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[FP-SA-42] New Technology
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Day
Apr 05 (Sat)
Time
13:30 - 15:00
Room
Room 13 - Tokyo International Forum 4F G402
Topic
New Technology and Alternative Medicine
Chair/Coordinator
Chair)Yuko Seko、Chair)Stephan Kaminski
 
 
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FP-SA-42-1

Duration 5min, Q&A 3min

Derivation of Human Differential Photoreceptor-Like Cells from Iris Cells by Defined Combinations of Transcription Factors

【Speaker】
Yuko Seko
【Author】
Yuko Seko Noriyuki Azuma Makoto Kaneda Kei Nakatani Akihiro Umezawa


Objective/Purpose
Redirecting differentiation of somatic cells by overexpression of transcription factors is a promising approach for regenerative medicine, elucidation of pathogenesis and development of new therapies. The process is called "direct reprogramming" or "direct conversion" and has been shown in ß cells, cardiomyocytes, neurons and platelets. The purpose of this study is to define the transcription factor combinations that can determine human photoreceptor cell fate. Neural retina and iris-pigmented epithelium (IPE) share a common developmental origin, leading us to test whether human iris cells could differentiate to retinal neurons.

Materials/Patients
The iris tissues were obtained from enucleated eye balls as a surgical treatment for retinoblastoma. IPE cells and iris-stromal cells were separated and cultivated. Human dermal fibroblasts were obtained from Promo Cell™.

Methods
We selected several transcription factors as candidates that may contribute to inducing photoreceptor-specific phenotypes. Full length of these transcription factor genes were amplified by PCR, cloned into pENTR11 and recombined with pMXs. Human iris cells were retrovirally infected and were examined for inducible expression of photoreceptor-specific phenotypes in those cells using RT-PCR, immunocytochemistry and microarray analyses. For functional assessment of infected cells, electrical recordings were made in the whole-cell patch-clamp configuration. The membrane current before and after the light stimulation was recorded and analyzed. We further tested whether human dermal fibroblasts, which are less specialized than iris cells, could be converted into photoreceptors.

Results and Conclusion
 Expression of rhodopsin, blue opsin and green/red opsin in induced photoreceptor-like cells were dependent on combinations of transcription factors: A combination of CRX and NEUROD induced rhodopsin and blue opsin, but did not induce green opsin; a combination of CRX and RX induced blue opsin and green/red opsin, but did not induce rhodopsin. Global gene expression data by microarray analysis and RT-PCR showed that phototransduction-related genes, such as recoverin, S-antigen, CNGB3 and PDE6C, were significantly increased in induced photoreceptor-like cells from iris cells and dermal fibroblasts. Both the NRL gene and the NR2E3 gene were endogenously up-regulated in induced photoreceptor-like cells, implying that exogenous CRX, RX, and NEUROD, but not NRL, are sufficient to generate rod photoreceptor cells. Functional analysis; i.e. patch clamp recordings, clearly revealed that photoreceptor-directed iris cells, induced by CRX, RX and NEUROD, responded to light. These data suggest that photosensitive photoreceptor-like cells can be generated by combinations of transcription factors from human iris cells.

[ Keyword ]
somatic cells / differentiation / photoreceptor-like cell / transcription factor

[ Conflict of Interest ]
No

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