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Stem Cells Transl Med. 2014 Aug;3(8):911-22. doi: 10.5966/sctm.2013-0192. Epub 2014 May 29.

A multiplex high-throughput gene expression assay to simultaneously detect disease and functional markers in induced pluripotent stem cell-derived retinal pigment epithelium.

Author information

1
NIH Center for Advancing Translational Sciences, NIH, Bethesda, MD, USA.
2
Neural Stem Cell Institute, Rennselaer, New York, NY, USA.
3
Unit on Ocular and Stem Cell Translational Research, National Eye Institute, NIH, Bethesda, MD, USA.
4
Section on Epithelial and Retinal Physiology and Disease, National Eye Institute, NIH, Bethesda, MD, USA.
5
Section on Epithelial and Retinal Physiology and Disease, National Eye Insitute, NIH, Bethesda, MD, USA.
6
Affymetrix, Santa Clara, CA, USA.
7
Neural Stem Cell Institute, Rensselaer, New York, NY, USA.
8
Section on Epithelial and Retinal Physiology, National Eye Institute, NIH, Bethesda, MD, USA.
9
Unit on Ocular and Stem Cell Translational Research, National Eye Institute, Bethesda, MD, USA. kapilbharti@nei.nih.gov

Abstract

There is continuing interest in the development of lineage-specific cells from induced pluripotent stem (iPS) cells for use in cell therapies and drug discovery. Although in most cases differentiated cells show features of the desired lineage, they retain fetal gene expression and do not fully mature into "adult-like" cells. Such cells may not serve as an effective therapy because, once implanted, immature cells pose the risk of uncontrolled growth. Therefore, there is a need to optimize lineage-specific stem cell differentiation protocols to produce cells that no longer express fetal genes and have attained "adult-like" phenotypes. Toward that goal, it is critical to develop assays that simultaneously measure cell function and disease markers in high-throughput format. Here, we use a multiplex high-throughput gene expression assay that simultaneously detects endogenous expression of multiple developmental, functional, and disease markers in iPS cell-derived retinal pigment epithelium (RPE). We optimized protocols to differentiate iPS cell-derived RPE that was then grown in 96- and 384-well plates. As a proof of principle, we demonstrate differential expression of eight genes in iPS cells, iPS cell-derived RPE at two different differentiation stages, and primary human RPE using this multiplex assay. The data obtained from the multiplex gene expression assay are significantly correlated with standard quantitative reverse transcription-polymerase chain reaction-based measurements, confirming the ability of this high-throughput assay to measure relevant gene expression changes. This assay provides the basis to screen for compounds that improve RPE function and maturation and target disease pathways, thus providing the basis for effective treatments of several retinal degenerative diseases.

KEYWORDS:

Gene expression; Induced pluripotency; Reprogramming; Retina; Retinal pigmented epithelium; Stem cell; iPS

PMID:
24873859
PMCID:
PMC4116245
DOI:
10.5966/sctm.2013-0192
[Indexed for MEDLINE]
Free PMC Article

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