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.
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Method Article
MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium
In This Article
Summary
Abstract
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.
Introduction
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 prob....
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Protocol
1. Preparation of Culture Reagents and Culture Plates
- mTeSR1 media: Prepare mTeSR1 media according to the manufacturer’s directions. Thaw 100 ml of mTeSR1 5x supplement overnight at 4 °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 °C for up to 2 weeks and at -20 °C for 6 months.
- Differentiation media: Prepare differentiation media in DMEM/F12 to yield 0.1mM β-mercaptoethanol, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 10% knockout serum, and 10 µg/ml gentamicin.
- iPS-RPE media: Prepare iPS-RPE media in MEM to yield 1x N1 supplemen....
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Results
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.......
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Discussion
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.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
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 Muñiz, 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 ....
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| mTeSR1 media + 5X supplement | Stem Cell Technologies | 5850 | |
| DMEM/F12 | Life Technologies | 11330-032 | |
| 2-β-mercaptoethanol | Sigma | M-7154 | |
| Non essential amino acids | Hyclone(Fisher) | SH30853.01 | |
| Knockout serum replacement | Life Technologies | 10828-028 | |
| Gentamicin | Life Technologies | 15750-060 | |
| MEM media | Life Technologies | 10370-021 | |
| N1 supplement | Sigma | N-6530-5ML | |
| Taurine | Sigma | T-8691-25G | |
| Hydrocortisone | Sigma | H0888-1G | |
| Fetal bovine serum | Hyclone(Fisher) | SH3008803HI | |
| Triiodo-l-thyronine sodium salt | Sigma | T6397 | |
| Sodium hydroxide | Sigma | S5881 | |
| Fetal RPE media RTEGM kit | Life Technologies | 195406 | |
| Dispase | Life Technologies | 17105-041 | |
| Matrigel | BD Biosciences | 354277 | |
| Phosphate buffered saline | Hyclone(Fisher) | 10010-023 | |
| Trypsin | Hyclone(Fisher) | 25200-072 | |
| Miltenyi Biotec washing buffer | StemGent | 130-092-987 | |
| Miltenyi Biotec rinsing buffer | StemGent | 130-092-222 | |
| Anti-TRA-1-60 microbead kit | StemGent | 130-095-816 | |
| Miltenyi Biotec cell sorter column | StemGent | 130-021-101 | |
| RNeasy micro kit | Qiagen | 74004 | |
| QIA shredder | Qiagen | 79654 | |
| RNA 6000 Pico LabChip kit | Agilent | G2938-90046 | |
| Human miRNA microarray v16 | Agilent | G4471A |
References
- Yu, Z., et al. miRNAs regulate stem cell self-renewal and differentiation. Front Genet. 3, 191 (2012).
- Romano, G., et al. A commentary on iPS....
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