Name: endothelial cell co-culture mediates maturation of human embryonic stem cell to pancreatic insulin producing cells in a directed differentiation approach
Uploaded: 2012-03-27T13:00:00
Description: 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

Vi erkjenner støtte fra NIH New Innovator Award DP2 116520 og ORAU Ralph Powe Junior fakultet Enhancement Award.

1. Cell Vedlikehold <ol><li> H1 hESC (WiCell) ble opprettholdt på hESC kvalifiserte matrigel belagt brønner med mTeSR1 media, med media endres hver dag. Brønnene ble belagt med fortynnet matrigel løsning, utarbeidet ved å legge til 300 mL av hESC matrigel i 25 ml DMEM: F12. 1 ml av denne matrigel løsningen ble lagt til hver brønn av en seks brønn plate, eller 400 mL lagt til hver brønn av en 12 brønn plate og lov til pels i 1 time ved romtemperatur. Celler ble mekanisk passaged ved en splitt forholdet 1:04 v...

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	<li>De Vos, J., Assou, S., Tondeur, S., Dijon, M., Hamamah, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Les+cellules+souches+embryonnaires+humaines+:+de+la+transgression+de+l'embryon+humain+&#225;+la+m&#233;decine+r&#233;g&#233;n&#233;ratrice+de+demain.">Les cellules souches embryonnaires humaines : de la transgression de l'embryon humain &#225; la m&#233;decine r&#233;g&#233;n&#233;ratrice de demain.</a> Gyn&#233;cologie Obst&#233;trique &#38; Fertilit&#233;. 37, 620-626 (2009).</li><li>Murtaugh, L. C., 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=Genes,+Signals,+and+Lineages+in+Pancreas+Development.">Genes, Signals, and Lineages in Pancreas Development.</a> Annual Review of Cell and Developmental Biology. 19, 71-89 (2003).</li><li>Kubo, A., Shinozaki, K., Shannon, J. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marked+differences+in+differentiation+propensity+among+human+embryonic+stem+cell+lines.">Marked differences in differentiation propensity among human embryonic stem cell lines.</a> Nat. Biotech. 26, 313-315 (2008).</li><li>Banerjee, I., Sharma, N., Yarmush, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+co-culture+on+pancreatic+differentiation+of+embryonic+stem+cells.">Impact of co-culture on pancreatic differentiation of embryonic stem cells.</a> J. Tissue Eng. Regen. Med. 5, 313-323 (2010).</li></ol>

Under bukspyttkjertelen utvikling, differensierende bukspyttkjertelen celler er i umiddelbar nærhet med endotelceller fra aorta, og videre, bukspyttkjertel holmer er tett vascularized å fremme rask utveksling av blodsukker og holmen hormoner. Gitt disse fakta, er det ikke overraskende at endotelceller spiller en viktig rolle i prosessen med bukspyttkjertelen organogenesen. Mens betydningen av endotelceller under bukspyttkjertelen utvikling blir i økende grad verdsatt, er dens rolle i in vitro differensiering...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Konsentrasjoner </td></tr><tr><td> mTeSR1 (med supplement) </td><td> Stamceller Technologies </td><td> 5850 </td><td></td></tr><tr><td> hESC kvalifisert matrigel </td><td> BD Biosciences </td><td> 354277 </td><td></td></tr><tr><td> DMEM: F12 </td><td> Invitrogen </td><td> 11330-032 </td><td></td></tr><tr><td> MCDB-131 </td><td> Invitrogen </td><td> 10372019 </td><td></td></tr><tr><td> MCDB-131 (Complete) </td><td> VEC Technologies </td><td> MCDB-131 </td><td></td></tr><tr><td> B27 Supplement </td><td> Invitrogen </td><td> 17504044 </td><td></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> 1μM </td></tr><tr><td> KAAD-Cyclopamie </td><td> Sigma-Aldrich </td><td> C4116 </td><td> 0.2μM </td></tr><tr><td> All-Trans retinsyre </td><td> Sigma-Aldrich </td><td> R2625 </td><td> 2μM </td></tr><tr><td> DAPT </td><td> Sigma-Aldrich </td><td> D5942 </td><td> 30μM </td></tr><tr><td> Nicotinamide </td><td> Sigma-Aldrich </td><td> N0636 </td><td> 10 mM </td></tr><tr><td> Sodium selenitt </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 mikrogram / ml </td></tr><tr><td> Transferrin </td><td> Sigma-Aldrich </td><td> T8158 </td><td> 50 mikrogram / 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> 90μg/ml </td></tr><tr><td> Hydrokortison </td><td> Sigma-Aldrich </td><td> H0888 </td><td> 1μg/ml </td></tr><tr><td> NucleoSpin RNA II </td><td> Macherey Nagel </td><td> 740955 </td><td></td></tr><tr><td> ImProm II revers transkripsjon System </td><td> Promega </td><td> A3800 </td><td></td></tr><tr><td> Brilliant II SYBR Grønn QPCR mester mix </td><td> Straragene </td><td> 600548 </td><td></td></tr><tr><td> Sox17 geit polyklonale IgG </td><td> Santa Cruz </td><td> sc-17355 </td><td> 1/500 </td></tr><tr><td> PDX1 geit polyklonale IgG </td><td> Santa Cruz </td><td> sc-14662 </td><td> 1/500 </td></tr><tr><td> C-peptid Rabbit polyklonale </td><td> Cellesignalisering </td><td> 4593 </td><td> 1/500 </td></tr><tr><td> Alexa Fluor 488 esel anti-kanin IgG </td><td> Invitrogen </td><td> A-21206 </td><td> 1/1000 </td></tr><tr><td> Alexa Fluor 647 esel anti-geit IgG </td><td> Invitrogen </td><td> A-21447 </td><td> 1/1000 </td></tr></tbody></table> Tabell 2. Reagenser og kits.

endothelial cell co-culture mediates maturation of human embryonic stem cell to pancreatic insulin producing cells in a directed differentiation approach

Embryonale stamceller (ESC) har to viktigste kjennetegnene: de kan være på ubestemt tid dyrket in vitro i en udifferensiert tilstand og de ​​er pluripotent, og dermed ha potensial til å differensiere i flere linjene. Slike egenskaper gjør ESCs svært attraktivt for celle-basert terapi og regenererende behandling applikasjoner en. Men for sitt fulle potensial til å bli realisert cellene må differensieres til modne og funksjonelle fenotyper, som er en skremmende oppgave. En lovende tilnærming i inducing cellulær differensiering er å nøye etterligne banen organogenesen i in vitro-innstillingen. Bukspyttkjertelen utvikling forekommer i bestemte stadier 2, starter med endoderm, som kan utvikle seg til flere organer, blant annet lever og bukspyttkjertel. Endoderm induksjon kan oppnås ved modulering av nodal vei gjennom tilførsel av activin A 3 i kombinasjon med flere vekstfaktorer 4-7 </sup&gt;. Definitive endoderm cellene deretter gjennomgå bukspyttkjertelen engasjement ved hemming av sonic hedgehog hemming, som kan oppnås in vitro ved tilsetting av cyclopamine 8. Bukspyttkjertelen modning er mediert av flere parallelle arrangementer, inkludert hemming av hakk signalering, aggregering av pankreas progenitors inn 3-dimensjonale klynger; induksjon av vascularization, for å nevne noen. Den klart mest vellykkede in vitro modning av MGP avledet bukspyttkjertelen stamceller har blitt oppnådd gjennom hemming av hakk signalisere ved DAPT tilskudd ni. Selv vellykket, dette resulterer i lav avkastning på den modne fenotype med redusert funksjonalitet. En mindre studert område er effekten av endotelial celle signalisering i bukspyttkjertelen modning, noe som blir i økende grad verdsatt som en viktig medvirkende faktor i in-vivo bukspyttkjertelen holmen modning 10,11. Den nåværende studien utforsker slik effekt av endothelIAL celle signalisering i modning av menneskelige MGP avledet bukspyttkjertelen stamceller til insulinproduserende holme-lignende celler. Vi rapporterer en flertrinns rettet differensiering protokoll hvor de menneskelige ESCs er første indusert mot endoderm av activin A langs med hemming av PI3K veien. Bukspyttkjertelen spesifikasjon av endoderm celler oppnås ved hemming av sonic hedgehog signalering ved Cyclopamine sammen med retinoid induksjon ved tilsetting av retinsyre. Den siste etappen av modning er indusert av endotelceller signalering oppnås ved en co-kultur konfigurasjon. Mens flere endotelceller har blitt testet i co-kulturen, her vi presentere våre data med rotte hjerte mikrovaskulære endotelceller (RHMVEC), primært for enkel analyse.

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

Den nåværende studien beskriver en rettet differensiering tilnærming i inducing bukspyttkjertelen differensiering av humane embryonale stamceller. Av stor betydning er det faktum at endotelceller co-kulturen formidler modning av humane embryonale stamceller hentet bukspyttkjertelen stamfedre til insulin som uttrykker cellene.

Endotelcelle Co-kulturen formidler Modning av Human embryonale stamceller til Bukspyttkjertelen insulinproduserende celler i en Regissert Differensiering Approach

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

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