Name: efficient differentiation of pluripotent stem cells to nkx6-1+ pancreatic progenitors
Uploaded: 2017-03-07T12:30:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors at JoVE.com

This manuscript was supported by funding from the Toronto General and Western Foundation and the Banting &#38; Best Diabetes Centre-University Health Network Graduate Award.

1. Preparation of Solutions and Media
NOTE: Prepare all media for cell culture in a sterile environment. Media has to be made and used immediately. Reagent details are provided in the Materials Table.
<ol>
	<li>Differentiation Media
	<ol>
		<li>Prepare Day 0 Differentiation Media: RPMI Medium with 1% Glutamine, 2 &#181;M CHIR 99021, 100 ng/ml Activin A, 104 M MTG.</li>
		<li>Prepare Day 1-2 Differentiation Media: RPMI Medium with 1% Glutamine, 100 ng/ml Activin A, 104 M MTG, 5 ng/ml bFGF, 50 &#181;g/ml Ascorbic Acid.</li>
		<li>Prepare Day 3-5 Differentiation Media: RPMI Medium with 1% Glutamine, 1% B27, 104 M MTG, 0.75 &#181;M Dorsomorphin, 50 ng/ml FGF10.</li>
		<li>Prepare Day 6-7 Differentiation Media: DMEM with 1% Glutamine, 1% B27, 50 &#181;g/ml Ascorbic Acid, 50 ng/ml FGF10, 0.25 &#181;M SANT-1, 2 &#181;M Retinoic Acid, 50 ng/ml NOGGIN. 
		NOTE: Retinoic Acid should be added to the media last and media should be protected from light to prevent oxidative degradation15.</li>
		<li>Prepare Day 8-12 Differentiation Media: DMEM with 1% Glutamine, 1% B27, 50 &#181;g/ml Ascorbic Acid, 50 ng/ml NOGGIN, 100 ng/ml hEGF, 10 mM Nicotinamide.</li>
	</ol>
	</li>
	<li>Prepare FACS buffer: 10% FBS in PBS, minus calcium and magnesium.</li>
</ol>
2. HESC Differentiation
NOTE: HESC are thawed, passaged and expanded on irradiated mouse embryonic fibroblasts in the presence of a KOSR-based media supplemented with bFGF16. HESCs are ready for differentiation when the cells reach 80-95% confluency. At this time the colonies should be large with defined borders and a &#39;domelike&#39; structure (Figure 1A). All cell culture is performed in flat-bottom, tissue culture treated plates coated with 0.1% gelatin. Typically, the cells are grown in 6 or 12-well plates, in media volumes of 2 ml or 1 ml, respectively.
<ol>
	<li>On day 0, begin differentiation by replacing the KOSR-based media with Day 0 Differentiation Media.</li>
	<li>On days 1 and 2, gently shake the plate to remove any dead cells from the monolayer before aspirating. Replace with Day 1-2 Differentiation Media.</li>
	<li>On day 3, harvest cells for flow cytometry. If the cells express more than 90% CXCR4+/CD117+, proceed to step 2.4.</li>
	<li>On days 3 and 5, gently shake the plate to remove any dead cells from the monolayer prior to aspirating. Replace with Day 3-5 Differentiation Media.</li>
	<li>On days 6 and 7, replace with Day 6-7 Differentiation Media.</li>
	<li>On days 8, 10 and 12, replace with Day 8-12 Differentiation Media.</li>
	<li>On day 13, harvest cells for flow cytometry to determine PDX1 and NKX6-1 expression.</li>
</ol>
3. Harvesting Cells for Flow Cytometry Analysis
<ol>
	<li>Dissociate cells with 1x commercial trypsin solution according to manufacturer&#39;s protocol and incubate at 37 &#176;C for 3 min.</li>
	<li>Remove commercial trypsin solution and resuspend single cells in 1,000 &#181;l FACS Buffer with 30 &#181;l DNase I.</li>
	<li>Filter cells using a 35 &#181;m nylon mesh cell strainer and transfer to a microcentrifuge tube.</li>
	<li>Centrifuge cells at 455 x g for 5 min. Prepare cells for either live or fixed staining.</li>
</ol>
4. Staining for Flow Cytometry
<ol>
	<li>Flow preparation for live staining on day 3
	<ol>
		<li>Resuspend cells in 1,000 &#181;l FACS Buffer. Transfer 200 &#181;l (approximately 1-3 x 105 cells) into two wells of a 96-well plate (for both unstained and stained samples).</li>
		<li>Centrifuge the plate at 931 x g for 2 min. Remove the supernatant by inverting the plate.</li>
		<li>Stain with primary-conjugated antibodies, CXCR4:APC and CD117:PE (see Materials Table) for 30 min at room temperature protected from light.</li>
		<li>Centrifuge the plate at 931 x g for 2 min. Remove the supernatant by inverting the plate.</li>
		<li>Resuspend samples in 100 &#181;l FACS Buffer.</li>
		<li>Centrifuge the plate at 931 x g for 2 min. Remove the supernatant by inverting the plate.</li>
		<li>Resuspend each sample and transfer in a total volume of 300-500 &#181;l FACS Buffer to 1 ml micro test tubes or 5 ml round-bottom tubes.</li>
		<li>Run samples on the flow cytometer17. If samples cannot be run immediately, store at 4 &#176;C protected from light.</li>
	</ol>
	</li>
	<li>Flow preparation for fixed staining on day 13
	<ol>
		<li>Resuspend the cell pellet in commercial fixation/permeabilization solution for 24 hr at 4 &#176;C.</li>
		<li>Centrifuge the sample at 455 x g for 5 min. Resuspend in 400 &#181;l Perm/Wash solution.</li>
		<li>Transfer 200 &#181;l of the sample (approximately 0.5-1 x 106 cells) to two wells of a 96-well plate (for IgG control and PDX1/NKX6-1 staining). Centrifuge at 931 x g for 2 min.</li>
		<li>Resuspend the cell pellets in 100 &#181;l Perm/Wash solution containing anti-PDX1 and anti-NKX6-1 primary or isotype control antibodies (see Materials Table). Incubate overnight at 4 &#176;C.</li>
		<li>Centrifuge the plate at 931 x g for 2 min. Remove the supernatant by inverting the plate. Resuspend samples in 100 &#181;l Perm/Wash solution.</li>
		<li>Centrifuge the plate at 931 x g for 2 min. Remove the supernatant by inverting the plate.</li>
		<li>Resuspend the samples in 100 &#181;l Perm/Wash containing secondary antibodies at room temperature for 1 hr protected from light.</li>
		<li>Centrifuge the plate at 931 x g for 2 min. Remove the supernatant by inverting the plate.</li>
		<li>Resuspend samples in 100 &#181;l Perm/Wash Solution.</li>
		<li>Centrifuge the plate at 931 x g for 2 min. Remove the supernatant by inverting the plate.</li>
		<li>Repeat step 4.2.9 and 4.2.10.</li>
		<li>Resuspend each sample and transfer in a total volume of 300-500 &#181;l FACS Buffer to 1 ml micro test tubes or 5 ml round-bottom tubes.</li>
		<li>Run samples on the flow cytometer17. If samples cannot be run immediately, store at 4 &#176;C protected from light.</li>
	</ol>
	</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+differentiation+of+human+embryonic+stem+cells+to+definitive+endoderm.">Efficient differentiation of human embryonic stem cells to definitive endoderm.</a> Nat Biotechnol. 23 (12), 1534-1541 (2005).</li><li>Gouon-Evans, V., 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=BMP-4+is+required+for+hepatic+specification+of+mouse+embryonic+stem+cell-derived+definitive+endoderm.">BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm.</a> Nat Biotechnol. 24 (11), 1402-1411 (2006).</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>Pan, F. 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B., 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=Nkx6+transcription+factors+and+Ptf1a+function+as+antagonistic+lineage+determinants+in+multipotent+pancreatic+progenitors.">Nkx6 transcription factors and Ptf1a function as antagonistic lineage determinants in multipotent pancreatic progenitors.</a> Dev Cell. 18 (6), 1022-1029 (2010).</li><li>Zhou, Q., 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+multipotent+progenitor+domain+guides+pancreatic+organogenesis.">A multipotent progenitor domain guides pancreatic organogenesis.</a> Dev Cell. 13 (1), 103-114 (2007).</li><li>Sander, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeobox+gene+Nkx6.1+lies+downstream+of+Nkx2.2+in+the+major+pathway+of+beta-cell+formation+in+the+pancreas.">Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas.</a> Development. 127 (24), 5533-5540 (2000).</li><li>Sharow, K. A., Temkin, B., Asson-Batres, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+stability+in+stem+cell+cultures.">Retinoic acid stability in stem cell cultures.</a> Int J Dev Biol. 56 (4), 273-278 (2012).</li><li>Korytnikov, R., Nostro, M. 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The authors have nothing to disclose.

Successfully generating NKX6-1+ pancreatic progenitors from hPSCs in vitro relies on the use of high quality cultures of hPSCs and directed differentiation involving the precise regulation of specific signaling pathways that govern key developmental stages during pancreatic development. Although this protocol can be used to induce robust expression of NKX6-1 across a variety of hPSC lines as previously shown3, to ensure efficient NKX6-1 generation the following considerations should be made. It is important that all cell culture and media preparation is performed in a sterile environment in order to prevent contamination. It is important that the hESC colonies have reached appropriate confluency and have not begun to differentiate prior to beginning day 0 of differentiation. Starting differentiation with hESCs that are not optimal can result in inefficient induction of the definitive endoderm and proceeding developmental stages. This is why it is critical to analyze cells at day 3 by flow cytometry to ensure efficient induction of definitive endoderm. The percentage of double positive CXCR4+CD117+ cells at day 3 should be greater than 90% for efficient pancreatic development3. If the percentage of cells expressing both CXCR4 and CD117 is lower than 90%, efficient induction to pancreatic progenitors will likely be compromised (results not shown). Furthermore, contrary to what was previously published, it is not necessary to wash cells with RPMI prior to feeding with stage 1 media16. Gentle shaking of the plate prior to aspirating the media is sufficient to remove the dead cells from the monolayer.
The addition of dorsomorphin at stage 2 has been shown to only be required for specific hPSC lines18. Thus, it is important to determine if dorsomorphin is necessary when working with different hPSC lines. Additionally, the duration of Stage 3 has been shown to be critical for specifying either monohormonal or polyhormonal progenitor cells and is cell line-dependent3. Thus, when working with a new hPSC line it is important to determine the duration that the cells should be cultured in Stage 3 media to determine the optimal conditions for NKX6-1 induction. To determine this, cells should be cultured in Stage 3 media for 1-4 days and analyzed 5 days later by flow cytometry for NKX6-1 expression.
Efficiently generating populations enriched in NKX6-1+ cells is important, as Nkx6-1 is critical for &#946;cell development14. While others have developed protocols that can differentiate hESC to NKX6-1+ cells (approximately 60% H1-derived NKX6-1+ cells and 55% HUES8-derived PDX+/NKX6-1+ cells)4,5, our method consistently generates &#62;80% H1-derived PDX1+/NKX6-1+ cells. However, our differentiation protocol relies on the use of mouse embryonic fibroblasts, which limits its use to research-only, as we have yet to optimize the method in Good Manufacturing Practices. Although we have not attempted to cryopreserve our stage-4 derived PDX1+/NKX6-1+ cells, this approach has been successfully performed by Schulz et al., who demonstrated that cryopreserved pancreatic aggregates were capable of retaining their function in vivo19.
In this paper we describe a 4-stage protocol to efficiently generate PDX1+/NKX6-1+ pancreatic progenitors from a variety of hPSC lines. By differentiating hPSC through four key stages of pancreatic development, this protocol provides a model to study the development of the human pancreas in vitro. Furthermore, since the PDX1+/NKX6-1+ cells have the potential to generate insulin-producing cells both in vivo and in vitro (data not shown)3,4,5, the hESC-derived PDX1+/NKX6-1+ pancreatic progenitors provide a possible source for the treatment of diabetes.

The prevalence of diabetes is increasing and according to the Canadian Diabetes Association, it is estimated that over 11 million individuals in Canada are diabetic or prediabetic, with 5-10% of these individuals having type 1 diabetes (T1D)1. T1D is an autoimmune disease that is caused by the destruction of the insulin producing &#946; cells that are located within the Islets of Langerhans. Currently, individuals living with T1D require exogenous sources of insulin2. Despite advances in insulin therapy, T1D patients continue to have a difficult time regulating their blood glucose levels and continue to suffer both hypo- and hyperglycemia. A promising form of treatment to restore normoglycemia in T1D is the use of human embryonic stem cells (hESCs), which could be used to generate an unlimited supply of insulin producing &#946; cells both in vivo and in vitro3,4,5,6,7. Differentiating hESCs to &#946;-like cells could make it possible to study diabetes in vitro, allowing for the identification of new therapeutic targets for type 2 diabetes and provide cells for transplantation into T1D patients.
The most successful attempt at generating insulin producing cells from hESCs in vitro is to recapitulate the embryonic events that occur during pancreatic development4,5. This involves the manipulation of distinct signaling pathways to accurately model key stages of the developing pancreas. Pancreatic development begins with the induction of the definitive endoderm, which is characterized by the expression of CXCR4 and CD117 (c-KIT)8,9. Precise regulation of definitive endoderm organization is required for the formation of the gut tube, which then undergoes anterior-to-posterior and ventral-dorsal patterning. The dorsal and ventral pancreatic buds emerge from the region of the posterior foregut that expresses the pancreatic and duodenal homeobox gene (Pdx1), which is necessary for pancreatic development10. The dorsal and ventral buds fuse to form the pancreas, which then undergoes extensive epithelial remodeling and expansion11. Commitment to the endocrine and exocrine lineage is accompanied by the generation of multipotent progenitor cells (MPCs) that express, among others, the transcription factors Pdx1, Nkx6.1 and Ptf1a12,13. MPCs that will become endocrine and ductal cells continue to express Nkx6-1 while decreasing Ptf1a expression. Contrary to this, exocrine lineage cells will lose expression of Nkx6-1 and maintain Ptf1a expression12.
The transcription factor Nkx6-1 has a key role in pancreatic development, particularly during the differentiation of endocrine progenitor cells to &#946; cells. As previously described, deletion of Nkx6-1 results in impaired formation of &#946; cells during pancreatic development14. Therefore, generating insulin-producing &#946; cells both in vitro and in vivo requires the efficient induction of Nkx6-1.
We recently developed a protocol to efficiently generate PDX1+/NKX6-1+ pancreatic progenitors from hPSCs. These hPSC-derived pancreatic progenitors generate mature &#946; cells upon transplantation into immunodeficient mice3. The differentiation protocol can be divided into 4 stages characteristic of: 1) definitive endoderm induction, 2) posterior foregut patterning, 3) pancreatic specification and 4) NKX6-1 induction. Here we provide a detailed description of each step of the directed differentiation.

<table><tbody><tr><td>&lt;strong&gt;Media and cytokines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1-Thioglycerol (MTG)</td><td>Sigma</td><td>M6145</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D</td><td>338-AC/CF&amp;nbsp;</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma</td><td>A4544</td><td></td></tr><tr><td>B-27 Supplement</td><td>Life Technologies</td><td>12587-010&amp;nbsp;</td><td></td></tr><tr><td>BD Cytofix/Cytoperm Buffer</td><td>BD Bioscience</td><td>554722</td><td></td></tr><tr><td>BD Perm/Wash buffer, 1x</td><td>BD Bioscience</td><td>554723</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D</td><td>233-FB</td><td></td></tr><tr><td>CHIR990210</td><td>Tocris</td><td>4423a</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>11995</td><td></td></tr><tr><td>DNase I</td><td>VWR</td><td>80510-412&amp;nbsp;</td><td></td></tr><tr><td>Dorsomorphin</td><td>Sigma</td><td>P5499</td><td></td></tr><tr><td>EGF</td><td>R&amp;amp;D</td><td>236-EG</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Wisent</td><td>88150</td><td></td></tr><tr><td>FGF10</td><td>R&amp;amp;D</td><td>345-FG</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma</td><td>G1890</td><td></td></tr><tr><td>Glutamine</td><td>Life Technologies</td><td>25030</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>NO636</td><td></td></tr><tr><td>NOGGIN</td><td>R&amp;amp;D</td><td>3344-NG</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma</td><td>R2625</td><td></td></tr><tr><td>RPMI Medium 1640</td><td>Life Technologies</td><td>11875</td><td></td></tr><tr><td>SANT-1</td><td>Tocris</td><td>1974</td><td></td></tr><tr><td>TrypLE Express Enzyme (1x), phenol red</td><td>Life Technologies</td><td>12605-010</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalogue Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies for flow cytometry (working dilutions)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD117 PE (1:100)</td><td>Life Technologies</td><td>CD11705</td><td></td></tr><tr><td>CXCR4 APC (1:50)</td><td>BD&amp;nbsp; Bioscience</td><td>551966</td><td></td></tr><tr><td>Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 conjugate (1:400)</td><td>Life Technologies</td><td>A-31571</td><td></td></tr><tr><td>Donkey Anti-Goat IgG (H+L), Alexa Fluor 488 (1:400)&amp;nbsp;</td><td>Jackson ImmunoResearch Laboratories Inc.</td><td>705-546-147</td><td></td></tr><tr><td>Isotype Control Mouse IgG&amp;nbsp;</td><td>Jackson ImmunoResearch Laboratories Inc.&amp;nbsp;</td><td>015-000-003</td><td></td></tr><tr><td>Isotype Control Goat IgG</td><td>R&amp;amp;D&amp;nbsp;&amp;nbsp;</td><td>AB-108-C</td><td></td></tr><tr><td>NKX6-1 (1:2,000)</td><td>DSHB</td><td>F55A10</td><td></td></tr><tr><td>PDX1 (1:100)</td><td>R&amp;amp;D</td><td>AF2419</td><td></td></tr></tbody></table>

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 generation of pancreatic progenitors relies on the proper growth and maintenance of undifferentiated cells followed by the precise addition of specific signaling molecules during the differentiation protocol, as illustrated in the schematic in Figure 1A. On day 0, undifferentiated cells should be 80-95% confluent and colonies should have defined edges (Figure 1A). During Stage 1, the media will likely appear cloudy since cell death is quite common at this stage. By the end of Stage 1, cells should co-express the definitive endoderm markers, CXCR4 and CD117, at a proportion greater than 90% (Figure 1B).
As cells differentiate throughout the four-stage protocol, their morphology changes from an elongated, oval-like appearance (observed at Stages 1 and 2) to smaller, rounder cells (as seen at Stages 3 and 4) (Figure 1A). By the end of Stage 4, efficient induction of PDX1+/NKX6-1+ is confirmed by flow cytometry. In the representative figure shown in Figure 1C, the expression of NKX6-1+ at day 13 is approximately 88%. As illustrated, all of the Stage 4-derived NKX6-1+ cells co-express PDX-1+, a characteristic of pancreatic progenitor cells. However, NKX6-1 expression varies across different cell lines, and a previous report showed that NKX6-1+ expression ranges from 30.5-91.4% in the 8 different hPSC lines tested3.
<img alt="Figure 1" src="/files/ftp_upload/55265/55265fig1.jpg" /> 
Figure 1: Schematic of pancreatic progenitor differentiation from hESCs and supporting data. (A) Schematic of the induction of pancreatic progenitor from hESCs. Day of differentiation, developmental stage, base media, and the cytokines added at every stage are included. Below, phase contrast images at the key stages of pancreatic differentiation are shown. In all representative figures, H1 cells were differentiated according to the 4-stage differentiation protocol depicted above. Day 0, undifferentiated hESCs; Stage 1, Definitive Endoderm; Stage 2, Posterior Foregut; Stage 3, PDX1+ Endoderm; Stage 4, NKX6-1+ endoderm/pancreatic progenitors. Scale bar = 200 &#181;m. (B) Day 3 representative flow cytometry plots for CXCR4:PE and CD117:APC antibody staining. Unstained sample on left, stained sample on right. (C) Day 13 representative flow cytometry plot for stage-4 derived pancreatic progenitor cells expressing NKX6-1 and PDX1. IgG control on left, stained sample on right. hESC - human embryonic stem cell, Act A - Activin A; bFGF - basic fibroblast growth factor, FGF10 - fibroblast growth factor 10, DM - dorsomorphin, NOG - NOGGIN, RA - retinoic acid, EGF - epidermal growth factor, NA - nicotinamide. <a href="http://cloudflare.jove.com/files/ftp_upload/55265/55265fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors at JoVE.com

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.

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 ...

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Nanyang Technological University

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Developmental Biology

7.8K Views.  University Health Network. 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.

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

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 ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

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 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 Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Biology

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 ...

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

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...

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

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Chapters in this video

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

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

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

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

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

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

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

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

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

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System