Xu Wei-Wei, Huang Li, Chong Kelvin K L, Leung Doreen S Y, Li Benjamin F L, Yin Zheng-Qin, Huang Yi-Fei, Pang Chi Pui
Department of Ophthalmology and Visual Sciences, the Chinese University of Hong Kong, Hong Kong 999077, China; Department of Ophthalmology, Chinese People's Liberation Army General Hospital, Beijing 100853, China.
Department of Ophthalmology and Visual Sciences, the Chinese University of Hong Kong, Hong Kong 999077, China.
Int J Ophthalmol. 2017 Jan 18;10(1):23-29. doi: 10.18240/ijo.2017.01.04. eCollection 2017.
To investigate the retinal photoreceptor differentiation potential of human orbital adipose tissue-derived stem cells (ADSCs) generated by enzyme (EN) and explant (EX) culture methods.
We investigated potentials of human orbital ADSCs to differentiate into photoreceptors through EN and EX culture methods. EN and EX orbital ADSCs were obtained from the same donor during rehabilitative orbital decompression, and then were subject to a 3-step induction using Noggin, DKK-1, IGF-1 and b-FGF at different time points for 38d. Stem cell, eye-field and photoreceptor-related gene and protein markers were measured by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescent (IMF) staining.
Both EX and EN orbital ADSCs expressed CD133, a marker of cell differentiation. Moreover, PAX6 and rhodopsin, markers of the retinal progenitor cells, were detected from EX and EN orbital ADSCs. In EX orbital ADSCs, PAX6 mRNA was detected on the 17 day and then the rhodopsin mRNA was detected on the 24 day. In contrast, the EN orbital ADSCs expressed PAX6 and rhodopsin mRNA on the 31 day. EX orbital ADSCs expressed rhodopsin protein on the 24 day, while EN orbital ADSCs expressed rhodopsin protein on the 31 day.
Orbital ADSCs isolated by direct explants culture show earlier and stronger expressions of markers towards eye field and retinal photoreceptor differentiation than those generated by conventional EN method.
研究通过酶（EN）消化法和组织块（EX）培养法获得的人眼眶脂肪组织来源干细胞（ADSCs）的视网膜光感受器分化潜能。
我们通过EN和EX培养法研究人眼眶ADSCs分化为光感受器的潜能。EN和EX眼眶ADSCs来自同一供体的眼眶减压修复手术，然后在不同时间点使用Noggin、DKK-1、IGF-1和b-FGF进行三步诱导，共38天。通过逆转录聚合酶链反应（RT-PCR）和免疫荧光（IMF）染色检测干细胞、眼场和光感受器相关基因及蛋白质标志物。
EX和EN眼眶ADSCs均表达细胞分化标志物CD133。此外，在EX和EN眼眶ADSCs中检测到视网膜祖细胞标志物PAX6和视紫红质。在EX眼眶ADSCs中，第17天检测到PAX6 mRNA，随后在第24天检测到视紫红质mRNA。相比之下，EN眼眶ADSCs在第31天表达PAX6和视紫红质mRNA。EX眼眶ADSCs在第24天表达视紫红质蛋白，而EN眼眶ADSCs在第31天表达视紫红质蛋白。
与传统的EN法相比，直接组织块培养分离的眼眶ADSCs在眼场和视网膜光感受器分化标志物的表达上更早且更强。
