Generation of Functional Lentoid Bodies from Human Induced Pluripotent Stem Cells Derived from Urinary Cells

Lens Generation of Functional Lentoid Bodies From Human Induced Pluripotent Stem Cells Derived From Urinary Cells

Qiuli Fu,1,2 Zhenwei Qin,1,2 Xiuming Jin,1,2 Lifang Zhang,1,2 Zhijian Chen,3 Jiliang He,4 Junfeng Ji,5,6 and Ke Yao1,2

1Eye Center of the Second Afﬁliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China 2Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, Zhejiang Province, China 3Department of Environmental and Occupational Health, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang Province, China 4Institute of Environmental Medicine, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China 5Center of Stem Cell and Regenerative Medicine, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China 6Zhejiang Provincial Key Laboratory of Tissue Engineering and Regenerative Medicine, Hangzhou, China

Correspondence: Ke Yao, Eye Center PURPOSE. The pathological mechanisms underlying cataract formation remain largely unknown of the 2nd Affiliated Hospital, School on account of the lack of appropriate in vitro cellular models. The aim of this study is to of Medicine, Zhejiang University, develop a stable in vitro system for human lens regeneration using pluripotent stem cells. Zhejiang Provincial Key Lab of Oph- thalmology, Jiefang Road 88#, Hang- METHODS. Isolated human urinary cells were infected with four Yamanaka factors to generate zhou, Zhejiang Province, China; urinary human induced pluripotent stem cells (UiPSCs), which were induced to differentiate [email protected]. into lens progenitor cells and lentoid bodies (LBs). The expression of lens-specific markers Junfeng Ji, Center of Stem Cell and was examined by real-time PCR, immunostaining, and Western blotting. The structure and Regenerative Medicine, School of magnifying ability of LBs were investigated using transmission electron microscopy and Medicine, Zhejiang University, Zhe- observing the magnification of the letter ‘‘X,’’ respectively. jiang Provincial Key Laboratory of Tissue Engineering and Regenerative RESULTS. We developed a ‘‘fried egg’’ differentiation method to generate functional LBs from Medicine, Yuhangtang Road 866#, UiPSCs. The UiPSC-derived LBs exhibited crystalline lens-like morphology and a transparent Hangzhou, Zhejiang Province, China; structure and expressed lens-specific markers aA-, aB-, b-, and c-crystallin and MIP. During LB [email protected]. differentiation, the placodal markers SIX1, EYA1, DLX3, PAX6, and the specific early lens QF and ZQ contributed equally to the markers SOX1, PROX1, FOXE3, aA-, and aB-crystallin were observed at certain time points. work presented here and should Microscopic examination revealed the presence of lens epithelial cells adjacent to the lens therefore be regarded as equivalent capsule as well as both immature and mature fiber-like cells. Optical analysis further authors. demonstrated the magnifying ability (1.73) of the LBs generated from UiPSCs.

Submitted: August 10, 2016 CONCLUSIONS. Our study provides the ﬁrst evidence toward generating functional LBs from Accepted: December 11, 2016 UiPSCs, thereby establishing an in vitro system that can be used to study human lens Citation: Fu Q, Qin Z, Jin X, et al. development and cataractogenesis and perhaps even be useful for drug screening. Generation of functional lentoid bod- Keywords: lentoid bodies, induced pluripotent stem cells, cataract, lens, urinary cells ies from human induced pluripotent stem cells derived from urinary cells. Invest Ophthalmol Vis Sci. 2017;58:517–527. DOI:10.1167/ iovs.16-20504

ataract, an eye disease in which the lens becomes to be elucidated because of the lack of appropriate cellular C opacified, is the cause of more than half of all cases of models. blindness worldwide. Surgical removal of cataractous lenses is The lens is a transparent biconvex structure that develops thought to be the only effective treatment. Interestingly, two from the surface ectoderm. Lens development begins as the recent studies have reported that lanosterol and a small presumptive lens ectoderm thickens to form the lens placode, chemical compound can prevent the aggregation of lens which then invaginates and subsequently pinches off to form protein in both in vitro systems and animal models; this finding the lens vesicle.5–7 Lens genesis is characterized by two key is the basis for a new strategy of cataract prevention and events in its onion structure organization: the sorting of lens- treatment.1,2 However, further investigation of the usefulness of fated cells and spatial restriction of the lens epithelial cells and these compounds for cataract treatment in humans is impeded commitment of fiber cells to the lens genesis process. In vitro by the lack of appropriate human cataract disease models. recreation of these developmental events is important from the There has been remarkable progress in research on the process research perspective, and organoid technology may have of lens development during the past few decades, and genetic potential in this regard. studies have provided important insights into this.3,4 However, Human pluripotent stem cell (PSC)–derived organoids are the molecular mechanisms of human lens development remain organ-like tissues that exhibit multiple organ-specific cell types

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and self-organize to form a structure that resembles the organ cells. Finally, mature LBs exhibiting a lens-like morphology and in vivo. These tissues are believed to have potential for transparent structure were obtained on D25. Images of the LBs studying the in vivo functions of organs such as the gut,8,9 were captured at various time points with an Olympus IX71 kidney,10–12 brain,13,14 and retina.15,16 Several studies have microscope (Olympus, Tokyo, Japan) equipped with the DP2- reported various methods for the development of lentoid BSW software (Olympus) and/or a digital camera. Please see bodies (LBs).17–21 Recently, a group developed a method to the details of the other methods in the Supplementary induce the differentiation of human PSCs into lens progenitor Materials and Methods. All primers used in the present study cells and LBs under experimental conditions.22 Although the are listed in Supplementary Table S1. LBs generated exhibited similar chemical and biological characteristics to the human lens, their ultrastructural and optical properties were not representative of the human lens,22 RESULTS which restricted further use of this differentiation method. In this study, we have established a method for the Generation of UiPSCs and Differentiation of differentiation of human induced PSCs (iPSCs) into LBs by UiPSCs Into LBs by the ‘‘Fried Egg’’ Method isolation of lens-fated cells at the early stage of differentiation and manipulation of the microenvironment, which exhibits a We collected urine samples from three healthy donors and isolated urine cells (UCs) as previously reported (Supplemen- ‘‘fried egg’’ morphology at certain time points. Our results tary Fig. S1A).23 Both type I and type II UC colonies were showed that iPSCs derived from urinary cells (UiPSCs) were observed (Supplementary Fig. S1B). At 2 to 3 weeks after viral able to differentiate into LBs that had a human lens-like infection of UCs with human , , , and , transparent structure, expressed lens-specific markers, and OCT4 SOX2 KLF4 c-MYC iPSC colonies started to appear (Supplementary Fig. S1C). Four exhibited basic optical characteristics in vitro. Our study colonies were picked and cultured on Matrigel for more than therefore presents a method for the derivation of functional 30 passages. They were able to form embryonic bodies and LBs from human iPSCs and thereby lays the foundation for were positive for alkaline phosphatase (Supplementary Fig. future studies on human lens development, cataract mecha- S1D) and for the ESC antigens NANOG, SSEA4, SOX2, and nisms, and drug screening. TRA1-81 (Supplementary Fig. S2A). Gene expression analysis also confirmed that, similar to ESCs, they highly expressed SOX2, OCT4, and NANOG in contrast to the human lens MATERIALS AND METHODS epithelial cells (Supplementary Fig. S2B). Teratoma analysis Donors and Clinical Data Collection revealed the capacity of the cells to develop into tissues representative of the three germ layers (Supplementary Figs. The ethics committee of Zhejiang University approved of all S2C, S2D). Our findings indicate that the cells generated from the procedures performed in this study. Written informed human UCs were indeed iPSCs. consent was obtained from the participants. The study To induce the differentiation of iPSCs into LBs, we protocol adhered to the principles of the Declaration of developed a new protocol called the ‘‘fried egg’’ method Helsinki. Three healthy participants who had no eye-related because the formation of cell clusters that had the appearance disorders or urinary diseases were recruited. of a fried egg was a unique feature during the stepwise differentiation process (Fig. 1A). The method involved two Differentiation of UiPSCs, iPSCs Derived From sequential isolation steps. The first step was performed on D6 Fibroblasts, and Human Embryonic Stem Cells of the differentiation process and involved the isolation of lens- fated cells located at the periphery of individual UiPSC colonies UiPSCs, iPSCs from fibroblasts, and human embryonic stem (Fig. 1C), at which time point the epithelial-like cells lost cells (hESCs) were subjected to the ‘‘fried egg’’ method of LB NANOG expression (Fig. 1D). This was followed by the second formation, which was partly modified from a previous study.22 isolation step on D11, when the ‘‘fried egg’’–like differentiated A diagram of the key steps and the growth factor treatment colonies appeared, at which stage the colonies that did not schedule are shown in Figure 1A. The formation of LBs was exhibit this characteristic feature were removed from the induced in feeder-free conditions. First, approximately 80 PSC culture to avoid their negative effect on LB generation (Fig. colonies with approximately 20 cells each were seeded and 1C). E-cadherin and FOXE3 are widely accepted as lens cultured in mTesR medium (Stemcell, Vancouver, Canada) on a epithelial cell markers. We found that differentiating colonies Matrigel-coated (BD Biosciences, Bedford, MA, USA) 35-mm with the ‘‘fried egg’’ appearance consisted of the following two dish. Four hours after plating, the PSCs were triggered via 100 types of cells: E-cadherinþ differentiating cells (D-Cells) with ng/ml of the bone morphogenetic protein (BMP) inhibitor compact arrangement of multiple cell layers in the center of noggin and induced to differentiate into the ectoderm/ the colony, which eventually differentiated into LBs, and E- neuroectoderm until epithelial-like cells first appeared on the cadherin- supporting cells (S-Cells) with loose arrangement at periphery of the colonies on D6. Second, approximately 50 the periphery surrounding the D-Cells (Figs. 1C, 1D). At D14 of differentiating PSCs together with the surrounding epithelial- the differentiation process, FOXE3 small and immature LBs like cells, as shown in Figure 1A, were mechanically isolated, with FOXE3 fiber cells in the center and with three- and 30 to 50 differentiating PSC colonies were selected and dimensional structures appeared at the center of the colonies reseeded in Matrigel-coated 35-mm dishes. BMP signaling was (Figs. 1C, 1D), and they progressively matured into transparent then reactivated through its agonist BMP4 and BMP7 (20 ng/ LBs that expressed the lens marker c-crystallin at D25 (Figs. ml). Fibroblast growth factor signaling was activated through 1B–D). bFGF (100 ng/ml) at the same time. Third, cell clusters with a To examine whether the initial isolation step was critical for ‘‘fried egg’’ structure were observed on D11; cell clusters that LB generation, we compared LB generation from cultures with did not have ‘‘fried egg’’ morphologies were mechanically (selected cells) and without the initial isolation (nontreated discarded to avoid any negative effects during the following cells) and from cultures containing the remaining cells after the differentiation processes. Fourth, on D15, BMP4 and BMP7 was mechanical removal of lens-fated cells (Fig. 2A). Although both replaced with Wnt3a (20 ng/ml) to activate Wnt signaling and D-Cells and S-Cells were present in all conditions on D8, PAX6þ trigger the differentiation of lens epithelial cells into lens fiber D-Cells with an ordered arrangement only appeared in the

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FIGURE 1. Differentiation of UiPSCs into LBs. (A) Schematic diagram showing the stages of the ‘‘fried egg’’ method of LB generation. (B) Representative images of mature LBs (arrows) derived from UiPSCs after 25 days of differentiation. (C) Representative images at various time points showing UiPSCs (D0); differentiating UiPSCs before selection, when the epithelial-like cells appeared (arrows), with select parts of the cells indicated with red dotted frames (D6); differentiating UiPSCs with a ‘‘fried egg’’ morphology (D11), with the ratio of the diameters of the differentiating cells (D-Cells) to the supporting cells (S-Cells) being 1:2; and the ﬁrst appearance of immature LBs (D14; red square frame and arrow) and mature LBs (D25) during LB formation. The red square frame (D25) indicates a lens-like transparent structure. Magniﬁed images (1003

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and 2003) show the structure in better detail at every stage. (D) Immunoﬂuorescence staining (red) of NANOG on D0 in all iPSCs, loss of NANOG expression in epithelial-like cells (white arrow) on D6, expression of E-cadherin on D11 in D-Cells, loss of FOXE3 expression on D14 in ﬁber-like cells (white dotted frame and arrow), and expression of c-crystallin on D25 in mature LBs. Scale bars:(B) 3 mm; (C) 100 lm (403); 50 lm (1003); 30 lm (2003); (D) NANOG, 30 lm; (D) E-cadherin and FOXE3, 50 lm; (D) c-crystallin, 400 lm.

FIGURE 2. Effect of the initial isolation step on D6 on mature LB generation. (A) Schematic diagram showing the various conditions, with or without the initial isolation step, and the growth factor treatment protocol. (B) Immunoﬂuorescence examination of the PAX6 protein (red). Only the D- Cells in the selected cells condition were positive for PAX6 on D8. (C) On D8, the D-Cells and S-Cells in the nontreated cells and remaining cells conditions showed similar morphology and a disordered arrangement, which were different from those observed in the selected cells condition. Magniﬁed images (1003, 2003) showed the D-Cells and S-Cells in each condition in increased detail. (D) After 25 days of differentiation, only the LBs in the selected cells condition appeared transparent, whereas those in the other conditions were in the form of small cell clusters. Scale bars:(B)50 lm; (C) 100 lm (403), 50 lm (1003), 30 lm (2003); (D) 100 lm (403).

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selected cells condition (Figs. 2B, 2C). Moreover, unlike what expression at different time points of UiPSC differentiation. We was observed in the other conditions, under the selected cells first analyzed the expression of the placodal markers SIX1, condition, the ratio of D-Cells to S-Cells dynamically changed EYA1, DLX3 and PAX6, as the expression of these markers was over time, and the colonies exhibited ‘‘fried egg’’ morphology upregulated after mechanical isolation on D6 and decreased on D11. Transparent LBs exhibiting lens morphological afterward (Fig. 3A). Immunostaining further demonstrated the features eventually developed from the selected cells condition expression of SIX1 and PAX6 in the cells located in the central in contrast to the nontreated cells and remaining cells area of the colonies after 7 and 9 days of differentiation, conditions (Fig. 2D). We also found that cell clusters in which respectively (Fig. 3B); this is indicative of the differentiation of the ratio of the diameter of D-Cells to S-Cells was 1:2 generated UiPSCs into lens progenitor cells. We then examined expres- LBs with an ideal shape (Fig. 1C). These results indicate that sion of the specific early lens markers SOX1, PROX1, FOXE3, the microenvironment created by the D-Cells and S-Cells in the and aA- and aB-crystallin. Gene expression analysis showed ‘‘fried egg’’ cell cluster played an important role in LB that the highest expression of SOX1, FOXE3, and PROX1 was formation. observed between D12 and D14 of differentiation (Fig. 3C). The occurance of ‘‘fried egg’’–like cell clusters on D11 was However, the expression of aA- and aB-crystallin was a unique feature of our differentiation method. To determine detectable as early as D8, and it robustly increased after D12 whether the ‘‘fried egg’’ structure is essential for the eventual (Fig. 3C). FOXE3-positive, aA-crystallin-positive, aB-crystallin- generation of mature LBs that are transparent and have a lens- positive, and PROX1-positive cells were observed on D7, D12, like morphology, we studied the differentiation outcomes of and D13, respectively (Figs. 3D, 3E); this indicates that the lens cell clusters with ‘‘non–fried egg’’ (Supplementary Fig. S3A), progenitor cells further differentiated into fiber cells. Further- ‘‘single fried egg’’ (Fig.1C),and‘‘multiple fried egg’’ more, representative images of immature LBs at D17 with appearances on D11 (Supplementary Fig. S3A). We found that higher expressions of aA- and aB-crystallin than those at D12 LBs could only be generated from cell clusters with the ‘‘single are shown in Figure 3E. In addition, younger cultures that were fried egg’’ and ‘‘multiple fried egg’’ appearances (Figs. 1B, 1C; negative for the differentiation markers are shown in Figures Supplementary Figs. S3A, S3B). Moreover, multiple LBs with 3B, 3D, and 3E. unclear boundaries were often observed from cell clusters with Next, we characterized the expression of fiber cell markers the ‘‘multiple fried egg’’ morphology (Supplementary Figs. during the differentiation process. Our results showed that the S3A, S3B). expression level of aA-, aB-, b-, and c-crystallin and MIP We also applied our differentiation method to iPSCs derived gradually increased as differentiation progressed and peaked from fibroblasts and human ESCs. LBs with similar properties on D25 (Fig. 4A), which marks the time point of final were also generated from both fibroblast-derived iPSCs (data differentiation of the fiber cells. aA-, aB-, b-, and c-crystallin- not shown) and H9 human ESCs (Supplementary Figs. S4A, and MIP-positive cells were detected in LBs on D25 by S4B). Similarly, the nontreated cells and remaining cells from immunofluorescence examination (Fig. 4B). Moreover, a iPSCs derived from fibroblasts and human ESCs were unable to cluster of cells devoid of nuclei were observed to be form LBs with lens properties (Supplementary Fig. S4C). These specifically located in the central area of the LBs, which findings indicate that our ‘‘fried egg’’ method can be applied to indicated the terminal differentiation of fiber cells (Figs. 4B, other independent PSCs and that the ‘‘fried egg’’ structure is 4D). Younger cultures that were negative for the fiber cell required for the formation of mature LBs. markers are shown in Figure 4C. Western blot analysis further confirmed the high expression level of aA-, aB-, and b-crystallin Effect of Cell Density on LB Formation on D25 (Supplementary Fig. S6). In addition, examples of gels from PCR samples for each gene analyzed and the reference Gradient colony densities of 10, 15, 50, 100, and 150 gene at earlier versus late time points are provided in differentiating UiPSC colonies were seeded in 35-mm dishes, Supplementary Figure S7. and the number of LBs was calculated under a microscope. Our results showed that the lower the UiPSC colony seeding Characteristics of UiPSC-Derived LBs density, the higher was the percentage of LB formation (77.5% 6 14.3% at a seeding density of 15 versus 46.5% 6 0.7% at a Confocal microscopy showed that most cells in the LBs were seeding density of 100 colonies (Supplementary Fig. S5). A long and closely packed with limited extracellular space (Fig. seeding density of 30 to 50 UiPSC colonies was associated with 5A). The entire LB had a lower density of nuclei in the middle the highest LB formation rate. and was covered by epithelial-like cells (Figs. 5B–D). Next, the number of differentiating UiPSCs in a colony was Further analysis of LBs by transmission electron microscope estimated on D6 before further differentiation, and the (TEM) showed that the lens epithelial cells (Figs. 5E, 5I) with number of LBs was quantified on D25. The colonies were rectangle profiles located next to the very thin capsule (Figs. observed to contain 10 to more than 100 cells on D6, and the 5F, 5J) had a compact arrangement that consisted of regular colonies with only approximately 20 cells generated the nuclei and organelles. Immature fiber-like cells with degener- smallest LBs. The sizes of the LBs increased as the number of ating nuclei and organelles were frequently found adjacent to cells increased, but only up to a certain point. The biggest LBs the epithelial cells (Figs. 5G, 5K, 5L). Mature fiber cells without were derived from colonies containing approximately 50 nuclei and organelles were mainly observed at a distance from cells, and multiple LBs were observed in one colony. Thus, a the capsules of LBs (Fig. 5H). The expression of LC3B indicated density of 30 to 50 differentiating UiPSC colonies with the role of autophagy in LBs (Supplementary Fig. S8). approximately 50 cells per colony in a 35-mm dish was The lens capsules and some intercellular regions among the considered as the optimal condition for the ‘‘fried egg’’ lens epithelial cells were observed to be enriched in collagen method of LB formation. IV (Fig. 6A). The lens epithelial markers E-cadherin and FOXE3 were found to only be expressed in epithelial cells (Figs. 1D, Expression of Human Lens-Specific Genes in LBs 3D), which formed a monolayer in some mature LBs (Fig. 6A). Derived From UiPSCs Most cells in mature LBs were positive for aA-, aB-, b-, and c- crystallin and MIP (Fig. 6B). To further examine the LB differentiation process, we Finally, we examined whether the mature LBs possessed the performed real-time PCR analysis of human lens–specific gene ability to magnify a printed letter ‘‘X.’’ We compared the

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FIGURE 3. Expression of placodal and lens progenitor cell markers during the LB induction process. (A) qRT-PCR analysis of the placodal markers SIX1, PAX6, DLX3, and EYA1. The bar represents the mean 6 SEM values from five independent experiments. (B) Immunofluorescence examination of SIX1 (D5 and D7; red) and PAX6 (D5 and D9; red). (C) qRT-PCR analysis of the early lens-specific markers SOX1, PROX1, FOXE3, and aA- and aB-crystallin. The bar represents the mean 6 SEM values from five independent experiments. (D) Immunofluorescence detection of PROX1 (D8 and D13; red) and FOXE3 (D7 and D12). (E) Immunofluorescence detection of aA- and aB-crystallin (D7, D12, and D17; red). Scale bars:(B) and (D)50lm; (E) D7, 100 lm; (E) D12, 200 lm; (E) D17, 400 lm.

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FIGURE 4. Analysis of lens-speciﬁc markers during LB differentiation. (A) qRT-PCR analysis of lens differentiation markers. The expression level of ﬁve lens differentiation markers (aA-, aB-, b-, and c-crystallin, and MIP) were analyzed on D0, D7, D14, and D25 of LB induction. The bar represents the mean 6 SEM value from nine independent experiments. (B) Expression of aA-, aB-, b-, and c-crystallin and MIP in mature LBs on D25. aA-, aB-, b-, and c-crystallin and MIP signals (red) were only detected in LBs, and not in the surrounding cells; a cluster of cells devoid of nuclei was observed in the central area of the LBs (arrow) with less dense DAPI signals. (C) Expression of b- and c-crystallin and MIP in differentiating UiPSCs on D12 as negative controls. (D) Three-dimensional image of cells (D25) located in the middle of LB showed b-crystallin (red) expression in the absence of nuclei (40,6-diamidino-2-phenylindole [DAPI], blue). Scale bars:(B) 400 lm, (C)50lm, (D) 100 lm.

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FIGURE 5. Histochemical and ultrastructure analysis of LBs. (A) Confocal microscope using the membrane stain DiOC6 depicting the cellular arrangement within LBs at three different positions. (B–D) Methylene blue staining of the mid-sagittal section of the LBs on D25. The LB was surrounded by lens epithelial-like cells (B, C; arrows). (C) A magnified image of the lens epithelial cells (arrow). Cells with less dense nuclei were observed in some regions at a distance from the lens capsule (arrows in D). (E–L) Transmission electron microscopy (TEM) images of LBs show lens capsules (arrows in F, J), close-packed arrangement of lens epithelial-like cells (arrows in E, I) and differentiating fiber-like cells (arrows in G) with degenerating nuclei and organelles as the electron dense structure that the arrows in K, L pointed toward. A few fiber-like cells without nuclei and organelles were observed in some regions (arrows in H). Scale bars:(B) 200 lm, (A, C, D)50lm, (E–G)5lm, (H, I)2lm, (J, L)1lm, (K) 0.5 lm.

central width of the ‘‘X’’ measured from images of LBs, rat lens, cryTom mouse-derived iPSCs.24 Human ESC-/iPSC-derived LBs and culture medium only taken under a dissection microscope. have also been reported by several groups.22,25,26 However, We found that the magnification of LBs was similar to that of there are certain limitations with regard to the size and the rat lens and that they were able to amplify the ‘‘X’’ at a morphology of the LBs generated with the methods reported magnification factor of approximately 1.73 (Figs. 7A, 7B). so far. The maximum diameter of the LBs induced in our study In summary, our results demonstrate that our new ‘‘fried reached approximately 3 mm, which is, to our knowledge, egg’’ differentiation method could be used for the generation the largest LBs generated in vitro. Importantly, LBs with of LBs that express human lens-specific genes and possess functional optical properties were derived from human PSCs magnification capacity from UiPSCs in vitro. in vitro for the first time through the ‘‘fried egg’’ method. An important factor to be considered in the in vitro generation of LBs is their induction efficiency, which is also a prerequisite DISCUSSION for the use of LBs for further studies. LBs with the largest size and the ideal shape were usually derived from colonies in In the present study, we developed a method for the which the ratio of the diameter of compact cells and loose generation of LBs from human PSCs. Our results demonstrated cells was 1:2. This indicates that the supporting microenvi- that LBs derived from iPSCs by the ‘‘fried egg’’ method ronment plays an important role during the differentiation exhibited structural and functional properties that were process. Therefore, to our knowledge, our ‘‘fried egg’’ partially similar to those of the human lens. Therefore, this method currently seems to be the most suitable method for method forms the basis for future studies on human lens the generation of LBs of adequate size and optimal shape and development and cataractogenesis and may even find applica- optical properties. tions in drug screening. In our study, gene expression and immunostaining analysis Various LB formation methods have been developed indicated that the differentiation of UiPSCs into LBs by our during the past few decades.17–21 Recently, LBs expressing a method, to a certain extent, mimicked lens development in lens cell–specific fluorescent reporter were generated from vivo. Ultrastructural examination of the UiPSC-derived LBs

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FIGURE 6. Expression of lens capsule and epithelial and ﬁber cell markers in LBs. (A) Expression of collagen IV in lens capsules, and E-cadherin and FOXE3 in lens epithelial cells at the indicated time points. (B) Expression of aA-, aB-, b-, and c-crystallin and MIP in lens ﬁber cells in mature LB slices from D25. Scale bar:50lm.

revealed that they contained all the cellular components Two recent seminal studies have reported the treatment of present in the human lens (lens capsules, epithelial and fiber cataract with small compounds in both in vitro systems and cells); thus, there were some similarities with the human lens. animal models,1,2 which opens up a new nonsurgical approach Autophagy and mitophagy play a role in the degradation of to cataract treatment. However, the evaluation of the efficacy ocular organelles27; therefore, the expression of LC3B and the of these compounds in human cataract treatment is hampered observation of degenerating organelles indicated the potential by the lack of in vitro human tissue–derived cataract models. role of autophagy during organelle degradation in LBs. Besides, Although iPSCs have been previously generated from the lens denucleation, as a dominant phenomenon during fiber cell epithelial cells of cataract patients, no proper cataract model differentiation,28,29 was also indicated to be involved in LB has been established.25 Our study opens up the possibility of generation, as evidenced by the gradual loss of nuclei during generating patient-specific cataract models for the evaluation the differentiation process. Our results are consistent with of the efficacy of these compounds. previous studies about organelles and nuclei degradation in In summary, our study presents the ‘‘fried egg’’ method for lens development.27–29 Experiments on detailed analyses of the the derivation of LBs of approximately 3-mm diameter, the process surrounding organelle loss and denucleation in LBs are largest to our knowledge, from PSCs. The differentiation needed in the future. process was representative of the molecular events underlying

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FIGURE 7. Magnifying ability of the LBs. (A) Light microscopic images of the letter ‘‘X’’ observed under the culture medium alone (medium), under the rat lens, and under the LBs. (B) The central width of the letter ‘‘X’’ was calculated under the control condition, under the rat lens, and under the LBs. The magniﬁcation of the LBs/rat lens was indicated by the ratio of the central width of the ‘‘X’’ under the culture medium (white bar) only, the central width of the ‘‘X’’ under the rat lens (gray bar), or the width under the LBs (black bar) to the central width of the ‘‘X’’ under the culture medium only. Bars represent the mean 6 SEM values (n ¼ 30 [LBs]) from ﬁve independent experiments). **P < 0.01 versus control (medium). Scale bar: 1 mm.

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