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 arbejde blev finansieret af et tilskud fra Qatar National Research Fund (QNRF) (bevilling nr. NPRP10-1221-160041). Maryam Aghadi blev støttet af GSRA-stipendium fra Qatar National Research Fund (QNRF).

Undersøgelsen er godkendt af den relevante institutionelle forskningsetiske komité og udført i overensstemmelse med de etiske standarder, der er fastlagt i Helsingfors-erklæringen fra 1964 og dens senere ændringer eller sammenlignelige etiske standarder. Protokollen blev godkendt af Institutional Review Board (IRB) i HMC (nr. 16260/16) og QBRI (nr. 2016-003). Dette arbejde er også optimeret til hESC'er som H1 og H9. Blodprøver blev taget fra raske personer med fuldt informeret samtykke. IPSC'erne genereres fra mon...

<ol>
	<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 protokol har afgørende betydning på grund af dens evne til at levere MSC'er med højt udbytte og effektivitet. Denne masseproduktion af MSC'er blev muliggjort ved forbigående inkubation af iPSC'er-afledte EB'er med 10 μM RA14,15. Transient behandling med 10 μM RA forbedrede MSC-udbyttet med 11,2 til 1542 gange14,15, idet denne protokol kan anvendes på både iPSC'er og hPSC'er. Ved denne dosis ...

Mesenkymale stamceller (MSC'er) fungerer som en effektiv forbigående ressource til fremstilling af celler af mesodermal oprindelse som adipocytter, osteocytter og chondrocytter, som yderligere kan bruges til modellering af deres respektive genetiske lidelser. Tidligere tilgange var imidlertid afhængige af at opnå disse MSC'er fra voksent væv 1, hvilket pålagde udfordringen med at få dem i stort antal fra donorerne og begrænsningen ved at holde dem funktionelt levedygtige under suboptimale in vitro-dyrkningsbetingelser 1,2. Disse hindringer har skabt et stort behov for at have en protokol til generering af MSC'er in vitro. Human inducerede pluripotente stamceller (iPSC'er) kan anvendes som en værdifuld kilde til MSC'er og udviser MSC-karakteristika 3,4,5. iPSC'er-afledte MSC'er kan bruges som en terapeutisk mulighed i flere sygdomme. Også iPSC'er-afledte MSC'ers evne til at generere adipocytter gør dem til en værdifuld in vitro human model til at studere human adipogenese, fedme og adipocytassocierede lidelser.
Nuværende differentieringsprotokoller for adipocytter kan klassificeres i to grupper, hvor den ene involverer differentiering af adipocytter ved hjælp af kemiske eller proteinbaserede cocktails, hvilket giver et resulterende udbytte på 30%-60%6,7,8,9, mens den anden involverer genetisk manipulation til robust induktion af nøgletranskriptionsfaktorer, der styrer adipocytudviklingen, for at give et udbytte på 80%-90%10, 11. Imidlertid rekapitulerer genetisk manipulation ikke den naturlige proces med adipocytdifferentiering og maskerer ofte de subtile paradigmer, der ankommer under adipogenese, hvilket gør det ineffektivt til sygdomsmodelleringsformål12,13. Derfor præsenterer vi en måde at sortere kemisk afledte modne adipocytter fra umodne ved fluorescerende mærkning af lipidbærende adipocytter ved hjælp af Nilrød.
Her præsenterer vi en protokol, der involverer forbigående inkubation af iPSC'er afledte embryoidlegemer (EB'er) med all-trans retinsyre for at producere et stort antal hurtigt prolifererende MSC'er, som yderligere kan bruges til at generere adipocytter14. Vi præsenterer også en måde at sortere kemisk afledte modne adipocytter fra den heterogene differentieringspulje ved fluorescerende mærkning af deres lipiddråber ved hjælp af et lipofilt farvestof; Nilen rød. Dette ville muliggøre generering af en ren population af modne adipocytter med forbedret funktionalitet til nøjagtigt at modellere adipocytassocierede metaboliske lidelser.

<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 fremskridt inden for induceret pluripotent stamcelleteknologi (iPSC) har gjort det muligt at generere forskellige celletyper, herunder adipocytter. De nuværende differentieringsmetoder har imidlertid lav effektivitet og producerer ikke en homogen population af adipocytter. Her omgår vi dette problem ved at bruge en all-trans retinologisk-baseret metode til at producere mesenkymale stamceller (MSC'er) med højt udbytte. Ved at regulere veje, der styrer celleproliferation, overlevelse og vedhæftning, muliggør vores differentieringsstrategi effektiv generering af embryonale kroppe (EB'er), der differentierer sig til en ren population af multipotente MSC'er. Det store antal MSC'er, der genereres ved denne metode, giver en ideel kilde til generering af adipocytter. Imidlertid er prøveheterogenitet som følge af adipocytdifferentiering fortsat en udfordring. Derfor brugte vi en Nile red-baseret metode til rensning af lipidbærende modne adipocytter ved hjælp af FACS. Denne sorteringsstrategi gjorde det muligt for os at etablere en pålidelig måde at modellere adipocytassocierede metaboliske lidelser ved hjælp af en pulje af adipocytter med reduceret prøveheterogenitet og forbedret cellefunktionalitet.

Skematisk og morfologi af celler under mesenkymal differentiering: Differentiering af iPSC'er i MSC'er involverer forskellige udviklingsstadier, der spænder over EB-dannelse, MSC-differentiering og MSC-ekspansion (figur 1). I løbet af disse udviklingsstadier erhverver celler forskellige morfologier på grund af de forskellige stimulerende kemikalier, de udsættes for. Ved initiering af differentiering belægges celler i suspension og forventes at være runde med definerede cellekanter, men...

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 tillader generering af en ren adipocytpopulation fra inducerede pluripotente stamceller (iPSC'er). Retinsyre bruges til at differentiere iPSC'er til mesenkymale stamceller (MSC'er), der anvendes til fremstilling af adipocytter. Derefter anvendes en sorteringsmetode baseret på Nilrød farvning til at opnå rene adipocytter.

Robust differentiering af humane iPSC'er i en ren population af adipocytter for at studere adipocytassocierede lidelser

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

Tidligere tilgange var afhængige af meget invasive metoder til opnåelse af mesenkymale stamceller in vitro, men disse mesenkymale stamceller havde en begrænsning på at producere heterogen pulje på 30% med effektivitet fra 30 til 60%Her præsenterer vi en protokol til fremstilling af et stort antal hurtigt prolifererende mesenkymale stamceller, der kan producere en ren population af modne adipocytter ved sortering ved hjælp af Nilrød. Når iPSC'erne har nået 80% sammenløb, vaskes cellerne med DPBS og tilsættes dissociationsmedium indeholdende EDTA. Inkuber cellerne ved 37 grader Celsius i et minut.Efter et minut aspireres dissociationsreagenset og placeres cellerne ved 37 grader Celsius i yderligere et minut. Fjern derefter pladen fra inkubatoren. Tilsæt en milliliter StemFlex-medier pr. Brønd og saml forsigtigt cellerne i et 15 ml konisk rør.Centrifuger cellerne. Supernatanten fjernes, og den resuspenderes forsigtigt i tre ml MSC-differentieringsmedier indeholdende 10 mikromol stenhæmmer. Bland cellesuspensionen og fordel 500 mikroliter pr. Brønd i en 24-brønds ultra-lav fastgørelsesplade.Placer derefter pladen i en inkubator ved 37 grader Celsius. Efter 24 timer, for at fremkalde de opnåede embryonale kroppe, saml dem i et 15 ml rør og lad dem slå sig ned. Efter 15 minutter fjernes supernatanten, og der tilsættes MSC-differentieringsmedium suppleret med 10 mikromolær retinsyre.Resuspender embryonale kroppe forsigtigt og fordel 500 mikroliter pr. Brønd i den samme 24-brønds ultra-lave fastgørelsesplade. Placer derefter pladen i inkubatoren. Efter 48 timer samles embryonlegemerne i et 15 ml rør og lader dem slå sig ned i 15 minutter.Derefter fjernes supernatanten, og der tilsættes MSC-differentieringsmedium suppleret med 0,1 mikromolær retinsyre. Efter resuspendering af embryonale kroppe fordeles 500 mikroliter pr. Brønd i samme 24-brøndplade og placeres pladen i inkubatoren. Otteogfyrre timer efter den sidste retinsyrebehandling opsamles embryonlegemerne, supernatanten fjernes, og der tilsættes DMEM-substrat med lavt glukoseindhold uden cytokiner.Resuspender og fordel forsigtigt 500 mikroliter af cellesuspensionen pr. brønd i den samme 24-brønds ultralave fastgørelsesplade, og inkuber derefter pladen ved 37 grader Celsius. Efter 48 timer opsamles embryonlegemerne i et rør på 15 ml for at plade de iPSC-afledte embryoner, lader dem bundfælde sig, fjerner derefter supernatanten og resuspenderes i to ml frisk MSC-differentieringsmedium. Derefter overføres suspensionen til to brønde i en kældermembranmatrixbelagt seksbrøndplade og inkuberer cellerne ved 37 grader Celsius.Efter fem dage udskiftes de brugte medier med et frisk MSC-differentieringsmedium indeholdende 2,5 nanogram pr. milliliter basisk fibroblastvækstfaktor, og MCS-differentieringen fortsættes i op til 11 og 15 dage. Når de belagte embryonale legemer har nået 80 til 90% sammenløb, passage dem ved vask med DPBS, tilsætning af trypsin-EDTA og inkubation ved 37 grader Celsius i et minut. Fjern Trypsin-EDTA fra pladen og inkuber igen ved 37 grader Celsius i et minut.Derefter tilsættes en milliliter medier pr. Brønd. Efter tre minutter opsamles cellerne ved hjælp af MSC-differentieringsmedier i et 15 ml konisk rør og centrifugeres cellerne. Derefter resuspenderes cellerne i et MSC-differentieringsmedium, der indeholder basisk fibroblastvækstfaktor, og cellerne plades på kældermembranmatrixbelagte plader i et forhold på et til tre.Når MSC'erne har nået 90% sammenløb, skal du fortsætte med at dyrke dem i yderligere 48 timer for at give dem mulighed for at gennemgå en periode med vækststop. Fjern derefter mediet og vask cellerne med DBPS. Efter vasken tilsættes komplet adipocytdifferentieringsmedium til pladen og inkuberer cellerne ved 37 grader Celsius.Skift mediet hver anden dag i 14 dage. Differentier iPSC-afledte MSC'er i adipocytter. For at sortere adipocytterne ved hjælp af Nilen rød, skal du forberede Nilen rød arbejdsløsning i DMSO som beskrevet i tekstmanuskriptet.Før brug optøs stamopløsningen og rekonstitueres i DPBS for at opnå en koncentration på 300 nanomolær arbejdsopløsning. På eller efter dag 14 af adipocytdifferentiering kasseres mediet fra cellerne og vaskes ved hjælp af DPBS. Tilføj derefter Nilen rød arbejdsløsning.Dæk pladen og inkuber cellerne ved 37 grader Celsius. Efter 15 minutter udskiftes Nilens røde opløsning med Trypsin-EDTA og inkuberer cellerne ved 37 grader Celsius i fire minutter. Derefter samles cellerne ved hjælp af DMEM indeholdende 5% FBS i et 15 ml konisk rør og centrifugeres cellerne.Supernatanten fjernes og cellerne i DPBS resuspenderes med en massefylde på 1 million celler pr. ml. Brug derefter FACS-sortereren til at isolere Nilens rødpositive celler ved hjælp af FL-1-kanalen. Saml de sorterede celler og rekultur dem i adipocytdifferentieringsmedier eller fortsæt til RNA-ekstraktion efterfulgt af kvantitativ analyse af adipocytdifferentieringsmarkører.Ved initiering af differentiering er celler belagt i suspension runde med definerede cellekanter og er små til mellemstore i diameter. Embryonale legemers levedygtighed observeres af den hurtige spredningsadfærd, der giver anledning til flere MSC'er. Denne hurtige spredningsadfærd, sammen med deres ejendommelige og langstrakte morfologi, bevares, selv efter at MSC'erne er passeret på friske matrixbelagte plader.God differentiering giver pålidelige MSC'er med mere end 90% ekspressionseffektivitet af mesenkymale overflademarkører CD73, CD44 og CD90. Derudover viste vurdering af celler for fravær af overflademarkører, der skildrer den hæmatopoietiske fænotype CD14, CD34 og CD19, mindre end 1% ekspressionseffektivitet, Høj ekspression i cytoplasmatisk fordeling af FABP4, en markør for terminalt differentierede adipocytter, indikerer deres udviklingsmæssige modenhed. Derudover indikerer høj ekspression af adiponectin, en anden markør for adipocytmodenhed, at adipocytterne er funktionelle nok til at gennemgå lipidopbevaring og adipogenese som reaktion på glukosesignalering.Ved farvning binder Nilrød udelukkende til de lipidbærende modne adipocytter, hvilket gør det til et effektivt værktøj til sortering af modne adipocytter ved hjælp af fluorescerende aktiveret flowcytometri. Nilens rødpositive celler viser en signifikant opregulering af modningsmarkører med mindst to gange sammenlignet med usorterede celler. Du skal være meget forsigtig, mens du indsamler celler under embryonal kropsdannelse og mesenkymal celledissociation, da det i høj grad ville påvirke overlevelseseffektiviteten efter dissociation af mesenkymale stamceller.Ren population af adipocytter opnået fra mutationsbærende patienter kan underkastes funktionel og hele transkriptomsekventering for at bestemme regulatoriske og signalveje, der påvirkes af en bestemt mutation, uden at dataene påvirkes af prøveheterogenitet. Denne teknik vil muliggøre skalerbar generering af mesenkymale stamceller in vitro, hvilket fjerner behovet for at høste dem fra menneskelige donorer for let at differentiere dem til adipocytter, chondrocytter og osteocytter til forståelse af vævsrelateret sygdomspatogenese.

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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 af menneskelige fedt-stamceller gennem dedifferentiering af Ældre Adipocytter i Loft Cultures

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.

Differentiering af SH-SY5Y human neuroblastomcellelinje

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 differentiering af humane pluripotente stamceller til trofoblastceller

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.

Generation af iPSC-afledte menneskelige hjerne organoider til Model Tidlig forstyrrelser i nervesystemets

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 differentieringen af ​​humane inducerede pluripotente stamceller i blandede kulturer af neuroner og glia til neurotoksicitetstestning

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 Differentiering af humane mesenkymale stamceller i funktionel 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 af Pro-fibrotisk signalering Potentiates faktor-medieret omprogrammering af musen embryonale fibroblaster til induceret 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 differentiering af humane pluripotente 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 neurale differentiering ved hjælp af enkelt cellekultur af humane 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 differentiering af hæmogene 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 ...