Generation of Retinal Organoids with Mature Rods and Cones from Urine-Derived Human Induced Pluripotent Stem Cells

Hindawi Stem Cells International Volume 2018, Article ID 4968658, 12 pages https://doi.org/10.1155/2018/4968658 Research Article Generation of Retinal Organoids with Mature Rods and Cones from Urine-Derived Human Induced Pluripotent Stem Cells Guilan Li,1 Bingbing Xie,1 Liwen He,1 Tiancheng Zhou,2 Guanjie Gao,1 Shengxu Liu,1 Guangjin Pan,2 Jian Ge ,1 Fuhua Peng ,3 and Xiufeng Zhong 1 1 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China 3 Department of Neurology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China 2 Correspondence should be addressed to Fuhua Peng; and Xiufeng Zhong; Received 2 March 2018; Accepted 7 May 2018; Published 13 June 2018 Academic Editor: Leonard M. Eisenberg Copyright © 2018 Guilan Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Urine cells, a body trash, have been successfully reprogrammed into human induced pluripotent stem cells (U-hiPSCs) which hold a huge promise in regenerative medicine. However, it is unknown whether or to what extent U-hiPSCs can generate retinal cells so far. With a modified retinal differentiation protocol without addition of retinoic acid (RA), our study revealed that U-hiPSCs were able to differentiate towards retinal fates and form 3D retinal organoids containing laminated neural retina with all retinal cell types located in proper layer as in vivo. More importantly, U-hiPSCs generated highly mature photoreceptors with all subtypes, even red/ green cone-rich photoreceptors. Our data indicated that a supplement of RA to culture medium was not necessary for maturation and specification of U-hiPSC-derived photoreceptors at least in the niche of retinal organoids. The success of retinal differentiation with U-hiPSCs provides many opportunities in cell therapy, disease modeling, and drug screening, especially in personalized medicine of retinal diseases since urine cells can be noninvasively collected from patients and their relatives. 1. Introduction Retinal degenerative diseases such as retinal pigmentosa and age-related macular degeneration (AMD) are the major causes of vision loss due to cell death or functional loss of photoreceptor cells (PRCs) and/or retinal pigment epithelium (RPE) [1]. The underlying mechanisms are largely unknown because of lacking human disease model and limited diseased tissues. Hence, there is no effective treatment for these diseases so far [2]. In the past decade, human induced pluripotent stem cell (hiPSC) technology has been established through somatic cell reprogramming approach and provides a huge promise for study and treatment of these kinds of degenerative diseases since hiPSCs have a capacity to differentiate all body cells like human embryonic stem cells (hESCs) [3, 4]. Especially, compared to hESCs, derivatives from hiPSCs hold subject’s personal genetic information, facilitating personalized medicine. With rapid advancement of hiPSC technology, much progress has been acquired in retinal regeneration field with hiPSCs. Many studies have demonstrated that human pluripotent stem cells (hPSCs) (hESCs and hiPSCs) are able to differentiate into not only retinal cells including PRCs and RPE cells, but also retinal organoids with architecture under specific differentiation conditions, such as two-dimensional (2D) adherent culture, 3D suspension culture, or combined 2D and 3D cultures [5–11]. More importantly, these retinal organoids could achieve a high degree of maturation with formation of outer segment discs, functional structures of light-sensing photoreceptors, which was first reported by Zhong et al. [10]. These achievements would greatly facilitate the basic and translational studies of retinal 2 degenerative diseases. In a molecular level, mature photoreceptors in human retina consist of three subtypes, rhodopsin + rods, L/M opsin + red/green cones, and S opsin + blue cones. The cones are responsible for color vision, and daytime vision human activities depend on more. So far, generation of red/green cone-rich photoreceptors with hPSCs was hardly reported. Many types of somatic cells, such as skin fibroblasts, cord or peripheral blood cells, keratinocytes, hair follicle cells, adipose cells, and urine cells, have been used to do reprogramming to produce hiPSCs [12–19]. Some of them have demonstrated that they can be guided to differentiate into retinal cells, even to form retinal organoids [10, 11]. Among these somatic cells, urine cells have been regarded as a preferred source for reprogramming since they can be noninvasively and routinely collected in clinical settings without any risks. Although previous studies have shown that the urine-derived hiPSCs (U-hiPSCs) can differentiate into neurons, hepatocytes, tooth, and cardiomyocytes as well [20–23], it is still unclear whether or to what extent U-hiPSCs are able to differentiate towards a retinal cell lineage. With a modified, multistep retinal differentiation protocol without addition of retinoic acid (RA), we differentiated U-hiPSCs into 3D retinal organoids which contained laminated neural retina with all major retinal cells located in corresponding layer as in vivo. Especially, highly mature photoreceptors with rods and cones were also acquired with expression of functional proteins and formation of rudimentary outer segment. Taking advantage of convenient, noninvasive acquisition of urine cells, our data suggested that U-hiPSCs could serve as a valuable source for retinal cell therapy, disease modeling, and drug screening in retinal degenerative diseases, especially in personalized medicine. 2. Materials and Methods 2.1. hiPSC Culture. Three U-hiPSC lines (UE017, UE022, and UC005), gifts of Professor Guangjin Pan (Chinese Academy of Sciences, China) were used in this study. They were generated from urine cells of three healthy human adults using episomal iPSC reprogramming vectors previously described by Xue et al. [24]. hiPSCs were routinely maintained on a feeder-free condition with mTeSR1 medium (Stem cell Technologies, USA) and MatriGel substrate (Corning, USA). Cells were passaged every 5–7 days on 80–90% confluency. Identifiable differentiated cells were removed before passage under inverted microscope. Pluripotent characteristics of U-hiPSCs were further confirmed in molecular and morphological levels. The cells without integration of exogenous reprogramming factors and vectors were also evaluated by RT-PCR. 2.2. Retinal Differentiation. Retinal differentiation was performed according to a published protocol with a slight modification [10]. Briefly, hiPSCs were dissociated into small clumps and cultured in suspen (...truncated)