation+of+a+human+induced+pluripotent+stem+cell+line+(QBRIi009-A)+from+a+patient+with+a+heterozygous+deletion+of+FOXA2.">Generation of a human induced pluripotent stem cell line (QBRIi009-A) from a patient with a heterozygous deletion of FOXA2.</a> Stem Cell Research. 42, 101705 (2020).</li><li>Davenport, C., Diekmann, U., Budde, I., Detering, N., Naujok, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior-posterior+patterning+of+definitive+endoderm+generated+from+human+embryonic+stem+cells+depends+on+the+differential+signaling+of+retinoic+acid,+Wnt-,+and+BMP-signaling.">Anterior-posterior patterning of definitive endoderm generated from human embryonic stem cells depends on the differential signaling of retinoic acid, Wnt-, and BMP-signaling.</a> Stem Cells. 34 (11), 2635-2647 (2016).</li><li>Schroeder, I. 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=Induction+and+selection+of+Sox17-expressing+endoderm+cells+generated+from+murine+embryonic+stem+cells.">Induction and selection of Sox17-expressing endoderm cells generated from murine embryonic stem cells.</a> Cells Tissues Organs. 195 (6), 507-523 (2012).</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>Elsayed, A. K., Younis, I., Ali, G., Hussain, K., Abdelalim, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+development+of+pancreatic+beta+cells+derived+from+human+iPSCs+with+FOXA2+deficiency.">Aberrant development of pancreatic beta cells derived from human iPSCs with FOXA2 deficiency.</a> Cell Death &amp; Disease. 12 (1), 103 (2021).</li><li>Mfopou, J. K., Chen, B., Mateizel, I., Sermon, K., Bouwens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noggin,+retinoids,+and+fibroblast+growth+factor+regulate+hepatic+or+pancreatic+fate+of+human+embryonic+stem+cells.">Noggin, retinoids, and fibroblast growth factor regulate hepatic or pancreatic fate of human embryonic stem cells.</a> Gastroenterology. 138 (7), 1-14 (2010).</li><li>Mfopou, J. K., De Groote, V., Xu, X., Heimberg, H., Bouwens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+and+other+soluble+factors+from+differentiating+embryoid+bodies+inhibit+pancreas+development.">Sonic hedgehog and other soluble factors from differentiating embryoid bodies inhibit pancreas development.</a> Stem Cells. 25 (5), 1156-1165 (2007).</li><li>Gattazzo, F., Urciuolo, A., Bonaldo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix:+A+dynamic+microenvironment+for+stem+cell+niche.">Extracellular matrix: A dynamic microenvironment for stem cell niche.</a> Biochimica et Biophysica Acta. 1840 (8), 2506-2519 (2014).</li><li>Watt, F. M., Huck, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+extracellular+matrix+in+regulating+stem+cell+fate.">Role of the extracellular matrix in regulating stem cell fate.</a> Nature Reviews Molecular Cell Biology. 14 (8), 467-473 (2013).</li><li>Shih, H. P., Panlasigui, D., Cirulli, V., Sander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECM+signaling+regulates+collective+cellular+dynamics+to+control+pancreas+branching+morphogenesis.">ECM signaling regulates collective cellular dynamics to control pancreas branching morphogenesis.</a> Cell Reports. 14 (2), 169-179 (2016).</li><li>Raza, A., Ki, C. S., Lin, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+matrix+properties+on+growth+and+morphogenesis+of+human+pancreatic+ductal+epithelial+cells+in+3D.">The influence of matrix properties on growth and morphogenesis of human pancreatic ductal epithelial cells in 3D.</a> Biomaterials. 34 (21), 5117-5127 (2013).</li><li>Lin, H. 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=Fibronectin+and+laminin+promote+differentiation+of+human+mesenchymal+stem+cells+into+insulin+producing+cells+through+activating+Akt+and+ERK.">Fibronectin and laminin promote differentiation of human mesenchymal stem cells into insulin producing cells through activating Akt and ERK.</a> Journal of Biomedical Science. 17, 56 (2010).</li><li>Boretti, M. I., Gooch, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+extracellular+matrix+and+3D+morphogenesis+on+islet+hormone+gene+expression+by+Ngn3-infected+mouse+pancreatic+ductal+epithelial+cells.">Effect of extracellular matrix and 3D morphogenesis on islet hormone gene expression by Ngn3-infected mouse pancreatic ductal epithelial cells.</a> Tissue Engineering Part A. 14 (12), 1927-1937 (2008).</li><li>Boretti, M. I., Gooch, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+cell+clustering+enhances+islet+beta+cell+formation+from+human+cultures+enriched+for+pancreatic+ductal+epithelial+cells.">Induced cell clustering enhances islet beta cell formation from human cultures enriched for pancreatic ductal epithelial cells.</a> Tissue Engineering. 12 (4), 939-948 (2006).</li><li>Beattie, G. M., Rubin, J. S., Mally, M. I., Otonkoski, T., Hayek, A. <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+proliferation+and+differentiation+of+human+fetal+pancreatic+islet+cells+by+extracellular+matrix,+hepatocyte+growth+factor,+and+cell-cell+contact.">Regulation of proliferation and differentiation of human fetal pancreatic islet cells by extracellular matrix, hepatocyte growth factor, and cell-cell contact.</a> Diabetes. 45 (9), 1223-1228 (1996).</li><li>Gage, B. K., Webber, T. D., Kieffer, T. 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The authors have nothing to disclose.

This work describes an enhanced protocol for generating pancreatic progenitors from hPSCs with a high co-expression of PDX1 and NKX6.1. Dissociation and replating of the hPSC-derived endoderm at half density on fresh matrix resulted in higher PDX1 and NKX6.1 in hPSC-derived pancreatic progenitors.
Although the growth factor cocktail for each stage is highly similar to P1-ND27, it has been shown that a more extended Stage 3 treatment including FGF and retinoid signaling and BMP and hedgehog inhibition increases NKX6.1 expression, which is in contrast to Nostro et al.&#39;s findings27. While PDX1 expression in the pancreas during embryonic development is positively regulated by FGF and retinoid signaling and BMP and hedgehog inhibition27,29,30, the present results demonstrated the extension of this signaling cocktail also upregulates NKX6.1. Nevertheless, the effect of an extended Stage 3 treatment was aided by dissociation and replating of endodermal cells that led to increased PDX1 and NKX6.1 expression. Furthermore, this method (P2-D) also increased SOX9-expressing cells in hPSC-derived pancreatic progenitors, in addition to the proportion of proliferative NKX6.1+ cells that co-express the proliferation marker Ki67.
Another crucial factor affecting the efficiency of differentiation was the availability of fresh membrane matrix to the dissociated cells. The extracellular matrix components have been previously demonstrated to regulate stem cell fate specification31,32. Overall, the favorable effects of extracellular matrix components on pancreatic development have been recorded33,34,35, particularly for the membrane matrix, which, along with laminin, was shown to have a pro-endocrine effect on pancreatic lineage cells36. Therefore, replating endodermal cells on fresh membrane matrix may have enhanced NKX6.1 expression in hPSC-derived pancreatic progenitors.
The cell density of differentiating cells also controls gene expression. Multiple studies have demonstrated the significance of cell-cell contact in regulating pancreatic development37,38,39. Cellular aggregation or embryoid body formation has been shown to induce higher pancreatic endocrine gene expression than 2D cultures19. However, the results demonstrate that higher pancreatic expression, especially NKX6.1, can be obtained by culturing cells in a 2D monolayer at half the endodermal density using our optimized protocol21. Interestingly, this method also inhibited hepatic cell fate (the alternative fate of hPSC-derived DE) as noticed by the decreased expression of hepatic genes AFP (Alpha-fetoprotein) and ALB (Albumin)21, indicating that physical factors play a role in lineage specification of hPSCs.
Overall, the results demonstrate that modulating the physical environment of the differentiating cells can enhance pancreatic gene expression21. Specifically, the dissociation of hPSC-derived endoderm and replating on fresh membrane matrix with a longer FGF and retinoid signaling and hedgehog and BMP inhibition can enhance the PDX1 and NKX6.1 co-expression in hPSC-derived pancreatic progenitors. However, the optimized protocol is at least 2 days longer than previously published protocols. Also, the length of Stage 4 may be extended beyond the minimum recommended, i.e., 4 days, based on the cell line being used. Multiple recombinant human growth factors are employed in the optimized protocol that can be substituted by their less expensive, small molecule compounds that perform the same action. Nevertheless, this optimized protocol can facilitate the scalable generation of pancreatic progenitors from hPSCs for cell therapy and disease modeling.

Diabetes is a complex metabolic disorder affecting millions of people globally. Supplementation of insulin is considered the only treatment option for diabetes. More advanced cases are treated with beta cell replacement therapy, achieved through transplantation of either whole cadaveric pancreas or islets1,2. Several issues surround transplantation therapy, such as limitation with the availability and quality of the tissue, invasiveness of transplantation procedures in addition to the continuous need for immunosuppressants. This necessitates the need for discovering novel and alternative options for beta cell replacement therapy2,3. Human pluripotent stem cells (hPSCs) have recently emerged as a promising tool for understanding human pancreas biology and as a non-exhaustive and potentially a more personalized source for transplantation therapy4,5,6,7. hPSCs, including human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs), have a high self-renewal capacity and give rise to any tissue type of the human body. hESCs are derived from the embryo&#39;s inner cell mass, and hiPSCs are reprogrammed from any somatic cell4,8.
Directed differentiation protocols are optimized to generate pancreatic beta cells from hPSCs that sequentially direct hPSCs through pancreatic developmental stages invitro. These protocols generate hPSC-derived islet organoids. While they have greatly improved at increasing the proportion of pancreatic beta cells therein, the efficiency of protocols is highly variable. It does not increase to more than ~40% of NKX6.1+/INSULIN+ or C-PEPTIDE + cells5,9,10,11,12,13. However, the generated beta cells are not entirely identical to the adult human beta cells in terms of their transcriptional and metabolic profiles and their response to glucose4,5,14. The hPSC-derived beta cells lack gene expression of key beta cell markers such as PCSK2, PAX6, UCN3, MAFA, G6PC2, and KCNK3 compared to adult humans islets5. Additionally, the hPSC-derived beta cells have diminished calcium signaling in response to glucose. They are contaminated with the co-generated polyhormonal cells that do not secrete appropriate amounts of insulin in response to increasing glucose levels5. On the other hand, hPSC-derived pancreatic progenitors, which are islet precursors, could be generated more efficiently in vitro compared to beta cells and, when transplanted in vivo, could mature into functional, insulin-secreting beta cells15,16. Clinical trials are currently focused on demonstrating their safety and efficacy upon transplantation in T1D subjects.
Notably, expression of the transcription factors PDX1 (Pancreatic and Duodenal Homeobox 1) and NKX6.1 (NKX6 Homeobox 1) within the same pancreatic progenitor cell is crucial for commitment towards a beta cell lineage5. Pancreatic progenitors that fail to express NKX6.1 give rise to polyhormonal endocrine cells or non-functional beta cells17,18. Therefore, a high co-expression of PDX1 and NKX6.1 in the pancreatic progenitor stage is essential for ultimately generating a large number of functional beta cells. Studies have demonstrated that an embryoid body or 3D culture enhances PDX1 and NKX6.1 in pancreatic progenitors where the differentiating cells are aggregated, varying between 40%-80% of the PDX1+/NKX6.1+ population12,19. However, compared to suspension cultures, 2D differentiation cultures are more cost-effective, feasible, and convenient for application on multiple cell lines5. We recently showed that monolayer differentiation cultures yield more than up to 90% of PDX1+/NKX6.1+ co-expressing hPSC-derived pancreatic progenitors20,21,22. The reported method conferred a high replicating capacity to the generated pancreatic progenitors and prevented alternate fate specifications such as hepatic lineage21. Therefore, herein, this protocol demonstrates a highly efficient method for the differentiation of hPSCs to pancreatic beta-cell precursors co-expressing PDX1 and NKX6.1. This method utilizes the technique of dissociating hPSC-derived endoderm and manipulating the cell density, followed by an extended FGF and Retinoid signaling as well as Hedgehog inhibition to promote PDX1 and NKX6.1 co-expression (Figure 1). This method can facilitate a scalable generation of hPSC-derived pancreatic beta-cell precursors for transplantation therapy and disease modeling.

<table><tbody><tr><td>15 mL, conical, centrifuge tubes</td><td>Thermo Scientific</td><td>339651</td><td></td></tr><tr><td>20X TBS Tween 20</td><td>Thermo Scientific</td><td>28360</td><td></td></tr><tr><td>24-well culture plates, flat bottom with lid</td><td>Costar</td><td>3524</td><td></td></tr><tr><td>50 mL, conical, centrifuge tubes</td><td>Thermo Scientific</td><td>339652</td><td></td></tr><tr><td>6- well culture plates, multidish</td><td>Thermo Scientific</td><td>140685</td><td></td></tr><tr><td>Accutase</td><td>Stem Cell Technologies</td><td>0-7920</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D</td><td>338-AC</td><td>Reconstituted in 4 mM HCl</td></tr><tr><td>Anti NKX6.1 antibody, mouse monoclonal</td><td>DSHB</td><td>F55A12-C</td><td>Diluted to 1:100 for flow-cytometry and 1:2000 for immunostaining</td></tr><tr><td>Anti-PDX1 antibody, guinea pig polyclonal</td><td>Abcam</td><td>ab47308</td><td>Diluted to 1:100 for flow-cytometry and 1:1000 for immunostaining</td></tr><tr><td>B27 minus Vit A</td><td>ThermoFisher</td><td>12587010</td><td></td></tr><tr><td>Bovine serum albumin, heat shock fraction, fatty acid free</td><td>Sigma</td><td>A7030</td><td></td></tr><tr><td>CHIR 99021</td><td>Tocris</td><td>4423</td><td>Reconstituted in DMSO</td></tr><tr><td>DMEM, high glucose</td><td>ThermoFisher</td><td>41965047</td><td></td></tr><tr><td>Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td><td>Invitrogen</td><td>A10037</td><td></td></tr><tr><td>Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td></td><td>A-21206</td><td></td></tr><tr><td>DPBS 1X</td><td>ThermoFisher</td><td>14190144</td><td></td></tr><tr><td>EGF</td><td>ThermoFisher</td><td>PHG0313</td><td>Reconstituted in 0.1% BSA in PBS</td></tr><tr><td>FGF10</td><td>R&amp;amp;D</td><td>345-FG</td><td>Reconstituted in PBS</td></tr><tr><td>Glucose</td><td>Sigma Aldrich</td><td>G8644</td><td></td></tr><tr><td>Hoechst 33258</td><td>Sigma</td><td>23491-45-4</td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX73</td><td></td></tr><tr><td>KnockOut DMEM/F-12 (1X)</td><td>Gibco</td><td>12660-012</td><td></td></tr><tr><td>KnockOut SR serum replacement</td><td>Gibco</td><td>10828-028</td><td></td></tr><tr><td>L-Ascorbic acid (vitamin C)</td><td>Sigma</td><td>A92902</td><td>Reconstituted in distilled water</td></tr><tr><td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td><td>Corning</td><td>354230</td><td>Aliquot the thawed stock and freeze at -20C.</td></tr><tr><td>MCDB131</td><td>ThermoFisher</td><td>10372019</td><td></td></tr><tr><td>Mouse anti-SOX17</td><td>ORIGENE</td><td>CF500096</td><td>Diluted to 1:100 for flow-cytometry and 1:2000 for immunostaining</td></tr><tr><td>mTeSR Plus</td><td>Stem Cell Technologies</td><td>85850</td><td>Mix the basal media with supplement. Aliquot and store at -20 &amp;deg;C for longer time or at 4 &amp;deg;C for instant use</td></tr><tr><td>Nalgene filter units, 0.2 &amp;micro;m PES</td><td>ThermoFisher</td><td>566-0020</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>72340</td><td>Reconstituted in distilled water</td></tr><tr><td>NOGGIN</td><td>R&amp;amp;D</td><td>6057-NG</td><td>Reconstituted in 0.1% BSA in PBS</td></tr><tr><td>Paraformaldehyde solution 4% in PBS</td><td>ChemCruz</td><td>sc-281692</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>ThermoFisher</td><td>15140122</td><td></td></tr><tr><td>Portable vacuum aspirator</td><td></td><td></td><td></td></tr><tr><td>Rabbit anti-FOXA2</td><td>Cell signaling technology</td><td>3143</td><td>Diluted to 1:100 for flow-cytometry and 1:500 for immunostaining</td></tr><tr><td>Retinoic Acid</td><td>Sigma Aldrich</td><td>R2625</td><td>Reconstituted in DMSO</td></tr><tr><td>Rock inhibitor (Y-27632)</td><td>ReproCell</td><td>04-0012-02</td><td>Reconstituted in DMSO</td></tr><tr><td>Round Bottom Polystyrene FACS Tubes with Caps, STERILE</td><td>Stellar Scientific</td><td>FSC-9010</td><td></td></tr><tr><td>SANT-1</td><td>Sigma Aldrich</td><td>S4572</td><td>Reconstituted in DMSO</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761-500G</td><td></td></tr><tr><td>StemFlex</td><td>ThermoFisher</td><td>A3349401</td><td>Mix the basal media with supplement. Aliquot and store at -20 &amp;deg;C for longer time or at 4 &amp;deg;C for instant use</td></tr><tr><td>TALI Cellular Analysis Slide</td><td>Invitrogen</td><td>T10794</td><td></td></tr><tr><td>Tali image-based cytometer automated cell counter</td><td>Invitrogen</td><td>T10796</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>9002-93-1</td><td></td></tr><tr><td>TrypLE 100 mL</td><td>ThermoFisher</td><td>12563011</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P2287</td><td></td></tr><tr><td>UltraPure 0.5 M EDTA, pH 8.0</td><td>Invitrogen</td><td>15575-038</td><td></td></tr></tbody></table>

differentiation of human pluripotent stem cells into pancreatic beta-cell precursors in a 2d culture system

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

The results show that optimized protocol P2-D (Figures 1A) enhanced pancreatic progenitor differentiation efficiency by upregulating PDX1 and NKX6.1 co-expression (Figure 2A,B, and Figure 3A). In particular, the results showed that dissociation of endodermal cells and their replating on fresh membrane matrix along with a longer duration of Stage 3 enhanced NKX6.1 expression in hPSC-derived pancreatic progenitors (optimized protocol, P2-D) (Figure 2 and Figure 3A), in comparison to the non-dissociated protocol (P1-ND) that was modified from a previously published study27. The present enhanced protocol also generated the highest proportion of PDX1+/NKX6.1+ progenitors compared to &#34;P1-D&#34; (Protocol 1, S3 = 2 days, dissociated) and &#34;P2-ND&#34; (Protocol 2, S3 = 4 days, non-dissociated), and the detailed results have been previously published21. Pancreatic progenitors generated using P2-D also have increased numbers of SOX9+ cells compared to the non-dissociated P1-ND (Figure 3B). The optimized method also generated a higher proportion of proliferative NKX6.1+ cells that co-express the proliferation marker Ki67 (Figure 3C).
The representative results presented here are from iPSCs generated from peripheral blood mononuclear cells (PBMCs) of control, healthy individual. However, we have reproducibly applied the current enhanced protocol on multiple hPSC lines such as H1-hESCs, H9-hESCs, HUES8-hESCs, and several other control iPSC lines to yield ~90% PDX1 and NKX6.1 co-expressing pancreatic progenitors20,21,28.
Following DE induction in hPSCs, mild levels of cell death are expected. However, if a high cell death rate was observed, the experiment was stopped and started again with a fresh batch of cells. The efficiency of DE induction should be higher than 70%-80% to ensure a high proportion of DE cells in the culture that will serve as an ideal starting source for pancreatic progenitor induction (Figure 1C).
The density of replating endodermal cells following dissociation can be manipulated based on the growth rate of that particular cell. For example, slow-growing cells may be replated at a higher density than recommended. This will minimize the chances of obtaining irrelevant pancreatic populations at Stage 4. A high co-expression of PDX1 and NKX6.1 can be obtained following dissociation after Stage 1 and replating at half density (Figure 1A, Figure 2A,B, and Figure 3A). If a high expression of PDX1 is observed but only a moderate expression of NKX6.1, Stage 4 can be extended by 2 days to enhance NKX6.1 expression in PDX1-expressing cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63298/63298fig01.jpg" /> 
Figure 1: Timeline for the addition of cytokines and growth factors during pancreatic progenitor differentiation. (A) Schematic representation of the optimized protocol (P1-D) for generation of PDX1 and NKX6.1 from hPSCs. hPSC-derived definitive endoderm (DE) is dissociated and replated at half density and then sequentially directed towards a pancreatic progenitor fate. (B) Images of hPSCs in brightfield before starting differentiation at Day 0. (C) Immunostaining analysis for expression of endodermal markers SOX17 and FOXA2 in hPSC-derived endoderm (Stage 1) cells. SOX17, green; FOXA2, red. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63298/63298fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63298/63298fig02.jpg" /> 
Figure 2: Generation of hPSC-derived pancreatic progenitors, co-expressing PDX1 and NKX6.1. Immunostaining analysis for comparison of PDX1 and NKX6.1 expression in hPSC-derived pancreatic progenitors using the enhanced protocol (P2-D) (A) and a non-optimized, previously published protocol (P1-ND) (B). The enhanced protocol achieved the highest co-expression of PDX1 and NKX6.1. PDX1, green; NKX6.1, red. Magnified images are provided in the second panel for each protocol. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63298/63298fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63298/63298fig03.jpg" /> 
Figure 3: Quantification of pancreatic markers in pancreatic progenitors generated using the enhanced protocol. Flow-cytometry analysis in hPSC-derived pancreatic progenitors yielded using P2-D in comparison to the non-dissociated P1-ND. (A) Histograms for PDX1 and NKX6.1 expression and double-positive staining graphs. (B) The histograms for SOX9 expression and (C) co-expression of NKX6.1 with the proliferation marker Ki67. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63298/63298fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stage</td>
			<td colspan="4">Media</td>
			<td colspan="4">Cytokines</td>
			<td>Days</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="4">MCDB 131 media + 0.5% fatty acid-free bovine serum albumin (FFA-BSA), 1.5 g/L sodium bicarbonate (NaHCO3), 10 mM of glucose</td>
			<td colspan="4">Day 1: 2 &#956;M CHIR99021, 100 ng/mL Activin A, 10 &#956;M Rock inhibitor (Y-27632), 0.25 mM Vitamin C. Day 2 onwards: 100 ng/mL Activin A, 0.25 mM Vitamin C</td>
			<td>3 or 4</td>
		</tr>
		<tr>
			<td>2</td>
			<td colspan="4">MCDB 131 media +&#160; 0.5% fatty acid-free bovine serum albumin (FFA-BSA), 1.5 g/L sodium bicarbonate (NaHCO3), 10 mM glucose</td>
			<td colspan="4">Day 1 (Dissociation): 50 ng/mL FGF10, 50 ng/mL NOGGIN, 0.25 &#956;M CHIR99021, 10 &#956;M Y-27632 (Rock inhibitor) , 0.25 mM Vitamin C. Day 2: 50 ng/mL FGF10, 50 ng/mL NOGGIN, 0.25 &#956;M CHIR99021, 0.25 mM Vitamin C.</td>
			<td>2</td>
		</tr>
		<tr>
			<td>3</td>
			<td colspan="4">DMEM + 4.5 g/L glucose</td>
			<td colspan="4">2 &#956;M Retinoic acid, 0.25 &#956;M SANT-1, 50 ng/mL FGF10, 50 ng/mL NOGGIN, 0.25 mM Vitamin C, 1% B27 supplement without vitamin A</td>
			<td>4</td>
		</tr>
		<tr>
			<td>4</td>
			<td colspan="4">DMEM + 4.5 g/L glucose</td>
			<td colspan="4">100 ng/mL EGF, 10 mM Nicotinamide, 50 ng/mL NOGGIN, 0.25 mM Vitamin C, 1% B27 supplement without vitamin A.</td>
			<td>4</td>
		</tr>
	</tbody>
</table>
Table 1: The compositions of the different differentiation media used in the study.

Watch this Scientific Journal Video about Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System at JoVE.com

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

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

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

differentiation-human-pluripotent-stem-cells-into-pancreatic-beta

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

2.7K Views.  Hamad Bin Khalifa University (HBKU), Qatar Foundation. Dit geoptimaliseerde protocol verbeterde de generatie van pancreas bètacelvoorlopers uit menselijke pluripotente stamcellen, die kunnen worden gebruikt voor celtherapie voor diabetes en het onderzoeken van moleculaire mechanismen die de biogenese van diabetes onderstrepen. Dit is een haalbare techniek omdat het wordt gebruikt voor 2D-monolaagculturen. Het is gemakkelijk aan te brengen en is consistent over meerdere controle- en patiënt-afgeleide pluripotente stamcellijnen.Deze bètac...

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

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

Genetics

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

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

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

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

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 ...

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 ...

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

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

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

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

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

Differentiation of Human Pluripotent Stem Cells into 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.

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 ...

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

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

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

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

Biology

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

Bioengineering

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

Differentiating Human Pluripotent Stem Cells into Pancreatic β-Cells

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

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

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

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

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

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