Name: efficient differentiation of pluripotent stem cells to nkx6-1+ pancreatic progenitors
Uploaded: 2017-03-07T12:30:00
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...

<ol>
	<li>Cogger, K., Nostro, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+cell+replacement+therapies+for+the+treatment+of+type+1+diabetes.">Recent advances in cell replacement therapies for the treatment of type 1 diabetes.</a> Endocrinology. 156 (1), 8-15 (2015).</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>Pagliuca, F. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Functional+Human+Pancreatic+beta+Cells+In+Vitro.">Generation of Functional Human Pancreatic beta Cells In Vitro.</a> Cell. 159 (2), 428-439 (2014).</li><li>Rezania, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+diabetes+with+insulin-producing+cells+derived+in+vitro+from+human+pluripotent+stem+cells.">Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.</a> Nat Biotechnol. , (2014).</li><li>Kroon, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+endoderm+derived+from+human+embryonic+stem+cells+generates+glucose-responsive+insulin-secreting+cells+in+vivo.">Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo.</a> Nat Biotechnol. 26 (4), 443-452 (2008).</li><li>Rezania, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+human+embryonic+stem+cell-derived+pancreatic+progenitors+into+functional+islets+capable+of+treating+pre-existing+diabetes+in+mice.">Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice.</a> Diabetes. 61 (8), 2016-2029 (2012).</li><li>D'Amour, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+differentiation+of+human+embryonic+stem+cells+to+definitive+endoderm.">Efficient differentiation of human embryonic stem cells to definitive endoderm.</a> 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. C., Wright, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+organogenesis:+from+bud+to+plexus+to+gland.">Pancreas organogenesis: from bud to plexus to gland.</a> Dev Dyn. 240 (3), 530-565 (2011).</li><li>Schaffer, A. E., Freude, K. K., Nelson, S. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+polyhormonal+and+multipotent+pancreatic+progenitor+lineages+from+human+pluripotent+stem+cells.">Generation of polyhormonal and multipotent pancreatic progenitor lineages from human pluripotent stem cells.</a> Methods. , 56-64 (2016).</li><li>Baumgarth, N., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+multicolor+flow+cytometry+for+immunophenotyping.">A practical approach to multicolor flow cytometry for immunophenotyping.</a> J Immunol Methods. 243 (1-2), 77-97 (2000).</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=Stage-specific+signaling+through+TGFbeta+family+members+and+WNT+regulates+patterning+and+pancreatic+specification+of+human+pluripotent+stem+cells.">Stage-specific signaling through TGFbeta family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells.</a> Development. 138 (5), 861-871 (2011).</li><li>Schulz, T. 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=A+scalable+system+for+production+of+functional+pancreatic+progenitors+from+human+embryonic+stem+cells.">A scalable system for production of functional pancreatic progenitors from human embryonic stem cells.</a> PLoS One. 7 (5), e37004 (2012).</li></ol>

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 s...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media and cytokines</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&#38;D</td>
			<td>338-AC/CF&#160;</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&#160;</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&#38;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&#8217;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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Sigma</td>
			<td>P5499</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>R&#38;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&#38;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&#38;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>Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies for flow cytometry (working dilutions)</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&#160; 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)&#160;</td>
			<td>Jackson ImmunoResearch Laboratories Inc.</td>
			<td>705-546-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotype Control Mouse IgG&#160;</td>
			<td>Jackson ImmunoResearch Laboratories Inc.&#160;</td>
			<td>015-000-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotype Control Goat IgG</td>
			<td>R&#38;D&#160;&#160;</td>
			<td>AB-108-C</td>
			<td></td>
		</tr>
		<tr>
			<td>NKX6-1 (1:2000)</td>
			<td>DSHB</td>
			<td>F55A10</td>
			<td></td>
		</tr>
		<tr>
			<td>PDX1 (1:100)</td>
			<td>R&#38;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 comm...

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 ...

The overall goal of this procedure is to efficiently generate NKX6.1 expressing pancreatic progenitors from human pluripotent stem cells in vitro by differentiating the cells through four key stages of pancreatic development. By differentiating human pluripotent stem cells through four key stages of pancreatic devleopment, this protocol provides a model to study human pancreas development in vitro. The main advantage of this technique over existing protocols is that we are able to generate high percentages of NKX6.1 positive pancreatic progenitor cells using a variety of human pluripotent stem cell lines.Given that pancreatic progenitors have the potential to generate insulin producing beta like cells, this technique will give us the opportunity to develop cells for diabetes treatment, for drug discovery and for disease modeling. On day zero, begin differentiation by replacing the KnockOut serum replacement media with day zero differentiation media. Then one days one and two, gently shake the plate to remove any dead cells from the monolayer before aspirating the media.Afterward, replace it with day one to two differentiation media. On day three, harvest the cells for flow cytometry. If the cells express more than 90%of CXCR4 and CD117, the efficiency has been reached.Then gently shake the plate to remove any dead cells from the monolayer prior to aspiration and replace it with day three to five differentiation media. It is important to ensure efficient induction of the definitive under derm by day three by flow cytometry. If the percentage of cells expressing both CXCR4 and CD117 is lower than 90%differentiation to pancreatic progenitors will likely be compromised.On days six and seven, replace the medium with day six to seven differentiation media. On days eight, 10 and 12, replace the medium with day eight to 12 differentiation media. Then on day 13, harvest the cells for flow cytometry to determine PDX1 and NKX6.1 expression.To harvest the cells for flow cytometry analysis, disassociate the cells with commercial Trypsin solution according to the manufacturer's protocol and incubate the cells at 37 degrees Celsius for three minutes. After three minutes, remove the Trypsin solution and re-suspend the single cells in 1, 000 microliters of fax buffer with 30 microliters of DNA One. Then filter the cells using a 35 micrometer nylon mesh cell strainer and transfer them to a micro centrifuge tube.Centrifuge the cells at 455 times g for five minutes before preparing the cells for live or fixed staining. Now re-suspend the cells in 1, 000 microliters of fax buffer. Transfer 200 microliters into two wells of a 96 well plate for both unstained and stained samples.Afterward, centrifuge the plate at 931 times g for two minutes, afterward remove the supernatant by inverting the plate, stain the cells with primary conjugated antibody in a final volume of 100 microliters for 30 minutes at room temperature and protect them from light. Subsequently, centrifuge the plate at 931 times g for two minutes, after two minutes remove the supernatant by inverting the plate. Following that, re-suspend the samples in 100 microliters of fax buffer.Then centrifuge the plate 931 times g for two minutes. And remove the supernatant by inverting the plate. Next re-suspend the samples in 300 to 500 microliters of fax buffer, transfer them to the one milliliter micro test tubes or the five milliliter round bottom tubes and run the samples on flow cytometor.If the samples cannot be run immediately, store them at four degrees Celsius and protect it from light. In this procedure, re-suspend the cell pellet in commercial fixation and permeable Isation solution for 24 hours at four degrees Celsius. After 24 hours, centrifuge the sample at 455 times g for five minutes, then re-suspend it in 400 microliters of commercial perm wash solution.Next, transfer 200 microliters of the sample in two wells of a 96 well plate for IGG control and PDX1, NKX6.1 staining, centrifuge the plate at 931 times g for two minutes. Then re-suspend the cell pellets in 100 microliters of commercial perm wash solution containing anti-PDX1 and anti-NKX61 primary or isotope control antibodies. Incubate them overnight at four degrees Celsius.The next day, centrifuge the plate at 931 times g for two minutes, remove the supernatant by inverting the plate and re-suspend the samples in 100 microliters of the commercial perm wash solution. Next, centrifuge the plate at 931 times g for two minutes. Remove the supernatant by inverting the plate.Then re-suspend the samples in 100 microliters of perm wash solution containing secondary antibody at room temperature for one hour and protect it from light. After an house, centrifuge the plate 931 times g for two minutes, then remove the supernatant by inverting the plate, following that, re-suspend the samples in 100 microliters of commercial perm wash solution, centrifuge the plate at 931 times g for two minutes, remove the supernatant by inverting the plate. Then, re-suspend the sample in 100 microliters of commercial perm wash solution and centrifuge the plate at 931 times g for two minutes again before removing the supernatant.Next, re-suspend the samples in 300 to 500 microliters of fax buffer and transfer them into the separate one milliliter test tubes or five milliliter round bottom tubes for running flow cytometry. Shown here is a sematic of the induction of pancreatic progenitor from human embryonic stem cells which indicates day of differentiation, develop mental stage, base media and the cytokines added at every stage. And here are the phase contrast images at the key stages of pancreatic differentiation.H1 cells were differentiated according to the four stage differentiation protocol depicted. With undifferentiated human embryonic stem cells at day zero, definitive endo derm at stage one, posterior four gut at stage two, pancreatic endo derm at stage three and NKX6.1 positive endo derm pancreatic progenitors at stage four. This figure shows the day three representative flow cytometry plots for CXCR4 PE and CD117 APC antibody staining.Here is the unstained sample and here is the stained sample. And this figure shows day 13 representative flow cytometry plots for stage four derived pancreatic progenitor cells expressing NKX61 and PDX1. This is the IGG control and this is the stained sample.Once mastered, this technique can be done in 13 days. However this may vary depending on the cell line used. While attempting this procedure, it is important to test all agents due to batch to batch variability.After following the procedure, the NKX6.1 positive pancreatic progenitor cells can be further differentiated to insulin producing beta like cells using other published protocols. This provides a platform for studying beta cell maturation and development in vitro. After watching this video, you should have a good understanding of how to efficiently differentiate human pluripotent stem cells to pancreatic progenitors by taking the cells through four key stages of development.

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Developmental Biology

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

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

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

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

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

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 ...

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

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...