The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
This research was performed while the authors Whitney A. Greene, Alberto Mu&#241;iz, and Ramesh R. Kaini held a National Research Council Postdoctoral Research Associateship at the USAISR.
Microarray assays were performed by the Greehey Children's Cancer Research Institute Microarray Core Facility and Bioinformatics Department at UT Health Science Center San Antonio.
This work was supported by U.S. Army Clinical Rehabilitative Medicine Research Program (CRMRP) and Military Operational Medicine Research Program (MOMRP).

1. Preparation of Culture Reagents and Culture Plates
<ol>
	<li>mTeSR1 media: Prepare mTeSR1 media according to the manufacturer&#8217;s directions. Thaw 100 ml of mTeSR1 5x supplement overnight at 4 &#176;C. Add the 100 ml of mTeSR1 5x supplement to 400 ml of mTeSR1 basal media and mix well. This media is stable at 4 &#176;C for up to 2 weeks and at -20 &#176;C for 6 months.</li>
	<li>Differentiation media: Prepare differentiation media in DMEM/F12 to yield 0.1mM &#946;-mercaptoethanol, 0.1 mM nonessential amino acids...

<ol>
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K., Rizzino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+of+microRNAs+in+the+control+of+embryonic+stem+cells+and+the+generation+of+induced+pluripotent+stem+cells.">Emerging roles of microRNAs in the control of embryonic stem cells and the generation of induced pluripotent stem cells.</a> Dev Biol. 344, 16-25 (2010).</li><li>Guo, 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=Mammalian+microRNAs+predominantly+act+to+decrease+target+mRNA+levels.">Mammalian microRNAs predominantly act to decrease target mRNA levels.</a> Nature. 466, 835-840 (2010).</li><li>Adijanto, 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=Microphthalmia-associated+transcription+factor+(MITF)+promotes+differentiation+of+human+retinal+pigment+epithelium+(RPE)+by+regulating+microRNAs-204/211+expression.">Microphthalmia-associated transcription factor (MITF) promotes differentiation of human retinal pigment epithelium (RPE) by regulating microRNAs-204/211 expression.</a> J Biol Chem. 287, 20491-20503 (2012).</li><li>Hu, 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=Identification+of+miRNA+signatures+during+the+differentiation+of+hESCs+into+retinal+pigment+epithelial+cells.">Identification of miRNA signatures during the differentiation of hESCs into retinal pigment epithelial cells.</a> PLoS One. 7, (2012).</li><li>Wilson, K. D., 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=MicroRNA+profiling+of+human-induced+pluripotent+stem+cells.">MicroRNA profiling of human-induced pluripotent stem cells.</a> Stem Cells Dev. 18, 749-758 (2009).</li><li>Neveu, P., 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=MicroRNA+profiling+reveals+two+distinct+p53-related+human+pluripotent+stem+cell+states.">MicroRNA profiling reveals two distinct p53-related human pluripotent stem cell states.</a> Cell Stem Cell. 7, 671-681 (2010).</li><li>Maminishkis, 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=Confluent+monolayers+of+cultured+human+fetal+retinal+pigment+epithelium+exhibit+morphology+and+physiology+of+native+tissue.">Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.</a> Invest Ophthalmol Vis Sci. 47, 3612-3624 (2006).</li><li>Qiagen, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNeasy+Mini+Handbook.">miRNeasy Mini Handbook.</a> Sample and Assay Technologies. , (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+System+with+miRNA+Complete+Labeling+and+Hyb+Kit.">Microarray System with miRNA Complete Labeling and Hyb Kit.</a> Agilent Technologies. , (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+hESCs+and+hiPSCs+in+mTESR1+and+mTESR2.">Maintenance of hESCs and hiPSCs in mTESR1 and mTESR2.</a> Stem Cell Technologies. , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Analysis+of+DNA+or+RNA+using+its+wavelengths:+230+nm,+260+nm,+280+nm.">The Analysis of DNA or RNA using its wavelengths: 230 nm, 260 nm, 280 nm.</a> About Biotechnology. , (2013).</li></ol>

In conclusion, this report describes the methods used to culture iPS cells, iPS-RPE, and fetal RPE. RPE derived from iPS are morphologically and functionally similar to fetal RPE. The iPS-RPE also expresses characteristic RPE genes including RPE65, CRALBP, PEDF, and LRAT16. To further characterize these cells, RNA was extracted and used to perform microarray analysis to identify differentially expressed miRNA. Analysis of the signal intensities revealed that the greatest number of differentially expressed miRN...

Stem cells have the capacity to replicate without limit and the potential to differentiate into any somatic cell type. The development of techniques to reprogram somatic cells into pluripotent stem cells has elicited great excitement in the research community and among clinicians, as the advent of personalized tissue regeneration is on the horizon1. Induced-pluripotent stem (iPS) cells exhibit the same features of unlimited replicative potential and pluripotency as embryonic stem (ES) cells while circumventing the ethical dilemmas associated with ESCs. In addition, patient-derived stem cells will not stimulate an immune response, greatly increasing the probability of successful therapeutic applications2-3. Under specific culture conditions, iPS cells have been shown to differentiate into several different cell types in vitro, including cardiomyocytes, neurons, pancreatic beta cells, hepatocytes, and retinal pigmented epithelium (RPE)4-12.
The RPE is a specialized layer of pigmented epithelial cells located at the back of the retina that performs several functions that are essential for visual health and function such as absorption of stray light, phagocytosis of the photoreceptor outer segments, and processing of retinoids for the production of visual chromophore. Dysfunction of the RPE due to damage or disease profoundly affects photoreceptor health and visual function as evidenced by the blinding diseases that are the result of underlying RPE pathology such as age-related macular degeneration (AMD), Stargardt&#8217;s disease, and retinitis pigmentosa (RP)13. Truly effective treatments that can restore vision have not been achieved, and replacement of diseased RPE with healthy RPE may be the best option to prevent loss of vision14-15. RPE derived from iPS (iPS-RPE) is a possible source of cells to replace the damaged RPE. iPS-RPE expresses characteristic RPE proteins LRAT, CRALBP, PEDF, and RPE65; displays the classical highly pigmented hexagonal RPE morphology; and performs RPE functions such as phagocytosis, retinoid processing, and secretion of 11-cis retinal5,16. However, before iPS-RPE can be used therapeutically, the iPS-RPE must be thoroughly characterized. Understanding the factors that govern RPE differentiation is necessary to improve the yield and purity of the cells to be used for clinical applications.
Cell differentiation is the result of highly regulated gene expression. Epigenetic remodeling of the genome and coordination of transcription factors are required for cell fate decisions that occur during differentiation and development17. Regulation of message RNA (mRNA) translation by microRNA (miRNA) presents yet another level of regulation that affects cell fate1. MiRNAs are short, ~22 nt, lengths of nucleotides that either suppress translation by binding to the 3&#8217; UTR of mRNA or target the mRNA for degradation. MiRNAs have been detected in virtually all tissues and to date over 2,000 unique human microRNAs have been registered in the miRBase database. Since miRNAs require only partial complementarity to bind to the target mRNA, a single miRNA can potentially bind to tens or hundreds of targets, and vice versa; i.e. a single mRNA can be targeted by several different miRNAs. This promiscuous binding characteristic dramatically increases the level of complexity of regulation as well as the level of difficulty of determining the functions of individual miRNAs and the role each plays in cellular functions18-21. Nevertheless, studies have shown that miRNA refines gene expression during differentiation by affecting DNA methylation status17. In a study more specific to the RPE, miR-204/211 was shown to promote the epithelial phenotype of the RPE22. Another group analyzed the miRNA profile of RPE during differentiation from ES cells and revealed distinct sets of miRNA are expressed during the differentiation process23. In fact, miRNA profiles can unambiguously distinguish cell types, including ES cells, precursor cells, and terminally differentiated cells24,25. Over 250 miRNAs are expressed in the retina. Based upon these studies, we hypothesize that miRNAs play an important role during the differentiation of RPE from iPS.
The objective of this report is to describe the protocols for the differentiation of RPE from IMR90-4 iPS cells, collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types, iPS cells, iPS-RPE and fetal RPE. Total RNA was extracted from cultures of each cell type and hybridized to a miRNA microarray, containing probes specific for 1,205 human miRNAs and 144 viral miRNAs. The microarray results were compared to determine which miRNAs were differentially expressed among the different cell types. miRNAs with 2 fold or greater fold change in expression were selected for further analysis. A miRNA analysis software program was used to identify potential targets of the differentially expressed miRNAs and to generate cellular networks regulated by the selected miRNAs.

<table border="0" cellpadding="0" cellspacing="0" width="676">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">mTeSR1 media + 5X supplement</td>
			<td style="width:156px;">Stem Cell Technologies</td>
			<td style="width:119px;">5850</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">M-7154</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:235px;">Non essential amino acids</td>
			<td style="width:156px;">Hyclone(Fisher)</td>
			<td>SH30853.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Knockout serum replacement</td>
			<td style="width:156px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Gentamicin&#160;</td>
			<td style="width:156px;">Life Technologies</td>
			<td style="width:119px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">MEM media</td>
			<td style="width:156px;">Life Technologies</td>
			<td style="width:119px;">10370-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">N1 supplement</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">N-6530-5ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Taurine</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">T-8691-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Hydrocortisone</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">H0888-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Fetal bovine serum</td>
			<td style="width:156px;">Hyclone(Fisher)</td>
			<td>SH3008803HI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triiodo-l-thyronine sodium salt</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">T6397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Sodium hydroxide</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">S5881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal RPE media RTEGM kit</td>
			<td style="width:156px;">Life Technologies</td>
			<td style="width:119px;">195406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Dispase</td>
			<td style="width:156px;">Life Technologies</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Matrigel</td>
			<td style="width:156px;">BD Biosciences</td>
			<td style="width:119px;">354277</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Phosphate buffered saline</td>
			<td style="width:156px;">Hyclone(Fisher)</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Trypsin</td>
			<td style="width:156px;">Hyclone(Fisher)</td>
			<td style="width:119px;">25200-072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Miltenyi Biotec washing buffer</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-092-987</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Miltenyi Biotec rinsing buffer</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-092-222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Anti-TRA-1-60 microbead kit</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-095-816</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Miltenyi Biotec cell sorter column</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-021-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">RNeasy micro kit</td>
			<td style="width:156px;">Qiagen</td>
			<td style="width:119px;">74004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">QIA shredder</td>
			<td style="width:156px;">Qiagen</td>
			<td style="width:119px;">79654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">RNA 6000 Pico LabChip kit</td>
			<td style="width:156px;">Agilent</td>
			<td style="width:119px;">G2938-90046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Human miRNA microarray v16</td>
			<td style="width:156px;">Agilent</td>
			<td style="width:119px;">G4471A</td>
			<td></td>
		</tr>
	</tbody>
</table>

microrna expression profiles of human ips cells, retinal pigment epithelium derived from ips, and fetal retinal pigment epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

iPS cells (Figure 1A) were grown in differentiation conditions to induce differentiation into iPS-RPE cells. The iPS-RPE exhibited classic RPE phenotype of hexagonal pigmented cell morphology (Figure 1B) similar to fetal RPE (Figure 1C).
To better understand the role that miRNA may play during the process of differentiation from iPS to iPS-RPE, microarray analysis of miRNA expression was conducted. Total RNA was collected from iPS cells, fetal...

Watch this Scientific Journal Video about MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium at JoVE.com

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

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Research

JoVE Journal

Biology

11.3K Aufrufe.  JBSA Fort Sam Houston.  Die microRNA (miRNA)-Profile der menschlichen induzierten pluripotenten Stammzellen (iPS-Zellen), retinale Pigmentepithel (RPE) aus menschlichen induzierten pluripotenten Stammzellen (iPS-Zellen) (iPS-RPE) und fetale RPE abgeleitet, wurden verglichen. 