*Contributed equally
Induced pluripotent stem (iPS) cells are widely used as a research tool in regenerative medicine and embryology. In studies related to lens regeneration in the eye, iPS cells have been reported to differentiate into lens epithelial cells (LECs); however, to the best of our knowledge, no study to date has described their formation of three-dimensional cell aggregates. Notably,
The development of the lens in the eye begins with the PAX6-expressing epidermal ectoderm making contact with the optic vesicle. Subsequently, SOX2 is expressed in the ectoderm region in contact with the optic vesicle, and the coordinated action of PAX6 as a partner factor for SOX2 causes cells in contact with the optic vesicle to thicken and form lens placodes, which are depressed inward by the formation of the optic cup (
The human lens is 9-10 mm in diameter and is surrounded by a lens capsule, which is rich in type IV collagen. On the corneal side, the lens epithelium is composed of a single layer of lens epithelial cells (LECs). Around the equatorial region of the lens, LECs separate from the capsule and begin to elongate toward the anterior and posterior poles of the lens, where the LECs differentiate into lens fiber cells (LFCs). Eventually, the organelles inside the LFCs disappear (
The lens is composed of approximately 90% crystallin, a water-soluble protein. Crystallin in vertebrates is mainly classified into α-, β-, and γ-crystallin. α-crystallin has 2 subunits of αA- and αB-, β-crystallin has 7 subunits of βA1-βA4 and βB1-βB3, and γ-crystallin has 5 subunits of γA-γD and γS (
After the lens of a newt is removed, the pigmented epithelial cells (PECs) located on the dorsal side of the iris first start to dedifferentiate, a process during which their pigment is degranulated, and then differentiate into LECs to regenerate a new lens (
Induced pluripotent stem (iPS) cells, first described by Takahashi
The tissues examined in the study were collected from patients with glaucoma during treatment with partial iris resection, and pieces of the collected human iris tissue were fixed in SUPER FIX™ rapid fixative solution (cat. no. KY-500; Kurabo Industries Ltd.) (
Human iris tissue was processed as described (
Human iris-derived iPS (H-iris iPS) cells were prepared through cell reprogramming using H-iris cells, as described previously (
The composition of the used LEC medium (with the STEP-4 culture condition being the same;
The common differentiation culture conditions were based on reports describing the differentiation of human embryonic stem (ES) cells into lens progenitor cells and lentoid bodies (
Distinct culture environments were used in our experiments, Experiments 1-5 (Ex.1-Ex.5), as described below (summarized in
As reported by Hayashi
Total cellular RNA was extracted using a TaqMan® Gene Expression Cells-to-CT™ Kit (cat. no. A25603; Thermo Fisher Scientific), and RNA concentrations were measured using a spectrophotometer (NanoVue™; GE Healthcare) (
Cell aggregates were collected from suspension cultures and treated using the cell-block method (
Immunofluorescence staining was performed as previously described (
Each experiment was performed in triplicate and repeated at least thrice. Data are presented as means ± standard deviation (SD) and were analyzed using repeated measures analysis of variance with Tukey's post hoc test. Statistical Package for Social Science (SPSS) Statistics 24 (IBM Corporation) was used for statistical analyses.
Human iris tissue was enzymatically treated and decomposed into small pieces (
When iPS cells cultured in iPS medium (
The expression of PAX6, SOX2, and αA-crystallin proteins at each step was confirmed through immunostaining performed under identical conditions. The expression of αA-crystallin was strongest at STEP-3, but the expression was not uniform, with certain cells expressing the protein more strongly than others, and the expression tended to be stronger in aggregated cells than in non-aggregated cells (
The Ex.2 method was the same as the Ex.1 method until the end of STEP-2, but in STEP-3, starting from 7 days after initiation of the culture, the cells were cultured on a rotary culture device (
In the Ex.3 method, cell aggregates were formed starting from the STEP-1 stage, with differentiation occurring in the cell aggregates, and several days after the start of culture in STEP-3, numerous sac-like structures were observed around the cell aggregates (
In the Ex.4 method, cells were cultured using the static-suspension method to allow the formation of cell-cell junctions for differentiation. Subsequently, a step involving rotational suspension culture, STEP-4, was included for an additional 2 weeks. Expression analysis of five genes revealed that the expression levels in STEP-3 and STEP-4 were significantly higher than the changes between STEP-0 and STEP-1 and STEP-2 (
Visual examination of cell aggregates obtained at the end of culture (
H-iris iPS cells (
In this study, we cultured human iris-derived tissue cells and iPS cells using various culture methods and generated three-dimensional cell aggregates expressing αA-crystallin, a lens-specific protein.
The history of research on lens regeneration began with lens regeneration in newts, which was discovered in the 1890s (
First, we cultured H-iris iPS cells to induce their differentiation into lens epithelial progenitor cells based on a report that ES cells can differentiate into these progenitor cells (
The maturation of cartilage cells has been reported to be promoted by the application of a mechanical stimulation or load to cells, and cellular aggregates generated using rotational culture have been proposed to represent an essential component for creating artificial cartilage through tissue engineering (
Considering the aforementioned results, we next cultured cells by employing the cell aggregates from STEP-1 and subjecting them to tilt rotation and horizontal rotation (Ex.3). Translucent sac-like structures formed by two layers of cell membranes were observed. In the sac-like structures, type IV collagen was strongly expressed outside the bilayer, mimicking the type IV collagen-rich lens capsule present on the outer surface of the lens (
As the next method, STEP-4 using LEC medium was performed (Ex.4). All measured gene expression increased during STEP-3 and further increased in STEP-4. The cell aggregates grew to ~2 mm in size, with the surrounding cells, including LECs, arranged in the same manner as in the lens in certain areas, and a few vacuolated areas were present in the interior.
Lastly, in Ex.5, the areas where LECs formed in differentiated SEAM were collected and cultured. The SEAM method is a two-dimensional adhesion-culture method, and the concentric SEAMs formed by cells differentiating on their own through cell-cell interaction without changing the medium conditions mimic the development of the whole eye: the location of cells in different zones shows lineages spanning the superficial ectoderm, lens, neural retina, and retinal pigment epithelium of the eye. When cells in the areas where superficial ectoderm differentiate under the SEAM method are collected and further differentiated using the air-lift method, a three-dimensional cell sheet expressing proteins characteristic of the corneal and conjunctival epithelium is formed (
The lens nucleus is located at the center of the lens, where organelles are lost during development. A recent study on organelle degradation inside the lens reported that organelle degradation by phospholipases of the PLAAT family leads to the achievement of optimal transparency and refractive function of the lens (
In the case of cataracts, a disease that causes opacity of the lens, several artificial lenses (intraocular lenses; IOLs) have been developed through micro-incisions and are being used for clinical treatment. Regeneration of the lens
In conclusion, we have reported that H-iris cells and iris tissue-derived iPS cells can be used to generate a variety of three-dimensional cell aggregates expressing αA-crystallin, a protein specific to the lens. However, all the cell aggregates formed in this study were opaque, and we were thus unable to regenerate a transparent lens. In the future, we will investigate the degradation of organelles necessary to achieve transparency of the interior of the generated cell aggregates expressing lens-specific proteins by creating transgenic cells that induce the disappearance of organelles, and we will conduct research on the regeneration of transparent lenses. In addition, we aim to utilize this cell aggregate model for various applications, such as studying ciliary body regeneration by co-culture and creating an
The authors would like to thank Ms. Chieko Nishikawa (Fujita Health University) for help with experiments and Ms. Mari Seto (Kanazawa Medical University) for English editing and technical support.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
NH and NY designed the study. NH, NY and YK were responsible for the data collection and manuscript writing. NH, NY, YK, NN, SI and KI participated in the experiments. NH, YK, NN and SI were responsible for data acquisition and analysis. NY and NN were responsible for statistical analysis. YK and NN were responsible for literature searches. NH, NY, YK and SI reviewed and revised the manuscript. NY and KI confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
This study was approved by the Ethics Review Committee of Fujita Health University (approval no. 05-065, first approval date: 21 December 2005, followed by continued ethical approval; Aichi, Japan). The written informed consent was obtained from all subjects. The experiment was carried out with the approval of the Recombinant DNA Experiment Committee of Fujita Health University (approval no. DP16055, approval date: 17 November 2016; Aichi, Japan).
Not applicable.
The authors declare that they have no competing interests.
Schematic of cell-culture conditions in Ex. 1-5. iPS, induced pluripotent stem; LECs, lens epithelial cells; Rot·Sus, rotational suspension; Ex, experiment; 2D, 2-dimensions; 3D, 3-dimensions; SEAM, self-formed ectodermal autonomous multi-zone.
Formation of lentoid body-like cell aggregates from human iris tissue-derived cells. (A) H&E-stained specimen of human iris tissue. The pigment epithelial cell layer on the lens side is thick, and a thin pigment epithelial cell layer is also present on the corneal side. The iris parenchyma is composed of blood vessels, smooth muscle, and fibroblasts. (B) Fibroblasts and pigment epithelial cells have extended out from the enzyme-treated tissue. (C) Pigment epithelial cells proliferate while degranulating. (D) When only degranulated epithelial cells were passaged, the cells eventually became confluent with a cobblestone-like appearance. (E) Cells aggregated to form a lentoid body-like cell mass. (F) Morphology of the picked-up cell mass by micropipette. (G) DAPI staining of the cell mass in (F). (H) αA-crystallin staining of the cell mass in (G); the cytoplasm is stained here. Scale bar, 100 µm. H&E, hematoxylin and eosin.
Induction of differentiation using the Ex.1 method. (A) Colony of human iris-derived iPS cells. (B-D) Cell colony in differentiation culture: (B) 6th day in STEP-1 medium, (C) 18th day in STEP-2 medium, and (D) 25th day in STEP-3 medium. Scale bar in (A-D), 100 µm. (E) Fold-increase in
Immunostained specimens from each of the four steps in the Ex.1 method. The images show DAPI staining in the same area as that stained for PAX6, SOX2, and αA-crystallin in STEP-0, -1, -2, and -3. Scale bar, 100 µm. Ex, experiment.
Induction of differentiation using the Ex.2 method. (A) System used for rotational suspension culture. (B) Opaque cell aggregates formed during culture. Scale bar, 2 mm. (C) H&E-stained specimen of cell aggregate, showing inward extension of pericytes (arrowhead). (D) Cell aggregate immunostained for αA-crystallin; the cytoplasm is stained. (E) DAPI staining of specimen shown in (D). Scale bar in (C-E), 100 µm. (F) Illustration of the lens during development. Ex, experiment; H&E, hematoxylin and eosin.
Induction of differentiation using the Ex.3 method. (A) Cell aggregates formed by using rotational suspension culture. (B) A sac-like structure (†) is observed here around the cell aggregate. Scale bar, 2 mm. (C) The sac-like structure (†) is translucent, and thus the black crosshairs are visible through it (arrowhead). (D) H&E-stained specimen of the sac-like structure. (E) Immunostaining for αA-crystallin in the sac-like structure. (F) Immunostaining for type IV collagen in the sac-like structure. (G) H&E-stained specimen of cell aggregate. (H) Immunostaining for αA-crystallin in cell aggregate. (I) Immunostaining for type IV collagen in cell aggregate. (D-I) inset: magnified view of area indicated by the arrowhead. Scale bar in (D-I), 100 µm. Ex, experiment; H&E, hematoxylin and eosin.
Changes in gene expression levels at each step of differentiation induction using Ex.4 the method. (A)
Induction of differentiation using the Ex.4 method. (A) Gross observation of cell aggregate. Reduced opacity is observed in certain areas. Scale bar, 2 mm. (B) H&E-stained specimen of cell aggregate. Scale bar, 200 µm. (C) Enlarged view of H&E staining in Area 1 of the specimen in (B). Cells displaying epithelial-like morphology have infiltrated inside the cell aggregate (yellow arrowhead). (D-G and I-L) Immunostaining of cell aggregates for (D and I) SOX2, (E and J) p75NTR, (F and K) αA-crystallin, and (G and L) type IV collagen. Blue in immunostaining images: DAPI staining of nuclei. (H) Enlarged view of H&E staining of Area 2 in the specimen in (B), where the cells on the surface of the aggregate have transitioned from a multilayer to a monolayer (white arrowhead). Scale bar in (C-L), 50 µm. Ex, experiment; H&E, hematoxylin and eosin.
Induction of differentiation using the Ex.5 method. (A) iPS cell colony immediately before medium replacement with SEAM medium. (B) Colony after culture for 4 weeks in SEAM medium. Cell morphology can be distinguished into four layers from the center. At the boundary between the 2nd and 3rd zones, small cell aggregates were observed (arrowhead). (C) αA-crystallin immunostaining of a small cell mass indicated by arrowhead in (B). (D) H&E staining of cell aggregate after static and rotational suspension culture. (E-G) Immunostaining of cell aggregate for (E) αA-crystallin, (F) βB2-crystallin, and (G) type IV collagen. Blue: DAPI staining of nuclei. Scale bar, 100 µm. Ex, experiment; iPS cells, induced pluripotent stem cells; SEAM, self-formed ectodermal autonomous multi-zone; H&E, hematoxylin and eosin.