CN111733132A

CN111733132A - Culture method for inducing directional differentiation of human embryonic stem cells into corneal epithelial cells and application

Info

Publication number: CN111733132A
Application number: CN202010599044.6A
Authority: CN
Inventors: 李炜; 贺佳; 欧尚坤; 刘祖国; 何昕; 孙慧敏
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2020-06-28
Filing date: 2020-06-28
Publication date: 2020-10-02
Anticipated expiration: 2040-06-28
Also published as: CN111733132B

Abstract

The invention provides a culture method for inducing the directional differentiation of human embryonic stem cells into corneal epithelial cells, which comprises the following steps: s1, recovering human embryonic stem cells, culturing the recovered human embryonic stem cells in an E8 culture medium for 4-6 days, inoculating the recovered human embryonic stem cells in a low adsorption culture dish, and performing differentiation culture on the recovered human embryonic stem cells by using an NP culture medium for 6-10 days to obtain NP cells; s2, digesting the NP cells obtained in the step S1, inoculating the digested NP cells into an attached culture dish, and continuously differentiating for 4-7 days by using an epithelial differentiation medium; s3, digesting the cells cultured in the step S2, adding the mouse fibroblast cell line 3T 3cells or inoculating the cells on the surface of de-epithelial amnion, and performing mixed culture on the cells by using an epithelial differentiation culture medium for 7 to 14 days to obtain corneal epithelial cells.

Description

Culture method for inducing directional differentiation of human embryonic stem cells into corneal epithelial cells and application

Technical Field

The invention relates to a culture method for inducing the directional differentiation of human embryonic stem cells into corneal epithelial cells and application thereof, belonging to the technical field of cell culture.

Background

Corneal epithelial stem cells (Corneal epithelial stem cell CESCs), also known as Limbal stem cells (Corneal Limbal stem cells CLSCs) or Limbal progenitor cells (Corneal Limbal progenitor cells CLPCs), are the only source of constant replacement of Corneal epithelial cells, serve to maintain the dynamic flattening of the Corneal epithelium and maintain Corneal transparency, and also serve as "barriers" that prevent conjunctival epithelial cells from migrating to the Corneal surface.

Limbal Stem Cell Deficiency (LSCD). Is a disease in which corneal epithelial cells cannot be regenerated due to loss or abnormality of corneal limbal stem cells, and the corneal epithelium cannot maintain the number of cells that normally function. The lack of limbal stem cells easily causes continuous corneal epithelial defect, chronic inflammation, corneal epithelial epithelialization, invasion of new blood vessels and the like, further causes corneal opacity and transparency reduction, causes serious visual disturbance to patients, and leads to vision loss of the patients in serious cases.

The treatment of limbal stem cell deficiency will vary depending on the extent of limbal stem cell loss. Treatment for a mild partial limbal stem cell deficiency will need to be determined based on the individual condition of the patient. When the ocular surface epithelium of the patient is still intact, the vision is good, and no other symptoms exist, conservative treatment can be adopted, and local medication lubrication such as autologous serum is given to the eye, so that the function of preventing corneal epithelial erosion is achieved. When a patient has partial corneal-limbal-conjunctival epithelial defects, conjunctival epithelium invading the limbus can be scraped off by simple surgery, followed to observe the healing of the cornea and conjunctiva and the reconstruction of the corneal limbus as a 'barrier'. When the patient has partial corneal coverage with conjunctival epithelium or persistent corneal epithelial defects, surgical intervention is required. In patients with severe/total limbal stem cell deficiencies, limbal transplant surgery is required to enable corneal function. The goal of these procedures is to repair the original damaged corneal epithelium by transplanting new limbal stem cells after removing the diseased corneal epithelium or pannus from the patient.

However, these techniques are more or less faced with insurmountable problems such as recurrent/persistent epithelial defects, neovascular invasion, corneal conjunctival changes, corneal thinning/melting/perforation, rejection, bacterial infections, donor deficiencies, or transmission of some underlying disease from the donor. It is expected that other safe and reliable substitutes can be found, such as 'seed' cells like human embryonic stem cells (hESC) and the like to induce differentiation into corneal limbal epithelial stem cells, and further become reliable cell sources for differentiation into corneal epithelial cells.

However, there is no established method for inducing the directional differentiation of human embryonic stem cells into corneal epithelial cells in the prior art.

Disclosure of Invention

The invention provides a culture method for inducing the directional differentiation of human embryonic stem cells into corneal epithelial cells and application thereof, which can effectively solve the problems.

The invention is realized by the following steps:

a culture method for inducing the directional differentiation of human embryonic stem cells into corneal epithelial cells comprises the following steps:

s1, recovering human embryonic stem cells, culturing the recovered human embryonic stem cells in an E8 culture medium for 4-6 days, inoculating the recovered human embryonic stem cells in a low adsorption culture dish, and performing differentiation culture on the recovered human embryonic stem cells by using an NP culture medium for 6-10 days to obtain NP cells;

s2, digesting the NP cells obtained in the step S1, inoculating the digested NP cells into an attached culture dish, and continuously differentiating for 4-7 days by using an epithelial differentiation medium;

s3, digesting the cells cultured in the step S2, adding the mouse fibroblast cell line 3T 3cells or inoculating the cells on the surface of de-epithelial amnion, and performing mixed culture on the cells by using an epithelial differentiation culture medium for 7 to 14 days to obtain corneal epithelial cells.

As a further improvement, the NP culture medium is prepared by adding 18-22 wt% of serum substitute KSR, 0.5-1.5 wt% of sodium pyruvate, 0.5-1.5 wt% of nonessential amino acids, 0.5-1.5 wt% of glutamine, 8-12nM ROCK inhibitor Y27632 and 0.5-1.5 v/v% of double-antibody P/S into a basal culture medium DMEM/F12.

As a further improvement, the epithelial differentiation medium is prepared by mixing the SHEM medium and the D-KSFM medium according to the volume ratio of 1: 1.5-2.5, and then adding 0.5-1.5 wt% of sodium pyruvate, 0.5-1.5 wt% of nonessential amino acid, 0.5-1.5 wt% of glutamine and 8-12nM ROCK inhibitor Y27632; the preparation method of the SHEM culture medium is characterized in that 4-6 v/v% serum FBS, 0.4-0.6mg/ml hydrocortition, 4-6ml/L ITS, 0.4-0.6 v/v% DMSO, 8-12ng/ml human epithelial cell growth factor EGF and 0.5-1.5 v/v% double-antibody P/S are added into a basal medium DMEM/F12.

As a further improvement, the step S1 specifically includes the following steps:

s11, culturing in E8 culture medium for 4-6 days after human embryonic stem cell is recovered, discarding culture medium when cell is in mass of size of one-horn coin under 10 × microscope, adding CTS^TMTrypLE^TMSelect Enzyme was digested at 37 ℃ for 2.5-3.5 min;

s12, observing the cell mass edge to be loose and bright under a microscope, abandoning the digestive juice, and adding the E8 culture medium; the whole culture dish is scribed to lead the cells to be in a block shape and fall off to form cell block suspension;

s13, centrifuging the cell mass suspension for 4-6 minutes at the rotating speed of 80-120 r/min; discard the supernatant, suspend it evenly with NP medium, put it in 5% CO₂And culturing in an incubator at 37 ℃ and changing the culture solution every day.

As a further improvement, the step S2 specifically includes the following steps:

s21, centrifuging the NP cells obtained in the step S1 at the speed of 700-900 rpm for 8-12 minutes, discarding the supernatant, and adding CTS^TMTrypLE^TMDigesting the Select Enzyme at 37 ℃ for 4-6 minutes, stopping digestion by using NP medium, and slightly blowing to obtain a single cell suspension;

s22, centrifuging at 1400-1600 rpm for 8-12 min, discarding the supernatant, adding epithelial differentiation medium, and resuspending at (2.5-3.5) × 10⁴/cm²The inoculated density of (A) was inoculated into a coated attached culture dish and placed in 5% CO₂Culturing in 37 deg.C incubator for 4-7 days, and changing culture medium every day.

As a further improvement, the treatment of the mouse fibroblast cell line 3T 3cells comprises the steps of:

s31, when the mouse fibroblast line 3T 3cells grow to 70-80% of fusion degree, discarding the supernatant, adding a SHEM culture medium containing 8-12 mu g/mL mitomycin, incubating at 37 ℃ for 2.5-3 hours, discarding the supernatant, replacing the fresh SHEM culture medium, and putting into a 5% CO2 and 37 ℃ incubator for standby use every other day;

s32, taking the 3T 3cells obtained in the step S31, abandoning the supernatant of the culture medium, adding 0.20-0.30 wt% of Trypsin of EDTA to digest at 37 ℃ for 2.5-3.5 minutes, adding a differentiation culture medium to stop digesting when the cells are round under a microscope, slightly blowing and beating the cells to form single cell suspension, centrifuging at the rotating speed of 1400-1600 rpm for 4-6 minutes, abandoning the supernatant, and adjusting the cells to (4.5-5.5) multiplied by 105 cells/ml by using the differentiation culture medium for standby;

s33, adding the 3T 3cell suspension obtained in the S32 and the same amount of the digested suspension of the cells obtained in the step S2 into the mixture, uniformly mixing, placing the mixture into a 5% CO2 and 37 ℃ incubator for culturing for 7-14 days, and changing the culture solution every other day.

As a further improvement, the step S2 of cell digestion includes the following steps: taking the cells obtained in the step S2, removing the supernatant of the culture medium, adding 0.20-0.30 wt% of EDTA Trypsin for digesting for 2.5-3.5 minutes at 37 ℃, and adding an epithelial differentiation culture medium to stop the digestion when the cells are completely round and part of the cells are in a suspension state under a microscope; lightly blowing and beating the mixture into a single cell suspension, centrifuging the suspension for 4 to 6 minutes at the rotating speed of 1400-1600 revolutions per minute, discarding the supernatant, and adjusting the cells to (7.5 to 8.5) multiplied by 103cells/ml by using a differentiation culture medium for later use;

as a further improvement, the inoculation on the surface of the de-epithelialized amniotic membrane comprises the following steps:

s41, separating the amnion from the placenta under aseptic condition, rinsing with HBSS to remove bloodstain, tearing off the residual chorion on the amnion to obtain transparent amnion, and cleaning with HBSS to completely remove residual bloodstain;

s42, placing the amnion under a dissecting microscope, separating the epithelial surface and the stroma surface of the amnion, and tightly attaching the epithelial surface of the amnion to the inner ring of the sterilized amnion fixing ring; adding DispaseII into the amniotic ring to digest epithelial cells, repeatedly cleaning with DPBS to remove epithelial cells and sponge debris, adding a differentiation culture medium, and placing in a 37 ℃ culture box overnight;

s43, taking the differentiated cells obtained in the step S2, digesting the cells to form a cell suspension, inoculating the cell suspension to the inner ring of the amniotic membrane ring, and inoculating (2.5-3.5) × 10 to each amniotic membrane ring⁴Adding differentiation medium into individual cells, and culturing at 37 deg.C with 5% CO₂Culturing in an incubator for 7-14 days, and changing the culture solution every day.

A corneal epithelial cell and a corneal epithelial sheet prepared by the method.

An epithelial differentiation medium is prepared by mixing a SHEM medium and a D-KSFM medium according to the volume ratio of 1: 1.5-2.5, adding 0.5-1.5 wt% of sodium pyruvate, 0.5-1.5 wt% of nonessential amino acid, 0.5-1.5 wt% of glutamine and 8-12nM ROCK inhibitor Y27632; the preparation method of the SHEM culture medium comprises the steps of adding 4-6 v/v% serum FBS, 0.4-0.6mg/ml hydrocortition, 4-6ml/L ITS, 0.4-0.6 v/v% DMSO, 8-12ng/ml human epithelial cell growth factor EGF and 0.5-1.5 v/v% double-antibody P/S into a basic culture medium DMEM/F12.

The invention has the beneficial effects that:

the method successfully induces and differentiates the human embryonic stem cells into the corneal epithelial cells, and can be used for repairing the original damaged corneal epithelium.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.

FIG. 1 is a diagram showing the morphological changes of the differentiated embryonic stem cells provided in example 1 of the present invention. A: human embryonic stem cells, B: NP cell sphere, C: differentiation of NP cells towards corneal epithelial cells day 1D: NP cells were differentiated in the direction of corneal epithelial cells on day 7.

FIG. 2 is a graph showing the expression changes of Pax6 and P63 in NP cells differentiated at different times, which are provided in example 1 of the present invention.

FIG. 3 is a graph showing the expression of markers for embryonic stem cells and NP cells from cells at different differentiation stages, which are provided in example 1 of the present invention.

FIG. 4 is a graph of immunofluorescence mapping of cell phenotypes at different differentiation stages as provided in example 1 of the present invention.

FIG. 5 is a qPCR assay identification of cell phenotypes at different differentiation stages as provided in example 1 of the present invention.

FIG. 6 is an immunofluorescence mapping of differentiation stage cell phenotype provided in example 2 of the present invention.

FIG. 7 is a morphological observation chart of an hES 2-derived AM + ES-CE epithelial sheet provided in example 3 of the present invention. The sections of AM + ES-CE epithelial cells cultured in the amniotic membrane ring for 7 days were observed under an A.10 Xmicroscope for H & E staining.

FIG. 8 is a gene expression profile of different stages of differentiated cells or different vectors detected by Western Blot as provided in example 3 of the present invention.

FIG. 9 is a graphical representation of the phenotypic characterization of tissue engineered corneal epithelium as provided in example 3 of the present invention.

FIG. 10 is a graph showing the measurement of the cell dryness of the hES 2-derived AM + ES-CE epithelial sheet according to example 3 of the present invention.

FIG. 11 is a chart of the clone phenotype of hES 2-derived AM + ES-CE epithelial sheets provided in example 3 of the present invention. The scale length in the figure is 20 μm.

Fig. 12 is a detection map of neoplasia provided in example 3 of the present invention. A. Post-operative AM + ES-CE group, b. post-operative AM group (negative control), c. post-operative AM + hES2 group (positive control), subcutaneous material was taken from AM + ES-CE group after d.8 weeks, subcutaneous material was taken from empty amnion AM group (negative control) after E.8 weeks, subcutaneous material was taken from AM + hES2 group (positive control) after F.8 weeks.

FIG. 13 is a graph showing the results of HE staining of teratoma sections of the positive control group provided in example 3 of the present invention. A. Ectoderm (visceral epithelium-like), b. mesoderm (fibroblasts), c. endoderm (glandular ducts), d. endoderm (mucous glands).

FIG. 14 is a graph of cell clustering during differentiation as provided in example 3 of the present invention.

FIG. 15 is a diagram of an animal model of limbal stem cell deficiency provided in example 4 of the present invention. A. Limbal stem cell deficient animals were slit lamp photographs at D0 days post-surgery. B. Limbal stem cell deficient animals were photographed with fluorescein sodium staining at D0 days post-surgery. C. Limbal stem cell deficient animals were slit lamp photographs 8 weeks post-surgery. D. Limbal stem cells lack photographs of sodium fluorescein staining at 8 weeks post-surgery in animals.

FIG. 16 is a slit-lamp photograph of hES 2-derived AM + ES-CE epithelial sheets and an AM control group of cells provided in example 4 of the present invention after transplantation of a model lacking limbal stem cells. A1-8. slit-lamp photographs 1-8 weeks after AM + ES-CE epithelial sheet assembly. Photographs of sodium fluorescein staining 1-8 weeks post-operation of the AM + ES-CE epithelial sheet set B1-8. C1-8.AM group pictures of slit lamps 1-8 weeks after surgery. D1-8 photograph of sodium fluorescein staining 1-8 weeks after AM epithelial sheet set surgery. N is more than or equal to 3.

FIG. 17 is H & E staining of two sets of rabbit corneal cryosections after transplantation, shown at 20 μm in length, as provided in example 4 of the present invention.

FIG. 18 shows the cellular phenotype of two groups of rabbit central corneal epithelial cells observed by immunofluorescence staining as provided in example 4 of the present invention at 8 weeks post-transplantation. The scale length in the figure is 20 μm.

FIG. 19 is a graph of a control experiment provided in example 5 of the present invention without the addition of sodium pyruvate.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the embodiments of the present invention will be described in detail and completely with reference to the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

The culture medium was prepared as follows:

configuration of SHEM:

serum FBS (5 v/v%), Hydrocortision (0.5mg/ml), ITS (5ml/L), DMSO (0.5 v/v%), human epithelial cell growth factor EGF (10ng/ml), double anti-P/S (1 v/v%), and storage at 4 ℃ were added to a basal medium DMEM/F12.

Preparation of NP Medium:

serum replacement KSR (20 v/v%), sodium pyruvate (1 wt%), nonessential amino acids NEAA (1 wt%), glutamine Glutamin (1 wt%), ROCK inhibitor Y27632(10nM) and P/S (1 v/v%) were added to basal medium DMEM/F12 and stored at 4 ℃.

Preparation of epithelial differentiation medium:

SHEM medium was mixed with D-KSFM medium (Invitrogen, Carlsbad, USA) at a volume ratio of 1: 2, sodium pyruvate (1 wt%), the nonessential amino acid NEAA (1 wt%), glutamine Glutamine (1 wt%) and ROCK inhibitor Y27632(10nM) were added and freshly prepared at the time of use.

The antibodies used are shown in the table below.

Name of antibody	Manufacturer of the product	Goods number	Concentration of antibody used
				P63	Abcam	ab124762	1:300
K14	Abcam	ab181595	1:300
				K14	Abcam	ab7800	1:300
K3	Abcam	ab68260	1:300
				Pax6	Abcam	ab195045	1:300
Pax6	Abcam	ab735	1:300
				E-Cadherin	Abcam	ab78286	1:300
Oct3/4	Santacruz	sc5279	1:50
				Sox2	Abcam	ab92494	1:300
Pi67	Abcam	A11055	1:300
				Pan-ck	Abcam	ab80826	1:300
P75	Abcam	ab52987	1:300

Example 1 differentiation of embryonic Stem cells into corneal epithelial progenitor cells

Handling of culture dishes

Thawing 1ml of Vitronect (Gibco, Carlsbad, USA) in a refrigerator at 4 deg.C, and packaging into 200 μ l EP tubes at 25 μ l/tube and-20 deg.C; taking a piece of Vitronectin (25 mu l), melting at 4 ℃, adding into 3ml of embryonic stem cell culture medium E8, and mixing uniformly to prepare a coating solution; adding 1ml of coating solution into a 35mm culture plate, shaking up gently to cover the coating solution on the bottom of the whole culture dish, placing the culture dish into an incubator at 37 ℃, standing for 30 minutes, taking out and placing the culture dish on a super clean bench for later use; when in use, the coating liquid is discarded. (Note: the dishes need fresh coating, calculated time before each use.)

Resuscitation of human embryonic stem cells

10ml of embryonic stem cell culture medium E8(Gibco, Carlsbad, USA) was added into a 15ml centrifuge tube, and the tube was placed in a 37 ℃ incubator and re-warmed to 37 ℃; taking out the frozen cells (the human embryonic stem cells used in the embodiment are international universal human embryonic stem cell line H1) from a liquid nitrogen tank, immediately putting the cells into a water bath kettle at 37 ℃, and slightly shaking the cells to thaw the cells into cell suspension as soon as possible; remove 1ml of media from 15ml centrifuge tube and slowly add gently to the cell cryopreservation tube (add one drop of media)Then gently shaking and mixing uniformly, then adding the next drop), shaking and mixing uniformly; adding the diluted cell frozen suspension into a culture medium in a 15ml culture tube gently, mixing uniformly, placing in a centrifuge, centrifuging for 5 minutes at 1000 rpm, and discarding the supernatant; adding 2ml of rewarming culture medium to resuspend the cells, adding the cells into a culture dish from which the coating solution is removed, gently shaking and uniformly mixing to ensure that the cell suspension is uniformly spread in the culture dish, and adding 5% CO₂Culturing in an incubator at 37 ℃ and changing the culture solution every day.

Differentiation of embryonic Stem cells into NP cells (NP cells, i.e., neural Stem cells)

When the hES2 cells were grown for 5 days and the cells were in 1-cornered coin-sized clumps under a 10 Xmicroscope, the media was discarded and Ca-free media was used²⁺，Mg²⁺The DPBS is washed twice; adding 1ml of digestive juice CTS^TMTrypLE^TMSelecting Enzyme (Gibco, Carlsbad, USA), gently shaking and uniformly spreading the whole culture dish, placing the culture dish in an incubator at 37 ℃, taking out the culture dish after 3 minutes, and observing slight looseness and brightness of the edges of the clone blocks under a microscope; discarding the digestive juice, and adding 1.5ml of E8 culture medium; drawing two circles of a 5ml pipette along the periphery of a culture dish to enable cells close to the edge of the culture dish to fall off, and then carrying out Z-shaped scribing on the whole culture dish to enable the cells to be packed into blocks and fall off to form cell block suspension; transferring the cell mass suspension into a 15ml centrifuge tube, rotating at 100 rpm, and centrifuging for 5 minutes; discard the supernatant, re-suspend 6ml NP medium, mix well, divide into 2 low adsorption culture dishes (3 ml cell mass suspension per dish) of 60mm on average, place in 5% CO₂Culturing in an incubator at 37 ℃ and changing the culture solution every day.

Differentiation of NP cells toward corneal epithelial progenitors

After 8 days of NP cell differentiation, placing the culture medium containing NP cell balls in a 15ml centrifuge tube, centrifuging for 10 minutes at 800 rpm, placing the cell balls at the bottom of the centrifuge tube in a loose manner, and removing the supernatant; after the DPBS is resuspended, the mixture is centrifuged for 10 minutes at 800 rpm, and the supernatant is discarded; adding 1ml of digestive juice CTS^TMTrypLE^TMResuspending the Select Enzyme, placing the suspension in an incubator at 37 ℃ for 5 minutes, and gently blowing the suspension to obtain a single cell suspension; digestion was stopped by adding 4ml NP medium and 1500 rpmCentrifuging for 10 min, discarding supernatant, adding epithelial differentiation medium, and resuspending as 3 × 10⁴/cm²The inoculation density of (2) was inoculated into coated dishes, the solution was changed every day, and samples were collected on day 7 and day 14 for phenotypic assay.

Preparation of trophoblast cells

When the mouse fibroblast line 3T 3cells grow to 70-80% confluence, i.e. in logarithmic growth phase, discarding the supernatant, adding SHEM culture medium containing 10 μ g/mL mitomycin MMC, incubating at 37 deg.C for 2.5-3 hr, discarding the supernatant, washing DPBS once, replacing fresh SHEM culture medium, and adding 5% CO₂And the incubator is used every other day at 37 ℃ for standby.

Clonal culture of differentiated corneal epithelial progenitor cells

Collecting differentiated cells, discarding supernatant, washing with DPBS, sucking supernatant, adding 500 μ L of digestive juice (0.25% Trypsin/EDTA), digesting at 37 deg.C in incubator, taking out every 3 min, observing cell morphology under 10 × microscope, adding 5ml of culture medium to stop digestion when all cells become round and part become suspension, blowing with 1ml of gun head to make all cells on the culture dish become suspended single cells, transferring cell suspension into 15ml centrifuge tube, counting, 1500 rpm, centrifuging for 5 min, discarding supernatant, adjusting cells to 8 × 10 using differentiation medium³Digesting and differentiating corneal epithelial progenitor cells, digesting 3T 3cells treated by mitomycin MMC with 0.25% Trypsin/EDTA digestive juice, adding a differentiation medium to stop digestion when the cells are round under a cell lens, gently blowing and beating the cells to a single cell, transferring the cells to a 15ml centrifuge tube, rotating the tube at 1500 rpm, centrifuging the cells for 5 minutes, (the differentiated corneal epithelial progenitor cells and the 3T 3cells can be centrifuged at the same time), discarding supernatant, and adjusting the cells to 5 × 10 by using the differentiation medium⁵cell/ml for use 250. mu.l of the differentiated corneal epithelial progenitor cell suspension (2000 cells) and 250. mu.l of the MMC-treated 3T 3cell suspension (1.25 × 10)⁵Individual cells), mixed well and added to a 12-well plate, i.e. 500 μ l of cell suspension is added to each well of the 12-well plate. Culturing in 5% CO2, 37 deg.C incubator, and changing the culture medium every other day. And (6) collecting and detecting.The detection comprises cell immunofluorescence staining, Western Blot detection and real-time quantitative PCR.

The primer design for real-time quantitative PCR is shown in the following table:

the above primers were synthesized by Biotechnology engineering (Shanghai) Ltd.

As shown in FIG. 1, the embryonic stem cells hES2 in the logarithmic growth phase were digested into small cell masses, which were then cultured on a low-adsorption petri dish using NP culture, and the morphological changes of the cells were observed under a microscope. In the condition of hES2, the cells grow adherently in the clone state, the clone edges are smooth, the cells are very small, the nucleus-cytoplasm ratio is large, and the nucleus can be obviously seen under a mirror (figure 1A). After differentiation into NP cell spheres, the cells grew in suspension in a spherical morphology (fig. 1B). The NP cell balls were digested into single cells, seeded into a petri dish, and differentiated, and the morphology of the cells gradually changed to a typical epithelial cell morphology (fig. 1D).

As shown in fig. 2, this study tested the expression of Pax6 and P63 in NP cells differentiated at different times. Undifferentiated hES2 cells were collected as a control group and labeled as NP-D0, while NP cells at day 2, day 4, day 6, day 8, and day 10 of differentiation of hES2 cells into NP cells were collected and labeled as NP-D2, NP-D4, NP-D6, NP-D8, and NP-D10, respectively. Extracting RNA, and carrying out real-time quantitative PCR detection. With the increase of the induction time, the expression level of the epithelial cell proliferation marker P63 was gradually increased (fig. 2A), while the expression of the gene Pax6 closely related to corneal epithelial development was gradually increased in the early stage of differentiation, reached a peak by day 8, and then began to decrease by day 10 (fig. 2B). Thus, NP cells that differentiated for 8 days were selected for subsequent experiments.

As shown in FIG. 3, cells were cultured to different differentiation stages and immunofluorescence assay was performed on 4% paraformaldehyde fixed cells or RNA was harvested from cells to detect changes in gene expression associated with embryonic stem cell phenotype. The hES2 stage cells were a control group and labeled as group D0, the NP stage 8 days after hES2 differentiation was labeled as group D8, the NP cells were differentiated into corneal epithelial progenitor cells at day 7 as group D15, and at day 14 as group D22. Immunofluorescence results showed that at the hES2 stage, the embryonic stem cell marker Oct4 was positively expressed (fig. 3A), the embryonic stem cell and neural stem cell marker Sox2 was positively expressed (fig. 3E), at the NP stage, Oct4 was positively expressed (fig. 3B), Sox2 was weakly expressed (fig. 2-3F), and at the differentiated D15, D22 stage, Oct4, Sox2 were not expressed (fig. 3C D G H).

The qPCR results show that the embryonic stem cell markers Oct4, Nanog and Tra-1-81 are obviously increased in the NP stage of the intermediate state, and the expression level of the markers is obviously reduced compared with the hES2 stage along with the progress of the differentiation process. The expression of Sox2 gradually decreased with the differentiation time. Has significant difference compared with the control group. The experimental result shows that the cells are gradually differentiated along with the progress of the differentiation process, and the differentiation process is obviously different from the differentiation process of embryonic stem cells in the early stage.

As shown in fig. 4, the immunofluorescent staining results showed that the neural stem cell marker P75 was highly expressed at the intermediate NP cell stage, and was not expressed at the hES2, D15, and D22 stages (fig. 4A-D). The protein Pax6, which plays an important role in corneal epithelium, was not expressed at the hES2 stage, was expressed at the NP stage, was weakly expressed in the D15 stage partial cells, and was strongly expressed in the D22 stage partial cells (fig. 4E-F). The epithelial stem cell marker P63 is not expressed in the hES2 stage, is expressed in the NP stage, and is strongly expressed in partial cells in the D15 and D22 stages (FIGS. 4E-F), and the number of the expressed cells is consistent with the characteristics of stem cells, namely, partial cells have the characteristics of stem cells. The epithelial cell marker Pan-ck was not expressed at the hES2 stage, expressed at the NP stage, and expressed in most cells at the D15 stage, and in the D22 stage, the number of positive cells was less than that of D15, but the expression level of single cells was higher (FIG. 4M-P). The stratified epithelial stem cell marker K14 was not expressed at the hES2 stage, was expressed at the NP stage, and was expressed in most cells at the D15 stage, and in the D22 stage, the number of positive cells was drastically reduced compared to the D15 stage, but the expression level of individual cells was increased (fig. 4R-U).

As shown in FIG. 5, phenotypes inducing different stages of differentiated cells were examined at the transcriptome level, and genes examined included an important factor regulating corneal epithelial differentiation, Pax6, markers associated with epithelial cell proliferation, P63, K14, and a corneal epithelial tissue-specific marker, K12. The experiment results of the control group, which is the hES2(D0), show that the expression level of P63 is gradually increased along with the progress of induction, and the expression level is increased by 400 times of 300-fold in the later period of induction. The expression of Pax6 is sharply increased in NP stage (D8), then gradually decreased, and gradually increased again at day 15 of differentiation (D15) and day 22 (D22), the expression levels of the two stages are far higher than those of D0, and the expression levels of K14 and K12 are both increased obviously in the later stage of differentiation.

The above results show that the differentiation-inducing epithelial-like cells do not express embryonic stem cell markers, that the intermediate stage-inducing NP cells have the potential to differentiate in different directions, and that the differentiation-inducing epithelial-like cells express markers of corneal epithelial progenitor cells, and that the experiments successfully induced differentiation of embryonic stem cells into corneal epithelial cells.

Example 2

The same procedure as in example 1 was repeated except that a control group was prepared in which sodium pyruvate was not added to the differentiation medium. As shown in FIG. 6, the expression levels of the proteins Pax6, the epithelial stem cell marker P63, the epithelial cell marker Pan-ck and the cell tight junction protein E-cadherin which play important roles in corneal epithelium in the group with sodium pyruvate added are obviously higher than those in the group without sodium pyruvate added, which indicates that sodium pyruvate has an important role in inducing differentiation of embryonic stem cells into corneal epithelial cells.

Example 3 construction experiment of tissue engineered corneal epithelial sheet (AM + ES-CE epithelial sheet)

Obtaining amnion

Signing an informed consent with healthy caesarean section lying-in women (HIV-I, hepatitis B, hepatitis C and syphilis detection indexes are all negative), and obtaining the placenta. Separating amnion from placenta in sterile super clean bench, rinsing with 1 × Hanks' balanced salt solution (HBSS) for 2 times to remove bloodstain, tearing off residual chorion on amnion with forceps to obtain transparent amnion, cleaning with 1 × HBSS to completely remove residual bloodstain, transferring into 15ml sterile centrifuge tube or 50ml sterile centrifuge tube, and storing at-80 deg.C.

Installation of amniotic membrane ring

Amnion frozen at-80 deg.C was removed in advance and thawed at 4 deg.C (about 4-8 hours). The custom made amniotic membrane fixation ring Insert (containing large ring, inner ring) was soaked overnight in sterile deionized water containing 30% 84 sterile water. The Insert was soaked in 75% alcohol and sterilized for 15 minutes. The plate was washed 3 times with 10% P/S-added sterile water for 10 minutes each time. Soaked in 1XDPBS with 2% P/S added for use. Taking out the thawed amnion, placing under a dissecting microscope, and separating the epithelial surface and the stroma surface of the amnion. The epithelial surface of the amnion is tightly attached to the inner ring, the outer ring is arranged, the amnion is clamped between the inner ring and the outer ring to form an amnion carrier structure for culturing cells, and redundant amnion is cut off. 2mg/ml DispaseII is added into the amniotic ring, and the amniotic ring is incubated in an incubator at 37 ℃ for 10 minutes. The digested epithelial cells were rubbed off using a sterile sponge. Digestion was stopped using differentiation medium and repeated washing with 1 × DPBS to remove epithelial cells and sponge debris. Adding differentiation medium, placing in a 37 deg.C incubator overnight, and detecting whether contamination occurs. After determining no contamination, adding fresh differentiation medium, placing in 37 deg.C incubator for use (absorbable dry culture medium, placing in 35mm culture plate, sealing with sealing membrane, storing at-20 deg.C for use, and rewarming at room temperature). The procedure for differentiation of embryonic stem cells into NP cells and differentiation of NP cells into corneal epithelial progenitor cells was the same as in example 1.

Construction of hES 2-derived corneal epithelial sheet

After 7 days of NP cell differentiation in Petri dishes (D15), the epithelial cell differentiation medium was discarded, 1 × DPBS was washed 1 time, and 400. mu.l CTS was added^TMTrypLE^TMPlacing the selected Enzyme in an incubator at 37 ℃ for digesting for 3 minutes, adding an epithelial cell differentiation culture medium to terminate when the cell lens is round, slightly blowing and beating the cells to completely fall off, transferring the cells to a 10ml centrifuge tube at 1500 rpm, centrifuging the cells for 10 minutes, discarding supernatant, adding a fresh epithelial cell differentiation culture medium to resuspend the cells, counting the cells, inoculating the cells to the inner ring of the amniotic membrane ring, and inoculating 3 × 10 to each amniotic membrane ring⁴One cell (about one 35mm culture dish is inoculated with 3-4 rings), the culture medium in the inner ring is filled to 500 μ l, and 1.5ml of culture medium is added outside the amniotic membrane ringCulturing at 37 deg.C in 5% CO2 incubator for 7-14 days, and changing the culture solution every day. The detection method is the same as that of the example 1, and the detection results are as follows:

as shown in FIG. 7, NP cells were induced to differentiate by adherence in an epithelial cell induction medium for 4 to 7 days, then digested into individual cells, inoculated into prepared de-epithelialized amniotic membrane ring, and cultured at 37 ℃ with 5% CO₂Culturing in an incubator for 7 days. The surface structure of the epithelial sheet is compact when observed under a mirror. The HE staining result of the section of the cross section of the epithelial sheet shows that the epithelial sheet is composed of 3-4 layers of cells, the cells are arranged closely, the shapes of the cells on the near amniotic membrane side and the far amniotic membrane side are the same, and no obvious difference exists, namely the cell sheet does not form the polarity similar to that of corneal epithelial cells.

As shown in FIG. 8, in the second step of the second differentiation stage, we selected two different vectors, one for differentiation directly on the culture dish and one for differentiation on the amnion, and found that the differentiation on the different vectors by Western Blot results that the markers P63, Pax6, K14, K3+ K12 were expressed on both vectors, but in different amounts, and the expression level of the cell marker differentiated on the culture dish was higher than that of the cells differentiated on the amnion. In contrast, it is considered that induction on a culture dish or an amniotic membrane results in uniform differentiation direction and progression of cells, but the expression level varies.

As shown in fig. 9, the immunofluorescent staining result showed that Pax6, a protein that plays an important role in corneal epithelium, was expressed in cells of epithelial sheet group. The epithelial stem cell marker P63 is expressed in a part of cells, and the expression amount is consistent with the characteristics of stem cells, namely, the part of cells have the characteristics of stem cells. The molecule associated with intercellular tight junctions, E-Cadherin, is expressed in epithelial sheets in almost all cells. The epithelial cell marker Pan-ck is expressed in the whole layer of the epithelial sheet. The stratified epithelial stem cell marker K14 is expressed in partial cells, and the expression of K14 is not seen in partial cells on the amniotic membrane side. And K3 and K12, which are differentiation maturation markers specific to corneal epithelial cells, are expressed in epithelial sheet partial cells.

Since the replacement of tissue cells is carried out by stem cells in tissues, it is necessary to evaluate whether an AM + ES-CE epithelial sheet obtained by differentiation has the potential to stably repair limbal stem cells for a long period of time, which lack the ocular function of an animal model, and we need to test whether an hES 2-derived AM + ES-CE epithelial sheet contains tissue stem cells. Therefore, we adopted the mode of clone culture to detect.

As shown in fig. 10, it was found from the experimental results that the occurrence of clones was observed on the third day after seeding for cells differentiated for 15 days (fig. 10, a), some clones grew gradually with the increase of the culture time, and it was clearly seen under the mirror that the number of clones was much larger than that on the third day by the sixth day of culture (fig. 10, B), and some clones were pseudoclones, i.e., appeared on the third day but did not increase with the increase of the culture time. When the culture is carried out till the sixth day, the under-lens clones are divided into two types, wherein the middle cells of one type of clone are large, the arrangement is loose, the marginal cells are small and the arrangement is tight, the clone and the feeder layer 3T 3cells have obvious limits, and the boundary cells are gathered to form partitions similar to mountain peaks (figure 10, B). Distinct boundaries were also seen between the other cell type and the surrounding feeder cells, but the cells within the clone were not different at the center and edge, were smaller cells, and were closely spaced (FIG. 10, C). The colony formation rate of the whole cells was found to be about 0.5% to 1% in terms of (number of colony formed/number of seeded cells) × 100%, which is comparable to the stem cell rate of normal somatic cells (FIG. 10, D). Cells differentiated for 22 days and clones formed similarly to cells differentiated for 15 days, clones appeared at the beginning of the third day of inoculation (FIG. 10, E), some clones were pseudoclones, some clones grew into macroclones, and two similar different types of clones appeared at the 6 th day of inoculation (FIG. 10, F, G). However, the clone formation rate is much less than that of cells differentiated for 15 days, about 0.05% -0.1% (fig. 10, H), that is, 2000 cells are inoculated in each well of a 12-well plate, 1-2 clones are grown, and the conditions of different repeated wells of epithelial sheet cells of different differentiation batches are consistent.

As shown in FIG. 11, the phenotype of the cells in the clones was examined using immunofluorescence staining. The experimental result shows that Pax6 is expressed in almost all cell nuclei in the clone, P63 is expressed in the cell nuclei of most cells, and a small part of cells at the edge of the clone are not positively stained. K14, Pan-ck, was expressed in all cells in the clone. And the fluorescence staining of K3 and K12 as specific markers of corneal epithelial differentiation maturation is negative. Cells that differentiated for 15 days were consistent with the phenotype of clones formed by cells that differentiated for 22 days.

As shown in FIG. 12, in order to examine the biosafety of AM + ES-CE epithelial cell sheets (AM + ES-CE group), an immunodeficient BALB/c nude mouse in vivo tumor formation test was performed with an undifferentiated embryonic stem cell hES2 group (AM + hES2 control group) as a positive control and an acellular amniotic membrane group (AM group) as a negative control. The results showed that at 5 weeks after surgery, tumors of macroscopic size were formed under the skin of mice transplanted with the AM + hES2 group, whereas no tumor was formed under the skin of mice transplanted with the AM + ES-CE group and the AM control group, and at 8 weeks after surgery, no tumor was formed in the AM + ES-CE group and the AM control group.

As shown in fig. 13, three different germ layers, epithelial cells (ectoderm) (fig. 13A), fibroblasts (mesoderm) (fig. 13B), glands (endoderm) and mucous glands (endoderm) (fig. 13C, D) were clearly observed by HE staining of the tumor cryosections of the AM + hES2 group.

To examine the cell status at each differentiation stage, we performed single cell sequencing experiments. Samples were collected at four time points for submission. Sample 1, undifferentiated "seed" cell stage, embryonic stem cell hES 2; sample 2 differentiated intermediate state NP cells; sample 3, cells obtained after 7 days of differentiation of NP cells toward corneal epithelial cells on a petri dish, namely D15; and 4, the NP cells are differentiated towards the corneal epithelial cells, digested and passaged after being differentiated for 7 days in a culture dish and inoculated in an amniotic ring, and the cells are cultured for 7 days in the amniotic ring, namely the tissue engineering corneal epithelial sheet constructed in the research, namely D22.

As shown in fig. 14, after the test results, the raw data with four different time period tags are mixed. Data analysis was performed using monocle after mixing, and FIG. 14A is a two-dimensional plot after tsne dimensionality reduction, where sample 1 partially coincides with the cell population of sample 2 and sample 3 partially coincides with the cell population of sample 4, as shown. This phenomenon coincides with the differentiation process in this study, where we induced differentiation of embryonic stem cell hES2 into NP cells using NP medium, which is a dynamic process, so that a small number of cells at the NP stage may still be in the state of differentiated embryonic stem cells, thus allowing sample 1 to coincide with a partial cell population of sample 2. In the latter two stages of differentiation, the same epithelial differentiation medium was used for differentiation, and sample 3 and sample 4 were a continuous differentiation process, and there was a difference in the differentiation rate of the cells, resulting in a phenomenon that the two cell populations partially overlap. On the basis of fig. 14A, an attempt was made to cluster the mixed data of the four stages using different clusters according to the characteristics of gene expression of cells, as shown in fig. 14B, and it is apparent that, compared with fig. 14A, when the number of clusters is 4, the clustering result almost corresponds to and coincides with the tag results of the four stages one to one.

Example 4 tissue engineering corneal epithelial sheets in the treatment of limbal stem cell deficiency animal model experiment

Experimental animals: healthy male New Zealand white rabbits 35, 8 weeks old, healthy male yellow rabbits 14, 8 weeks old, were provided by the animal testing center, university of Xiamen and were responsible for rearing.

Construction of limbal stem cell deficiency animal model

The rabbits were fixed and anesthetized by intramuscular injection of 1% sodium pentobarbital (2mL/kg) in combination with hypnotic (0.1mL/kg), with the animals being placed under deep anesthesia approximately 10 minutes after injection. After the rabbit was fully anesthetized, it was placed in lateral decubitus with the right monocular exposed and the operating area was fully disinfected with iodophor. The anesthetized rabbit is placed on an operating table, a hole towel is laid, and after the eyelid retractor opens the eyelid, the surface of the eye is subjected to surface anesthesia by elkayin. After the limbal tissue was completely destroyed using a motorized epithelial spatula while the juxta-limbal conjunctiva was separated, the limbal tissue was excised approximately 2mm wide by 0.2mm deep. The central and peripheral corneal epithelial cells were scraped thoroughly using an epithelial spatula. The ocular surface and conjunctival sac were rinsed several times with 1 × DPBS to completely remove the remaining corneal epithelial fragments.

Transport of AM + ES-CE epithelial sheets

The fresh culture medium is replaced in advance on the same day of the transplantation operation, the culture dish is sealed by a sealing film and then placed into an aseptic transport box, the low temperature of about 10 ℃ is maintained in the box, and the oscillation is avoided in the transport process.

Transplantation operation of AM + ES-CE epithelial sheet

Limbal stem cell deficient animals were randomly divided into 2 groups and were transplanted with AM (amniotic membrane) and AM + ES-CE grafts, respectively. The grafts were gently rinsed with 1xDPBS and the limbal stem cells depleted of ocular surface excess DPBS of the animal model were wiped dry with a blood-sucking sponge. The implant is attached to the surface of the corneal stroma of a receptor in the epithelial-downward direction, and the iris restorer is used for gently and rapidly flattening the implant to avoid bubbles as much as possible. The implant is fixed on the surface of the peribulbar conjunctiva by using a 10-0 absorbable thread, and continuous suturing or intermittent suturing can be selected during suturing. The eye ointment (Dianbizhi) is coated with tobramycin and dexamethasone in conjunctival sac. To protect the ocular surface from the animal's scratching, the rabbit eyelids were sutured using an 8-0 suture at the middle and outer 1/3 eyelids. The same operation is completed by the same experienced operation operator, so that the influence on the experimental result due to the difference of the operation skills of different operators is reduced.

And (3) postoperative administration: the postoperative eye medicine is mainly anti-inflammatory, anti-infection and stem cell growth promoting medicine.

Week 1: deproteinized calf serum extract eye drops, 3 times a day; cyclosporin eye drops, 3 times a day; dianbihu eye drops, 3 times a day; ding Bian Yan Gao, 3 times a day.

Week 2: deproteinized calf blood extract eye drop, 3 times a day; cyclosporin eye drops, 2 times a day; dianbihu eye drops, 2 times a day; ding Bian Yan Gao, 2 times a day.

Week 3 to week 5: deproteinized calf blood extract eye drop, 1 time a day; cyclosporin eye drops, 1 time a day; dianbihu eye drops, 1 time a day; ding Bian Yan Gao, 1 time a day.

Week 6 to week 8: deproteinized calf blood extract eye drop, 1 time a day; cyclosporin eye drops, 1 time a day; dikeluo eye ointment, 1 time a day.

As shown in FIG. 15, the surface of the anesthetized rabbit eye was subjected to surface anesthesia by Iekain, and the limbal structure was deeply destroyed by using an electric epithelial spatula while isolating the juxta-limbal conjunctiva, and then a deep limbal tissue of about 2mm in width × 0.2mm was excised. The blank group was observed by slit lamp photography after surgery, and showed positive total corneal sodium fluorescein staining (fig. 15 AB). In addition, the postoperative medicine is the same as the experimental group, and the medicines for resisting inflammation, resisting infection and promoting the growth of epithelial cells are applied every day. The 8-week-post-operative slit-lamp results showed diffuse neovascularization of the corneal surface, severe opacity of the central cornea, unsmooth corneal epithelium, and sodium fluorescein staining, with lamellar staining of the central and peripheral cornea (fig. 15C D). Taken together, this result confirms that we have successfully constructed an animal model of limbal stem cell deficiency.

As shown in FIG. 16, we transplanted the constructed hES 2-derived AM + ES-CE epithelial sheets onto animal models deficient in pericyte-derived stem cells of whole cornea, and observed whether the AM + ES-CE epithelial sheets could reconstruct damaged ocular surface. After the transplantation, 1 week observation shows that the amnion of the AM + ES-CE group and the amnion of the AM group are not dissolved, no obvious difference exists between the two groups, the eyelid suture of the AM + ES-CE group is loosened, and the eyelid suture of the AM group is in a loosening state. At 2 weeks after operation, the amnion is still not completely dissolved, the AM + ES-CE group has no obvious difference with the AM group, at 3 weeks after operation, the amnion of the AM + ES-CE group is not dissolved, and the condition of the ocular surface of the AM + ES-CE group cannot be observed due to the amnion coverage. The amnion of AM group begins to dissolve, large pieces of amnion can be obviously observed by fluorescein sodium staining, and the corneal limbus epithelium position is exposed. In the AM group, although the ocular surface was covered with the amniotic membrane, the central corneal part was not observed, but invasion of new blood vessels into the cornea was observed in the peripheral cornea. At week 4, the amnion in the AM + ES-CE group began to dissolve slightly, exposing the limbal location, with a small amount of new blood vessel invasion. The amnion in the AM group was completely dissolved and a large number of new blood vessels were visible in the upper cornea, invading the limbus by about 4 mm. The inferior corneal neovascularization invaded the limbus by about 2mm, the temporal corneal neovascularization invaded the limbus by about 1mm, and no significant neovascularization invasion was seen in the nasal cornea. Large lamellar staining was seen in the central cornea with fluorescein sodium staining. At 5 weeks, the amnion in AM + ES-CE group began to dissolve, and a large block of the amnion about to drop was observed, while the new blood vessels were seen to invade the limbus about 3.5mm in the cornea above the nose, and the new blood vessels were seen to grow under the cornea, on the temporal side and on the nasal side. The AM group of new blood vessels continue to grow towards the pupil, the new blood vessels above the cornea invade the cornea for about 5mm, the new blood vessels below the cornea, on the nasal side and on the temporal side invade the corneal limbus for about 2mm, the whole cornea is turbid, the central cornea can be stained in large slices by fluorescein sodium staining, and compared with the 4 th week, the area is obviously increased. At week 6, the amnion of AM + ES-CE group was completely dissolved, the cornea was recovered to be transparent, the new blood vessels were significantly reduced compared with week 5, only a small amount of blood vessels invaded the limbus of cornea by about 2.5mm, and the amount was significantly reduced compared with the former, and a small amount of punctate coloration was seen by fluorescein sodium staining. The opacity of the cornea of the AM group is obviously deepened compared with the former cornea, the new blood vessels continue to grow towards the pupil direction, invade into the corneal limbus for about 6mm, reach the pupillary limbus, and simultaneously, the new blood vessels below the cornea, on the temporal side and on the nasal side continue to grow towards the pupil direction, invade into the corneal limbus for about 3mm, and large-piece coloration can still be seen after fluorescein sodium staining. At 7 weeks, the cornea of the AM + ES-CE group was transparent, the neovasculature above the eyeball was completely regressed, a small amount of neovasculature was visible below, the corneal limbus was about 2.5mm invasive, and no significant staining was seen with sodium fluorescein staining. And the corneal opacity of the AM group is further aggravated, a large amount of new blood vessels are diffused and grown, the new blood vessels above the cornea invade the corneal limbus for about 7mm, a half area of the pupil is covered by the new blood vessels, the new blood vessels below the eyeball are better rotated, the new blood vessels on the nasal side invade the corneal limbus for about 2.5mm, a large amount of new blood vessels on the temporal side invade the corneal limbus for about 2.5mm, and meanwhile, large-piece coloration can still be seen after fluorescein sodium staining. At week 8, the corneal layer of the AM + ES-CE group was clear, the neovasculature further regressed to the periphery of the limbus, and no significant staining was observed with fluorescein sodium staining. The cornea in the AM group was further turbid, full corneal neovascular coverage, and large lamellar staining was still visible with sodium fluorescein staining.

As shown in fig. 17, the animals were sacrificed at 8 weeks after transplantation, and cryo-section H & E staining was performed on rabbit corneal specimens, and as a result, it was found that: the tissue structure of the AM + ES-CE transplanted group is similar to that of the normal control group, and in the corneal limbus area, the AM + ES-CE transplanted group can form a good palisade structure 8 weeks after transplantation, and is consistent with the structure of the normal control group, the corneal limbus comprises 6-7 layers of corneal limbus epithelial cells, and stroma is not obviously infiltrated by inflammatory cells. The AM transplantation group can not form the limbal palisade structure after 8 weeks of transplantation, only forms 2-3 layers of corneal epithelial cells, and contains a large amount of inflammatory cell infiltration in stroma. In the central corneal region, the AM + ES-CE transplanted group contained 4 to 5 layers of corneal epithelial cells 8 weeks after transplantation, and the cell growth thereof was polar, i.e., cells on the stroma-proximal side were arranged in a vertical manner and cells on the stroma-distal side were arranged in a horizontal manner, which was consistent with the normal control group. However, the thickness of corneal epithelium is slightly thinner than that of normal control group, and the central cornea of the normal control group can contain 5-6 layers of epithelial cells. And the central corneal epithelium of the AM transplantation group only forms 2-3 layers of corneal epithelial cells at 8 weeks after the transplantation, and the cells have no polarity.

To further observe the repair of rabbit central corneas after transplantation of h ES 2-derived AM + ES-CE and the expression of central corneal epithelial cell markers after transplantation, we performed immunofluorescent staining of sacrificed rabbit corneal specimens 8 weeks after transplantation with human nucleolei, Pax6, Pan-ck, K14 and K3+ K12, as shown in FIG. 18. human nucleolein (green) was expressed in both rabbit central corneal epithelium and in a portion of stromal cells near the epithelium in the AM + ES-CE group, whereas positive expression of human nucleolein was not seen in the AM control group and the Normal control group (FIGS. 18A-C). Pax6 was positively expressed in central corneal epithelial cells of the AM + ES-CE group, whereas positive staining was not seen in both the AM control group and the Normal control group (FIGS. 18D-F). The epithelial marker Pan-ck was positively expressed in all three groups (FIGS. 18G-I). The ocular surface epithelial stem cell marker K14 was highly expressed in the corneal epithelial basal layer of the AM + ES-CE group, in the epithelial cells of the AM control group, and in the central corneal epithelial full layer of the Normal control group (FIGS. 18J-L). The corneal epithelium specific marker K3+ K12 was highly expressed in the central corneal epithelial cells in the AM + ES-CE group and the Normal control group, and partially expressed in the AM control group (FIG. 18M-O). The experimental results show that the AM + ES-CE derived from hES2 can well restore the structure and function of the central cornea.

Example 5

AM + ES-CE epithelial sheets were prepared as in example 2, and tissue-engineered corneal epithelial sheets for limbal stem cell deficiency animal model experiments were performed as in example 4. The sodium pyruvate group is formed by adding sodium pyruvate into a differentiation culture medium to construct a tissue engineering corneal epithelial sheet for treating the limbal stem cell deficiency animal model; the sodium pyruvate-free control group is characterized in that sodium pyruvate is not added into a differentiation medium, and a tissue engineering corneal epithelial sheet is constructed for treating the limbal stem cell deficiency animal model; the amniotic membrane control group is an animal model for treating limbal stem cell deficiency with a blank amniotic membrane.

As shown in fig. 19, as can be seen from the slit lamp results, there was no significant difference between the amnion control group, the control group without addition of sodium pyruvate, and the control group with addition of sodium pyruvate at 2 weeks after transplantation; at 4 weeks post-transplantation, addition of sodium pyruvate was used to visualize corneal epithelial ingrowth in the central cornea, whereas the amniotic control group, without sodium pyruvate, showed no significant corneal epithelial ingrowth and visible corneal neovascularization. At 6 weeks post-transplantation, the sodium pyruvate-added group had essentially intact corneal epithelium, while the amniotic control group, without sodium pyruvate, had seen repeated corneal epithelial detachment. Similar phenotypes were maintained at 8 weeks post-transplantation.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> university of mansion

<120> culture method for inducing human embryonic stem cells to directionally differentiate into corneal epithelial cells and application thereof

<130>2020

<160>22

<170>PatentIn version 3.5

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Claims

1. A culture method for inducing the directional differentiation of human embryonic stem cells into corneal epithelial cells is characterized by comprising the following steps:

2. The method of claim 1, wherein the culture medium for inducing the directional differentiation of human embryonic stem cells into corneal epithelial cells is prepared by adding 18-22 wt% of a serum replacement KSR, 0.5-1.5 wt% of sodium pyruvate, 0.5-1.5 wt% of non-essential amino acids, 0.5-1.5 wt% of glutamine, 8-12nM of ROCK inhibitor Y27632, and 0.5-1.5 v/v% of double antibody P/S to DMEM/F12.

3. The method for inducing the directional differentiation of human embryonic stem cells into corneal epithelial cells according to claim 1, wherein the epithelial differentiation medium is prepared by mixing a SHEM medium and a D-KSFM medium in a volume ratio of 1: 1.5-2.5, and then adding 0.5-1.5 wt% of sodium pyruvate, 0.5-1.5 wt% of nonessential amino acid, 0.5-1.5 wt% of glutamine and 8-12nM ROCK inhibitor Y27632; the preparation method of the SHEM culture medium is characterized in that 4-6 v/v% serum FBS, 0.4-0.6mg/ml hydrocortition, 4-6ml/L ITS, 0.4-0.6 v/v% DMSO, 8-12ng/ml human epithelial cell growth factor EGF and 0.5-1.5 v/v% double-antibody P/S are added into a basal medium DMEM/F12.

4. The culture method for inducing the directional differentiation of human embryonic stem cells into corneal epithelial cells according to claim 1, wherein the step S1 specifically comprises the following steps:

s13, centrifuging the cell mass suspension for 4-6 minutes at the rotating speed of 80-120 r/min; discarding supernatant, suspending and mixing with NP medium, inoculating into low adsorption culture dish, and placing in 5% CO₂Culturing in 37 deg.C incubator for 6-8 days, and changing culture medium every day.

5. The culture method for inducing the directional differentiation of human embryonic stem cells into corneal epithelial cells according to claim 1, wherein the step S2 specifically comprises the following steps:

s21, centrifuging the NP cells obtained in the step S1 at the speed of 700-900 rpm for 8-12 minutes, discarding the supernatant, and adding CTS^TMTrypLE^TMDigesting the Select Enzyme at 37 ℃ for 4-6 minutes, stopping digestion with NP medium, and gently pipetting to obtain a single cell suspension;

s22, centrifuging at 1400-1600 rpm for 8-12 min, discarding the supernatant, adding epithelial differentiation medium, and resuspending at (2.5-3.5) × 10⁴/cm²The inoculated density of (A) was inoculated into a coated attached culture dish and placed in 5% CO₂Culturing at 37 deg.CCulturing in incubator for 4-7 days, and changing liquid every day.

6. The culture method for inducing the directional differentiation of human embryonic stem cells into corneal epithelial cells according to claim 1, wherein the treatment of the mouse fibroblast line 3T 3cells comprises the following steps:

s31, when the mouse fibroblast line 3T 3cells grow to 70% -80% confluency, discarding the supernatant, adding SHEM culture medium containing 8-12 μ g/mL mitomycin, incubating at 37 deg.C for 2.5-3 hr, discarding the supernatant, replacing fresh SHEM culture medium, and adding 5% CO₂And a 37 ℃ incubator for standby use every other day;

s32, taking the 3T 3cells obtained in the step S31, abandoning the supernatant of the culture medium, adding 0.20-0.30 wt% of Trypsin of EDTA to digest at 37 ℃ for 2.5-3.5 minutes, adding a differentiation culture medium to stop the digestion when the cells are round under a microscope, gently blowing and beating the cells into a single cell suspension, centrifuging the cells for 4-6 minutes at the rotating speed of 1400-1600 rpm, abandoning the supernatant, and regulating the cells to (4.5-5.5) × 10 by using the differentiation culture medium⁵cell/ml for standby;

s33, adding the 3T 3cell suspension obtained in S32 and the same amount of the digested suspension of the cells obtained in the step S2, uniformly mixing, and placing in 5% CO₂Culturing in 37 deg.C incubator for 7-14 days, and changing culture medium every other day.

7. The method of claim 1, wherein the step S2 comprises the steps of collecting the cells obtained in step S2, discarding the supernatant of the culture medium, adding 0.20-0.30 wt% EDTA Trypsin to the cells at 37 ℃ for 2.5-3.5 min, adding the epithelial differentiation medium to stop the digestion when the cells become round and partially suspended under a microscope, gently blowing the mixture into a single cell suspension, centrifuging the mixture at 1400-1600 rpm for 4-6 min, discarding the supernatant, and adjusting the cells to (7.5-8.5) × 10 by using the differentiation medium³cells/ml for use.

8. The method for inducing the directional differentiation of human embryonic stem cells into corneal epithelial cells according to claim 1, wherein said seeding on the surface of de-epithelialized amniotic membrane comprises the following steps:

9. A corneal epithelial cell and a corneal epithelial sheet prepared by the method according to any one of claims 1 to 8.

10. An epithelial differentiation medium is characterized in that the preparation method comprises the following steps of mixing a SHEM medium and a D-KSFM medium according to the volume ratio of 1: 1.5-2.5, adding 0.5-1.5 wt% of sodium pyruvate, 0.5-1.5 wt% of nonessential amino acid, 0.5-1.5 wt% of glutamine and 8-12nM ROCK inhibitor Y27632; the preparation method of the SHEM culture medium comprises the steps of adding 4-6 v/v% serum FBS, 0.4-0.6mg/ml hydrocortition, 4-6ml/L ITS, 0.4-0.6 v/v% DMSO, 8-12ng/ml human epithelial cell growth factor EGF and 0.5-1.5 v/v% double-antibody P/S into a basal medium DMEM/F12.