Methods for transdifferentiation of in vitro purified limbal stem cells into lens spherules

Abstract

The present application provides a method for in vitro purified corneal limbal stem cells to be transdifferentiated into lens spherules, specifically, the present application inoculates corneal limbal stem cells onto a culture plate containing a Matrigel glue layer, and cultures the corneal limbal stem cells in a culture system containing Y-27632 additives in LSCM culture solution, so that lens spherule organoids can be obtained. The method of the present application can realize the rapid transdifferentiation of corneal limbal stem cells into lens spherules, so that they can be implanted into the capsular bag as seed cells for in situ regeneration of the lens.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. Specifically, this invention relates to a method for transdifferentiating in vitro purified limbal stem cells into lens bodies. Background Technology

[0002] Cataracts are the leading cause of blindness worldwide, accounting for approximately 51% of all blindness cases. Currently, surgery is the only effective treatment for this disease, but its clinical application still has significant limitations, including potential complications such as postoperative endophthalmitis, secondary cataracts, and intraocular lens dislocation. Therefore, developing new treatment strategies has become an important research direction in this field.

[0003] With the development of regenerative medicine, lens regeneration is considered a promising new treatment approach that may restore vision, preserve accommodative function, and reduce postoperative risks. Current research focuses on two main directions: one is in vitro regeneration based on embryonic stem cells or induced pluripotent stem cells, which can form lens bodies in vitro, serving as a model for studying lens development and disease mechanisms. However, its clinical translation is limited due to the inability to simulate the in vivo microenvironment and the difficulty in intraocular transplantation; the other is in situ lens regeneration, which involves re-forming the lens within the capsule by proliferating and differentiating residual equatorial epithelial progenitor cells while preserving the lens capsule. This approach has been validated in various mammalian models and cases of congenital cataracts in infants, demonstrating considerable clinical potential.

[0004] However, existing in-situ regeneration techniques still have significant shortcomings: the regenerated lens differs from the normal lens in size, morphology, and transparency; the regeneration cycle is long; and the efficiency is low. More critically, this strategy is highly dependent on the activity and quantity of the patient's own lens epithelial progenitor cells, and the proliferative capacity of this cell population declines significantly with age, making this approach unsuitable for the vast majority of adult and elderly cataract patients, severely limiting its clinical applicability. Therefore, how to overcome age-related cell activity limitations and establish an efficient, stable, and widely applicable lens regeneration method remains a pressing technical challenge in this field. Previous studies have shown that in-situ lens regeneration within the capsular bag can be achieved by implanting iPSCs / ESCs to induce differentiation of seed cells. This suggests that effective seed cells are key to improving this situation. However, their growth rate is slow, and iPSC / ESC-derived cells still carry the risk of tumorigenesis.

[0005] In amphibians, corneal tissue can transdifferentiate after lens damage, suggesting that corneal cells have the potential to transdifferentiate into the lens and may serve as seed cells for lens regeneration. However, this transdifferentiation of corneal cells into the lens has not yet been observed in mammals. Limbal stem cells, as adult stem cells in the corneal tissue, possess the function of replenishing corneal epithelial cells and maintaining corneal homeostasis through proliferation and differentiation. Currently, no research suggests that limbal stem cells have the potential to transdifferentiate into the lens.

[0006] Therefore, there is an urgent need in this field to develop a method for transdifferentiating limbal stem cells purified in vitro into lens bodies. This method can enable the rapid transdifferentiation of limbal stem cells into lens bodies, allowing them to be implanted into the capsular bag as seed cells for in situ lens regeneration. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of the aforementioned background technology by providing a method for transdifferentiating limbal stem cells purified in vitro into lens bodies. This method enables the rapid transdifferentiation of limbal stem cells into lens bodies, thereby allowing them to be implanted into the capsular bag as seed cells for in situ lens regeneration.

[0008] The technical solution provided by this invention is:

[0009] A method for preparing lens bodies by transdifferentiation of purified limbal stem cells (LSCs) from vitro includes the following steps:

[0010] (1) Limbal tissue separation

[0011] After euthanizing the rats, the eyeballs were removed and immersed in limbal stem cell medium (LSCM). The eyeballs were rinsed twice with PBS containing gentamicin and amphotericin B. The limbal tissue was then mechanically separated under a stereomicroscope.

[0012] (2) Digestion of limbal epithelial tissue

[0013] After treating the limbal tissue with 10 mg / ml Dispase II at 37°C for 20 minutes, the limbal epithelium was peeled off under a stereomicroscope and transferred to trypsin. The limbal epithelium was then treated at 37°C for 15 minutes, followed by the addition of an equal volume of LSCM to neutralize the trypsin. The tissue was then centrifuged at 1000 rpm at room temperature for 5 minutes.

[0014] (3) Culture of limbal epithelial cells

[0015] Remove the supernatant, resuspend the cell pellet in LSCM containing 10 μM Y-27632, and seed the cells in 2% Matrigel-coated culture dishes. Incubate overnight at 37°C; this is passage P1. The next day, replace the medium with LSCM without Y-27632, and change the medium daily thereafter.

[0016] (4) LSC purification

[0017] When the cell density reaches 80-90%, digest the cells with trypsin and then passage 1 / 3 of the cells. The seeding method is as described in step (3). This is the P2 generation.

[0018] Change the medium once a day for P2 cells and culture them for 4 weeks without passage. After 4 weeks, digest the cells with trypsin and seed them at 500 cells / well in 6-well plates coated with 2% Matrigel, as described in step (3). This is the P3 generation.

[0019] When the cell density reaches 80-90%, digest the cells with trypsin and seed them in 96-well plates at 2 cells / well, as described in step (3). This is the P4 generation. Observe the 96-well plates daily for cell clone formation. When the cell density reaches 80-90%, pick the wells containing a single cell clone for digestion. This will allow for the stable expansion and passage of LSCs.

[0020] (5) In vitro induction of LSC transdifferentiation into lens bodies

[0021] Add 50% Matrigel diluted with DMEM / F12 to each well of a 96-well plate and incubate at 37°C for 30 minutes to form a gel. After digesting the cells with trypsin, seed 5000 LSCs per well onto the Matrigel gel in the 96-well plate. The culture medium is LSCM containing Y-27632.

[0022] The next day, the culture medium was replaced with LSCM without Y-27632. The culture medium was changed once a day thereafter. Fully morphologically purified LSC-derived lens bodies could be seen 5 days after inoculation.

[0023] In a first aspect of the present invention, a method for differentiating limbal stem cells into lens organoids is provided, the method comprising the steps of:

[0024] (S1) Provide a limbal stem cell, seed the limbal stem cell onto a culture plate containing Matrigel gel, culture the limbal stem cell in a culture system containing Y-27632 additive, after 1-2 days, preferably after 1 day, replace with culture medium without Y-27632 additive and continue culturing, and change the culture medium daily thereafter, i.e., culture medium without Y-27632 additive, to obtain lens small body organoids.

[0025] In another preferred embodiment, the culture plate includes a 96-well plate, a 24-well plate, a 12-well plate, and a 6-well plate.

[0026] In another preferred embodiment, the culture medium is selected from the group consisting of LSCM.

[0027] In another preferred embodiment, the concentration (volume ratio) of Matrigel in the Matrigel layer is 40-60%, preferably 50%.

[0028] In another preferred embodiment, the Matrigel layer is formed by dissolving Matrigel in DMEM / F12 and gelling it in a cell culture incubator.

[0029] In another preferred embodiment, the concentration of Y-27632 in the culture system is 8-15 μM, preferably 10 μM.

[0030] In another preferred embodiment, the limbal stem cells are limbal stem cells that have been enzymatically digested.

[0031] In another preferred embodiment, the enzyme includes pancreatin and EDTA.

[0032] In another preferred embodiment, the seeding density of limbal stem cells is 1,000-8,000 cells / well, more preferably 4,000-6,000 cells / well, and most preferably 5,000 cells / well.

[0033] In another preferred embodiment, the limbal stem cells are prepared using the following method:

[0034] (a) Provide a detached limbal tissue, remove the limbal epithelium, and obtain limbal epithelial cells by enzymatic digestion and centrifugation;

[0035] (b) Resuspend limbal epithelial cells in LSCM culture medium containing Y-27632 additive, seed the cells in Matrigel-coated culture dishes, and culture for 1-2 days. Preferably, after 1 day, replace the culture medium with LSCM culture medium without Y-27632 additive. Change the culture medium daily to obtain P1 generation limbal epithelial cells.

[0036] (c) The limbal epithelial cells cultured in step (b) are purified to obtain limbal stem cells.

[0037] In another preferred embodiment, step (c) includes:

[0038] (c1) When the cell density reaches 80-90%, after washing, enzyme digestion, centrifugation, and resuspending in LSCM culture medium containing Y-27632 additive, 1 / 3 of the cells are seeded in Matrigel-coated culture dishes and cultured for 1-2 days. Preferably, after 1 day, the culture medium is replaced with LSCM culture medium without Y-27632 additive. The culture medium is replaced daily with LSCM culture medium without Y-27632 additive to obtain P2 generation limbal epithelial cells.

[0039] (c2) Change the medium (LSCM culture medium without Y-27632 additive) of P2 generation limbal epithelial cells once a day for 3-5 weeks, preferably 4 weeks, without passage. Then digest with enzymes, resuspend in LSCM culture medium containing Y-27632 additive, and seed the cells in Matrigel-coated culture plates (preferably 6-well plates) and culture for 1-2 days, preferably 1 day. Then change the medium to LSCM culture medium without Y-27632 additive and culture daily to obtain P3 generation limbal epithelial cells.

[0040] (c3) When the cell density reaches 80-90%, after enzymatic digestion, the cells are resuspended (using LSCM culture medium containing Y-27632 additive). The cells are seeded in Matrigel-coated culture plates (preferably 96-well plates) and cultured for 1-2 days, preferably 1 day, then replaced with LSCM culture medium without Y-27632 additive. The culture medium is changed daily to obtain P4 generation limbal epithelial cells. When the cell density reaches 80-90%, the resulting cell clones are digested, resuspended in LSCM culture medium containing Y-27632 additive, and transferred to a new culture plate (preferably a 24-well plate). The medium is changed daily (LSCM culture medium without Y-27632 additive). When the cell density reaches 80-90%, the cells are digested and all are transferred to a new culture plate (preferably a 6-well plate) to obtain purified limbal stem cells.

[0041] In another preferred embodiment, step (c3) further includes the expansion and passage of limbal stem cells.

[0042] In another preferred embodiment, in steps (b) and (c1)-(c3), the concentration (volume ratio) of Matrigel is 0.8-10%, preferably 1-3%.

[0043] In another preferred embodiment, in step (c2), the cell seeding amount is 100-1000 cells / well, more preferably 400-600 cells / well, and most preferably 500 cells / well.

[0044] In another preferred embodiment, in step (c3), 100-300, preferably 200, cells are resuspended.

[0045] In another preferred embodiment, the concentration of Y-27632 in the LSCM culture medium containing the Y-27632 additive is 2-50 μM, preferably 8-15 μM.

[0046] In another preferred embodiment, the enzyme includes pancreatin and EDTA.

[0047] In another preferred embodiment, in step (S1), the inoculated organism is cultured for 4-6 days, preferably 5 days, to obtain a lens organoid.

[0048] In another preferred embodiment, the LSCM culture medium comprises 80-95% DMEM / F12, 8-15% fetal bovine serum, 0.1-1 μg / mL hydrocortisone, 2-8 μg / mL insulin, 2-8 μg / mL transferrin, 2-8 ng / mL sodium selenite, 20-30 μg / mL adenine, 5-10 ng / mL cholera toxin B subunit, 8-15 ng / mL epidermal growth factor, 1-5 nM 3,3′,5′-triiodothyronine, 40-60 μg / mL gentamicin, and 1-3 μg / mL amphotericin B.

[0049] In another preferred embodiment, the LSCM culture medium comprises 90% DMEM / F12, 10% fetal bovine serum, 0.4 μg / ml hydrocortisone, 5 μg / mL insulin, 5 μg / mL transferrin, 5 ng / mL sodium selenite, 25 μg / mL adenine, 8.4 ng / ml cholera toxin B subunit, 10 ng / ml epidermal growth factor, 2 nM 3,3′,5′-triiodothyronine, 50 μg / mL gentamicin, and 1.25 μg / mL amphotericin B.

[0050] In another preferred embodiment, the method is an in vitro method.

[0051] In another preferred embodiment, the method is for non-diagnostic and non-therapeutic purposes.

[0052] A second aspect of the present invention provides a lens organoid, which is obtained by means of the method described in the first aspect of the present invention.

[0053] The third aspect of this invention provides a use of the lens organoid described in the second aspect of this invention for research on the mechanism of limbal stem cell transdifferentiation into the lens, the mechanism of lens embryonic development, the pathogenesis of cataracts, and the screening of cataract-related drugs.

[0054] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0055] Figure 1 This is a morphological image of LSC purified in vitro under a microscope.

[0056] Figure 2 This is a schematic diagram of the preparation method of LSC purified in vitro.

[0057] Figure 3 Immunofluorescence staining images show the expression results of K10, K12, K13, and K15 in LSCs purified in vitro.

[0058] Figure 4 Immunofluorescence staining results of PAX6 and P63 expression in LSC purified in vitro.

[0059] Figure 5 This is a morphological image of LSCs purified in vitro that have been induced to transdifferentiate into lens bodies under a microscope.

[0060] Figure 6 This is a photographic image of a lens corpuscle.

[0061] Figure 7 This is a microscopic image of cell clusters without the removal of Y-27632 during the induction of transdifferentiation of LSCs purified in vitro.

[0062] Figure 8 Immunofluorescence staining results of expression of β-crystallin, γ-crystallin, and E-cadherin in LSC-derived lens bodies purified in vitro. Detailed Implementation

[0063] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that limbal stem cells can be seeded onto culture plates containing Matrigel gel and cultured in LSCM medium containing Y-27632 additive to obtain lens body organoids. The method of this invention enables rapid transdifferentiation of limbal stem cells into lens bodies, allowing them to be implanted into the capsular bag as seed cells for in situ lens regeneration. This invention was completed based on this discovery.

[0064] the term

[0065] Limbal stem cells

[0066] Limbal stem cells are adult stem cells located in the basal layer of epithelial cells at the limbus (the junction of the cornea and sclera). They possess the potential for proliferation and differentiation. Through continuous proliferation and differentiation, they generate new corneal epithelial cells, thus achieving the daily renewal, homeostasis maintenance, and post-injury repair functions of corneal epithelial cells.

[0067] Lens organoids

[0068] Lens organoids are miniature, simplified models of the human eye's lens that are cultivated in vitro using stem cell technology, mimicking its morphology, structure, development, and some physiological functions.

[0069] Basic culture medium

[0070] Basic cell culture medium is the fundamental nutrient medium used for cell culture, and it usually needs to be selected and replenished according to different cell types and experimental requirements. LSCM culture medium is a culture medium used for the expansion culture of limbal stem cells, containing 90% DMEM / F12, 10% fetal bovine serum, 0.4 μg / ml hydrocortisone, 5 μg / mL insulin, 5 μg / mL transferrin, 5 ng / mL sodium selenite, 25 μg / mL adenine, 8.4 ng / ml cholera toxin B subunit, 10 ng / ml epidermal growth factor, 2 nM 3,3′,5′-triiodothyronine, 50 μg / mL gentamicin, and 1.25 μg / mL amphotericin B.

[0071] In this invention, the LSCM culture medium preparation process is as follows:

[0072] Dissolve 4 mg of hydrocortisone powder in 1 ml of dimethyl sulfoxide to prepare hydrocortisone stock solution; dissolve insulin-transferrin-sodium selenite supplement (containing 25 mg insulin, 25 mg transferrin, and 25 μg sodium selenite) in 5 ml of double-distilled water to prepare insulin-transferrin-sodium selenite supplement stock solution; dissolve 25 mg of adenine powder in 100 μl of dimethyl sulfoxide to prepare adenine stock solution; dissolve 10 μg of cholera toxin B subunit in 100 μl of double-distilled water (containing 0.1% bovine serum albumin) to prepare cholera toxin B subunit stock solution; dissolve 10 μg of epidermal growth factor in 100 μl of double-distilled water (containing 0.1% bovine serum albumin) to prepare epidermal growth factor stock solution; dissolve 5 mg of... 3,3′,5′-Triiodothyronine powder was dissolved in 3,71 Methyl sulfoxide to prepare 3,3′,5′-Triiodothyronine stock solution. Gentamicin stock solution with a concentration of 15 mg / ml and amphotericin B stock solution with a concentration of 5 mg / ml were purchased.

[0073] Take 45 ml of DMEM / F12 medium, add 5 ml of fetal bovine serum, 5 μl of hydrocortisone stock solution, 50 μl of insulin-transferrin-sodium selenite supplement stock solution, 5 μl of adenine stock solution, 4.2 μl of cholera toxin B subunit stock solution, 5 μl of epidermal growth factor stock solution, 5 μl of 3,3′,5′-triiodothyronine stock solution, 166.6 μl of gentamicin stock solution, and 12.5 μl of amphotericin B stock solution. Mix thoroughly by inverting the container to obtain the LSCM culture medium.

[0074] Y-27632

[0075] Y-27632 is a small molecule inhibitor of Rho-associated protein kinase p160ROCK.

[0076] The structural formula of Y-27632 (CAS. NO: 146986-50-7) is shown below:

[0077] .

[0078] Culture system for differentiating limbal stem cells into lens organoids

[0079] As used in this article, "culture system for differentiating limbal stem cells into lens organoids" and "differentiation culture system for lens organoids" are interchangeable and both refer to the differentiation culture system for differentiating limbal stem cells into lens organoids.

[0080] This invention provides a differentiation and culture system for lens corpuscle organoids, comprising a basal culture medium and additives; wherein the basal culture medium is selected from the following group: LSCM, and the additives include: Y-27632. The lens corpuscle organoids obtained by differentiation and culture with the above-mentioned specially added components have a similar morphological structure to human lens corpuscles.

[0081] The preferred concentration of Y-27632 in the culture system is as described above, and the most preferred concentration is the Y-27632 concentration in the embodiments of the present invention.

[0082] Using the culture system and culture method of this invention, the lens organoids differentiated and cultured by this invention have similar morphology and structure to human lens organs, and can be used for research on the mechanism of limbal stem cell transdifferentiation into lens, the mechanism of lens embryonic development, the pathogenesis of cataracts, and the screening of cataract-related drugs.

[0083] Differentiation and culture methods for limbal stem cells to differentiate into lens organoids

[0084] This invention also provides a method for differentiating limbal stem cells into lens organoids, the method comprising the following steps:

[0085] A limbal stem cell is provided and seeded onto a culture plate containing a Matrigel layer. The limbal stem cell is cultured in a culture medium containing Y-27632 additive for 1-2 days, preferably 1 day. Then, the culture medium is replaced with a medium without Y-27632 additive and cultured for another day. The culture medium is then replaced daily thereafter, i.e., a culture medium without Y-27632 additive, to obtain a lens organoid.

[0086] The main advantages of this invention include:

[0087] (1) This invention is the first to discover that by seeding limbal stem cells onto a culture plate containing a Matrigel layer and culturing the limbal stem cells in an LSCM culture medium containing Y-27632 additive, lens body organoids can be obtained. The method of this invention enables the rapid transdifferentiation of limbal stem cells into lens bodies, thereby allowing them to be implanted into the capsular bag as seed cells for in situ lens regeneration.

[0088] (2) This invention provides a method for transdifferentiating limbal stem cells purified in vitro into lens bodies. This method can achieve rapid transdifferentiation of limbal stem cells into lens bodies, so that they can be implanted into the capsule as seed cells for in situ regeneration of the lens.

[0089] (3) During embryonic development, both LSCs and lens epithelial cells (i.e., seed cells) originate from the ectoderm on the surface of the eye. Using LSCs as adult stem cells to transdifferentiate into lens bodies is significantly faster than inducing differentiation from iPSCs / ESCs. Therefore, this method can be used for lens regeneration model research and suggests that LSCs can serve as seed cells for in situ lens regeneration, greatly reducing the risk of tumor formation.

[0090] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0091] Unless otherwise stated, all reagents and materials used in the embodiments of this invention are commercially available products.

[0092] Example 1: In vitro culture and purification of LSC

[0093] (1) Limbal tissue separation

[0094] After euthanizing rats (purchased from Shanghai Slack Laboratory Animal Co., Ltd.) with excessive anesthesia, the eyeballs were removed and immersed in limbal stem cell medium (LSCM) (DMEM / F12 purchased from Gibco, fetal bovine serum, hydrocortisone, cholera toxin B subunit, and epidermal growth factor purchased from MCE, insulin-transferrin-sodium selenite, adenine, and triiodothyronine purchased from Sigma Aldrich, and gentamicin and amphotericin B purchased from Sangon Biotech (Shanghai) Co., Ltd.). In a biosafety cabinet dedicated to primary cells, the eyeballs were rinsed twice with PBS containing gentamicin and amphotericin B, and limbal tissue was mechanically separated under a stereomicroscope.

[0095] (2) Digestion of limbal epithelial tissue

[0096] The removed limbal tissue was placed in 500 μL of 10 mg / ml Dispase II and treated at 37°C for 20 minutes. The limbal epithelium was then peeled off under a stereomicroscope and transferred to 500 μL of trypsin. The limbal epithelium was then treated at 37°C for 15 minutes. An equal volume of LSCM was then added to neutralize the trypsin. The cells were repeatedly pipetted and centrifuged at 1000 rpm for 5 minutes at room temperature.

[0097] (3) Culture of limbal epithelial cells

[0098] Remove the supernatant and resuspend the cell pellet in 5 ml of LSCM containing 10 μM Y-27632. Coat culture dishes with 2% Matrigel beforehand, and seed the cells into the dishes. Incubate overnight at 37°C in a 5% CO2 cell culture incubator; this is the P1 generation. The next day, replace the medium with LSCM without Y-27632, and change the medium daily thereafter.

[0099] (4) LSC purification

[0100] When the cell density reaches 80-90%, wash the cells once with PBS, add trypsin to digest the cells for 5 minutes, then add an equal volume of LSCM to neutralize the trypsin, centrifuge at 1000 rpm at room temperature for 5 minutes, remove the supernatant, and resuspend the cell pellet in 3 ml of LSCM containing 10 μM Y-27632. Coat culture dishes with 2% Matrigel beforehand, and seed 1 / 3 of the cells into the dishes. Incubate overnight at 37°C in a 5% CO2 cell culture incubator. The next day, replace the medium with LSCM without Y-27632. Change the culture medium daily thereafter; this is generation P2.

[0101] P2 cells were cultured daily with the medium changed (LSCM without 10 μM Y-27632) for 4 weeks without passage. After 4 weeks, cells were digested with trypsin, followed by neutralization with an equal volume of LSCM. The cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was removed, and the cell pellet was resuspended in 3 ml of LSCM containing 10 μM Y-27632. Cell counting was performed, and cells were seeded at 500 cells / well in culture dishes pre-coated with 2% Matrigel. The cells were incubated overnight at 37°C with 5% CO2. The medium was changed the next day with LSCM without Y-27632. The culture medium was changed daily thereafter. This was P3 generation.

[0102] When the cell density reaches 80-90%, digest the cells with trypsin and count them. Resuspend 200 cells (containing 10 μM Y-27632 LSCM) and seed them in all wells of a 96-well plate coated with 2% Matrigel. Incubate overnight at 37°C in a 5% CO2 cell culture incubator. The next day, replace the medium with LSCM without Y-27632. Change the culture medium once a day thereafter. This is the P4 generation.

[0103] Daily observation was conducted to check for cell clone formation in 96-well plates. Wells containing single-cell clones were continuously monitored. When the cell density reached 80-90%, the cells were digested, resuspended in LSCM medium containing 10 μM Y-27632, and transferred to 24-well plates. The medium was changed daily (without 10 μM Y-27632 LSCM). When the cell density reached 80-90%, the cells were digested again and transferred to 6-well plates. Subsequent expansion and cryopreservation were performed to obtain limbal stem cells purified from P4 generation limbal epithelial cells.

[0104] The results showed that cell morphology was as follows Figure 1 As shown in the diagram. In the P1 generation, there was still a considerable amount of mixed cell morphology; in the P2 generation, cell morphology became slightly more uniform; in the P3 generation, cell morphology was relatively uniform; and in the P4 generation, cell morphology was uniform, achieving purification. The purification flowchart is shown below. Figure 2 As shown.

[0105] Example 2: Identification of LSC-related markers by immunofluorescence staining

[0106] The purified LSCs were seeded onto 24-well plates. Once the cell density reached 30-50%, the culture medium was removed, and the cells were fixed with 4% paraformaldehyde for 10 minutes. The cells were then washed three times with PBS, 10 minutes each time. 0.3% Triton-100 (dissolved in PBS) was added to the cells for 15 minutes, followed by three more washes with PBS, 10 minutes each time. 10% goat serum (diluted with PBS) was added to the cells for 1 hour. The supernatant was then removed, and primary antibodies (K10 Rabbit mAb from Abcam, K12 mouse mAb from Santa Cruz, K13 Rabbit mAb from Abclonal, K15 Rabbit mAb from Proteintech, P63 mouse mAb from Abcam, PAX6 Rabbit mAb from Abclonal) (diluted with primary antibody dilution buffer) were added and incubated overnight (or more than 18 hours) at 4°C. The next day, the supernatant was removed, and the cells were washed three times with PBS, 10 minutes each time. Secondary antibodies (Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 555 Conjugate), Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate), all purchased from Cell Signaling Technology) (diluted with PBS) were added, and the cells were incubated at room temperature in the dark for 1 hour. The supernatant was then removed, and the cells were washed three times with PBS, 10 minutes each time. The cells were mounted using a DAPI-containing anti-fluorescence quenching mounting medium, and the staining was observed under an upright fluorescence microscope.

[0107] The results show that, Figure 3 As shown, the purified LSCs contained a very small number of K10+ (a marker of superficial limbal epithelial cells), but no K12 (corneal epithelial cell marker) or K13 (conjunctival epithelial cell marker) expression was observed. K15 (an LSC marker) was highly, extensively, and uniformly expressed. Figure 4 As shown, the purified LSCs highly co-expressed P63 and PAX6 (LSC markers). This indicates that the extracted LSCs possessed high purity.

[0108] Example 3: In vitro induction of LSC transdifferentiation into lens bodies

[0109] Add 50 μL of 50% (v / v) Matrigel diluted with DMEM / F12 to each well of a 96-well plate and incubate at 37°C for 30 minutes to form a gel. After digesting the cells with trypsin, seed 5000 LSCs per well onto the Matrigel gel in the 96-well plate. The culture medium is 100 μL of LSCM containing Y-27632 (10 μM).

[0110] The next day, the culture medium was replaced with LSCM without Y-27632. The culture medium was changed once a day thereafter, and the samples were observed and photographed using a white light microscope.

[0111] The results show that, Figure 5 As shown, the cells remained in clumps one day after inoculation. From the second to the fourth day, the clumps spread out to form a monolayer of cells, and a transparent tissue-like structure was observed in the center of the cells. On the fifth day after inoculation, well-defined, transparent, and fully shaped lens bodies were observed (indicated by arrows).

[0112] The obtained lens corpuscles exhibit a transparent 3D structure, such as Figure 6 As shown, it is a transparent circular or oval protrusion with a diameter of about 1 mm.

[0113] Example 4: Identification of lens cell-related markers in LSC transdifferentiation by immunofluorescence staining

[0114] On day 5 post-inoculation, after the appearance of lens bodies, the culture medium was removed, and the cells were fixed with 4% paraformaldehyde for 10 minutes. The cells were then gently washed three times with PBS for 10 minutes each time. 0.3% Triton-100 (dissolved in PBS) was added to the cells for 15 minutes, followed by three gentle washes with PBS for 10 minutes each time. 10% goat serum (diluted with PBS) was added to the cells for 1 hour, then the supernatant was removed, and primary antibodies (β-crystallin Mouse mAb and γ-crystallin Mouse mAb were purchased from Santa Cruz, and E-cadherin Rabbit mAb was purchased from Cell Signaling Technology) (diluted with primary antibody dilution buffer) were added and incubated overnight (at least 18 hours) at 4°C. The next day, the supernatant was removed, and the cells were gently washed three times with PBS for 10 minutes each time. Secondary antibodies (Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 555 Conjugate), Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate, all purchased from Cell Signaling Technology)) were added (diluted with PBS) and incubated at room temperature in the dark for 1 hour. The supernatant was then removed, and the cells were gently washed three times with PBS for 10 minutes each time. DAPI dilution buffer (diluted with PBS) was added for nuclear staining, and the cells were incubated at room temperature in the dark for 10 minutes. The supernatant was then removed, and the cells were gently washed three times with PBS for 10 minutes each time. The staining was observed under an upright fluorescence microscope.

[0115] The results show that, Figure 8 As shown, lens bodies derived from LSC transdifferentiation express lens-specific structural proteins, including β-crystallin and γ-crystallin. Expression of E-cadherin suggests the presence of lens epithelial cells. The structure is similar to that of a normal human lens.

[0116] Comparative Example

[0117] The method is the same as in Example 3, except that: 50 μL of 50% Matrigel diluted with DMEM / F12 was added to each well of a 96-well plate, and the plate was placed in a 37°C cell culture incubator for 30 minutes to form a gel. After digesting the cells with trypsin, 5000 LSCs were seeded per well onto the Matrigel layer of the 96-well plate. The culture medium was 100 μL of LSCM containing 10 μM Y-27632. The culture medium (LSCM containing 10 μM Y-27632) was changed once daily thereafter, and the cells were observed and photographed daily using a white light microscope.

[0118] The results show that, Figure 7 As shown, the cells remained in a clump for 1-5 days after inoculation, increasing in size daily, but unable to spread out and differentiate into lens bodies.

[0119] It should be understood that after reading the above teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for differentiating limbal stem cells into lens organoids in vitro, characterized in that, The method includes the following steps: A limbal stem cell (LSCM) was provided and seeded onto a culture plate containing a Matrigel layer. The LSCM was cultured in a medium containing Y-27632 additive for 1-2 days, then replaced with a medium without Y-27632 additive. The medium was then changed daily thereafter to obtain a lens organoid. The culture medium was selected from the following group: LSCM, containing 80-95% DMEM / F12, 8-15% fetal bovine serum, 0.1-1 μg / ml hydrocortisone, 2-8 μg / mL insulin, 2-8 μg / mL transferrin, 2-8 ng / mL sodium selenite, 20-30 μg / mL adenine, 5-10 ng / ml cholera toxin B subunit, 8-15 ng / ml epidermal growth factor, and 1-5 nM... 3,3′,5′-Triiodothyronine, 40-60 μg / mL Gentamicin, 1-3 μg / mL Amphotericin B.

2. The differentiation culture method as described in claim 1, characterized in that, The Matrigel layer contains 40-60% Matrigel.

3. The differentiation culture method as described in claim 1, characterized in that, In the culture system, the concentration of Y-27632 was 8-15 μM.

4. The differentiation culture method as described in claim 1, characterized in that, The seeding density of limbal stem cells is 1000-8000 cells / well.

5. The differentiation culture method as described in claim 4, characterized in that, The seeding density of limbal stem cells is 4000-6000 cells / well.

6. The differentiation culture method as described in claim 5, characterized in that, The seeding density of limbal stem cells was 5000 cells / well.

7. The differentiation culture method as described in claim 1, characterized in that, The limbal stem cells were prepared using the following method: (a) Provide a detached limbal tissue, remove the limbal epithelium, and obtain limbal epithelial cells by enzymatic digestion and centrifugation; (b) Resuspend limbal epithelial cells in LSCM culture medium containing Y-27632 additive, seed the cells in Matrigel-coated culture dishes, and culture for 1-2 days. Then replace the culture medium with LSCM culture medium without Y-27632 additive and culture daily to obtain P1 generation limbal epithelial cells. (c) The limbal epithelial cells cultured in step (b) are purified to obtain limbal stem cells.

8. The differentiation culture method as described in claim 7, characterized in that, Step (c) includes: (c1) When the cell density reaches 80-90%, after washing, enzyme digestion, centrifugation, and resuspending in LSCM culture medium containing Y-27632 additive, 1 / 3 of the cells are seeded in Matrigel-coated culture dishes and cultured for 1-2 days. Then, the culture medium is replaced with LSCM culture medium without Y-27632 additive. The culture medium without Y-27632 additive is replaced daily to obtain P2 generation limbal epithelial cells. (c2) P2 generation limbal epithelial cells were cultured in LSCM medium without Y-27632 additive once a day for 3-5 weeks without passage. After digestion with enzymes, the cells were resuspended in LSCM medium containing Y-27632 additive and seeded in Matrigel-coated culture plates. After 1-2 days of culture, the medium was replaced with LSCM medium without Y-27632 additive. The culture medium was changed daily to obtain P3 generation limbal epithelial cells. (c3) When the cell density reaches 80-90%, after enzymatic digestion, the cells are resuspended in LSCM culture medium containing Y-27632 additive, and the cells are seeded in Matrigel-coated culture plates. After culturing for 1-2 days, the culture medium is replaced with LSCM culture medium without Y-27632 additive. The culture medium is changed daily to obtain P4 generation limbal epithelial cells. When the cell density reaches 80-90%, the resulting cell clones are digested, resuspended in LSCM culture medium containing Y-27632 additive, and transferred to a new culture plate. The LSCM culture medium without Y-27632 additive is changed daily. When the cell density reaches 80-90%, the cells are digested and all are transferred to a new culture plate to obtain purified limbal stem cells.

9. The differentiation culture method as described in claim 1, characterized in that, The LSCM culture medium contained 90% DMEM / F12, 10% fetal bovine serum, 0.4 μg / ml hydrocortisone, 5 μg / mL insulin, 5 μg / mL transferrin, 5 ng / mL sodium selenite, 25 μg / mL adenine, 8.4 ng / ml cholera toxin B subunit, 10 ng / ml epidermal growth factor, 2 nM 3,3′,5′-triiodothyronine, 50 μg / mL gentamicin, and 1.25 μg / mL amphotericin B.

10. The differentiation culture method as described in claim 1, characterized in that, In step (S1), after inoculation, the lens organoids are cultured for 4-6 days to obtain small lens bodies.

Images