We acknowledge support from NIH New Innovator Award DP2 116520 and ORAU Ralph Powe Junior Faculty Enhancement Award.

1. Cell Maintenance
<ol>
 <li>H1 hESC (WiCell) were maintained on hESC qualified matrigel coated wells with mTeSR1 media, with media change every day. Wells were coated by dilute matrigel solution, prepared by adding 300 &mu;l of hESC matrigel in 25 ml of DMEM:F12. 1 ml of this matrigel solution was added to each well of a six well plate, or 400 &mu;l added to each well of a 12 well plate and allowed to coat for 1 hour at room temperature. Cells were mechanically passaged at a split ratio of 1:4 by scraping colonies on...

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M., Kouskoff, V., Kennedy, M., Woo, S., Fehling, H. J., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+definitive+endoderm+from+embryonic+stem+cells+in+culture.">Development of definitive endoderm from embryonic stem cells in culture.</a> Development. 131, 1651-1662 (2004).</li><li>D'Amour, K. A., Bang, A. G., Eliazer, S., Kelly, O. G., Agulnick, A. D., Smart, N. G., Moorman, M. A., Kroon, E., Carpenter, M. K., Baetge, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+pancreatic+hormone-expressing+endocrine+cells+from+human+embryonic+stem+cells.">Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.</a> Nat. Biotech. 24, 1392-1401 (2006).</li><li>Zhang, D., Jiang, W., Liu, M., Sui, X., Yin, X., Chen, S., Shi, Y., Deng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+differentiation+of+human+ES+cells+and+iPS+cells+into+mature+pancreatic+insulin-producing+cells.">Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells.</a> Cell Res. 19, 429-438 (2009).</li><li>Basma, H., Soto-Guti&#233;rrez, A., Yannam, G. R., Liu, L., Ito, R., Yamamoto, T., Ellis, E., Carson, S. D., Sato, S., Chen, Y., Muirhead, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+Transplantation+of+Human+Embryonic+Stem+Cell-Derived+Hepatocytes.">Differentiation and Transplantation of Human Embryonic Stem Cell-Derived Hepatocytes.</a> Gastroenterology. 136, 990-999 (2009).</li><li>Phillips, B. W., Hentze, H., Rust, W. L., Chen, Q. -. P., Chipperfield, H., Tan, E. -. K., Abraham, S., Sadasivam, A., Soong, P. L., Wang, S. T., Lim, R., Sun, W., Colman, A., Dunn, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+Differentiation+of+Human+Embryonic+Stem+Cells+into+the+Pancreatic+Endocrine+Lineage.">Directed Differentiation of Human Embryonic Stem Cells into the Pancreatic Endocrine Lineage.</a> Stem Cells and Development. 16, 561-578 (2007).</li><li>Kim, S. K., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+development+is+promoted+by+cyclopamine,+a+Hedgehog+signaling+inhibitor.">Pancreas development is promoted by cyclopamine, a Hedgehog signaling inhibitor.</a> Proceedings of the National Academy of Sciences. 95, 13036-13041 (1998).</li><li>Docherty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+development+of+the+islets+of+Langerhans:+implications+for+the+treatment+of+diabetes+mellitus.">Growth and development of the islets of Langerhans: implications for the treatment of diabetes mellitus.</a> Current Opinion in Pharmacology. 1, 641-649 (2001).</li><li>Nikolova, G., Jabs, N., Konstantinova, I., Domogatskaya, A., Tryggvason, K., Sorokin, L., F&#228;ssler, R., Gu, G., Gerber, H. -. P., Ferrara, N., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Vascular+Basement+Membrane:+A+Niche+for+Insulin+Gene+Expression+and+[beta]&gt;+Cell+Proliferation.">The Vascular Basement Membrane: A Niche for Insulin Gene Expression and [beta]&gt; Cell Proliferation.</a> Developmental Cell. 10, 397-405 (2006).</li><li>Yoshitomi, H., Zaret, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+interactions+initiate+dorsal+pancreas+development+by+selectively+inducing+the+transcription+factor+Ptf1a.">Endothelial cell interactions initiate dorsal pancreas development by selectively inducing the transcription factor Ptf1a.</a> Development. 131, 807-817 (2004).</li><li>Osafune, K., Caron, L., Borowiak, M., Martinez, R. J., Fitz-Gerald, C. S., Sato, Y., Cowan, C. A., Chien, K. R., Melton, D. 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During pancreatic development, differentiating pancreatic cells are in close proximity with endothelial cells from the aorta; furthermore, pancreatic islets are densely vascularized to promote rapid exchange of blood glucose and islet hormones. Given these facts, it is not surprising that endothelial cells play an important role in the process of pancreatic organogenesis. While the importance of endothelial cells during pancreatic development is increasingly being appreciated, its role in in vitro differentiatio...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Concentrations</td>
		</tr>
		<tr>
			<td>mTeSR1 (with supplement)</td>
			<td>Stem cell Technologies</td>
			<td>5850</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>hESC qualified matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Invitrogen</td>
			<td>11330-032</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MCDB-131</td>
			<td>Invitrogen</td>
			<td>10372019</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MCDB-131 (Complete)</td>
			<td>VEC Technologies</td>
			<td>MCDB-131</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>R&amp;D</td>
			<td><a href="http://www.rndsystems.com/pdf/338-ac.pdf">338-AC</a></td>
			<td>100ng/ml</td>
		</tr>
		<tr>
			<td>Wortmannin</td>
			<td>Invitrogen</td>
			<td>W3144</td>
			<td>1&mu;M</td>
		</tr>
		<tr>
			<td>KAAD-Cyclopamie</td>
			<td>Sigma-Aldrich</td>
			<td>C4116</td>
			<td>0.2&mu;M</td>
		</tr>
		<tr>
			<td>All-Trans Retinoic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td>2&mu;M</td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Sigma-Aldrich</td>
			<td>D5942</td>
			<td>30&mu;M</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>Sigma-Aldrich</td>
			<td>S5261</td>
			<td>30 nM</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I1882</td>
			<td>25 &mu;g/ml</td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>T8158</td>
			<td>50 &mu;g/ml</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>R&amp;D</td>
			<td><a href="http://www.rndsystems.com/pdf/236-eg.pdf">236-EG</a></td>
			<td>10ng/ml</td>
		</tr>
		<tr>
			<td>EndoGro</td>
			<td>VEC Technologies</td>
			<td>ENDOGRO</td>
			<td>10mg</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3149</td>
			<td>90&mu;g/ml</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0888</td>
			<td>1&mu;g/ml</td>
		</tr>
		<tr>
			<td>NucleoSpin RNA II</td>
			<td>Macherey Nagel</td>
			<td>740955</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ImProm II reverse transcription System</td>
			<td>Promega</td>
			<td>A3800</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Brilliant II SYBR Green QPCR master mix</td>
			<td>Straragene</td>
			<td>600548</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sox17 goat polyclonal IgG</td>
			<td>Santa Cruz</td>
			<td>sc-17355</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>PDX1 goat polyclonal IgG</td>
			<td>Santa Cruz</td>
			<td>sc-14662</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>C-Peptide Rabbit polyclonal</td>
			<td>Cell Signaling</td>
			<td>4593</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-21206</td>
			<td>1/1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 donkey anti-goat IgG</td>
			<td>Invitrogen</td>
			<td>A-21447</td>
			<td>1/1000</td>
		</tr>
	</tbody>
</table>
Table 2. Reagents and Kits.

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.

Watch this Scientific Journal Video about Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach at J

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

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 ...

endothelial-cell-co-culture-mediates-maturation-human-embryonic-stem

Research

JoVE Journal

Biology

16.2K vues.  University of Pittsburgh. La présente étude décrit une approche différenciation dirigée dans l'induction de la différenciation du pancréas de cellules souches embryonnaires humaines. D'une grande importance est le fait que des cellules endothéliales de co-culture de maturation de la médiation sur les cellules souches embryonnaires humaines dérivées des progéniteurs pancréatiques en cellules exprimant l'insuline.

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