Name: a novel culture model for human pluripotent stem cell propagation on gelatin in placenta-conditioned media
Uploaded: 2015-08-03T15:30:00
Description: Watch this Scientific Journal Video about A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media at JoVE.com

Forfatterne ønsker å takke Soon-Cheol Hong (MD, Ph.D., førsteamanuensis, Institutt for obstetrikk og gynekologi, Medical College Korea University) for å gi placenta vev. Dette arbeidet ble støttet delvis av tilskudd (R1211902) fra National Research Foundation of Korea (NRF), Republic of Korea.

Etikk uttalelse: The human placenta Studien ble gjennomført prospektivt, med godkjenning av Institutional Review Board for menneskelig forskning av Korea University (AN09085-001). Alle forsøkene ble utført i et rent Germ-Free Room anlegget på Korea University Medical Center. Den eksperimentelle design og prosedyrer ved hjelp hPSCs ble godkjent av Institutional Review Board of Korea University Medical Center (AN12277-003). 1. Utarbeidelse av Instruments, Culture Media og retter <ol><li>...

<ol>
	<li>Xu, 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=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nat Biotechnol. 19 (10), 971-974 (2001).</li><li>Amit, M. C., Shakiri, V., Margulets, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Itskovitz-Eldor+Feeder+layer+and+serum-free+culture+of+human+embryonic+stem+cells.">Itskovitz-Eldor Feeder layer and serum-free culture of human embryonic stem cells.</a> Biol Reprod. 70 (3), 837-845 (2004).</li><li>Nagaoka, M., Si-Tayeb, K., Akaike, T., Duncan, S. 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H., 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=Basic+FGF+and+suppression+of+BMP+signaling+sustain+undifferentiated+proliferation+of+human+ES+cells.">Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.</a> Nat Methods. 2 (3), 185-190 (2005).</li><li>Xu, 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=Basic+fibroblast+growth+factor+supports+undifferentiated+human+embryonic+stem+cell+growth+without+conditioned+medium.">Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium.</a> Stem Cells. 23 (3), 315-323 (2005).</li><li>Levenstein, M. 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=Basic+fibroblast+growth+factor+support+of+human+embryonic+stem+cell+self-renewal.">Basic fibroblast growth factor support of human embryonic stem cell self-renewal.</a> Stem Cells. 24 (3), 568-574 (2006).</li><li>Park, Y., 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=Human+feeder+cells+can+support+the+undifferentiated+growth+of+human+and+mouse+embryonic+stem+cells+using+their+own+basic+fibroblast+growth+factors.">Human feeder cells can support the undifferentiated growth of human and mouse embryonic stem cells using their own basic fibroblast growth factors.</a> Stem Cells Dev. 20 (11), 1901-1910 (2011).</li><li>Park, Y., 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+efficacy+of+human+placenta+as+a+source+of+the+universal+feeder+in+human+and+mouse+pluripotent+stem+cell+culture.">The efficacy of human placenta as a source of the universal feeder in human and mouse pluripotent stem cell culture.</a> Cell Reprogram. 12 (3), 315-328 (2010).</li><li>Park, Y., 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=Undifferentiated+propagation+of+the+human+embryonic+stem+cell+lines,+H1+and+HSF6,+on+human+placenta-derived+feeder+cells+without+basic+fibroblast+growth+factor+supplementation.">Undifferentiated propagation of the human embryonic stem cell lines, H1 and HSF6, on human placenta-derived feeder cells without basic fibroblast growth factor supplementation.</a> Stem Cells Dev. 19 (11), 1713-1722 (2010).</li><li>Seok, K., 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=Human+placenta-derived+feeders+support+prolonged+undifferentiated+propagation+of+a+human+embryonic+stem+cell+line,+SNUhES3:+comparison+with+human+bone+marrow-derived+feeders.">Human placenta-derived feeders support prolonged undifferentiated propagation of a human embryonic stem cell line, SNUhES3: comparison with human bone marrow-derived feeders.</a> Stem Cells Dev. 16 (3), 421-428 (2007).</li><li>Levenstein, M. 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=Basic+fibroblast+growth+factor+support+of+human+embryonic+stem+cell+self-renewal.">Basic fibroblast growth factor support of human embryonic stem cell self-renewal.</a> Stem Cells. 24 (3), 568-574 (2006).</li><li>Xu, R. H., Peck, R. M., Li, D. S., Feng, X., Ludwig, T., Thomson, J. 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=Basic+FGF+and+suppression+of+BMP+signaling+sustain+undifferentiated+proliferation+of+human+ES+cells.">Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.</a> Nat Methods. 2 (3), 185-190 (2005).</li><li>Mahmood, A., Harkness, L., Schrøder, H. D., Abdallah, B. 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Denne modell ble utviklet for å kunne forplante hPSCs, samtidig som deres egenskaper, humaniserte matefrie dyrkningsbetingelser uten tilsetning av eksogene rekombinante vekstfaktorer så som bFGF eller insulin, slik at manipulering av hPSCs i et humanisert mikromiljøet. Eksogent bFGF tilskudd er vanlig og anvendelse av substrater som Matrigel eller laminin er avgjørende for materen fritt dyrkning av hPSCs 14. Denne protokollen er enkel og lett å gjennomføre. Men noen tiltak e...

The goal of this protocol is the propagation of human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) in fully humanized ex vivo feeder-free conditions without requiring additional exogenous supplementation and synthetic substrates. To date, the development of ex vivo hPSC culture models, that enable the introduction of culture products to the clinic, has been a major concern in stem cell research. Specifically, two critical problems need to be addressed. First, a humanized ex vivo culture system for the propagation of hPSCs that obviates the risk of contamination by animal cell products is needed. Second, a feeder-free culture model is needed to facilitate easy and economic mass production of hPSCs. For therapeutic applications of hPSCs, it is necessary to identify the factors that regulate their self-renewal and differentiation.
Since Xu et al. initially reported the feasibility of using conditioned media (CM) derived from mouse embryonic fibroblasts (MEF) to grow hESCs on Matrigel1, many studies have examined optimal ex vivo propagation methods for hPSCs2-4. However, an ideal humanized ex vivo feeder-free hPSC propagation system has not been developed because current methods require additional exogenous substrates including bFGF and insulin, well-known hPSC-sustaining factors, in culture media5-7. Moreover, synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures.
The rationale behind the development and use of this protocol is based on our previous studies showing that human placenta chorion cells excellently support the propagation of hPSCs without bFGF supplementation8-11. This protocol has a number of advantages including its simplicity with respect to handling and its cost-effectiveness, and it is a near-perfect humanized culture that enables hPSC propagation without exogenous synthetic substrates. The application of human placenta-derived CM (hPCCM) for hPSCs involves 3 steps. First, chorion cells are isolated from the human placenta and cultured. Second, hPCCM is produced from cultured cells. Third, hPSCs are cultured using hPCCM and their characteristics are confirmed. 
This protocol will facilitate clinical applications of hPSCs and studies of the mechanisms of hPSC proliferation and attachment. In this paper, the protocol for the successful propagation of hPSCs in hPCCM on a gelatin-coated dish is presented.

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	<tbody>
		<tr>
			<td>Mitomycin-C</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>M4287</td>
			<td>10ug/ml</td>
		</tr>
		<tr>
			<td>Mycoplasma detection kit</td>
			<td>TaKaRa Bio Inc.</td>
			<td>#6601</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>#354277</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>STEMCELL Technologies Inc.</td>
			<td>#05850</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS02100</td>
			<td>1mg/ml</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>G2500</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>BM-Cyclin</td>
			<td>Roche</td>
			<td>799 050</td>
			<td>10ug/ml</td>
		</tr>
		<tr>
			<td>RNeasy mini kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop Spectrophotometer</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iQ SYBR Green qPCR Master Mix&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>#170-8882AP</td>
			<td></td>
		</tr>
		<tr>
			<td>ES Cell Characterization kit</td>
			<td>Chemicon International, Inc.,</td>
			<td>SCR001</td>
			<td></td>
		</tr>
		<tr>
			<td>Power cDNA Synthesis Kit&#160;</td>
			<td>iNtRON Biotechnology</td>
			<td>25011</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp&#174; DNA Micro kit</td>
			<td>Qiagen</td>
			<td>56304</td>
			<td></td>
		</tr>
		<tr>
			<td>AmpF/STR&#174; Identifiler&#174; PCR Amplification kit</td>
			<td>Applied Biosystems Inc.</td>
			<td>4322288</td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems&#174; 3130xl Genetic Analyzer&#160;</td>
			<td>Applied Biosystems Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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a novel culture model for human pluripotent stem cell propagation on gelatin in placenta-conditioned media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

En fordel med denne hPSC forplantning metoden er at den benytter komponenter som skilles fra humane celler. En mest ciritical trinn i protokollen er isolering og kulturen av human placenta-avledede celler. Dette krever nøyaktig og disseksjon av en nøyaktig del av HPC plate villi. Figur 1 viser fremgangsmåten for human placenta-avledet celleisolasjon. Denne prosessen er enkel og praktisk for isolering av celler, og kan lett utføres innen 1 time. Som beskrevet i figur 2, vev festet ti...

Watch this Scientific Journal Video about A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media at JoVE.com

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

A Novel Kultur Modell for Menneskelig pluripotent Stem Cell Formering på Gelatin i Placenta kondisjonerte Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

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