Deriving retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells by different sizes of embryoid bodies.
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.
Ocular Trauma, U.S. Army Institute of Surgical Research.
Alberto
A
Muñiz
Ocular Trauma, U.S. Army Institute of Surgical Research.
Kaini R
KR
Ramesh
Ocular Trauma, U.S. Army Institute of Surgical Research.
Whitney A
WA
Greene
Ocular Trauma, U.S. Army Institute of Surgical Research.
Jae-Hyek
JH
Choi
Ocular Trauma, U.S. Army Institute of Surgical Research; heuy-ching.h.wang.civ@mail.mil.
Heuy-Ching
HC
Wang
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D045744:Cell Line, Tumor; D019070:Cell Lineage; D058732:Embryoid Bodies / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015415:Biomarkers