CN106635955B - Method for obtaining corneal endothelial cells - Google Patents

Abstract

The invention relates to a method for obtaining corneal endothelial cells, and belongs to the technical field of cell biology. The corneal endothelial cells are obtained by obtaining, pre-differentiating and differentiating embryoid bodies. The corneal endothelial cells obtained by the method for differentiating the human pluripotent stem cells in vitro can be used as an ideal cell source for replacing donated corneal endothelial cells. The method of the present invention employs human embryonic stem cells that are commercialized, and thus the availability of corneal endothelial-like cells has an abundant source because of their ability to self-replicate and immortalize and their differentiation potential. The invention solves the problem of low differentiation rate of the corneal endothelial cells in vitro, improves the differentiation efficiency of the corneal endothelial cells in vitro, and the time from the embryoid body stage to the acquisition of the corneal endothelial cells is 11 days at the fastest. The method has simple technical scheme and easy operation.

Description

Method for obtaining corneal endothelial cells

Technical Field

The invention relates to a method for obtaining corneal endothelial cells, and belongs to the technical field of cell biology.

Background

the corneal endothelial cells are a single layer of cells arranged in a regular hexagon, are a physiological barrier separating the corneal stroma from the aqueous anterior chamber, and play a key role in maintaining corneal transparency, and the reduction or damage of the corneal endothelial cells is caused by (i) gradual reduction with age, (②) infection, inflammation, trauma (such as mechanical injury, chemical injury, birth injury, laser ray, etc.), eye surgery (such as cataract surgery, anti-glaucoma surgery, and other intraocular operations such as removal of foreign body from the anterior chamber), hereditary diseases, metabolic diseases, etc., all can cause the reduction and damage of the endothelial cells.

Clinically, corneal transplantation is the only effective therapy for treating human corneal endothelial cell diseases. However, the lack of the source of the cornea donor is a difficult problem facing the world, which greatly limits the development of the cornea transplantation operation, so that a plurality of patients with cornea diseases can not be cured in time, and only the cornea materials can be waited in the pain, thereby bringing great social burden. In recent years, with the progress of corneal transplantation technology, the development of Descemet membrane technology (DMEK), i.e., only the corneal endothelium and the Descemet membrane, has been developed to realize a good vision recovery technology. However, this therapy is significantly limited by the shortage of donated sources of corneal endothelial cells. Therefore, stem cell-based technology is considered to be a promising alternative to the treatment of diseases caused by the loss of human corneal endothelial cells.

Disclosure of Invention

The invention aims to overcome the defects and provides a method for obtaining corneal endothelial cells by differentiating human pluripotent stem cells in vitro.

The invention also provides an identification index of the corneal endothelial cells obtained by in vitro differentiation.

In order to achieve the technical purpose and achieve the technical effect, the invention is realized by the following technical scheme:

the method for obtaining the corneal endothelial cells comprises the following steps:

(1) obtaining an embryoid body: culturing human embryonic stem cells on mouse embryonic fibroblasts, and after fully paving a culture vessel with collagenase IV and DispaseII for digestion, adopting a serum-free culture medium for culture to obtain an embryoid body;

(2) pre-differentiation culture, namely culturing the embryoid bodies obtained in the step (1) in a differentiation medium A supplemented with noggin and a TGF-β 1 receptor inhibitor for 4-5 days or culturing the embryoid bodies in a differentiation medium B for 12-16 days to obtain pre-differentiation culture intermediate cells;

(3) differentiation culture: transferring the intermediate cells subjected to the pre-differentiation culture in the step (2) to a culture vessel coated with chondroitin sulfate and laminin, and culturing for 7-8 days by adopting a differentiation culture medium C or culturing for 25-40 days by adopting a differentiation culture medium D to obtain the corneal endothelial cells.

The proportion of the collagenase IV and the neutral protease DispaseII in the step (1) is 1: 1-5, wherein before configuration, the concentration of collagenase IV is 400-1000U/mL, and the concentration of neutral protease DispaseII is 1-6U/mL;

serum-free medium: the basic culture medium is DMEM/F12 culture medium, 10-20% of serum substitute, 10-20 ng/mL of alkaline fibroblast growth factor, 1 multiplied by non-essential amino acid and 2mM of GlutaMAX^TMmedium and additive, 100 units/mL of cyan-chain double antibody, 0.1mM of β -mercaptoethanol;

the concentration of the supplemented noggin in the step (2) is 300-1000 ng/mL, and the concentration of the supplemented TGF-β 1 receptor inhibitor is 2.5-15 mu M.

The differentiation medium A in the step (2): a basal medium DMEM/F12 culture medium added with 1 XN 2 additive, 10-20 ng/mL basic fibroblast growth factor, 1 Xnon-essential amino acid and 2mM GlutaMAX^TMA culture medium and an additive, 100 units/mL of a cyan-chain double antibody, and 2-10 ng/mL of recombinant human insulin;

differentiation medium B: a basal medium DMEM/F12 culture medium, which is added with 5 to 20 percent of serum substitute, 1 multiplied by non-essential amino acid and 2mM GlutaMAX according to the mass ratio^TMmedium and additives, 100 units/mL cyan-chain double antibody, 0.1mM β -mercaptoethanol.

The concentration of the chondroitin sulfate coated by the culture vessel in the step (3) is 10-50 mg/mL; the concentration of laminin is 1-10 mug/mL;

the differentiation medium C in the step (3): the basic culture medium is prepared by DMEM/F12 and M199 according to the proportion of 1-1.5: 1, and 5% -20% of fetal bovine serum, 1 × non-essential amino acid and 2mM GlutaMAX are added^TMCulture medium and additive, 100 units/mL of cyan-streptomycin cyan chain double antibody, 2-10 ng/mL of recombinant human insulin, 20-50 mg/mL of levorotatory vitamin C and 10-20 ng/mL of basic fibroblast growth factor.

the differentiation medium D in the step (3) is prepared by culturing bovine corneal endothelial cells with the differentiation medium C, collecting a conditioned medium for culturing the bovine corneal endothelial cells, and preparing the conditioned medium and a fresh differentiation medium C according to the proportion of 1: 1-1.5, and adding a chemokine composition consisting of 200-1000 nM recombinant human Dickkopf-related protein, 2.5-20 ng/mL platelet-derived growth factor and 2.5-10 ng/mL transforming growth factor β.

The method for obtaining the conditioned medium comprises the following steps: culturing ox horn membrane endothelial cells, taking out a freezing tube filled with the ox horn membrane endothelial cells from an ultra-low temperature refrigerator, quickly transferring the freezing tube to a 37 ℃ water bath kettle, quickly dissolving the tube, transferring cell suspension in the freezing tube to a 15mL centrifugal tube, adding 5-10 mL of a differentiation medium C, centrifuging for 5 minutes at 1000 rpm, discarding supernatant, adding 4-6 mL of the differentiation medium C for resuspending cell precipitation, finally adding the cells into a culture bottle coated with 1-10 mu g/mL laminin and 10-50 mu g/mL chondroitin sulfate, placing the culture bottle at 37 ℃ and 5% CO by volume concentration₂Culturing in the incubator, and changing the culture solution after overnight; changing the culture medium every two days, and collecting the culture medium for culturing ox horn endothelial cells every two days to obtain the conditioned medium.

Carrying out immunofluorescence staining on the intermediate cell markers HNK-1 and P75 obtained by pre-differentiation culture to show positive expression; the obtained markers of the corneal endothelial cells, namely sodium-potassium-ATPase, S100A4 protein, aquaporin-1, namely AQP-1 and zonulin ZO-1, are positively expressed by immunostaining.

The corneal endothelial cell-like cell is applied to diseases caused by human corneal endothelial cell loss.

The invention has the beneficial effects that:

(1) the corneal endothelial cells obtained by the method for differentiating the human pluripotent stem cells in vitro can be used as an ideal cell source for replacing donated corneal endothelial cells.

(2) The method of the present invention employs human embryonic stem cells that are commercialized, and thus the availability of corneal endothelial-like cells has an abundant source because of their ability to self-replicate and immortalize and their differentiation potential.

(3) The invention solves the problem of low differentiation rate of the corneal endothelial cells in vitro, improves the differentiation efficiency of the corneal endothelial cells in vitro, and the time from the embryoid body stage to the acquisition of the corneal endothelial cells is 11 days at the fastest.

(4) The method for identifying the corneal endothelial cells obtained by in vitro differentiation can be used for quality control of the corneal endothelial cells and paves a way for treating corneal endothelial cell deficiency diseases by using the corneal endothelial cells in the future.

(5) The corneal endothelial cells are obtained by the method, so that a foundation is laid for the development of Descemet membrane technology (DMEK), and the method has very important clinical significance and wide application prospect.

(6) The method has simple technical scheme and easy operation.

Drawings

FIG. 1 is a schematic representation of a cell. a. Diagram of embryoid body differentiation; b. example 4-2 schematic representation of corneal endothelial cells; c. example 4-3 schematic representation of corneal endothelial cells.

FIG. 2 is a schematic representation of the results of immunofluorescence staining. a. Example 5-1 immunofluorescence staining results; b. the result of immunofluorescence staining with sodium-potassium-ATP enzyme is shown schematically; c. the S100A4 protein immunofluorescence staining result is shown in a schematic diagram; d. a schematic diagram of the result of aquaporin-1 immunofluorescence staining; e. and (3) a schematic diagram of the result of the immunofluorescence staining of the zonulin ZO-1.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.

The media and their supplements used in the present invention are, unless otherwise specified, from the Gibco company and the Sigma company.

EXAMPLE 1 preparation of culture Medium

1. Preparation of serum-free medium

The basal medium is DMEM/F12 medium supplemented with 15% serum replacement, 15ng/mL basic fibroblast growth factor, 1 × non-essential amino acids, 2mM GlutaMAX^TMculture medium and additive, 100 units/mL of cyan-chain double antibody, 0.1mM of β -mercaptoethanol, preparing serum-free culture medium, and storing at 4 ℃ in dark place for later use.

2. Preparation of differentiation Medium A

The basic culture medium is DMEM/F12 culture medium, and 1 XN 2 additive, 12ng/mL basic fibroblastGrowth factor, 1 × non-essential amino acids, 2mM GlutaMAX^TMCulture medium and additive, 100 units/mL cyan-chain double antibody, 2ng/mL recombinant human insulin, prepared into differentiation culture medium A, and stored at 4 ℃ in dark place for later use.

3. Preparation of differentiation Medium B

The basic culture medium is DMEM/F12 culture medium, and is supplemented with 10% serum substitute, 1 × non-essential amino acids, 2mMGlutamax^TMculture medium and additive, 100 units/mL green-chain double antibody, 0.1mM β -mercaptoethanol, to prepare differentiation culture medium B, and storing at 4 deg.C in dark place.

4. Preparation of differentiation Medium C

The basal medium was prepared from DMEM/F12 and M199 at 1.2:1, supplemented with 5% fetal bovine serum, 1 × non-essential amino acids, 2mM GlutaMAX^TMCulture medium and additive, 100 units/mL cyan-chain double antibody, 2ng/mL recombinant human insulin, 30mg/mL levorotatory vitamin C and 12ng/mL basic fibroblast growth factor, and preparing into differentiation culture medium C, and storing at 4 deg.C in dark place for later use.

5. Acquisition and preparation of differentiation Medium D

(1) Conditioned Medium harvesting

Culturing ox horn membrane endothelial cell, taking out the freezing tube with ox horn membrane endothelial cell from the ultra-low temperature refrigerator, transferring to 37 deg.C water bath, dissolving rapidly, transferring cell suspension in the freezing tube to 15m1 centrifugal tube, adding 5mL differentiation culture medium C, centrifuging at 1000 rpm for 5 min, discarding supernatant, adding 4mL differentiation culture medium C for resuspending cell precipitation, adding cells into culture bottle coated with l0ug/mL laminin and l0ug/mL chondroitin sulfate, placing at 37 deg.C and 5% CO₂Culturing in an incubator, changing the culture solution after overnight, changing the culture solution every two days later, and collecting a culture medium for culturing bovine corneal endothelial cells every two days as a conditioned medium.

(2) Preparation of differentiation Medium D

the conditioned medium and the fresh differentiation medium C are prepared according to the proportion of 1:1.2, and a chemotactic factor composition consisting of 200nM recombinant human Dickkopf-related protein 2, 5ng/mL platelet-derived growth factor and 5ng/mL transforming growth factor β is added to prepare a differentiation medium D, and the differentiation medium D is stored at 4 ℃ in a dark place for later use.

EXAMPLE 2 methods for obtaining corneal endothelial cells

1. Culture of commercial human embryonic stem cells

Culturing mouse embryo fibroblast by conventional method, taking 3-4 generations thereof as feeder layer, inducing proliferation in culture medium containing Leukemia Inhibitory Factor (LIF), and maintaining undifferentiated state.

2. Obtaining embryoid bodies

The commercial human embryonic stem cells cultured as described above were treated with collagenase iv and DispaseII at a ratio of 1: 2, preparing and digesting, and culturing the human embryonic stem cells by adopting the prepared serum-free culture medium until the human embryonic stem cells are differentiated to form a chrysanthemum-shaped group, namely an embryoid body, as shown in a figure 1 a.

Example 3 Pre-differentiation culture

1. Operation of Pre-differentiation culture

the embryoid bodies obtained in example 2 were cultured for 4 days in differentiation medium A prepared in example 1 supplemented with 500ng/mL of noggin and 10. mu.M of TGF-β 1 receptor inhibitor;

2. operation two of pre-differentiation culture

the embryoid bodies obtained in example 2 were cultured for 2 weeks in differentiation medium B prepared in specific example 1 supplemented with 500ng/mL of noggin and 10. mu.M of TGF-. beta.1 receptor inhibitor;

EXAMPLE 4 class of corneal endothelial cell harvesting

1. Procedure for obtaining corneal endothelial cells

The intermediate cells after pre-differentiation culture in the first operation of example 3 were transferred to a T25 flask coated with chondroitin sulfate 25mg/mL andlaminin 1. mu.g/mL and cultured for 7 days, and the corneal endothelial-like cells were obtained using the differentiation medium C prepared in the specific example 1.

2. Operation two for obtaining corneal endothelial cells

(1) The intermediate cells after pre-differentiation culture in the first operation of example 3 were transferred to a T25 flask coated with chondroitin sulfate 25mg/mL andlaminin 1. mu.g/mL and cultured for 25 days, and the differentiation medium D prepared in the specific example 1 was used to obtain corneal endothelial cells.

(2) The intermediate cells after pre-differentiation culture in the first operation of example 3 were transferred to a T25 flask coated with chondroitin sulfate 25mg/mL andlaminin 1. mu.g/mL and cultured for 30 days, and the corneal endothelial-like cells were obtained using the differentiation medium D prepared in the specific example 1.

(3) The intermediate cells after pre-differentiation culture in the first operation of example 3 were transferred to a T25 flask coated with chondroitin sulfate 25mg/mL andlaminin 1. mu.g/mL and cultured for 40 days, and then the differentiation medium D prepared in the specific example 1 was used to obtain corneal endothelial cells, as shown in FIG. 1 b.

3. Operation three for obtaining corneal endothelial cells

Transferring the intermediate cells after the pre-differentiation culture in the second operation of the specific example 3 into a T25 culture flask coated with chondroitin sulfate 25mg/mL andlaminin 1 μ g/mL for culture for 7 days, and obtaining the corneal endothelial-like cells by using the differentiation medium C prepared in the specific example 1.

4. Operation four for obtaining corneal endothelial cells

(1) The intermediate cells after pre-differentiation culture in the second operation of example 3 were transferred to a T25 flask coated with chondroitin sulfate 25mg/mL andlaminin 1. mu.g/mL and cultured for 25 days, and the corneal endothelial-like cells were obtained using the differentiation medium D prepared in the specific example 1.

(2) The intermediate cells after pre-differentiation culture in the second operation of example 3 were transferred to a T25 flask coated with chondroitin sulfate 25mg/mL andlaminin 1. mu.g/mL and cultured for 28 days, and the corneal endothelial-like cells were obtained using the differentiation medium D prepared in the specific example 1.

(3) The intermediate cells after pre-differentiation culture in the second operation of example 3 were transferred to a T25 flask coated with chondroitin sulfate 25mg/mL andlaminin 1. mu.g/mL, and cultured for 38 days, and then the differentiation medium D prepared in the specific example 1 was used to obtain corneal endothelial cells, as shown in FIG. 1 c.

Example 5

1. The intermediate cells after the pre-differentiation culture in the first operation and the second operation of the embodiment 3 are subjected to immunostaining, and the specific operations are as follows:

(1) fixing the embryoid body with 0.1M PBS buffer solution containing 4% paraformaldehyde at room temperature for 30 minutes;

(2) permeabilization of the cells with 0.5% triton X-100 (in PBS) for 10 min;

(3) PBS washing for 5 minutes for three times;

(4) blocking with 2% BSA for 30 min, washing twice with PBS;

(5) adding a primary antibody: HNK-1(1:100), incubated for 1 hour at room temperature;

(6) PBS washing for 5 minutes for three times;

(7) adding secondary antibodies labeled with Cy2 and Cy3, and incubating for 30-45 minutes at room temperature;

(8) PBS washing for 5 minutes for four times;

(9) adding 0.5 mu g/mL DAPI (prepared by PBS) to stain for 10 minutes;

(10) washing with PBS for three times to remove excessive DAPI;

(11) add 20. mu.l of mounting medium to mount.

The immunofluorescence staining results show that: the intermediate cells obtained from the pre-differentiation culture showed positive expression of HNK-1 as a marker, as shown in FIG. 2 a.

2. The corneal endothelial cells obtained in the second step (1) of the operation of the specific example 4 are immunostained, and the specific operation is as follows:

(1) the day before the experiment, cells were seeded into six-well plates, each of which was plated with 22X 22mm coverslips.

(2) The next day, the staining procedure was started until the confluency of cells reached about 70%.

(3) Fixing the corneal endothelial cells with a 0.1M PBS buffer solution containing 4% paraformaldehyde at room temperature for 30 minutes;

(4) permeabilization of the cells with 0.5% triton X-100 (in PBS) for 10 min;

(5) PBS washing for 5 minutes for three times;

(6) blocking with 2% BSA for 30 min, washing twice with PBS;

(7) adding primary antibodies respectively: sodium-potassium-ATPase (1:100), S100A4 protein (1:100), AQP-1(1:100) and ZO-1(1:100) incubated at room temperature for 1 hour;

(8) PBS washing for 5 minutes for three times;

(9) adding secondary antibodies labeled with Cy2 and Cy3, and incubating for 30-45 minutes at room temperature;

(10) PBS washing for 5 minutes for four times;

(11) adding 0.5 mu g/mL DAPI (prepared by PBS) to stain for 10 minutes;

(12) washing with PBS for three times to remove excessive DAPI;

(13) add 20. mu.l of mounting medium to mount.

The immunofluorescence staining results show that: the obtained markers of the corneal endothelial cells, i.e., sodium-potassium-ATPase, S100A4 protein, aquaporin-1 (AQP-1) and zonulin ZO-1, were positively expressed by immunostaining, as shown in FIGS. 2b, 2c, 2d and 2 e.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. To those skilled in the art to which the invention relates, numerous changes, substitutions and alterations can be made without departing from the spirit of the invention, which should be deemed to be within the scope of the invention as defined by the appended claims.

Claims (3)

1. The method for obtaining the corneal endothelial cells is characterized by comprising the following steps:

(1) obtaining an embryoid body: culturing commercial human embryonic stem cells on mouse embryonic fibroblasts, digesting the mouse embryonic fibroblasts by using collagenase IV and DispaseII in a culture vessel, and culturing the mouse embryonic stem cells by using a serum-free culture medium to obtain an embryoid body;

(2) pre-differentiation culture, namely culturing the embryoid bodies obtained in the step (1) in a differentiation medium A supplemented with noggin and a TGF- β 1 receptor inhibitor for 4 days to obtain pre-differentiation culture intermediate cells;

(3) differentiation culture: transferring the intermediate cells subjected to the pre-differentiation culture in the step (2) to a culture vessel coated with chondroitin sulfate and laminin, and culturing for 7 days by adopting a differentiation culture medium C to obtain corneal endothelial cells;

the proportion of the collagenase IV and the neutral protease DispaseII in the step (1) is 1: 1-5, wherein before configuration, the concentration of collagenase IV is 400-1000U/mL, and the concentration of neutral protease DispaseII is 1-6U/mL;

serum-free medium: the basic culture medium is DMEM/F12 culture medium, 10-20% of serum substitute, 10-20 ng/mL of alkaline fibroblast growth factor, 1 multiplied by non-essential amino acid and 2mM of GlutaMAX^TMmedium and additive, 100 units/mL of cyan-chain double antibody, 0.1mM of β -mercaptoethanol;

the concentration of the supplemented noggin in the step (2) is 300-1000 ng/mL, and the concentration of the supplemented TGF- β 1 receptor inhibitor is 2.5-15 mu M;

the differentiation medium A in the step (2): a basal medium DMEM/F12 culture medium added with 1 XN 2 additive, 10-20 ng/mL basic fibroblast growth factor, 1 Xnon-essential amino acid and 2mM GlutaMAX^TMA culture medium and an additive, 100 units/mL of a cyan-chain double antibody, and 2-10 ng/mL of recombinant human insulin;

the differentiation medium C in the step (3): the basic culture medium is prepared from DMEM/F12 and M199 at a ratio of 1-1.5: 1, and 5-20% of fetal bovine serum, 1 × non-essential amino acids and 2mM GlutaMAX^TMCulture medium and additive, 100 units/mL of cyan-streptomycin cyan chain double antibody, 2-10 ng/mL of recombinant human insulin, 20-50 mg/mL of levorotatory vitamin C and 10-20 ng/mL of basic fibroblast growth factor.

2. The method for obtaining corneal endothelial cells according to claim 1, wherein: the concentration of the chondroitin sulfate coated by the culture vessel in the step (3) is 10-50 mg/mL; the concentration of laminin is 1-10 mug/mL.

3. The method for obtaining corneal endothelial cells according to claim 1, wherein: carrying out immunofluorescence staining on the intermediate cell markers HNK-1 and P75 obtained by pre-differentiation culture to show positive expression; the obtained markers of the corneal endothelial cells, namely sodium-potassium-ATPase, S100A4 protein, aquaporin-1, namely AQP-1 and zonulin ZO-1, are positively expressed by immunostaining.

Images