Lens regeneration using endogenous stem cells with gain of visual function

Abstract

The repair and regeneration of tissues using endogenous stem cells represents an ultimate goal in regenerative medicine. To our knowledge, human lens regeneration has not yet been demonstrated. Currently, the only treatment for cataracts, the leading cause of blindness worldwide, is to extract the cataractous lens and implant an artificial intraocular lens. However, this procedure poses notable risks of complications. Here we isolate lens epithelial stem/progenitor cells (LECs) in mammals and show that Pax6 and Bmi1 are required for LEC renewal. We design a surgical method of cataract removal that preserves endogenous LECs and achieves functional lens regeneration in rabbits and macaques, as well as in human infants with cataracts. Our method differs conceptually from current practice, as it preserves endogenous LECs and their natural environment maximally, and regenerates lenses with visual function. Our approach demonstrates a novel treatment strategy for cataracts and provides a new paradigm for tissue regeneration using endogenous stem cells.

Conflict of interest statement

The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper.

Figures

Extended Data Figure 1. Surgical methods and lens regeneration for congenital cataract

a, b, Slit-lamp photography of ‘doughnut-like’ lens regeneration at different time points after treatment using the current surgical method. Two years after surgery (a), the transparent regenerated lens tissue contained the sealed capsular opening with an opaque white scar at the centre. The regions between the dashed circles indicated by the red arrows are the regenerated lens tissues. Four years after surgery (b), the capsular opening was constricted compared to that seen at two years post-surgery, indicating continued growth of the regenerated lens. There was also the complication of iridolenticular synechiae. c, Schematic diagrams of the current surgical method for paediatric cataracts: the currently practiced paediatric ACCC creates an opening 6 mm in diameter at the centre of the anterior capsule, removing the LECs underneath it and leaving a relatively large wound area of 28 mm2. The scars formed often cause postoperative VAO. Additionally, PCCC and anterior vitrectomy are commonly performed at follow-up visits.

Extended Data Figure 2. BrdU pulse labelling of human LECs

a, Whole mount of a human lens capsule showing BrdU+ cells (brown) by enzymatic immunohistology and diaminobenzidine staining. b, High-magnification images of human donor lenses showing BrdU+ LECs. c, Bar graph showing quantification of BrdU+ cells. There was an age-dependent decrease in the number of BrdU+ cells (8 months, 39.9 ± 8.1; 30 years, 20.3 ± 7.3 and 40 years, 5.9 ± 2.9; 8 months versus 40 years, *P < 0.05). Six randomly chosen fields of each capsule were used for analysis, four samples in each group, (n = 24 fields, chosen over four samples). d, High-magnification images of whole-mount staining of human lens capsules with or without injury showed a marked increase in the number of BrdU+ cells after injury. e, Bar graph showing quantification of BrdU+ cells. The contralateral eyes from the respective donors were used as controls. There was a significant increase in number of BrdU+ cells. No injury, 1.5 ± 1.2; after injury, 18.4 ± 4.2; fold change after injury, 11.3 ± 2.5; *P < 0.05. Six randomly chosen fields within the germinative zone of each capsule were used for analysis, five samples in each group (n = 30 fields, chosen over five samples). Data shown as means ± s.d. f, Cultured human fetal LECs were positive for BMI-1 (green, right upper panel); co-staining of PAX6 (red) and Ki67 (green), middle panels; co-staining of SOX2 (red) and Ki67 (green), lower panels. g, Co-staining of PAX6 (red) and SOX2 (green) of human fetal LECs. All scale bars, 100 μm.

Extended Data Figure 3. Conditional deletion of Bmi-1 led to decrease in Pax6+ and Sox2+ cells and cataract formation

A, Loss of Bmi-1 reduced the Pax6+ and Sox2+ LECs population. a, Representative images of haematoxylin and eosin-stained lens sections from Bmi1fl/fl control mice and Nestin-cre;Bmi1fl/fl mice. b, Representative images of Bmi-1 (red) staining in LECs. c, Pax6 (red) and Sox2 (green) immunostaining. d, Percentage of Pax6+(Bmi1fl/fl, 88.5 ± 2.9%; Nestin-cre;Bmi1fl/fl, 2.4 ± 2.3%) and Sox2+(Bmi1fl/fl, 82.7 ± 3.9%; Nestin-cre;Bmi1fl/fl, 4.9 ± 1.5%) cells (n = 5 mice; 5 sections counted per mice, for a total of 25 sections across 5 mice), *P < 0.001. Data are shown as mean ± s.d. B, Conditional deletion of Bmi1 led to reduced LEC proliferation. The percentage of BrdU+ LECs per eye is shown (2M: Bmi1fl/fl, 2.6 ± 0.9%; Nestin-cre;Bmi1fl/fl, 3.0 ± 0.4%; n = 4 mice. 7M: Bmi1fl/fl,, 1.5 ± 0.2%; Nestin-cre;Bmi1fl/fl,, 0.6 ± 0.4%; n = 6 mice. 12M: Bmi1fl/fl, 1.8 ± 0.6%; Nestin-cre;Bmi1fl/fl, 0.2 ± 0.2%; n = 8 mice), two sections counted per eye. Statistical significance was assessed using a two-tailed Student’s t-test. *P < 0.05. Data are shown as mean ± s.d. C, Nestin (green) staining is shown in E13.5, E18.5, and 2-month-old wild-type mice. All scale bars, 100 μm. D, Representative images of lenses from Nestin-cre;Bmi1fl/fl and Bmi1fl/fl control mice. a, Cataracts are evident in 7- and 12-month-old Nestin-cre;Bmi1fl/fl mice (arrow). b, Deletion of Bmi-1 at 6 weeks of age with Nestn-creER did not recapitulate the cataract phenotype 10 months after tamoxifen treatment. Haematoxylin and eosin-stained sections of the same eyes are also shown. All scale bars, 100 μm.

Extended Data Figure 4. Loss of BMI-1 decreased the proliferative ability of LECs

a, Phase-contrast photographs of human LECs (upper panels) and quantification of Ki67+ proliferating human fetal LECs upon BMI1 knockdown (shBMI1) compared to controls (two shRNAs gave similar results; n = 5, P < 0.05). Data shown as mean ± s.d. Blue indicates DAPI staining. b, Loss of BMI-1 did not significantly affect expression of LEC or lens fibre cell makers in LECs. BMI1 was reduced by 3.3-fold (all n = 3, P < 0.05); gene expression changes of LEC markers were: 1.3-fold increase (PAX6), 1.1-fold increase (SOX2), 1.3-fold increase (C-MAF) and 1.1-fold increase (E-cadherin); gene expression changes of differentiated lens fibre cell markers were: 1.6-fold increase (Filensin), 0.9 fold increase (CP49) and 1.4-fold decrease (CRYBA2). Two different shRNAs gave similar results; n = 5, P < 0.05. Data shown as mean ± s.d.

Extended Data Figure 5. Higher expression levels of Bmi1, Sox2 and Ki67 in Pax6+ LECs

a, Pax6-GFP+ LECs were observed at the germinative zone. Left panel, a section of lens of a Pax6P0-3.9-GFPcre mouse at P1. Middle and right panels, higher magnification of the framed area in the left panel. Blue indicates DAPI staining. b, Upper panel, bright-field photograph showing flat-mount preparation of a lens capsule of a Pax6P0-3.9-GFPcre mouse at 6 months; lens capsule materials between two red circles were dissected to enrich Pax6-GFP+ LECs. Lower panel, fluorescence image of GFP+ LECs from the framed area in the upper panel. AC, anterior capsule; PC, posterior capsule. c, Comparison of gene expression levels in Pax6-GFP+ LECs versus GFP− LECs in anterior lens capsule in 6-month-old mice, increased expression of the following genes were observed: 10.1-fold in Pax6 (P < 0.005), 8.2-fold in Ki67 (P < 0.05), 4.3-fold in Bmi1 (P < 0.05), and 2.6-fold in Sox2 (P < 0.05), all n = 5. Data shown as mean ± s.d.

Extended Data Figure 6. Lens regeneration surgery in rabbits

a, A 3.2-mm keratome was used to make a limbus tunnel incision at the 11-12 o’clock position into the anterior chamber. b, The capsular opening was created by a capsulorhexis needle. c, A 1-2 mm diameter anterior capsulotomy was performed using the anterior continuous curvilinear capsulorhexis (ACCC) technique near the capsular opening area (yellow arrow). d, A blunt needle was used to inject balanced salt solution for hydrodissection of the cortex from the anterior capsule. e, The cortex was removed using a phacoemulsification device. f, The remaining cortex was removed using irrigation and aspiration. h, An elbow I/A handle was used to clear the equatorial cortex. i, j, The limbus wound was sutured with an interrupted 10-0 nylon suture. The wound was found to be watertight.

Extended Data Figure 7. Lens regeneration in rabbits

a, Haematoxylin and eosin staining of regenerated lenses at different time points after surgery. At postoperative day 1, a monolayer of LECs between the anterior and posterior capsules was visible (arrowheads). At postoperative day 4, LECs proliferated and covered the posterior capsule. At postoperative day 7, LECs in the posterior capsule began to elongate and differentiate. b, At postoperative day 28, LECs in the posterior capsule further elongated, forming primary lens fibres. c, Transparency and shape of regenerated lenses in rabbits. Upper panel, slit-lamp photography of a regenerated lens at different time points after surgery. Lower panel, schematic diagram of slit-lamp photographs in the upper panel. At day 1 after surgery, the capsular opening was clearly seen in the peripheral anterior capsule, and the area of LEC loss during surgery is indicated. At 7 weeks after surgery, loss of LECs led to adhesion between the anterior and the posterior capsule and inhibition of lens regeneration in this area.

Extended Data Figure 8. Human lens regeneration

a, A clinical trial consort flowchart. b, Comparison of visual acuity mean response profiles in two groups. A non-parallel pattern of mean responses between two groups was observed largely due to the vision loss at 3 months before laser surgery in the control group (left panel), whereas a parallel pattern of mean responses between two groups was observed using time points including 3 months after laser surgery (right panel); n = 25 control, n = 12 experimental. Data are shown as mean ± s.d. c, Lens thickness increased after surgery. Pentacam showed that 3 months after surgery, the regenerating lens tissue grew from the periphery of the capsular bag to the centre. The sealed capsular bag was only partially filled, appearing spindle-shaped on cross-sectional scan. The fundus was clearly visible on ophthalmoscopy. Arrowheads indicate the regenerated lens structure. d, Six months after surgery, the capsular bag was filled with regenerated lens tissue and appeared biconvex on cross-sectional scan by Pentacam. The anterior-posterior capsular adhesion disappeared. The fundus could be seen clearly using an ophthalmoscope with an 18-dioptre lens. e, Minimally invasive capsulorhexis preserved LECs for lens regeneration in human infants. Top panel, slit-lamp exam demonstrating human infant’s eye visual axis transparency 6 months after minimally invasive surgery compared to baseline (before cataract surgery). Bottom panel, retro-illumination demonstrating the reduced size of the capsulorhexis (white arrowheads).

Figure 1. Lineage tracing of Pax6+ LECs in mice

a, Pax6-directed GFP was expressed in mouse LEC nuclei at post-natal days P1, P14 and P30; a sagittal section of a P0-3.9-GFPcre mouse lens is shown. Blue and green represent DAPI and GFP, respectively. b, Lineage tracing of Pax6+ LECs in ROSAmTmG; P0-3.9-GFPcre mice at P1, P14 and P30 reveals that lens fibre cells express membrane GFP fluorescence; hence, PAX6+ LECs were able to generate lens fibre cells. c, As an additional control, the ROSAmTmG allele alone exhibits Tomato (red) staining at sites of non-recombination. All scale bars, 100 μm.

Figure 2. Characterization and differentiation of rabbit LECs

a, LECs were positive for PAX6 (green) and SOX2 (red). b, Lentoid formation (green arrows) with positive αA-crystallin and β-crystallin staining on day 15 of LEC differentiation. c, Left panel, phase-contrast photograph of a lentoid body on day 30; middle panel, a lentoid body demonstrating magnifying properties; right panels, photograph from western blot analysis and quantification showing a dramatic increase in expression relative to pre-differentiation expression of mature lens fibre markers αA-crystallin (2.6, 3.1, 2.2), β-crystallin (11.51, 9.0, 10.2) and γ-crystallin (2.2, 2.0, 2.8) (numbers in parentheses represent fold change after differentiation). n = 3 biological replicates. All scale bars, 100 μm.

Figure 3. Lens regeneration in rabbits

a, New minimally invasive surgical method. The capsulorhexis size was decreased to 1.0—1.5 mm in diameter, resulting in a reduced wound area of 1.2 mm2, and moved to the periphery of the lens. b, Slit-lamp microscopy showing the progress of lens regeneration after minimally invasive surgery in a rabbit eye. c, Fundus examination of rabbit eyes 7 weeks post-surgery demonstrated a clearly visible retina. Normal healthy lens shown for comparison. d, Measurements of refractive dioptres in rabbit eyes at different time points post-surgery (M, month; D, dioptres). Refractive dioptres of the eyes increased with time after surgery, demonstrating the functionality of the regenerated lenses (ANOVA, *P < 0.01). The refractive power immediately after surgery was defined as zero, 1 month = 0.0 dioptre, 3 months = 11.0 ± 0.8 dioptres and 5 months = 15.8 ± 2.2 dioptres, n = 6 at each time point, data shown as means ± s.d. e, f, Ki67 staining in the germinative zone of normal rabbit lens (e) and regenerated rabbit lens 7 weeks post-surgery (f). Lower panels show higher magnification. g, PAX6 (red) and BrdU (green) staining at the germinative zone of regenerated rabbit lens 7 weeks post-surgery. Scale bars, 100 μm.

Figure 4. Lens regeneration in macaque models after minimally invasive surgery

a, Slit-lamp microscopy showed regenerating lens tissue grew from the peripheral to the central lens in a circular symmetrical pattern 2-3 months after surgery, reaching the centre at 5 months post-surgery. Five months after surgery, direct illumination showed that the visual axis remained translucent. b, Pentacam cross-sectional scanning showed formation of a biconvex structure 5 months after surgery (yellow arrowheads). Direct illumination and fundus photography showed that the visual axis remained transparent and the retina was clearly visible (n = 6).

Figure 5. Functional characteristics of regenerated human lenses

a, Lens thickness increased significantly 6 and 8 months after surgery (1.9 ± 0.3 and 3.7 ± 0.3 mm, respectively, *P < 0.01), n = 24. b, Lens refractive power increased significantly 6 and 8 months after surgery (5.1 ± 0.5 and 19.0 ± 0.6 dioptres, respectively, *P < 0.01). n = 24. c, Visual acuity improved after surgery. logMAR, logarithm of the minimum angle of resolution. Pairwise analysis was performed to compare visual acuity before and after surgery (P < 0.05) OD (oculus dexter, right eye) n = 25; OS (oculus sinister, left eye) n = 12, OU (oculus uterque, both eyes) n = 12. d, Accommodative power increased significantly from 1 week (control OD, control OS, OD and OS, all 0.1 ± 0.1 dioptres) to 8 months (control OD and control OS, 0.2 ± 0.1 dioptres; OD and OS, 2.5 ± 0.2 dioptres) postoperatively (*P < 0.001). Control OD, n = 25; control OS, n = 12; OD, n = 12; OS, n = 12. e, Visual acuity was measured preoperatively and at 1 week, 3 months, and 6 months postoperatively. The majority of eyes in the control group underwent additional laser capsulotomy at 3 months after surgery, with visual acuity measured before and after the procedure. There was no significant difference in visual acuity between eyes that received minimally invasive surgery (n = 24) and those that received the current surgical technique (n = 50), except at 3 months before the control group underwent laser capsulotomy (t-test, ***P < 0.001). Data are shown as mean ± s.d. f, Visual axis transparency was achieved in nearly all cataractous infant eyes after minimally invasive surgery (95.8%). The scar tissue of the wound on the anterior capsule was <1.5 mm in diameter and located in the periphery, away from the visual axis. The scars were not visible unless the pupils were dilated. No disorganized tissue regeneration was observed. Compared with the current standard surgical method, the new surgical technique decreased VAO by >20-fold.