Name: small-scale propagation of human ipscs in serum-free conditions for routine immunocytochemical characterization
Uploaded: 2017-02-18T13:00:00
Description: Watch this Scientific Journal Video about Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization at JoVE.com

Funding Sources: The University of Arizona, The Jim Himelic Foundation, and the Arizona Center for the Biology of Complex Diseases.

hiPSCs were derived from human dermal fibroblasts isolated from 4 mm skin punch biopsies and reprogrammed in house via Sendai virus-mediated reprogramming11. The University of Arizona Institutional Review Board approved all procedures for subject recruitment and biopsy collection.
1. Preparation of Extracellular Matrix Coated Surface for iPSC Culture
<ol>
	<li>One day prior to the confluence of hiPSC cultures growing on iMEFs, prepare extracellular matrix (Ma...

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Park, I. 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=Disease-specific+induced+pluripotent+stem+cells.">Disease-specific induced pluripotent stem cells.</a> Cell. 134, 877-886 (2008).</li><li>Hallett, P. J., 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=Successful+function+of+autologous+iPSC-derived+dopamine+neurons+following+transplantation+in+a+non-human+primate+model+of+Parkinson's+disease.">Successful function of autologous iPSC-derived dopamine neurons following transplantation in a non-human primate model of Parkinson's disease.</a> Cell stem cell. 16, 269-274 (2015).</li><li>Haston, K. M., Finkbeiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Trials+in+a+Dish:+The+Potential+of+Pluripotent+Stem+Cells+to+Develop+Therapies+for+Neurodegenerative+Diseases.">Clinical Trials in a Dish: The Potential of Pluripotent Stem Cells to Develop Therapies for Neurodegenerative Diseases.</a> Annu rev pharm toxicol. 56, 489-510 (2016).</li><li>Zhang, L., 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+and+high+engraftment+of+patient-specific+cardiomyocyte+sheet+using+induced+pluripotent+stem+cells+generated+from+adult+cardiac+fibroblast.">Derivation and high engraftment of patient-specific cardiomyocyte sheet using induced pluripotent stem cells generated from adult cardiac fibroblast.</a> Circ heart fail. 8, 156-166 (2015).</li><li>Byrne, J. A., Nguyen, H. N., Reijo Pera, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+generation+of+induced+pluripotent+stem+cells+from+a+subpopulation+of+human+fibroblasts.">Enhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblasts.</a> PloS one. 4, 7118 (2009).</li><li>Sivapatham, R., Zeng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+Characterization+of+Patient-Specific+Induced+Pluripotent+Stem+Cell+for+Disease+Modeling.">Generation and Characterization of Patient-Specific Induced Pluripotent Stem Cell for Disease Modeling.</a> Methods mol bio. 1353, 25-44 (2016).</li><li>Park, I. 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=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, 141-146 (2008).</li><li>Ruff, D., Lieu, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+stem+cells+using+quantitative+PCR+protein+assays.">Profiling stem cells using quantitative PCR protein assays.</a> Methods mol bio. 997, 225-236 (2013).</li><li>Pripuzova, N. 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=Development+of+a+protein+marker+panel+for+characterization+of+human+induced+pluripotent+stem+cells+(hiPSCs)+using+global+quantitative+proteome+analysis.">Development of a protein marker panel for characterization of human induced pluripotent stem cells (hiPSCs) using global quantitative proteome analysis.</a> Stem cell res. 14, 323-338 (2015).</li><li>Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+induction+of+transgene-free+human+pluripotent+stem+cells+using+a+vector+based+on+Sendai+virus,+an+RNA+virus+that+does+not+integrate+into+the+host+genome.">Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.</a> Proc jpn acad ser B phys biol sci. 85, 348-362 (2009).</li><li>Bigdeli, 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=Adaptation+of+human+embryonic+stem+cells+to+feeder-free+and+matrix-free+culture+conditions+directly+on+plastic+surfaces.">Adaptation of human embryonic stem cells to feeder-free and matrix-free culture conditions directly on plastic surfaces.</a> J Biotechnol. 133, 146-153 (2008).</li><li>Stover, A. E., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+human+pluripotent+stem+cells+to+feeder-free+conditions+in+chemically+defined+medium+with+enzymatic+single-cell+passaging.">Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging.</a> Methods mol bio. 767, 137-146 (2011).</li></ol>

The systematic protocol presented here offers a timesaving and cost-effective method, in the form of a scaled-down culture technique, specifically designed to support effective pluripotency analysis via immunocytochemistry.
The main advantages of the described methodology are as follows. Traditionally more than 3 to 4 passages are needed to transition iPSCs from feeder layers to feeder-free culture conditions in order to eliminate residual iMEFs remaining after the use of bulk dissociation tec...

Reprogramming human adult somatic cells into induced pluripotent stem cells (iPSCs) provides a way to obtain a potentially unlimited supply of patient-specific cells to study disease1,2. Recapitulating a disease phenotype in vitro would make it plausible to examine cellular and molecular mechanisms associated with disease, and enhance drug discovery and personalized medicine3. In addition, human iPSCs (hiPSCs) offer the possibility of deriving specific cell types which can be used as a unique resource to replace dead or dysfunctional cells and restore function in the context of several disorders4,5.
An important prerequisite to using iPSCs in the above applications is ensuring that their pluripotent and undifferentiated state is maintained during expansion in culture. Typically, techniques such as flow cytometry, western blotting, polymerase chain reaction and functional assays, which require large quantities of cells and specialized equipment, are used for the detailed analysis of iPSC pluripotency6,7,8,9,10. However, routine assessment of the iPSCs&#39; undifferentiated state might effectively be achieved through the limited propagation of these cells specifically for immunocytochemistry (ICC), thus involving reduced time and resources.
Recent advances allow for the growth of iPSCs in defined serum-free conditions, which is a significant improvement over conventional culture systems that require murine fibroblast feeder layers and serum containing media. However, the current literature does not include clear stepwise protocols that describe how to transition iPSCs from feeder layer to feeder-free systems.
In this context, the present protocol systematically details how hiPSCs grown on irradiated mouse embryonic fibroblast (iMEF) feeder layers can be (1) adapted to propagate in serum-free medium, and (2) cultured on a small-scale to specifically support robust immunocytochemical analysis. Overall, this methodology represents a timely and cost-effective procedure for propagating human iPSCs in serum-free conditions for confirming their pluripotency on a routine basis using immunocytochemistry.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM-F12/HEPES</td>
			<td>Life Technologies</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Life Technologies</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM-NEAA</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-Basic</td>
			<td>Cell Sciences</td>
			<td>CRF001B</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 ROCK Inhibitor</td>
			<td>R&#38;D</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Life Technologies</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel hESC-qualified Matrix</td>
			<td>Corning</td>
			<td>354277</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 Basal Medium</td>
			<td>StemCell Technologies</td>
			<td>05850</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 5X Supplement</td>
			<td>StemCell Technologies</td>
			<td>05850</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Buffer</td>
			<td>StemCell Technologies</td>
			<td>07174</td>
			<td></td>
		</tr>
		<tr>
			<td>0.1M PO4 Buffer</td>
			<td>In-House</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldeyde, prill</td>
			<td>Electron Microscopy Sciences</td>
			<td>19202</td>
			<td></td>
		</tr>
		<tr>
			<td>1X Phoshate Buffered Saline</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Life Technologies</td>
			<td>16210072</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Oct-4A (C30A3) Rabbit mAb, 
			Sox2 (D6D9) Rabbit mAb, 
			SSEA4 (MC813) Mouse mAb, 
			TRA-1-60(S) (TRA-1-60(S)) Mouse mAb</td>
			<td>Cell Signaling 
			Cell Signaling 
			Cell Signaling 
			Cell Signaling</td>
			<td>2840 
			3579 
			4755 
			4746</td>
			<td>Alternatively, a combination of 6 pluripotency primary antibodies can be purchased together as a kit in Catalog #9656</td>
		</tr>
		<tr>
			<td>Goat anti-Ms IgM Alexa Fluor 488</td>
			<td>Life Technologies</td>
			<td>A21042</td>
		</tr>
		<tr>
			<td>Goat anti-Ms IgG3 Alexa Fluor 488</td>
			<td>Life Technologies</td>
			<td>A21151</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rb IgG Alexa Fluor 594</td>
			<td>Life Technologies</td>
			<td>A11037</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell Cell Culture Plates</td>
			<td>Fisher Scientific&#160;</td>
			<td>0720080/0720081</td>
			<td>Available in 6, 12, 24, 48, 96 well sizes</td>
		</tr>
		<tr>
			<td>Chamber Slides</td>
			<td>Fisher Scientific&#160;</td>
			<td>12 565 21</td>
			<td>Available in Glass or Permanox Plastic in 1, 2, 4, 8, 16 well sizes</td>
		</tr>
		<tr>
			<td>Coverglass for growth</td>
			<td>Fisher Scientific&#160;</td>
			<td>12 545 82</td>
			<td>Available in 12, 15, 18, 22 and 25mm sizes</td>
		</tr>
	</tbody>
</table>

small-scale propagation of human ipscs in serum-free conditions for routine immunocytochemical characterization

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

This protocol provides a step-wise description of how human iPSCs can be transferred from feeder layer to feeder-free conditions, and subsequently propagated in a limited manner to specifically enable cost-effective immunocytochemistry for confirming pluripotency maintenance. Figure 1 shows a schematic representation of this protocol. Figure 2A shows hiPSC colonies growing on iMEFs in 6-well plates. These colonies exhibit typical morphology with defined b...

Watch this Scientific Journal Video about Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization at JoVE.com

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient ...

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