Name: robust differentiation of human ipscs into a pure population of adipocytes to study adipocyte-associated disorders
Uploaded: 2022-02-09T14:45:00
Description: Watch this Scientific Journal Video about Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders at JoVE.com

Dette arbeidet ble finansiert av et tilskudd fra Qatar National Research Fund (QNRF) (Grant No. NPRP10-1221-160041). Maryam Aghadi ble støttet av GSRA-stipend fra Qatar National Research Fund (QNRF).

Studien er godkjent av vedkommende institusjonelle forskningsetiske komité og utført i henhold til de etiske standarder som er fastsatt i Helsinkideklarasjonen av 1964 og senere endringer eller sammenlignbare etiske standarder. Protokollen ble godkjent av Institutional Review Board (IRB) av HMC (nr. 16260/16) og QBRI (nr. 2016-003). Dette arbeidet er også optimalisert for hESC-er som H1 og H9. Blodprøver ble innhentet fra friske personer med fullt informert samtykke. iPSCene genereres fra mononukleære celler i perif...

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	<li>Hass, R., Kasper, C., Bohm, S., Jacobs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+populations+and+sources+of+human+mesenchymal+stem+cells+(MSC):+A+comparison+of+adult+and+neonatal+tissue-derived+MSC.">Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC.</a> Cell Communication and Signaling: CCS. 9, 12 (2011).</li><li>Wagner, 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=Aging+and+replicative+senescence+have+related+effects+on+human+stem+and+progenitor+cells.">Aging and replicative senescence have related effects on human stem and progenitor cells.</a> PLoS One. 4 (6), 5846 (2009).</li><li>Brown, P. T., Squire, M. W., Li, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+evaluation+of+mesenchymal+stem+cells+derived+from+human+embryonic+stem+cells+and+bone+marrow.">Characterization and evaluation of mesenchymal stem cells derived from human embryonic stem cells and bone marrow.</a> Cell and Tissue Research. 358 (1), 149-164 (2014).</li><li>Trivedi, P., Hematti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+immunological+characterization+of+mesenchymal+stromal+cells+from+human+embryonic+stem+cells.">Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells.</a> Experimental Hematology. 36 (3), 350-359 (2008).</li><li>Barberi, T., Willis, L. M., Socci, N. D., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+multipotent+mesenchymal+precursors+from+human+embryonic+stem+cells.">Derivation of multipotent mesenchymal precursors from human embryonic stem cells.</a> PLoS Medicine. 2 (6), 161 (2005).</li><li>Xiong, 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=Derivation+of+adipocytes+from+human+embryonic+stem+cells.">Derivation of adipocytes from human embryonic stem cells.</a> Stem Cells and Development. 14 (6), 671-675 (2005).</li><li>Cuaranta-Monroy, I., 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=Highly+efficient+differentiation+of+embryonic+stem+cells+into+adipocytes+by+ascorbic+acid.">Highly efficient differentiation of embryonic stem cells into adipocytes by ascorbic acid.</a> Stem Cell Research. 13 (1), 88-97 (2014).</li><li>van Harmelen, 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=Differential+lipolytic+regulation+in+human+embryonic+stem+cell-derived+adipocytes.">Differential lipolytic regulation in human embryonic stem cell-derived adipocytes.</a> Obesity (Silver Spring). 15 (4), 846-852 (2007).</li><li>Noguchi, 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=In+vitro+characterization+and+engraftment+of+adipocytes+derived+from+human+induced+pluripotent+stem+cells+and+embryonic+stem+cells.">In vitro characterization and engraftment of adipocytes derived from human induced pluripotent stem cells and embryonic stem cells.</a> Stem Cells and Development. 22 (21), 2895-2905 (2013).</li><li>Ahfeldt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+human+pluripotent+stem+cells+into+white+and+brown+adipocytes.">Programming human pluripotent stem cells into white and brown adipocytes.</a> Nature Cell Biology. 14 (2), 209-219 (2012).</li><li>Lee, Y. K., Cowan, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+white+and+brown+adipocytes+from+human+pluripotent+stem+cells.">Differentiation of white and brown adipocytes from human pluripotent stem cells.</a> Methods in Enzymology. 538, 35-47 (2014).</li><li>Abdelalim, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+different+types+of+diabetes+using+human+pluripotent+stem+cells.">Modeling different types of diabetes using human pluripotent stem cells.</a> Cellular and Molecular Life Sciences: CMLS. 78 (6), 2459-2483 (2021).</li><li>Abdelalim, E. M., Bonnefond, A., Bennaceur-Griscelli, A., Froguel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cells+as+a+potential+tool+for+disease+modelling+and+cell+therapy+in+diabetes.">Pluripotent stem cells as a potential tool for disease modelling and cell therapy in diabetes.</a> Stem Cell Reviews and Reports. 10 (3), 327-337 (2014).</li><li>Karam, M., Younis, I., Elareer, N. R., Nasser, S., Abdelalim, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+Generation+of+mesenchymal+stem+cells+and+adipocytes+from+human+pluripotent+stem+cells.">Scalable Generation of mesenchymal stem cells and adipocytes from human pluripotent stem cells.</a> Cells. 9 (3), (2020).</li><li>Karam, M., Abdelalim, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+and+highly+efficient+protocol+for+differentiation+of+human+pluripotent+stem+cells+into+mesenchymal+stem+cells.">Robust and highly efficient protocol for differentiation of human pluripotent stem cells into mesenchymal stem cells.</a> Methods in Molecular Biology. , (2020).</li><li>Li, L., Bennett, S. A., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+E-cadherin+and+other+cell+adhesion+molecules+in+survival+and+differentiation+of+human+pluripotent+stem+cells.">Role of E-cadherin and other cell adhesion molecules in survival and differentiation of human pluripotent stem cells.</a> Cell Adhesion &amp; Migration. 6 (1), 59-70 (2012).</li><li>Lai, L., Bohnsack, B. L., Niederreither, K., Hirschi, K. K. <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+regulates+endothelial+cell+proliferation+during+vasculogenesis.">Retinoic acid regulates endothelial cell proliferation during vasculogenesis.</a> Development. 130 (26), 6465-6474 (2003).</li><li>Chanchevalap, S., Nandan, M. O., Merlin, D., Yang, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-trans+retinoic+acid+inhibits+proliferation+of+intestinal+epithelial+cells+by+inhibiting+expression+of+the+gene+encoding+Kruppel-like+factor+5.">All-trans retinoic acid inhibits proliferation of intestinal epithelial cells by inhibiting expression of the gene encoding Kruppel-like factor 5.</a> FEBS Letters. 578 (1-2), 99-105 (2004).</li><li>di Masi, 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=Retinoic+acid+receptors:+from+molecular+mechanisms+to+cancer+therapy.">Retinoic acid receptors: from molecular mechanisms to cancer therapy.</a> Molecular Aspects of Medicine. 41, 1 (2015).</li><li>Simandi, Z., Balint, B. L., Poliska, S., Ruhl, R., Nagy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+retinoic+acid+receptor+signaling+coordinates+lineage+commitment+of+spontaneously+differentiating+mouse+embryonic+stem+cells+in+embryoid+bodies.">Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies.</a> FEBS Letters. 584 (14), 3123-3130 (2010).</li><li>De Angelis, M. T., Parrotta, E. I., Santamaria, G., Cuda, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+retinoic+acid+treatment+sustains+pluripotency+and+suppresses+differentiation+of+human+induced+pluripotent+stem+cells.">Short-term retinoic acid treatment sustains pluripotency and suppresses differentiation of human induced pluripotent stem cells.</a> Cell Death &amp; Disease. 9 (1), 6 (2018).</li><li>Li, L., Dong, L., Wang, Y., Zhang, X., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lats1/2-mediated+alteration+of+hippo+signaling+pathway+regulates+the+fate+of+bone+marrow-derived+mesenchymal+stem+cells.">Lats1/2-mediated alteration of hippo signaling pathway regulates the fate of bone marrow-derived mesenchymal stem cells.</a> BioMed Research International. 2018, 4387932 (2018).</li><li>Moldes, 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=Peroxisome-proliferator-activated+receptor+gamma+suppresses+Wnt/beta-catenin+signalling+during+adipogenesis.">Peroxisome-proliferator-activated receptor gamma suppresses Wnt/beta-catenin signalling during adipogenesis.</a> The Biochemical Journal. 376, 607-613 (2003).</li><li>Ross, S. 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=Inhibition+of+adipogenesis+by+Wnt+signaling.">Inhibition of adipogenesis by Wnt signaling.</a> Science. 289 (5481), 950-953 (2000).</li><li>Wang, Y. K., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+and+mechanical+stimulation+in+the+regulation+of+mesenchymal+stem+cell+differentiation.">Cell adhesion and mechanical stimulation in the regulation of mesenchymal stem cell differentiation.</a> Journal of Cellular and Molecular Medicine. 17 (7), 823-832 (2013).</li><li>Mohsen-Kanson, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+induced+pluripotent+stem+cells+into+brown+and+white+adipocytes:+role+of+Pax3.">Differentiation of human induced pluripotent stem cells into brown and white adipocytes: role of Pax3.</a> Stem Cells. 32 (6), 1459-1467 (2014).</li><li>Billon, N., 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=The+generation+of+adipocytes+by+the+neural+crest.">The generation of adipocytes by the neural crest.</a> Development. 134 (12), 2283-2292 (2007).</li><li>Li, N., Kelsh, R. N., Croucher, P., Roehl, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neural+crest+cell+fate+by+the+retinoic+acid+and+Pparg+signalling+pathways.">Regulation of neural crest cell fate by the retinoic acid and Pparg signalling pathways.</a> Development. 137 (3), 389-394 (2010).</li><li>Ussar, S., 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=ASC-1,+PAT2,+and+P2RX5+are+cell+surface+markers+for+white,+beige,+and+brown+adipocytes.">ASC-1, PAT2, and P2RX5 are cell surface markers for white, beige, and brown adipocytes.</a> Science Translational Medicine. 6 (247), (2014).</li><li>Festy, F., 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=Surface+protein+expression+between+human+adipose+tissue-derived+stromal+cells+and+mature+adipocytes.">Surface protein expression between human adipose tissue-derived stromal cells and mature adipocytes.</a> Histochemistry and Cell Biology. 124 (2), 113-121 (2005).</li><li>Cai, L., Wang, Z., Ji, A., Meyer, J. M., vander Westhuyzen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scavenger+receptor+CD36+expression+contributes+to+adipose+tissue+inflammation+and+cell+death+in+diet-induced+obesity.">Scavenger receptor CD36 expression contributes to adipose tissue inflammation and cell death in diet-induced obesity.</a> PLoS One. 7 (5), 36785 (2012).</li><li>Mesuret, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neuronal+role+of+the+Alanine-Serine-Cysteine-1+transporter+(SLC7A10,+Asc-1)+for+glycine+inhibitory+transmission+and+respiratory+pattern.">A neuronal role of the Alanine-Serine-Cysteine-1 transporter (SLC7A10, Asc-1) for glycine inhibitory transmission and respiratory pattern.</a> Scientific Reports. 8 (1), 8536 (2018).</li><li>Silverstein, R. L., Febbraio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD36,+a+scavenger+receptor+involved+in+immunity,+metabolism,+angiogenesis,+and+behavior.">CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior.</a> Science Signaling. 2 (72), (2009).</li><li>Brooimans, R. A., van Wieringen, P. A., van Es, L. A., Daha, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+roles+of+decay-accelerating+factor,+membrane+cofactor+protein,+and+CD59+in+the+protection+of+human+endothelial+cells+against+complement-mediated+lysis.">Relative roles of decay-accelerating factor, membrane cofactor protein, and CD59 in the protection of human endothelial cells against complement-mediated lysis.</a> European Journal of Immunology. 22 (12), 3135-3140 (1992).</li><li>Davies, 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=CD59,+an+LY-6-like+protein+expressed+in+human+lymphoid+cells,+regulates+the+action+of+the+complement+membrane+attack+complex+on+homologous+cells.">CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells.</a> The Journal of Experimental Medicine. 170 (3), 637-654 (1989).</li><li>Lapid, K., Graff, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form(ul)ation+of+adipocytes+by+lipids.">Form(ul)ation of adipocytes by lipids.</a> Adipocyte. 6 (3), 176-186 (2017).</li><li>Aldridge, 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=Assay+validation+for+the+assessment+of+adipogenesis+of+multipotential+stromal+cells--a+direct+comparison+of+four+different+methods.">Assay validation for the assessment of adipogenesis of multipotential stromal cells--a direct comparison of four different methods.</a> Cytotherapy. 15 (1), 89-101 (2013).</li><li>Schaedlich, K., Knelangen, J. M., Navarrete Santos, A., Fischer, B., Navarrete Santos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+sort+ESC-derived+adipocytes.">A simple method to sort ESC-derived adipocytes.</a> Cytometry A. 77 (10), 990-995 (2010).</li><li>Costa, L. 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=Functional+heterogeneity+of+mesenchymal+stem+cells+from+natural+niches+to+culture+conditions:+implications+for+further+clinical+uses.">Functional heterogeneity of mesenchymal stem cells from natural niches to culture conditions: implications for further clinical uses.</a> Cellular and Molecular Life Sciences: CMLS. 78 (2), 447-467 (2021).</li></ol>

Denne protokollen er svært viktig på grunn av dens evne til å levere MSC-er i høy ytelse og effektivitet. Denne masseskalaproduksjonen av MSC-er ble gjort mulig ved forbigående inkubasjon av iPSCs-avledede EB-er med 10 μM RA14,15. Forbigående behandling med 10 μM RA forbedret MSC-utbyttet med 11,2 til 1542 fold14,15, med denne protokollen som gjelder på både iPSCs og hPSCs. Ved denne dosen og be...

Mesenkymale stamceller (MSC) fungerer som en effektiv forbigående ressurs for å produsere celler av mesodermal opprinnelse som adipocytter, osteocytter og kondrocytter, som videre kan brukes til å modellere deres respektive genetiske lidelser. Tidligere tilnærminger baserte seg imidlertid på å oppnå disse MSC-ene fra voksent vev 1, noe som medførte utfordringen med å skaffe dem i høyt antall fra donorene, og begrensningen av å holde dem funksjonelt levedyktige under suboptimale in vitro-kulturforhold 1,2. Disse hindringene har gitt stor etterspørsel etter å ha en protokoll for generering av MSC-er in vitro. Humaninduserte pluripotente stamceller (iPSCs) kan brukes som en verdifull kilde til MSC, med MSC-egenskaper 3,4,5. iPSCs-avledede MSCer kan brukes som et terapeutisk alternativ i flere sykdommer. Også evnen til iPSCs-avledede MSCer til å generere adipocytter, gjør dem til en verdifull in vitro menneskelig modell for å studere human adipogenese, fedme og adipocytt-assosierte lidelser.
Nåværende differensieringsprotokoller av adipocytter kan klassifiseres i to grupper, med en som involverer differensiering av adipocytter ved bruk av kjemiske eller proteinbaserte cocktailer som gir et resulterende utbytte på 30% -60% 6,7,8,9, mens den andre involverer genetisk manipulasjon for robust induksjon av viktige transkripsjonsfaktorer som styrer adipocytterutvikling for å gi et utbytte på 80% -90%10, 11. Genetisk manipulasjon rekapitulerer imidlertid ikke den naturlige prosessen med adipocytdifferensiering, og maskerer ofte de subtile paradigmene som kommer under adipogenese, noe som gjør den ineffektiv for sykdomsmodelleringsformål12,13. Derfor presenterer vi en måte å sortere kjemisk avledede modne adipocytter fra umodne ved fluorescerende merking av lipidbærende adipocytter ved bruk av Nilrød.
Her presenterer vi en protokoll som involverer forbigående inkubasjon av iPSCs-avledede embryoidlegemer (EB) med all-trans retinsyre for å produsere et høyt antall raskt prolifererende MSC, som videre kan brukes til å generere adipocytter14. Vi presenterer også en måte å sortere kjemisk avledede modne adipocytter fra det heterogene differensieringsbassenget ved fluorescerende merking av lipiddråpene ved hjelp av et lipofilt fargestoff; Nilen rød. Dette vil tillate generering av en ren populasjon av modne adipocytter med forbedret funksjonalitet for nøyaktig modellering av adipocytassosierte metabolske forstyrrelser.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adiponectin</td>
			<td>Abcam</td>
			<td>ab22554</td>
			<td>Adipocyte maturation marker</td>
		</tr>
		<tr>
			<td>anti-CD105</td>
			<td>BD Pharmingen</td>
			<td>560839</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD14</td>
			<td>BD Pharmingen</td>
			<td>561712</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD19</td>
			<td>BD Pharmingen</td>
			<td>555415</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD34</td>
			<td>BD Pharmingen</td>
			<td>555824</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD44</td>
			<td>abcam</td>
			<td>ab93758</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD45</td>
			<td>BD Pharmingen</td>
			<td> 
			560975</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD73</td>
			<td>BD Pharmingen</td>
			<td>550256</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD90</td>
			<td>BD Pharmingen</td>
			<td>555596</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D</td>
			<td>233-FP</td>
			<td>MSC culture media supplement</td>
		</tr>
		<tr>
			<td>C/EBPA</td>
			<td>Abcam</td>
			<td>ab40761</td>
			<td>Adipocyte maturation marker</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Torics</td>
			<td>1126</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>FABP4</td>
			<td>Abcam</td>
			<td>ab93945</td>
			<td>Adipocyte maturation marker</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ThermoFisher</td>
			<td>10082147</td>
			<td>MSC culture media supplement</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>ThermoFisher</td>
			<td>35050-061</td>
			<td>MSC culture media supplement</td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Sigma Aldrich</td>
			<td>I5879</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma Aldrich</td>
			<td>I7378</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma Aldrich</td>
			<td>91077C</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>ThermoFisher</td>
			<td>12660012</td>
			<td>Basal media for preparing matrigel</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>ThermoFisher</td>
			<td>11885084</td>
			<td>MSC culturing media</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Coating matrix</td>
		</tr>
		<tr>
			<td>MEM-alpha</td>
			<td>ThermoFisher</td>
			<td>12561056</td>
			<td>Adipocyte differentiation media</td>
		</tr>
		<tr>
			<td>Nilered</td>
			<td>Sigma Aldrich</td>
			<td>19123</td>
			<td>Sorting marker for adipocyte</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td>MSC/Adipocyte media supplement</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>ThermoFisher</td>
			<td>14190144</td>
			<td>wash buffer</td>
		</tr>
		<tr>
			<td>Pierce&#8482; 20X TBS Buffer</td>
			<td>Thermo Fisher</td>
			<td>28358</td>
			<td>wash buffer</td>
		</tr>
		<tr>
			<td>PPARG</td>
			<td>Cell Signaling Technology</td>
			<td>2443</td>
			<td>Adipocyte maturation marker</td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stem Cell Technologies</td>
			<td>5872</td>
			<td>Dissociation reagent</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma Aldrich</td>
			<td>R2625</td>
			<td>MSC differentiation media supplement</td>
		</tr>
		<tr>
			<td>Rock inhibitor</td>
			<td>Tocris</td>
			<td>1254/10</td>
			<td>hPSC culture media supplement</td>
		</tr>
		<tr>
			<td>Roziglitazone</td>
			<td>Sigma Aldrich</td>
			<td>R2408</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>StemFlex</td>
			<td>ThermoFisher</td>
			<td>A334901</td>
			<td>hPSC culture media</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Thermo Fisher</td>
			<td>28314</td>
			<td>Permebealization reagent</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>ThermoFisher</td>
			<td>25200072</td>
			<td>Dissociation reagent</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>P7942</td>
			<td>Wash buffer</td>
		</tr>
	</tbody>
</table>

robust differentiation of human ipscs into a pure population of adipocytes to study adipocyte-associated disorders

Nylige fremskritt innen indusert pluripotent stamcelleteknologi (iPSC) har gjort det mulig å generere forskjellige celletyper, inkludert adipocytter. Imidlertid har de nåværende differensieringsmetodene lav effektivitet og produserer ikke en homogen populasjon av adipocytter. Her omgår vi dette problemet ved å bruke en all-trans retinoic-basert metode for å produsere mesenkymale stamceller (MSC) i høyt utbytte. Ved å regulere veier som styrer celleproliferasjon, overlevelse og adhesjon, tillater vår differensieringsstrategi effektiv generering av embryonale legemer (EB) som skiller seg ut til en ren populasjon av multipotente MSC-er. Det høye antallet MSC generert av denne metoden gir en ideell kilde for å generere adipocytter. Imidlertid er prøveheterogenitet som følge av adipocytdifferensiering fortsatt en utfordring. Derfor brukte vi en Nilrødbasert metode for rensing av lipidbærende modne adipocytter ved bruk av FACS. Denne sorteringsstrategien tillot oss å etablere en pålitelig måte å modellere adipocytassosierte metabolske forstyrrelser ved å bruke en pool av adipocytter med redusert prøveheterogenitet og forbedret cellefunksjonalitet.

Skjematisk og morfologi av celler under mesenkymal differensiering: Differensiering av iPSC til MSC involverer ulike utviklingsstadier som spenner over EB-dannelse, MSC-differensiering og MSC-utvidelse (figur 1). I løpet av disse utviklingsstadiene får cellene forskjellig morfologi på grunn av de forskjellige stimulerende kjemikaliene de blir utsatt for. Ved initiering av differensiering blir cellene belagt med suspensjon og forventes å være runde, med definerte cellekanter, samtidig so...

Watch this Scientific Journal Video about Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders at JoVE.com

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

Protokollen tillater generering av en ren adipocytpopulasjon fra induserte pluripotente stamceller (iPSCs). Retinsyre brukes til å differensiere iPSCs til mesenkymale stamceller (MSC) som brukes til å produsere adipocytter. Deretter brukes en sorteringsmetode basert på Nilrødfarging for å oppnå rene adipocytter.

Robust differensiering av humane iPSCs til en ren populasjon av adipocytter for å studere adipocytt-assosierte lidelser

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

Tidligere tilnærminger stod på svært invasive metoder for å oppnå mesenkymale stamceller in vitro, men de mesenkymale stamcellene hadde en begrensning av å produsere heterogen pool på 30% med effektivitet fra 30 til 60% Her presenterer vi en protokoll for å produsere høyt antall raskt prolifererende mesenkymale stamceller som kan produsere en ren populasjon av modne adipocytter ved sortering ved hjelp av Nilrød. Når iPSCene har nådd 80 % sammenløp, vask cellene med DPBS og tilsett dissosiasjonsmedium som inneholder EDTA. Inkuber cellene ved 37 grader Celsius i ett minutt.Etter ett minutt, aspirer dissosiasjonsreagenset og plasser cellene ved 37 grader Celsius i ytterligere ett minutt. Fjern deretter platen fra inkubatoren. Legg til en milliliter StemFlex-medier per brønn og samle cellene forsiktig i et 15 milliliter konisk rør.Sentrifuge cellene. Fjern supernatanten, og resuspender dem forsiktig i tre milliliter MSC-differensieringsmedier som inneholder 10 mikromol av en steinhemmer. Bland celleopphenget og fordel 500 mikroliter per brønn i en 24-brønns ultra-lav festeplate.Legg deretter platen i en inkubator ved 37 grader Celsius. Etter 24 timer, for å indusere de oppnådde embryonale legemene, samle dem i et 15 milliliter rør og la dem slå seg ned. Etter 15 minutter, fjern supernatanten og tilsett MSC-differensieringsmedium supplert med 10 mikromolar retinsyre.Resuspender de embryonale kroppene forsiktig og fordel 500 mikroliter per brønn i samme 24-brønns ultra-lave festeplate. Legg deretter platen i inkubatoren. Etter 48 timer, samle de embryonale legemene i et 15 milliliter rør og la dem slå seg ned i 15 minutter.Fjern deretter supernatanten og tilsett MSC-differensieringsmedium supplert med 0,1 mikromolar retinsyre. Etter resuspendering av embryonale legemer, fordel 500 mikroliter per brønn i samme 24-brønnplate og plasser platen i inkubatoren. Førtiåtte timer etter den siste retinsyrebehandlingen, samle embryonale legemer, fjern supernatanten og tilsett DMEM lavglukosemedium uten cytokiner.Forsiktig resuspendere og distribuere 500 mikroliter av cellesuspensjonen per brønn i samme 24-brønns ultra-lave festeplate, og inkuber deretter platen ved 37 grader Celsius. Etter 48 timer, for å plate de iPSC-avledede embryonale legemene, samle de embryonale legemene i et 15 milliliter rør, la dem slå seg ned, fjern deretter supernatanten og resuspender i to milliliter ferskt MSC-differensieringsmedium. Deretter overfører du suspensjonen til to brønner i en kjellermembranmatrisebelagt seksbrønnsplate og inkuberer cellene ved 37 grader Celsius.Etter fem dager, erstatt det brukte mediet med et nytt MSC-differensieringsmedium som inneholder 2,5 nanogram per milliliter basisk fibroblastvekstfaktor og fortsett MCS-differensieringen i opptil 11 og 15 dager. Når de belagte embryonale kroppene har nådd 80 til 90% samløp, passerer du dem ved å vaske med DPBS, tilsette Trypsin-EDTA og inkubere ved 37 grader Celsius i ett minutt. Fjern Trypsin-EDTA fra platen og inkuber igjen ved 37 grader Celsius i ett minutt.Deretter legger du til en milliliter media per brønn. Etter tre minutter samler du cellene ved hjelp av MSC-differensieringsmedier i et 15 milliliter konisk rør og sentrifugerer cellene. Deretter resuspenderer du cellene i et MSC-differensieringsmedium som inneholder basisk fibroblastvekstfaktor og plater cellene på kjellermembranmatrisebelagte plater i forholdet en til tre.Etter at MSC-ene har nådd 90% samløp, fortsett å dyrke dem i ytterligere 48 timer for å la dem gjennomgå en periode med vekstarrest. Fjern deretter mediet og vask cellene med DBPS. Etter vasken, tilsett fullstendig adipocytdifferensieringsmedium til platen og inkuber cellene ved 37 grader Celsius.Bytt medium annenhver dag i 14 dager. Differensiere iPSC-avledede MSCer til adipocytter. For å sortere adipocytter ved hjelp av Nilrød, klargjør Nilrød arbeidsløsning i DMSO som beskrevet i tekstmanuskriptet.Før bruk, tine stamløsningen og rekonstituere den i DPBS for å oppnå en 300-nanomolar arbeidsløsningskonsentrasjon. På eller etter dag 14 av adipocytdifferensiering, kast mediet fra cellene og vask med DPBS. Deretter legger du til Nilens røde arbeidsløsning.Dekk platen og inkuber cellene ved 37 grader Celsius. Etter 15 minutter, erstatt Nilens røde løsning med Trypsin-EDTA og inkuber cellene ved 37 grader Celsius i fire minutter. Deretter samler du cellene ved hjelp av DMEM som inneholder 5% FBS i et 15 milliliter konisk rør og sentrifugerer cellene.Fjern supernatanten og resuspender cellene i DPBS med en tetthet på 1 million celler per milliliter. Bruk deretter FACS-sortereren, isoler Nilens røde positive celler ved hjelp av FL-1-kanalen. Samle de sorterte cellene og rekultur dem i adipocytdifferensieringsmedier eller fortsett til RNA-ekstraksjon etterfulgt av kvantitativ analyse av adipocytdifferensieringsmarkører.Ved initiering av differensiering er celler belagt i suspensjon runde med definerte cellegrenser og er små til middels store i diameter. Levedyktigheten til embryonale legemer observeres av den raske spredningsatferden som gir opphav til flere MSC-er. Denne raske spredningsoppførselen, sammen med deres særegne og langstrakte morfologi, beholdes selv etter å ha passert MSC-ene på ferske matriksbelagte plater.God differensiering gir pålitelige MSC-er med mer enn 90 % ekspresjonseffektivitet av mesenkymale overflatemarkører CD73, CD44 og CD90. I tillegg viste vurdering av celler for fravær av overflatemarkører som viser hematopoietisk fenotype CD14, CD34 og CD19 mindre enn 1% ekspresjonseffektivitet, Høyt uttrykk i cytoplasmatisk distribusjon av FABP4, en markør for terminalt differensierte adipocytter, indikerer deres utviklingsmodenhet. I tillegg indikerer høyt uttrykk av adiponektin, en annen markør for adipocyttmodenhet, at adipocytter er funksjonelle nok til å gjennomgå lipidlagring og adipogenese som respons på glukosesignalering.Ved farging binder Nilrød seg utelukkende til de lipidbærende modne adipocytter, noe som gjør den til et effektivt verktøy for sortering av modne adipocytter ved bruk av fluorescerende aktivert flowcytometri. Nilens rødpositive celler viser en signifikant oppregulering av modningsmarkører med minst to ganger sammenlignet med usorterte celler. Du må være veldig forsiktig mens du samler celler under embryonal kroppsdannelse og mesenkymal celledissosiasjon, da det vil ha stor innvirkning på overlevelseseffektiviteten etter dissosiasjon av mesenkymale stamceller.Ren populasjon av adipocytter oppnådd fra mutasjonsbærende pasienter kan bli utsatt for funksjonell og hele transkriptomsekvensering for å bestemme regulatoriske og signalveier påvirket av bestemt mutasjon uten at dataene påvirkes av prøveheterogenitet. Denne teknikken vil tillate skalerbar generering av mesenkymale stamceller in vitro, og fjerner behovet for å høste dem fra menneskelige givere for lett å skille dem fra adipocytter, kondrocytter og osteocytter for å forstå vevsrelatert sykdomspatogenese.

robust-differentiation-human-ipscs-into-pure-population-adipocytes-to

Research

JoVE Journal

Developmental Biology

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Generation of Human adipose stamceller gjennom Dedifferentiation av Mature Adipocytter i Ceiling kulturer

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

Differensiering av SH-SY5Y human cellelinje Neuroblastom

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

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.

In Vitro Differensiering av menneskelige pluripotent stamceller til 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 ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

Generering av IPSC-avledet menneskelige hjerne Organoids til Model Tidlig nevrologiske lidelser

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Protokol for differensiering av humane inducerte pluripotente stamceller i blandede kulturer av neuroner og glia for test av neurotoxicitet

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

In Vitro Differensiering av menneskelige stamceller i funksjonelle Cardiomyocyte-lignende celler

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Undertrykkelse av Pro-fibrotiske signalering Potentiates faktor-mediert omprogrammering av mus embryonale fibroblaster til indusert Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

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.

Effektiv differensiering av Human Pluripotent stamceller i leverceller

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

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.

Effektiv neural differensiering bruker single-Cell kultur av Human embryonale stamceller

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

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Rettet differensiering av hemogene endotelceller fra humane pluripotente stamceller

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...