인간 유도만능 줄기세포로부터 유래된 신경세포구를 이용한 망막전구세포의 분화 및 생존에 관한 연구

 
저자 윤철민
형태사항 40 ; 26 cm
일반주기 지도교수: 허걸
학위논문사항 학위논문(박사)-- 고려대학교 대학원 : 의학과 안과학 전공 2016. 2
발행국 서울
언어 한국어
출판년 2016
소장기관 고려대학교 도서관

 
초록
Purpose: To differentiate neural retinal progenitor cells (RPCs) from human induced pluripotent stem cells (hiPSCs) - derived spherical neural masses (SNMs) and investigate whether the RPCs survive and differentiate in a mouse retina.
 Methods: SNMs were derived from hiPSCs by going through embryoid body formation, neural precursors selection, neural precursors expansion and dissection of neural structures. RPCs were differentiated from SNMs with noggin/fibroblast growth factor-basic (bFGF)/Dickkopf-1(Dkk-1)/Insulin-like growth factor 1 (IGF-1)/fibroblast growth factor 9 (FGF-9) protocol for three weeks. Differentiation profiles of human RPCs in vitro were analyzed by immunocytochemistry and quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Human RPCs were transplanted into the mouse (adult 8-12 weeks old C57BL/6) retina. Transplanted eye was examined post-operatively at three months and analyzed with immunohistochemistry.
 Results: Human RPCs from hiPSCs - derived SNMs expressed eye field markers (Paired box 6, PAX6) and early neural retinal markers (Ceh-10 homeodomain containing homolog, CHX10), except for a photoreceptor marker (S-cone opsin [OPN1SW]). RT-PCR revealed that early neural retinal markers (achaete-scute complex homolog 1 [MASH], mouse atonal homolog 5 [MATH5], neurogenic differentiation 1 [NEUROD1]) and retinal fate markers (brain-specific homeobox/POU domain transcription factor 3B [BRN3B], recoverin) were up-regulated, while the marker of retinal pigment epithelium (microphthalmia-associated transcription factor [MITF]) only showed a slight upregulation. The transplanted cells in the retina of mouse were matured to express markers of mature retinal cells (OPN1SW) and human nuclei (HNu) on immunohistochemistry at three months after transplantation.
 Conclusions: Human RPCs with neural retinal fate were differentiated from the hiPSCs-derived SNMs with noggin/bFGF/Dkk-1/IGF-1/FGF-9 protocol in vitro. Intraocular transplanted RPCs survived and differentiated into retinal photoreceptor cells in a mouse retina. Development of RPCs using SNMs may be a fast and useful method for differentiation of neural retinal cells.

 
목차

I. 서론 ‥‥‥‥‥‥‥‥‥‥‥‥1
II. 방법 ‥‥‥‥‥‥‥‥‥‥‥‥3
III. 결과 ‥‥‥‥‥‥‥‥‥‥‥‥11
IV. 고찰 ‥‥‥‥‥‥‥‥‥‥‥‥14
V. 결론 ‥‥‥‥‥‥‥‥‥‥‥‥20
VI. 참고문헌 ‥‥‥‥‥‥‥‥‥‥21

주제어
transplantation, retinal progenitor cell, retinal photoreceptor, human induced pluripotent stem cells, spherical neural mass