The objective of this report is to describe the protocols to derive the retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells using different sizes of embryoid bodies.
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Method Article
Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies
In This Article
Summary
Abstract
Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.
Introduction
Induced pluripotent stem (iPS) cells are a type of pluripotent stem cell derived by reprogramming adult cells with extrinsic factors1. In contrast, embryonic stem cells (ESCs), another type of pluripotent stem cell, are generated from the inner cell mass of the blastocyst2-3. Despite their different origins, iPS cells and ESCs are comparable in their unlimited capacity to replicate in vitro and in their capacity to differentiate into any cell type4-5. These characteristics of iPS cells make them ideal candidates for applications in personalized regenerative medicine. Recent research efforts are focused on developing robust dif....
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Protocol
1. Preparation of Culture Reagents and Culture Plates
- Prepare feeder-free stem cell culture medium by adding 100 ml of 5x serum-free supplement to 400 ml of stem cell basal medium. The medium is stable at 4 °C for up to 2 weeks and at -20 °C for 6 months.
- Add 10 µM solution of rho-associated, coiled-coil containing protein kinase (Rock) inhibitor (Y-27362) to commercially available embryoid body (EB) formation medium.
- Prepare differentiation medium by adding 0.1 mM β-mercaptoethanol, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 10% knockout serum replacement (KSR) and 10 µg/ml gentamicin to Dulbecco's ....
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Results
In this experiment, iPS cells were cultured and differentiated into the RPE lineage from EBs. EBs of controlled sizes were formed using microwell plates. As seen in Figure 1 EB formation was homogenous in the microwell plates. These EBs were then collected and plated on 6-well plates (Figure 2).
RPE can be identified by their classical hexagonal morphology, pigmentation, and expression of RPE markers. After 12 weeks of culture, the 200-cell EBs had developed a.......
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Discussion
To realize the full promise of pluripotent stem cells for cell therapy, it is necessary to regulate their differentiation in a consistent and reproducible way. This report describes protocols to form size-controlled EBs using microwell plate technology, initiate differentiation toward RPE and identify protein and gene markers of RPE. To synchronize the in vitro differentiation, homogenous sizes of EBs were formed by known numbers of iPS cells centrifuged in microwell plates by forced aggregation. Immunocytochemi.......
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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 Alberto Muñiz, Ramesh R Kaini, Whitney A Greene and Jae-Hyek Choi held a National Research Council Postdoctoral Research Associateship at the USAISR. This work was supported by U.S. Army Clinical Rehabilitative Medicine Research Program (CRMRP) and Military Operational Medicine Research Program (MOMRP).
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| mTeSR1 media + 5x supplement | Stem Cell Technologies | 5850 | |
| Y-27632 (Rock Inhibitor) | Stem Cell Technologies | 72304 | |
| DMEM/F12 | Life Technologies | 11330-032 | |
| 2-Mercaptoethanol | Sigma | M-7154 | |
| Non essential amino acids | Hyclone(Fisher) | SH30853.01 | |
| Knockout serum replacement | Life Technologies | 10828-028 | |
| Gentamicin | Life Technologies | 15750-060 | |
| L-Glutamine | Life Technologies | 25030-081 | |
| 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 | |
| Dispase | Life Technologies | 17105-041 | |
| Matrigel | BD Biosciences | 354277 | |
| Phosphate buffered saline | Hyclone(Fisher) | 10010-023 | |
| Aggrewell 400 plate | Stem Cell Technologies | 27940 | |
| AggreWell medium | Stem Cell Technologies | 5893 | |
| Accutase | Stem Cell Technologies | 7920 | |
| BD Cytofix/Cytoperm Fixation/Permeabilization Kit | BD Biosciences | 554714 | |
| Mouse Anti-PAX6 antibody | Developmental Studies Hybridoma Bank | ||
| Rabbit Anti-RX antibody | Abcam | Ab23340 | |
| Mouse Anti-MITF antibody | Thermo Scientific | MS-772-P | |
| Rabbit Anti-ZO-1 antibody | Invitrogen | 40-2200 | |
| RNeasy plus mini kit | Qiagen | 74134 | |
| PCR master mix | Promega | M7502 | |
| High capacity RNA to cDNA kit | Life Technologies | 4387406 |
References
- Takahashi, K., et al. Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc. 2 (12), 3081-3089 (2007).
- Reubinoff, B. E., et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol
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