January 2017
Volume 58, Issue 1
Open Access
Lens  |   January 2017
Generation of Functional Lentoid Bodies From Human Induced Pluripotent Stem Cells Derived From Urinary Cells
Author Affiliations & Notes
  • Qiuli Fu
    Eye Center of the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
    Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, Zhejiang Province, China
  • Zhenwei Qin
    Eye Center of the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
    Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, Zhejiang Province, China
  • Xiuming Jin
    Eye Center of the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
    Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, Zhejiang Province, China
  • Lifang Zhang
    Eye Center of the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
    Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, Zhejiang Province, China
  • Zhijian Chen
    Department of Environmental and Occupational Health, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang Province, China
  • Jiliang He
    Institute of Environmental Medicine, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
  • Junfeng Ji
    Center of Stem Cell and Regenerative Medicine, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
    Zhejiang Provincial Key Laboratory of Tissue Engineering and Regenerative Medicine, Hangzhou, China
  • Ke Yao
    Eye Center of the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
    Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, Zhejiang Province, China
  • Correspondence: Ke Yao, Eye Center of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Lab of Ophthalmology, Jiefang Road 88#, Hangzhou, Zhejiang Province, China; xlren@zju.edu.cn
  • Junfeng Ji, Center of Stem Cell and Regenerative Medicine, School of Medicine, Zhejiang University, Zhejiang Provincial Key Laboratory of Tissue Engineering and Regenerative Medicine, Yuhangtang Road 866#, Hangzhou, Zhejiang Province, China; jijunfeng@zju.edu.cn
  • Footnotes
     QF and ZQ contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science January 2017, Vol.58, 517-527. doi:10.1167/iovs.16-20504
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Qiuli Fu, Zhenwei Qin, Xiuming Jin, Lifang Zhang, Zhijian Chen, Jiliang He, Junfeng Ji, Ke Yao; Generation of Functional Lentoid Bodies From Human Induced Pluripotent Stem Cells Derived From Urinary Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(1):517-527. doi: 10.1167/iovs.16-20504.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: The pathological mechanisms underlying cataract formation remain largely unknown on account of the lack of appropriate in vitro cellular models. The aim of this study is to develop a stable in vitro system for human lens regeneration using pluripotent stem cells.

Methods: Isolated human urinary cells were infected with four Yamanaka factors to generate urinary human induced pluripotent stem cells (UiPSCs), which were induced to differentiate into lens progenitor cells and lentoid bodies (LBs). The expression of lens-specific markers was examined by real-time PCR, immunostaining, and Western blotting. The structure and magnifying ability of LBs were investigated using transmission electron microscopy and observing the magnification of the letter “X,” respectively.

Results: We developed a “fried egg” differentiation method to generate functional LBs from UiPSCs. The UiPSC-derived LBs exhibited crystalline lens-like morphology and a transparent structure and expressed lens-specific markers αA-, αB-, β-, and γ-crystallin and MIP. During LB differentiation, the placodal markers SIX1, EYA1, DLX3, PAX6, and the specific early lens markers SOX1, PROX1, FOXE3, αA-, and αB-crystallin were observed at certain time points. Microscopic examination revealed the presence of lens epithelial cells adjacent to the lens capsule as well as both immature and mature fiber-like cells. Optical analysis further demonstrated the magnifying ability (1.7×) of the LBs generated from UiPSCs.

Conclusions: Our study provides the first evidence toward generating functional LBs from UiPSCs, thereby establishing an in vitro system that can be used to study human lens development and cataractogenesis and perhaps even be useful for drug screening.

Cataract, an eye disease in which the lens becomes opacified, is the cause of more than half of all cases of blindness worldwide. Surgical removal of cataractous lenses is thought to be the only effective treatment. Interestingly, two recent studies have reported that lanosterol and a small chemical compound can prevent the aggregation of lens protein in both in vitro systems and animal models; this finding is the basis for a new strategy of cataract prevention and treatment.1,2 However, further investigation of the usefulness of these compounds for cataract treatment in humans is impeded by the lack of appropriate human cataract disease models. There has been remarkable progress in research on the process of lens development during the past few decades, and genetic studies have provided important insights into this.3,4 However, the molecular mechanisms of human lens development remain to be elucidated because of the lack of appropriate cellular models. 
The lens is a transparent biconvex structure that develops from the surface ectoderm. Lens development begins as the presumptive lens ectoderm thickens to form the lens placode, which then invaginates and subsequently pinches off to form the lens vesicle.57 Lens genesis is characterized by two key events in its onion structure organization: the sorting of lens-fated cells and spatial restriction of the lens epithelial cells and commitment of fiber cells to the lens genesis process. In vitro recreation of these developmental events is important from the research perspective, and organoid technology may have potential in this regard. 
Human pluripotent stem cell (PSC)–derived organoids are organ-like tissues that exhibit multiple organ-specific cell types and self-organize to form a structure that resembles the organ in vivo. These tissues are believed to have potential for studying the in vivo functions of organs such as the gut,8,9 kidney,1012 brain,13,14 and retina.15,16 Several studies have reported various methods for the development of lentoid bodies (LBs).1721 Recently, a group developed a method to induce the differentiation of human PSCs into lens progenitor cells and LBs under experimental conditions.22 Although the 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 which restricted further use of this differentiation method. 
In this study, we have established a method for the differentiation of human induced PSCs (iPSCs) into LBs by isolation of lens-fated cells at the early stage of differentiation and manipulation of the microenvironment, which exhibits a “fried egg” morphology at certain time points. Our results showed that iPSCs derived from urinary cells (UiPSCs) were able to differentiate into LBs that had a human lens-like transparent structure, expressed lens-specific markers, and exhibited basic optical characteristics in vitro. Our study therefore presents a method for the derivation of functional LBs from human iPSCs and thereby lays the foundation for future studies on human lens development, cataract mechanisms, and drug screening. 
Materials and Methods
Donors and Clinical Data Collection
The ethics committee of Zhejiang University approved of all the procedures performed in this study. Written informed consent was obtained from the participants. The study protocol adhered to the principles of the Declaration of Helsinki. Three healthy participants who had no eye-related disorders or urinary diseases were recruited. 
Differentiation of UiPSCs, iPSCs Derived From Fibroblasts, and Human Embryonic Stem Cells
UiPSCs, iPSCs from fibroblasts, and human embryonic stem cells (hESCs) were subjected to the “fried egg” method of LB formation, which was partly modified from a previous study.22 A diagram of the key steps and the growth factor treatment schedule are shown in Figure 1A. The formation of LBs was induced in feeder-free conditions. First, approximately 80 PSC colonies with approximately 20 cells each were seeded and cultured in mTesR medium (Stemcell, Vancouver, Canada) on a Matrigel-coated (BD Biosciences, Bedford, MA, USA) 35-mm dish. Four hours after plating, the PSCs were triggered via 100 ng/ml of the bone morphogenetic protein (BMP) inhibitor noggin and induced to differentiate into the ectoderm/neuroectoderm until epithelial-like cells first appeared on the periphery of the colonies on D6. Second, approximately 50 differentiating PSCs together with the surrounding epithelial-like cells, as shown in Figure 1A, were mechanically isolated, and 30 to 50 differentiating PSC colonies were selected and reseeded in Matrigel-coated 35-mm dishes. BMP signaling was then reactivated through its agonist BMP4 and BMP7 (20 ng/ml). Fibroblast growth factor signaling was activated through bFGF (100 ng/ml) at the same time. Third, cell clusters with a “fried egg” structure were observed on D11; cell clusters that did not have “fried egg” morphologies were mechanically discarded to avoid any negative effects during the following differentiation processes. Fourth, on D15, BMP4 and BMP7 was replaced with Wnt3a (20 ng/ml) to activate Wnt signaling and trigger the differentiation of lens epithelial cells into lens fiber cells. Finally, mature LBs exhibiting a lens-like morphology and transparent structure were obtained on D25. Images of the LBs were captured at various time points with an Olympus IX71 microscope (Olympus, Tokyo, Japan) equipped with the DP2-BSW software (Olympus) and/or a digital camera. Please see the details of the other methods in the Supplementary Materials and Methods. All primers used in the present study are listed in Supplementary Table S1
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 (100× and 200×) 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 γ-crystallin on D25 in mature LBs. Scale bars: (B) 3 mm; (C) 100 μm (40×); 50 μm (100×); 30 μm (200×); (D) NANOG, 30 μm; (D) E-cadherin and FOXE3, 50 μm; (D) γ-crystallin, 400 μm.
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 (100× and 200×) 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 γ-crystallin on D25 in mature LBs. Scale bars: (B) 3 mm; (C) 100 μm (40×); 50 μm (100×); 30 μm (200×); (D) NANOG, 30 μm; (D) E-cadherin and FOXE3, 50 μm; (D) γ-crystallin, 400 μm.
Results
Generation of UiPSCs and Differentiation of UiPSCs Into LBs by the “Fried Egg” Method
We collected urine samples from three healthy donors and isolated urine cells (UCs) as previously reported (Supplementary Fig. S1A).23 Both type I and type II UC colonies were observed (Supplementary Fig. S1B). At 2 to 3 weeks after viral infection of UCs with human OCT4, SOX2, KLF4, and c-MYC, iPSC colonies started to appear (Supplementary Fig. S1C). Four colonies were picked and cultured on Matrigel for more than 30 passages. They were able to form embryonic bodies and were positive for alkaline phosphatase (Supplementary Fig. S1D) and for the ESC antigens NANOG, SSEA4, SOX2, and 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 epithelial cells (Supplementary Fig. S2B). Teratoma analysis revealed the capacity of the cells to develop into tissues representative of the three germ layers (Supplementary Figs. S2C, S2D). Our findings indicate that the cells generated from human UCs were indeed iPSCs. 
To induce the differentiation of iPSCs into LBs, we developed a new protocol called the “fried egg” method because the formation of cell clusters that had the appearance of a fried egg was a unique feature during the stepwise differentiation process (Fig. 1A). The method involved two sequential isolation steps. The first step was performed on D6 of the differentiation process and involved the isolation of lens-fated cells located at the periphery of individual UiPSC colonies (Fig. 1C), at which time point the epithelial-like cells lost NANOG expression (Fig. 1D). This was followed by the second isolation step on D11, when the “fried egg”–like differentiated colonies appeared, at which stage the colonies that did not exhibit this characteristic feature were removed from the culture to avoid their negative effect on LB generation (Fig. 1C). E-cadherin and FOXE3 are widely accepted as lens epithelial cell markers. We found that differentiating colonies with the “fried egg” appearance consisted of the following two types of cells: E-cadherin+ differentiating cells (D-Cells) with compact arrangement of multiple cell layers in the center of the colony, which eventually differentiated into LBs, and E-cadherin- supporting cells (S-Cells) with loose arrangement at the periphery surrounding the D-Cells (Figs. 1C, 1D). At D14 of the differentiation process, FOXE3 small and immature LBs with FOXE3 fiber cells in the center and with three-dimensional structures appeared at the center of the colonies (Figs. 1C, 1D), and they progressively matured into transparent LBs that expressed the lens marker γ-crystallin at D25 (Figs. 1B–D). 
To examine whether the initial isolation step was critical for LB generation, we compared LB generation from cultures with (selected cells) and without the initial isolation (nontreated cells) and from cultures containing the remaining cells after the mechanical removal of lens-fated cells (Fig. 2A). Although both D-Cells and S-Cells were present in all conditions on D8, PAX6+ D-Cells with an ordered arrangement only appeared in the selected cells condition (Figs. 2B, 2C). Moreover, unlike what was observed in the other conditions, under the selected cells condition, the ratio of D-Cells to S-Cells dynamically changed over time, and the colonies exhibited “fried egg” morphology on D11. Transparent LBs exhibiting lens morphological features eventually developed from the selected cells condition in contrast to the nontreated cells and remaining cells conditions (Fig. 2D). We also found that cell clusters in which the ratio of the diameter of D-Cells to S-Cells was 1:2 generated LBs with an ideal shape (Fig. 1C). These results indicate that the microenvironment created by the D-Cells and S-Cells in the “fried egg” cell cluster played an important role in LB formation. 
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 (100×, 200×) 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 μm; (C) 100 μm (40×), 50 μm (100×), 30 μm (200×); (D) 100 μm (40×).
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 (100×, 200×) 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 μm; (C) 100 μm (40×), 50 μm (100×), 30 μm (200×); (D) 100 μm (40×).
The occurance of “fried egg”–like cell clusters on D11 was a unique feature of our differentiation method. To determine whether the “fried egg” structure is essential for the eventual generation of mature LBs that are transparent and have a lens-like morphology, we studied the differentiation outcomes of cell clusters with “non–fried egg” (Supplementary Fig. S3A), “single fried egg” (Fig. 1C), and “multiple fried egg” appearances on D11 (Supplementary Fig. S3A). We found that LBs could only be generated from cell clusters with the “single fried egg” and “multiple fried egg” appearances (Figs. 1B, 1C; Supplementary Figs. S3A, S3B). Moreover, multiple LBs with unclear boundaries were often observed from cell clusters with the “multiple fried egg” morphology (Supplementary Figs. S3A, S3B). 
We also applied our differentiation method to iPSCs derived from fibroblasts and human ESCs. LBs with similar properties were also generated from both fibroblast-derived iPSCs (data not shown) and H9 human ESCs (Supplementary Figs. S4A, S4B). Similarly, the nontreated cells and remaining cells from iPSCs derived from fibroblasts and human ESCs were unable to form LBs with lens properties (Supplementary Fig. S4C). These findings indicate that our “fried egg” method can be applied to other independent PSCs and that the “fried egg” structure is required for the formation of mature LBs. 
Effect of Cell Density on LB Formation
Gradient colony densities of 10, 15, 50, 100, and 150 differentiating UiPSC colonies were seeded in 35-mm dishes, and the number of LBs was calculated under a microscope. Our results showed that the lower the UiPSC colony seeding density, the higher was the percentage of LB formation (77.5% ± 14.3% at a seeding density of 15 versus 46.5% ± 0.7% at a seeding density of 100 colonies (Supplementary Fig. S5). A seeding density of 30 to 50 UiPSC colonies was associated with the highest LB formation rate. 
Next, the number of differentiating UiPSCs in a colony was estimated on D6 before further differentiation, and the number of LBs was quantified on D25. The colonies were observed to contain 10 to more than 100 cells on D6, and the colonies with only approximately 20 cells generated the smallest LBs. The sizes of the LBs increased as the number of cells increased, but only up to a certain point. The biggest LBs were derived from colonies containing approximately 50 cells, and multiple LBs were observed in one colony. Thus, a density of 30 to 50 differentiating UiPSC colonies with approximately 50 cells per colony in a 35-mm dish was considered as the optimal condition for the “fried egg” method of LB formation. 
Expression of Human Lens-Specific Genes in LBs Derived From UiPSCs
To further examine the LB differentiation process, we performed real-time PCR analysis of human lens–specific gene expression at different time points of UiPSC differentiation. We first analyzed the expression of the placodal markers SIX1, EYA1, DLX3 and PAX6, as the expression of these markers was upregulated after mechanical isolation on D6 and decreased afterward (Fig. 3A). Immunostaining further demonstrated the expression of SIX1 and PAX6 in the cells located in the central area of the colonies after 7 and 9 days of differentiation, respectively (Fig. 3B); this is indicative of the differentiation of UiPSCs into lens progenitor cells. We then examined expression of the specific early lens markers SOX1, PROX1, FOXE3, and αA- and αB-crystallin. Gene expression analysis showed that the highest expression of SOX1, FOXE3, and PROX1 was observed between D12 and D14 of differentiation (Fig. 3C). However, the expression of αA- and αB-crystallin was detectable as early as D8, and it robustly increased after D12 (Fig. 3C). FOXE3-positive, αA-crystallin-positive, αB-crystallin-positive, and PROX1-positive cells were observed on D7, D12, and D13, respectively (Figs. 3D, 3E); this indicates that the lens progenitor cells further differentiated into fiber cells. Furthermore, representative images of immature LBs at D17 with higher expressions of αA- and αB-crystallin than those at D12 are shown in Figure 3E. In addition, younger cultures that were negative for the differentiation markers are shown in Figures 3B, 3D, and 3E. 
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 ± 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 αA- and αB-crystallin. The bar represents the mean ± SEM values from five independent experiments. (D) Immunofluorescence detection of PROX1 (D8 and D13; red) and FOXE3 (D7 and D12). (E) Immunofluorescence detection of αA- and αB-crystallin (D7, D12, and D17; red). Scale bars: (B) and (D) 50 μm; (E) D7, 100 μm; (E) D12, 200 μm; (E) D17, 400 μm.
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 ± 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 αA- and αB-crystallin. The bar represents the mean ± SEM values from five independent experiments. (D) Immunofluorescence detection of PROX1 (D8 and D13; red) and FOXE3 (D7 and D12). (E) Immunofluorescence detection of αA- and αB-crystallin (D7, D12, and D17; red). Scale bars: (B) and (D) 50 μm; (E) D7, 100 μm; (E) D12, 200 μm; (E) D17, 400 μm.
Next, we characterized the expression of fiber cell markers during the differentiation process. Our results showed that the expression level of αA-, αB-, β-, and γ-crystallin and MIP gradually increased as differentiation progressed and peaked on D25 (Fig. 4A), which marks the time point of final differentiation of the fiber cells. αA-, αB-, β-, and γ-crystallin- and MIP-positive cells were detected in LBs on D25 by immunofluorescence examination (Fig. 4B). Moreover, a cluster of cells devoid of nuclei were observed to be specifically located in the central area of the LBs, which indicated the terminal differentiation of fiber cells (Figs. 4B, 4D). Younger cultures that were negative for the fiber cell markers are shown in Figure 4C. Western blot analysis further confirmed the high expression level of αA-, αB-, and β-crystallin on D25 (Supplementary Fig. S6). In addition, examples of gels from PCR samples for each gene analyzed and the reference gene at earlier versus late time points are provided in Supplementary Figure S7
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 (αA-, αB-, β-, and γ-crystallin, and MIP) were analyzed on D0, D7, D14, and D25 of LB induction. The bar represents the mean ± SEM value from nine independent experiments. (B) Expression of αA-, αB-, β-, and γ-crystallin and MIP in mature LBs on D25. αA-, αB-, β-, and γ-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 β- and γ-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 β-crystallin (red) expression in the absence of nuclei (4′,6-diamidino-2-phenylindole [DAPI], blue). Scale bars: (B) 400 μm, (C) 50 μm, (D) 100 μm.
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 (αA-, αB-, β-, and γ-crystallin, and MIP) were analyzed on D0, D7, D14, and D25 of LB induction. The bar represents the mean ± SEM value from nine independent experiments. (B) Expression of αA-, αB-, β-, and γ-crystallin and MIP in mature LBs on D25. αA-, αB-, β-, and γ-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 β- and γ-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 β-crystallin (red) expression in the absence of nuclei (4′,6-diamidino-2-phenylindole [DAPI], blue). Scale bars: (B) 400 μm, (C) 50 μm, (D) 100 μm.
Characteristics of UiPSC-Derived LBs
Confocal microscopy showed that most cells in the LBs were long and closely packed with limited extracellular space (Fig. 5A). The entire LB had a lower density of nuclei in the middle and was covered by epithelial-like cells (Figs. 5B–D). 
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. (BD) 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). (EL) 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 μm, (A, C, D) 50 μm, (EG) 5 μm, (H, I) 2 μm, (J, L) 1 μm, (K) 0.5 μm.
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. (BD) 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). (EL) 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 μm, (A, C, D) 50 μm, (EG) 5 μm, (H, I) 2 μm, (J, L) 1 μm, (K) 0.5 μm.
Further analysis of LBs by transmission electron microscope (TEM) showed that the lens epithelial cells (Figs. 5E, 5I) with rectangle profiles located next to the very thin capsule (Figs. 5F, 5J) had a compact arrangement that consisted of regular nuclei and organelles. Immature fiber-like cells with degenerating nuclei and organelles were frequently found adjacent to the epithelial cells (Figs. 5G, 5K, 5L). Mature fiber cells without nuclei and organelles were mainly observed at a distance from the capsules of LBs (Fig. 5H). The expression of LC3B indicated the role of autophagy in LBs (Supplementary Fig. S8). 
The lens capsules and some intercellular regions among the lens epithelial cells were observed to be enriched in collagen IV (Fig. 6A). The lens epithelial markers E-cadherin and FOXE3 were found to only be expressed in epithelial cells (Figs. 1D, 3D), which formed a monolayer in some mature LBs (Fig. 6A). Most cells in mature LBs were positive for αA-, αB-, β-, and γ-crystallin and MIP (Fig. 6B). 
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 αA-, αB-, β-, and γ-crystallin and MIP in lens fiber cells in mature LB slices from D25. Scale bar: 50 μm.
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 αA-, αB-, β-, and γ-crystallin and MIP in lens fiber cells in mature LB slices from D25. Scale bar: 50 μm.
Finally, we examined whether the mature LBs possessed the ability to magnify a printed letter “X.” We compared the central width of the “X” measured from images of LBs, rat lens, and culture medium only taken under a dissection microscope. We found that the magnification of LBs was similar to that of the rat lens and that they were able to amplify the “X” at a magnification factor of approximately 1.7× (Figs. 7A, 7B). 
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 ± SEM values (n = 30 [LBs]) from five independent experiments). **P < 0.01 versus control (medium). Scale bar: 1 mm.
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 ± SEM values (n = 30 [LBs]) from five independent experiments). **P < 0.01 versus control (medium). Scale bar: 1 mm.
In summary, our results demonstrate that our new “fried egg” differentiation method could be used for the generation of LBs that express human lens-specific genes and possess magnification capacity from UiPSCs in vitro. 
Discussion
In the present study, we developed a method for the generation of LBs from human PSCs. Our results demonstrated that LBs derived from iPSCs by the “fried egg” method exhibited structural and functional properties that were partially similar to those of the human lens. Therefore, this method forms the basis for future studies on human lens development and cataractogenesis and may even find applications in drug screening. 
Various LB formation methods have been developed during the past few decades.1721 Recently, LBs expressing a lens cell–specific fluorescent reporter were generated from cryTom mouse-derived iPSCs.24 Human ESC-/iPSC-derived LBs have also been reported by several groups.22,25,26 However, there are certain limitations with regard to the size and morphology of the LBs generated with the methods reported so far. The maximum diameter of the LBs induced in our study reached approximately 3 mm, which is, to our knowledge, the largest LBs generated in vitro. Importantly, LBs with functional optical properties were derived from human PSCs 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 for the use of LBs for further studies. LBs with the largest size and the ideal shape were usually derived from colonies in which the ratio of the diameter of compact cells and loose cells was 1:2. This indicates that the supporting microenvironment plays an important role during the differentiation process. Therefore, to our knowledge, our “fried egg” method currently seems to be the most suitable method for the generation of LBs of adequate size and optimal shape and optical properties. 
In our study, gene expression and immunostaining analysis indicated that the differentiation of UiPSCs into LBs by our method, to a certain extent, mimicked lens development in vivo. Ultrastructural examination of the UiPSC-derived LBs revealed that they contained all the cellular components present in the human lens (lens capsules, epithelial and fiber cells); thus, there were some similarities with the human lens. Autophagy and mitophagy play a role in the degradation of ocular organelles27; therefore, the expression of LC3B and the observation of degenerating organelles indicated the potential role of autophagy during organelle degradation in LBs. Besides, denucleation, as a dominant phenomenon during fiber cell differentiation,28,29 was also indicated to be involved in LB generation, as evidenced by the gradual loss of nuclei during the differentiation process. Our results are consistent with previous studies about organelles and nuclei degradation in lens development.2729 Experiments on detailed analyses of the process surrounding organelle loss and denucleation in LBs are needed in the future. 
Two recent seminal studies have reported the treatment of cataract with small compounds in both in vitro systems and animal models,1,2 which opens up a new nonsurgical approach to cataract treatment. However, the evaluation of the efficacy of these compounds in human cataract treatment is hampered by the lack of in vitro human tissue–derived cataract models. Although iPSCs have been previously generated from the lens epithelial cells of cataract patients, no proper cataract model has been established.25 Our study opens up the possibility of generating patient-specific cataract models for the evaluation of the efficacy of these compounds. 
In summary, our study presents the “fried egg” method for the derivation of LBs of approximately 3-mm diameter, the largest to our knowledge, from PSCs. The differentiation process was representative of the molecular events underlying lens development. Importantly, the generated LBs not only expressed human lens–specific genes but also exhibited optical functions. Therefore, our study lays the foundation for investigations into the molecular mechanism of lens development and cataractogenesis in humans and has potential for application in drug screening methods for cataract treatment. 
Acknowledgments
Supported by the Key Program of National Natural Science Foundation of China (81130018), the National Key Basic Research Program of the Ministry of Science and Technology of China (2015CB964901), National Natural Science Foundation of China (81371001, 81300641, 81570822, 31271594, 81670833), National Twelfth Five-Year Plan Foundation of China (2012BAI08B01), Zhejiang Key Laboratory Fund of China (2011E10006), Program of Zhejiang Medical technology (2015KYA109), the International S&T Cooperation Program of the Ministry of Science and Technology of China (2014DFG32790), and a Technology Development Project (CXZZ20130320172336579) from the Science Technology and Innovation Committee of Shenzhen Municipality. 
Disclosure: Q. Fu, None; Z. Qin, None; X. Jin, None; L. Zhang, None; Z. Chen, None; J. He, None; J. Ji, None; K. Yao, None 
References
Zhao L, Chen XJ, Zhu J, et al. Lanosterol reverses protein aggregation in cataracts. Nature. 2015; 523: 607–611.
Makley LN, McMenimen KA, DeVree BT, et al. Pharmacological chaperone for alpha-crystallin partially restores transparency in cataract models. Science. 2015; 350: 674–677.
Cvekl A, Ashery-Padan R. The cellular and molecular mechanisms of vertebrate lens development. Development. 2014; 141: 4432–4447.
Cvekl A, Duncan MK. Genetic and epigenetic mechanisms of gene regulation during lens development. Prog Retin Eye Res. 2007; 26: 555–597.
Charlton-Perkins M, Brown NL, Cook TA. The lens in focus: a comparison of lens development in Drosophila and vertebrates. Mol Genet Genomics. 2011; 286: 189–213.
Martinez G, de Iongh RU. The lens epithelium in ocular health and disease. Int J Biochem Cell Biol. 2010; 42: 1945–1963.
Ogino H, Ochi H, Reza HM, et al. Transcription factors involved in lens development from the preplacodal ectoderm. Dev Biol. 2012; 363: 333–347.
Sato T, Vries RG, Snippert HJ, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009; 459: 262–265.
Spence JR, Mayhew CN, Rankin SA, et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature. 2011; 470: 105–109.
Taguchi A, Kaku Y, Ohmori T, et al. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells. Cell Stem Cell. 2014; 14: 53–67.
Takasato M, Er PX, Becroft M, et al. Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat Cell Biol. 2014; 16: 118–126.
Xia Y, Nivet E, Sancho-Martinez I, et al. Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells. Nat Cell Biol. 2013; 15: 1507–1515.
Lancaster MA, Renner M, Martin CA, et al. Cerebral organoids model human brain development and microcephaly. Nature. 2013; 501: 373–379.
Lancaster MA, Knoblich JA. Organogenesis in a dish: modeling development and disease using organoid technologies. Science. 2014; 345: 1247125.
Eiraku M, Takata N, Ishibashi H, et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature. 2011; 472: 51–56.
Nakano T, Ando S, Takata N, et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell. 2012; 10: 771–785.
Hirano M, Yamamoto A, Yoshimura N, et al. Generation of structures formed by lens and retinal cells differentiating from embryonic stem cells. Dev Dyn. 2003; 228: 664–671.
O'Connor MD, McAvoy JW. In vitro generation of functional lens-like structures with relevance to age-related nuclear cataract. Inves Ophthalmol Vis Sci. 2007; 48: 1245–1252.
Ooto S, Haruta M, Honda Y, et al. Induction of the differentiation of lentoids from primate embryonic stem cells. Inves Ophthalmol Vis Sci. 2003; 44: 2689–2693.
Simonneau L, Crisanti P, Lorinet AM, et al. Crystallin gene expression and lentoid body formation in quail embryo neuroretina cultures transformed by the oncogenic retrovirus Mill Hill 2 or Rous sarcoma virus. Mol Cell Biol. 1986; 6: 3704–3710.
Wagner LM, Takemoto DJ. PKCalpha and PKCgamma overexpression causes lentoid body formation in the N/N 1003A rabbit lens epithelial cell line. Mol Vis. 2001; 7: 138–144.
Yang C, Yang Y, Brennan L, et al. Efficient generation of lens progenitor cells and lentoid bodies from human embryonic stem cells in chemically defined conditions. FASEB J. 2010; 24: 3274–3283.
Zhou T, Benda C, Dunzinger S, et al. Generation of human induced pluripotent stem cells from urine samples. Nat Protoc. 2012; 7: 2080–2089.
Anand T, Talluri T, Kumar D, et al. Differentiation of induced pluripotent stem cells to lentoid bodies expressing a lens cell-specific fluorescent reporter. PLoS One. 2016; 11: e0157570.
Qiu X, Yang J, Liu T, et al. Efficient generation of lens progenitor cells from cataract patient-specific induced pluripotent stem cells. PLoS One. 2012; 7: e32612.
Mengarelli I, Barberi T. Derivation of multiple cranial tissues and isolation of lens epithelium-like cells from human embryonic stem cells. Stem Cells Transl Med. 2013; 2: 94–106.
Costello M, Brennan L, Basu S, et al. Autophagy and mitophagy participant in ocular lens organelle degradation. Inves Ophthalmol Vis Sci. 2013; 116: 141–150.
Maria A, Bassnett S. DNase IIβ Distribution and activity in the mouse lens. Invest Ophthalmol Vis Sci. 2007; 48: 5638–5646.
Costello M, Brennan L, Mohamed A, et al. Identification and ultrastructural characterization of a novel nuclear degradation complex in differentiating lens fiber cells. PLoS One. 2016; 11: e0160785.
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 (100× and 200×) 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 γ-crystallin on D25 in mature LBs. Scale bars: (B) 3 mm; (C) 100 μm (40×); 50 μm (100×); 30 μm (200×); (D) NANOG, 30 μm; (D) E-cadherin and FOXE3, 50 μm; (D) γ-crystallin, 400 μm.
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 (100× and 200×) 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 γ-crystallin on D25 in mature LBs. Scale bars: (B) 3 mm; (C) 100 μm (40×); 50 μm (100×); 30 μm (200×); (D) NANOG, 30 μm; (D) E-cadherin and FOXE3, 50 μm; (D) γ-crystallin, 400 μm.
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 (100×, 200×) 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 μm; (C) 100 μm (40×), 50 μm (100×), 30 μm (200×); (D) 100 μm (40×).
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 (100×, 200×) 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 μm; (C) 100 μm (40×), 50 μm (100×), 30 μm (200×); (D) 100 μm (40×).
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 ± 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 αA- and αB-crystallin. The bar represents the mean ± SEM values from five independent experiments. (D) Immunofluorescence detection of PROX1 (D8 and D13; red) and FOXE3 (D7 and D12). (E) Immunofluorescence detection of αA- and αB-crystallin (D7, D12, and D17; red). Scale bars: (B) and (D) 50 μm; (E) D7, 100 μm; (E) D12, 200 μm; (E) D17, 400 μm.
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 ± 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 αA- and αB-crystallin. The bar represents the mean ± SEM values from five independent experiments. (D) Immunofluorescence detection of PROX1 (D8 and D13; red) and FOXE3 (D7 and D12). (E) Immunofluorescence detection of αA- and αB-crystallin (D7, D12, and D17; red). Scale bars: (B) and (D) 50 μm; (E) D7, 100 μm; (E) D12, 200 μm; (E) D17, 400 μm.
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 (αA-, αB-, β-, and γ-crystallin, and MIP) were analyzed on D0, D7, D14, and D25 of LB induction. The bar represents the mean ± SEM value from nine independent experiments. (B) Expression of αA-, αB-, β-, and γ-crystallin and MIP in mature LBs on D25. αA-, αB-, β-, and γ-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 β- and γ-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 β-crystallin (red) expression in the absence of nuclei (4′,6-diamidino-2-phenylindole [DAPI], blue). Scale bars: (B) 400 μm, (C) 50 μm, (D) 100 μm.
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 (αA-, αB-, β-, and γ-crystallin, and MIP) were analyzed on D0, D7, D14, and D25 of LB induction. The bar represents the mean ± SEM value from nine independent experiments. (B) Expression of αA-, αB-, β-, and γ-crystallin and MIP in mature LBs on D25. αA-, αB-, β-, and γ-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 β- and γ-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 β-crystallin (red) expression in the absence of nuclei (4′,6-diamidino-2-phenylindole [DAPI], blue). Scale bars: (B) 400 μm, (C) 50 μm, (D) 100 μm.
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. (BD) 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). (EL) 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 μm, (A, C, D) 50 μm, (EG) 5 μm, (H, I) 2 μm, (J, L) 1 μm, (K) 0.5 μm.
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. (BD) 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). (EL) 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 μm, (A, C, D) 50 μm, (EG) 5 μm, (H, I) 2 μm, (J, L) 1 μm, (K) 0.5 μm.
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 αA-, αB-, β-, and γ-crystallin and MIP in lens fiber cells in mature LB slices from D25. Scale bar: 50 μm.
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 αA-, αB-, β-, and γ-crystallin and MIP in lens fiber cells in mature LB slices from D25. Scale bar: 50 μm.
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 ± SEM values (n = 30 [LBs]) from five independent experiments). **P < 0.01 versus control (medium). Scale bar: 1 mm.
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 ± SEM values (n = 30 [LBs]) from five independent experiments). **P < 0.01 versus control (medium). Scale bar: 1 mm.
Supplement 1
Supplement 2
Supplement 3
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×