Name: efficient generation of pancreas/duodenum homeobox protein 1+ posterior foregut/pancreatic progenitors from hpscs in adhesion cultures
Uploaded: 2019-03-27T12:30:00.000+00:00
Duration: 8 min 32 s
Description: 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

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>

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(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 efficient induction to PDX1+ cells is likely to be compromised.&#160;
Undifferentiated states of hPSCs are maintained as colonies or aggregates of compacted cells. However, methods that start the differentiation from colonies or aggregates may suffer from heterogeneity, because of different cell adhesion and density in the colony, such as in the center and periphery22, and because cells are at different stages in the cell cycle25. On the other hand, our method starts with dissociated single cells, which enables a relatively homogenous state of cell adhesion of single cells or cell density in every single cell. In terms of homogenous handling for each cell, our methods might be easier than others that start with colony or aggregation cultures.&#160;
Although our protocol can be used to induce PDX1+ cells from multiple hPSC lines, the differentiation could still be inefficient. In such cases, differences in adhesion state right after seeding among the hPSC lines could be the cause, and modification of the seeding density could be a solution33. Indeed, Figure 3 shows that inappropriate seeding cell density compromises differentiation into definitive endoderm and PDX1+ cells. Interestingly, the optimal cell density for PDX1+ cell induction was different among the cell populations that achieved &#62;90% definitive endoderm. Another possibility for inefficient differentiation is the poor maintenance condition of the undifferentiated hPSCs, which compromises the quality of pluripotency despite the expression of markers for the undifferentiated state. In this case, the hPSC expansion culture should be recommenced from early passage frozen stocks or sub cloning should be performed to obtain hPSCs in suitable conditions. In the case of inefficient differentiation on days 8 (Stage 2) or 11 (Stage 3), the duration of these steps should be optimized. Supporting this idea, the duration of Stage 3 has been shown critical for acquiring later stage cell characteristics and is cell line-dependent in pancreatic lineage34.&#160;
The generation of PDX1+ cells is crucial for the in vitro generation of pancreatic cells. PDX1 is functionally essential for pancreatic development based on knowledge from Pdx1 null mice, which are apancreatic35. Consistently, in vitro and in vivo implantation studies showed hPSC-derived PDX1+ cells have the potential to develop into all pancreatic components, including exocrine and endocrine cells such as pancreatic &#946; cells3,16,36,37. Thus, the efficient generation of PDX1+ cells from hPSCs leads to a stable pancreatic cell supply for the establishment of &#946; cell therapy against diabetes and the understanding of human pancreas development and pancreatic diseases.&#160;
The limitations of this method are related to the two-dimensional (2D) monolayer culture format, which is not suitable for some cell types and cell processing. In recent years, three-dimensional (3D) cultures have been shown to promote the generation of mature cells and tissues, possibly due to mimicking the in vivo microenvironment. For example, &#946; cells generated in 3D cultures but not 2D monolayer cultures could attain the ability to secrete insulin in response to extracellular glucose levels38.&#160;
To use PDX1+ cells for the generation of developmentally later cell types, it is important to shift to 3D cultures, such as suspension cultures of aggregates embedded in an extracellular matrix and aggregate cultures on an air-liquid interface3,16,37. In addition, 2D monolayer cultures require more surface area for culturing than suspension cultures, limiting scalability. The processing of large amounts of cells for commercial use requires modifications such as the use of microbeads. At the same time, the present method is suitable for the screening of differentiation-inducing factors and the exploration of molecular mechanisms by gene transfer.&#160;

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><tbody><tr><td>3-Keto-N-aminoethyl-N&amp;prime;-aminocaproyldihydrocinnamoyl cyclopamine</td><td>Toronto Research Chemicals</td><td>K171000</td><td>CYC</td></tr><tr><td>4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]-benzoic acid</td><td>Santa Cruz Biotechnology</td><td>SC-203303</td><td>TTNPB</td></tr><tr><td>50 mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>Thermo Fisher Scientific</td><td>339652</td><td></td></tr><tr><td>Anti-CDX2 antibody [EPR2764Y]</td><td>Abcam</td><td>Ab76541</td><td>Anti-CDX2, &amp;times; 1/1000 dilution</td></tr><tr><td>B-27 Supplement (50 &amp;times;)</td><td>Thermo Fisher Scientific</td><td>17504-044</td><td>Serum-free supplement</td></tr><tr><td>BD FACSAria II Cell Sorter</td><td>BD Biosciences</td><td></td><td>For flow cytometry</td></tr><tr><td>Biomedical freezer</td><td>SANYO</td><td>MDF-U538</td><td>For -30 &amp;deg;C storing</td></tr><tr><td>Cell Counting Slides for TC10/TC20 Cell Counter, Dual-Chamber</td><td>BIO-RAD</td><td>1450011</td><td>Counting slide glass</td></tr><tr><td>CELL CULTURE MULTIWELL PLATE, 6 WELL, PS, CLEAR</td><td>Greiner bio-one</td><td>657165</td><td>For differentiation culture/6-well plate</td></tr><tr><td>Centrifuge</td><td>TOMY</td><td>AX-310</td><td>For cell culturing</td></tr><tr><td>Centrifuge</td><td>TOMY</td><td>MX-305</td><td>For RT-qPCR</td></tr><tr><td>CHIR99021</td><td>Axon Medchem</td><td>Axon 1386</td><td></td></tr><tr><td>CLEAN BENCH</td><td>SHOWA KAGAKU&amp;nbsp;</td><td>S-1601PRV</td><td>Clean bench</td></tr><tr><td>Corning CellBIND 6-well plate</td><td>Corning</td><td>3335</td><td>For feeder-free culture of hPSCs/6-well plate</td></tr><tr><td>Corning Matrigel Basement Membrane Matrix Growth Factor Reduced</td><td>Corning</td><td>354230</td><td>Basement membrane matrix</td></tr><tr><td>Corning Synthemax II-SC Substrate</td><td>Corning</td><td>3535</td><td>For feeder-free culture of hPSCs/synthetic surface material for hPSCs</td></tr><tr><td>Cryostat</td><td>Leica</td><td>Leica CM1510 S</td><td>For immunostaining of aggregates.</td></tr><tr><td>Cytofix/Cytoperm Kit</td><td>Becton Dickinson</td><td>554714</td><td>Perm/Wash buffer is&amp;nbsp; Permeabilization/Wash buffer. Cytofix/Cytoperm buffer is fixation and permeabilization buffer.</td></tr><tr><td>Dako pen</td><td>Dako</td><td>S2002</td><td>For immunostaining of aggregates</td></tr><tr><td>dNTP mix (10 mM)</td><td>Thermo Fisher Scientific</td><td>18427-088</td><td>For RT-qPCR</td></tr><tr><td>Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A11055</td><td>Secondary antibody, &amp;times; 1/500 dilution</td></tr><tr><td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td><td>Thermo Fisher Scientific</td><td>A10036</td><td>Secondary antibody, &amp;times; 1/500 dilution</td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td><td>Thermo Fisher Scientific</td><td>A10040</td><td>Secondary antibody, &amp;times; 1/500 dilution</td></tr><tr><td>Donkey Serum</td><td>Merck Millipore</td><td>S30</td><td>Donkey serum</td></tr><tr><td>D-PBS(-) without Ca or Mg</td><td>Nacalai tesque</td><td>14249-95</td><td>DPBS</td></tr><tr><td>Essential 8 Medium</td><td>Thermo Fisher Scientific</td><td>A1517001</td><td>For feeder-free culture of hPSCs/hPSC maintenance medium</td></tr><tr><td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td><td>Corning</td><td>352235</td><td>5 mL round bottom polystyrene tube with cell strainer</td></tr><tr><td>Filter Tip, 1,000 &amp;micro;L</td><td>Watoson</td><td>124-1000S</td><td>Use together with pipettes</td></tr><tr><td>Filter Tip, 20 &amp;micro;L</td><td>Watoson</td><td>124-P20S</td><td>Use together with pipettes</td></tr><tr><td>Filter Tip, 200 &amp;micro;L</td><td>Watoson</td><td>124-P200S</td><td>Use together with pipettes</td></tr><tr><td>Fluorescence Microscope</td><td>Keyence</td><td>BZ-X700</td><td>For immunostaining</td></tr><tr><td>Forma Steri-Cycle CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Fisher Scientific</td><td>370A</td><td>Incubator</td></tr><tr><td>HNF-1&amp;beta; Antibody (C-20)</td><td>Santa Cruz Biotechnology</td><td>sc-7411</td><td>Anti-HNF1&amp;beta;, &amp;times; 1/200 dilution</td></tr><tr><td>HNF-4&amp;alpha; Antibody (H-171)</td><td>Santa Cruz Biotechnology</td><td>sc-8987</td><td>Anti-HNF4&amp;alpha;, &amp;times; 1/200 dilution</td></tr><tr><td>Hoechst 33342</td><td>Thermo Fisher Scientific</td><td>H3570</td><td>For nucleus staining, &amp;times; 1/200 dilution</td></tr><tr><td>Human Pancreas Total RNA</td><td>Ambion</td><td>AM7954</td><td>For RT-qPCR</td></tr><tr><td>Human PDX-1/IPF1 Antibody</td><td>R&amp;amp;D Systems</td><td>AF2419</td><td>Anti-PDX1, goat IgG, &amp;times; 1/200 dilution</td></tr><tr><td>Human SOX17 Antibody</td><td>R&amp;amp;D Systems</td><td>AF1924</td><td>Anti-SOX17, &amp;times; 1/200 dilution</td></tr><tr><td>Improved MEM Zinc Option medium</td><td>Thermo Fisher Scientific</td><td>10373-017</td><td>iMEM</td></tr><tr><td>Incubation chamber</td><td>Cosmo Bio</td><td>10DO</td><td>For immunostaining of aggregates</td></tr><tr><td>Latex Examination Gloves</td><td>Adachi</td><td></td><td></td></tr><tr><td>MAS coated slide glass</td><td>Matsunami Glass</td><td>83-1881</td><td>For immunostaining of aggregates</td></tr><tr><td>MicroAmp Fast 96-well Reaction Plate</td><td>Applied Biosystems/Thermo Fisher Scientific</td><td>4346907</td><td>For RT-qPCR</td></tr><tr><td>Microscope</td><td>Olympus</td><td>CKX41N-31PHP</td><td>For cell culturing</td></tr><tr><td>Microtube</td><td>Watoson</td><td>131-515CS</td><td></td></tr><tr><td>Monoclonal Anti-&amp;alpha;-Fetoprotein</td><td>SIGMA</td><td>A8452</td><td>Anti-AFP, &amp;times; 1/200 dilution</td></tr><tr><td>Nanodrop 8000</td><td>Thermo Fisher Scientific</td><td></td><td>For RT-qPCR</td></tr><tr><td>Oligo dT</td><td>FASMAC</td><td>Custom made Oligo&amp;nbsp;</td><td>For RT-qPCR of sequence is &quot;TTTTTTTTTTTTTTTTTTTT&quot;</td></tr><tr><td>Paraformaldehyde, powder</td><td>Nacalai tesque</td><td>26126-54</td><td>PFA, fixative, diluted in DPBS</td></tr><tr><td>Pharmaceutical refrigerator</td><td>SANYO</td><td>MPR-514</td><td>For 4 &amp;deg;C storing</td></tr><tr><td>PIPETMAN P&amp;nbsp;</td><td>GILSON</td><td></td><td>Pipette</td></tr><tr><td>Recombinant Human KGF/FGF-7</td><td>R&amp;amp;D Systems</td><td>251-KG</td><td>KGF</td></tr><tr><td>Recombinant Human Noggin</td><td>PeproTech</td><td>120-10C</td><td>NOGGIN</td></tr><tr><td>Recombinant Human/Mouse/Rat Activin A</td><td>R&amp;amp;D Systems</td><td>338-AC</td><td>Activin A</td></tr><tr><td>ReverTra Ace (100 U/&amp;mu;L)</td><td>TOYOBO</td><td>TRT-101</td><td>For RT-qPCR</td></tr><tr><td>RNase-free DNase Set (50)</td><td>QIAGEN</td><td>79254</td><td>For RT-qPCR</td></tr><tr><td>RNeasy Mini Kit</td><td>QIAGEN</td><td>74104</td><td>For RT-qPCR</td></tr><tr><td>RPMI 1640 with L-Gln</td><td>Nacalai tesque</td><td>30264-85</td><td>RPMI 1640</td></tr><tr><td>Sealing Film for Real Time</td><td>Takara</td><td>NJ500</td><td>For RT-qPCR</td></tr><tr><td>Serological pipettes 10 mL</td><td>Costar/Corning</td><td>4488</td><td>For cell culturing</td></tr><tr><td>Serological pipettes 25 mL</td><td>Costar/Corning</td><td>4489</td><td>For cell culturing</td></tr><tr><td>Serological pipettes 5 mL</td><td>Costar/Corning</td><td>4487</td><td>For cell culturing</td></tr><tr><td>Sox2 (D6D9) XP Rabbit mAb</td><td>Cell signaling</td><td>3579S</td><td>Anti-SOX2, &amp;times; 1/200 dilution</td></tr><tr><td>StepOnePlus</td><td>Applied Biosystems/Thermo Fisher Scientific</td><td></td><td>For RT-qPCR</td></tr><tr><td>Sucrose</td><td>Nacalai tesque</td><td>30406-25</td><td>For immunostaining of aggregates</td></tr><tr><td>TB Green&lt;br/&gt; Premix Ex Taq II&amp;nbsp;</td><td>Takara</td><td>RR820B</td><td>For RT-qPCR</td></tr><tr><td>TC20 Automated Cell Counter</td><td>BIO-RAD</td><td>1450101J1</td><td>Automatic cell counter</td></tr><tr><td>Tissue-Tek OCT compound 4583&amp;nbsp;</td><td>Sakura Finetechnical</td><td>4583</td><td>For immunostaining of aggregates</td></tr><tr><td>Tissue-Tek Cryomold Molds/Adapters</td><td>Sakura Finetechnical</td><td>4566</td><td>For immunostaining of aggregates</td></tr><tr><td>Triton X-100</td><td>Nacalai tesque</td><td>35501-15</td><td></td></tr><tr><td>Trypan Blue</td><td>BIO-RAD</td><td>1450021</td><td></td></tr><tr><td>Ultracold freezer</td><td>SANYO</td><td>MDF-U33V</td><td>For -80 &amp;deg;C storing</td></tr><tr><td>UltraPure 0.5M EDTA, pH 8.0</td><td>Thermo Fisher Scientific</td><td>15575-038</td><td>Dilute with DPBS to prepare 0.5 mM EDTA</td></tr><tr><td>Veriti Thermal Cycler</td><td>Applied Biosystems/Thermo Fisher Scientific</td><td></td><td>For RT-qPCR</td></tr><tr><td>Y-27632</td><td>Wako</td><td>251-00514</td><td></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 surface area. During Stage 1B, the media appear cloudy due to dead cells. The removal of dead cells is critical for highly efficient differentiation since dead cells likely disturb the survival and differentiation of cells lying underneath. On days 3-4, cells form a homogenous monolayer sheet that can be described as a cobblestone appearance. At this point, most cells stop expressing sex determining region Y-box 2 (SOX2), a marker for undifferentiated cells, and instead express a definitive endoderm marker, SOX17, at more than 90% (Figure 2A and B). Most SOX17+ cells express FOXA2 (Figure 2A). Starting the differentiation with an inappropriate cell density compromises the differentiation efficiency at this step (Figure 3). At Stages 2 and 3, cell death relaxes, and the used medium is not as cloudy as it is in Stage 1B. Cells express the primitive gut tube markers HNF1&#946; and HNF4&#945; (Figure 2C) and eventually express a posterior foregut/pancreatic progenitor marker, PDX1, at more than 90% (Figure 2D and E). The PDX1+ cell induction is reproducible in another hiPSC line, 1231A3 31, and an hESC line, KhES-3 32 (Figure 4). qRT-PCR results of the mRNA expression of stage markers were consistent with immunostaining (Figure 5A). The mRNA expression of PDX1 is evident at Stage 3 and substantially increases afterword.&#160;
PDX1+ cells at early developmental stages have the potential to differentiate into not only pancreatic cells but also gastric antrum, duodenum, extrahepatic bile duct and a part of the intestine 9. The differentiation potential of in vitro generated PDX1+ cells to pancreatic cells can be assessed by extended culture with reported protocols for pancreatic endoderm and pancreatic endocrine cells3,16,26. The expressions of a pancreatic endoderm marker, NKX6.1, and two pancreatic endocrine markers, INSULIN, and GLUCAGON, were observed on days 19 (Stage 4) and 31 (Stage 5), respectively (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57641/57641fig1.jpg" /> 
Figure 1: Representative appearance of cells under stepwise differentiation. (A) A scheme of directed differentiation from hPSCs to pancreatic lineages. Numbers in parentheses indicate concentrations (units are written below). (B) Representative bright field micrographs of hiPSCs at key steps of the differentiation culture. 585A1 hiPSCs were dissociated as single cells and induced to differentiate into definitive endoderm, primitive gut tube and posterior foregut/pancreatic progenitor. The lower panels are enlarged views of the upper panels. RPMI, RPMI 1640; AA, activin A (ng/mL); CH, CHIR99021 (&#956;M); KGF (ng/mL); NOG, NOGGIN (ng/mL); CYC, 3-Keto-N-aminoethyl-N&#8217;-aminocaproyldihydrocinnamoyl cyclopamine (&#956;M); TTNPB, 4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]-benzoic acid (nM). Scale bars = 300 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57641/57641fig1large.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/57641/57641fig2.jpg" /> 
Figure 2: Representative induction toward pancreatic lineages from hiPSCs. (A) The proportion of SOX2-SOX17+ cells analyzed by flow cytometry before differentiation (Stage 0) and on day 4 (Stage 1B). Undifferentiated hiPSCs (SOX2+SOX17-) were differentiated into definitive endoderm (SOX2-SOX17+). Most SOX17+ cells co-expressed FOXA2. (B) Representative immunofluorescent micrographs on day 4 (Stage 1B). The fixed cells were stained for SOX17 (green), SOX2 (red), and nuclei (blue). (C) Representative immunofluorescent micrographs on days 4 (Stage 1B) and 8 (Stage 2). The fixed cells were stained for HNF1&#946; (green), HNF4&#945; (red) and nuclei (blue). (D) The proportion of cells positive for PDX1, as analyzed by flow cytometry on days 4 (Stage 1B) and 11 (Stage 3). (E) Representative immunofluorescent micrographs of PDX1 (green) and nuclei (blue) on day 11 (Stage 3). Scale bars = 100 &#956;m.&#160; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57641/57641fig2large.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/57641/57641fig3.jpg" /> 
Figure 3: Representative induction toward definitive endoderm initiated from different cell densities. (A) Representative bright field micrographs of cells 1 h after differentiation. hiPSCs were dissociated as single cells and induced to differentiate into definitive endoderm at different cell densities (1-50 x 104/cm2). The lower panels are enlarged views of the upper panels. (B) The proportion of SOX2-SOX17+ cells analyzed by flow cytometry before differentiation (Stage 0) and on day 4 (Stage 1B). (C) The proportion of PDX1+ cells analyzed by flow cytometry on days 8 (Stage 2) and 11 (Stage 3). Scale bars = 300 &#956;m.&#160; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57641/57641fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57641/57641fig4.jpg" /> 
Figure 4: Representative induction toward PDX1+ cells in hiPCSs and hESCs. An hiPSC line, 1231A3 (A), and an hESC line, KhES-3 (B), were differentiated to definitive endoderm and PDX1+ cells. The cell composition was analyzed by flow cytometry. The proportion of SOX2-SOX17+ cells was analyzed before differentiation (Stage 0) and on day 4 (Stage 1B). The proportion of PDX1+ cells was analyzed on days 8 (Stage 2) and 11 (Stage 3).&#160; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57641/57641fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57641/57641fig5.jpg" /> 
Figure 5: Representative induction toward pancreatic endoderm and endocrine cells. hiPSCs (585A1) were differentiated into PDX1+ cells. The cells were further differentiated into pancreatic endoderm and pancreatic endocrine cells with reported protocols3,16,26. (A) mRNA expressions of stage markers were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The data were normalized to GAPDH expression and presented as the fold-change in gene expression relative to the peak value. Note that PDX1 expression was increased at &#62;20-fold from days 8 (Stage 2) to 11 (Stage 3) and at &#62;100-fold from days 11 (Stage 3) to 19 (Stage 4). The expression in the adult human pancreas is shown as Panc. SOX2, black; SOX17, purple; HNF1&#946;, brown; HNF4&#945;, orange; PDX1, light green; NKX6.1, green; INSULIN, blue; GLUCAGON, red. (B) Representative immunofluorescent micrographs of a pancreatic endoderm marker, NKX6.1 (red), on days 11 (Stage 3) and 19 (Stage 4). PDX1 (green) and nuclei (blue) were co-stained. (C) Representative immunofluorescent micrographs of two pancreatic endocrine makers, insulin (INS, green) and glucagon (GCG, red), on days 19 (Stage 4) and 31 (Stage 5). Nuclei (blue) were co-stained.&#160; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57641/57641fig5large.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>Gene name</td>
			<td>Gene symbol</td>
			<td>Forward primer</td>
			<td>Reverse primer</td>
		</tr>
		<tr>
			<td>glyceraldehyde-3-phospha 
			te dehydrogenase</td>
			<td>GAPDH</td>
			<td>GAAGGTGAAGGTCGGAGTC&#160;</td>
			<td>GAAGATGGTGATGGGATTTC</td>
		</tr>
		<tr>
			<td>SRY-box 2</td>
			<td>SOX2</td>
			<td>AGTCTCCAAGCGACGAAAAA</td>
			<td>TTTCACGTTTGCAACTGTCC</td>
		</tr>
		<tr>
			<td>SRY-box 17</td>
			<td>SOX17</td>
			<td>CGCACGGAATTTGAACAGTA</td>
			<td>TTAGCTCCTCCAGGAAGTGTG</td>
		</tr>
		<tr>
			<td>HNF1 homeobox B</td>
			<td>HNF1&#946;</td>
			<td>CCTCTCCTCCAAACAAGCTG</td>
			<td>TGTTGCCATGGTGACTGATT</td>
		</tr>
		<tr>
			<td>hepatocyte nuclear factor 4 alpha</td>
			<td>HNF4&#945;</td>
			<td>GAGCTGCAGATCGATGACAA</td>
			<td>TACTGGCGGTCGTTGATGTA</td>
		</tr>
		<tr>
			<td>pancreatic and duodenal homeobox 1</td>
			<td>PDX1</td>
			<td>AGCAGTGCAAGAGTCCCTGT</td>
			<td>CACAGCCTCTACCTCGGAAC</td>
		</tr>
		<tr>
			<td>NK6 homeobox 1</td>
			<td>NKX6.1</td>
			<td>ATTCGTTGGGGATGACAGAG</td>
			<td>TGGGATCCAGAGGCTTATTG</td>
		</tr>
		<tr>
			<td>glucagon</td>
			<td>GCG</td>
			<td>GAATTCATTGCTTGGCTGGT</td>
			<td>CGGCCAAGTTCTTCAACAAT</td>
		</tr>
		<tr>
			<td>insulin</td>
			<td>INS</td>
			<td>CTACCTAGTGTGCGGGGAAC</td>
			<td>GCTGGTAGAGGGAGCAGATG</td>
		</tr>
	</tbody>
</table>
Table 1: Primers for qPCR.

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 ...

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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

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

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 Differentiation of Human Pluripotent Stem Cells into Liver Cells

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

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

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

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

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

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

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

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 ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

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.

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

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