阅读说明：本技术 体外诱导类角膜缘干细胞的方法及试剂盒 (Method and kit for inducing limbal stem cells in vitro ) 是由 孙长斌 张曦 于 2020-05-27 设计创作，主要内容包括：本发明提供了一种体外诱导类角膜缘干细胞的方法及试剂盒。所提供的方法包括(1)利用第一诱导剂对多能干细胞进行第一培养,获得表面外胚层细胞,第一诱导剂包括第一小分子抑制剂和第一蛋白,第一小分子抑制剂包括TGF-beta/Smad抑制剂,第一蛋白包括bFGF或BMP4；(2)利用第二诱导剂对表面外胚层细胞进行第二培养,以便获得类角膜缘干细胞,第二诱导剂包括第二蛋白,第二蛋白包括BMP4。所提供的试剂盒包括小分子抑制剂、蛋白和培养基,小分子抑制剂包括SB505124或SB431542；蛋白包括bFGF或BMP4。利用该方法或试剂盒能够快速获得类角膜缘干细胞,而且稳定性好。(The invention provides a method and a kit for inducing limbal stem cells in vitro. The provided methods include (1) subjecting pluripotent stem cells to a first culture using a first inducer to obtain surface ectodermal cells, the first inducer including a first small molecule inhibitor including a TGF-beta/Smad inhibitor and a first protein including bFGF or BMP 4; (2) performing a second culture on the surface ectodermal cells using a second inducing agent to obtain limbal-like stem cells, the second inducing agent comprising a second protein, the second protein comprising BMP 4. The provided kit comprises a small molecule inhibitor, a protein and a culture medium, wherein the small molecule inhibitor comprises SB505124 or SB 431542; the protein comprises bFGF or BMP 4. The method or the kit can be used for quickly obtaining the limbal-like stem cells and has good stability.)

1. A method of inducing limbal-like stem cells in vitro comprising:

(1) subjecting pluripotent stem cells to a first culture using a first inducing agent comprising a first small molecule inhibitor comprising a TGF-beta/Smad inhibitor and a first protein comprising at least one selected from bFGF or BMP4, to obtain surface ectodermal cells;

(2) performing a second culture of the surface ectodermal cells using a second inducing agent to obtain limbal-like stem cells, the second inducing agent comprising a second protein comprising BMP 4.

2. The method of claim 1, wherein the TGF-beta/Smad inhibitor comprises at least one selected from SB505124 or SB 431542.

3. The method of claim 1, wherein step (1) further comprises:

(1-1) subjecting the pluripotent stem cells to a first induction culture using a first surface ectoderm induction medium comprising a first small molecule inhibitor and bFGF;

(1-2) replacing a second surface ectoderm induction medium containing a first small molecule inhibitor and BMP4 to perform a second induction culture on the product of step (1-1) to obtain the surface ectoderm cells.

4. The method of claim 1, wherein step (2) further comprises:

(2-1) subjecting the surface ectodermal cells to a third induction culture using a first limbal stem cell induction medium comprising BMP 4;

(2-2) replacement of the second limbal stem cell-inducing medium without BMP4 the product of step (2-1) is subjected to a fourth induction culture to obtain the limbal stem cell-like cells.

5. The method of claim 3, wherein the first superficial ectoderm induction medium comprises at least one selected from DMEM/F12 medium or E6 medium, further comprising at least one selected from fetal bovine serum, platelet lysate, or substitutes;

optionally, the second surface ectoderm-inducing medium comprises at least one selected from DMEM/F12 medium or E6 medium, further comprising at least one selected from fetal bovine serum, platelet lysate, or a substitute.

6. The method of claim 4, wherein the first limbal stem cell induction medium comprises at least one selected from the group consisting of DMEM/F12 medium or corneal epithelial medium, further comprising at least one selected from the group consisting of fetal bovine serum, platelet lysate, or a substitute;

optionally, the second limbal stem cell induction medium comprises at least one selected from DMEM/F12 medium or corneal epithelial medium, further comprising at least one selected from fetal bovine serum, platelet lysate, or a substitute.

7. The method according to claim 3, wherein the first small molecule inducer is used at a concentration of 5 to 15 μ M in the first induction culture and 0 to 15 μ M in the second induction culture.

8. The method according to claim 1, wherein the first protein is used at a concentration of 5 to 50 ng/mL;

optionally, the second protein is used at a concentration of 5-15 ng/mL.

9. The method of claim 3, further comprising: culturing pluripotent stem cells to a confluency of 60-80%, and performing first induction culture on the pluripotent stem cells by using a first surface ectoderm induction culture medium containing a first small molecule inhibitor and bFGF for 1-2 days;

optionally, the culture time of the second induction culture is 1-3 days;

optionally, the pluripotent stem cells are cultured with a feeder-free cell culture medium comprising at least one selected from mTeSR media, E8 media;

optionally, the pluripotent stem cells comprise embryonic stem cells or induced pluripotent stem cells;

optionally, prior to the first culturing of the pluripotent stem cells with the first inducing agent, further comprising:

coating the culture dish with a first Matrigel, wherein the first Matrigel comprises at least one selected from Matrigel and Vitronectin Matrigel;

optionally, the concentration of the Matrigel is 8-12 mg/mL;

optionally, the concentration of the Vitronectin matrigel is 0.35-0.75 μ g/mL;

optionally, prior to the second culturing of the surface ectodermal cells with the second inducing agent, further comprising:

coating the culture dish with a second matrigel, wherein the second matrigel comprises at least one selected from type IV collagen and laminin;

preferably, the method further comprises culturing the surface ectoderm cells inoculated to the coated culture dish in a second surface ectoderm induction medium containing Y27632 for 10-12 hours.

10. A kit for inducing limbal-like stem cells in vitro comprising:

a small molecule inhibitor comprising a TGF-beta/Smad inhibitor;

a protein comprising at least one member selected from the group consisting of bFGF, BMP 4; and

a culture medium;

optionally, the TGF-beta/Smad inhibitor comprises at least one selected from SB505124 or SB 431542;

optionally, the culture medium comprises at least one selected from DMEM/F12 medium, E6 medium, corneal epithelial medium, further comprises at least one selected from fetal bovine serum, platelet lysate, or substitutes;

optionally, the kit further comprises:

a petri dish coated with Matrigel comprising at least one selected from Matrigel, Vitronectin Matrigel, type IV collagen, laminin.

Technical Field

The invention relates to the technical field of biology, in particular to a method and a kit for inducing limbal stem cells in vitro.

Background

The limbal stem cells are located in the limbal tissue at the junction of the cornea and sclera, have a strong proliferative capacity and are effective in corneal regeneration and repair. Patients with global visual impairment are estimated to be 4 million people, 4 million of them are blind. Among these people, the number of blindness caused by corneal (cornea) dysfunction is about 1 million or more, and 1 million or more new blindness patients are newly added every year. In addition, the corneal Limbal Stem Cell Deficiency (LSCD) occurs due to the abnormal functioning of the limbal function caused by genetic, chemical, burn, infection, etc., and the normal corneal epithelium homeostasis is destroyed, which is one of the important causes of poor vision and blindness. At present, the treatment modes of LSCD include autologous or allogeneic corneal limbus transplantation, transplantation of LSCs cultured in vitro, transplantation of oral mucosal cells cultured in vitro, autologous or allogeneic conjunctival transplantation, and the like. Although the limbal transplantation has a certain curative effect, the success rate of the operation is often influenced by the shortage of donor materials, chronic inflammation and immune rejection, and the risk of corneal exfoliation, infection and the like exists in the acquisition of autologous limbal tissue. And for bilateral LSCD patients, autologous limbal tissue is not available.

The cornea develops from the surface ectoderm, and the small molecular compound can simulate the development process by regulating extracellular signal channels, induce the development of the pluripotent stem cells to the surface ectoderm and further form the limbal stem cells. 2014, Mikhalivova et al developed a limbal stem cell small molecule induction technology, and utilized small molecule inhibitors to inhibit TGF-b and Wnt signaling pathways, simulating the early developmental stage of the cornea. The cells obtained express typical corneal markers that can be separated into two distinct layers, only the top layer expressing the K12 gene. This study describes for the first time the efficient induction of LSC by small molecules under serum-free, non-trophoblast conditions. Recently, the research team reported improved techniques for this approach, further utilizing animal-derived, serum-free methods for induction of LSC. The method for inducing the pluripotent stem cells to be differentiated into the LSC by using the small molecular compounds is favorable for the stability of the induction technology, does not add serum and feeder cells, improves the safety, and is bound to become the main direction for the research of the in vitro induction technology of the pluripotent stem cells. Although there are some progress in inducing with small molecule substances, there are few reports, and further improvement is needed to obtain limbal stem cells in vitro using small molecule compounds for corneal regeneration and repair.

Disclosure of Invention

The present invention solves at least one of the technical problems of the prior art to at least a certain extent. Therefore, the invention provides a method and a kit for stably obtaining the limbal-like stem cells in vitro.

Pluripotent Stem Cells (PSC) have pluripotency and unlimited expansion capacity and are capable of inducing the formation of limbal stem cells in vitro. Although methods for inducing pluripotent stem cells into limbal stem cells have been developed in recent years, there are still many challenges to the formation of limbal stem cells induced in vitro by pluripotent stem cells. For example, induction methods involve the use of materials or agents that require trophoblast cells, undefined components, such as conditioned medium, PA6 trophoblast cells, Bowman's membrane or amniotic membrane, and the like. The trophoblast is easy to introduce into pathogens of animal origin, and the type, passage number and growth state of the trophoblast have a large influence on the induction efficiency, while there may be large differences between batches of materials or reagents with undefined compositions, which influence the stability and reproducibility of the induction technology. In addition, some methods do not report whether the induced cells can be further differentiated into mature corneal epithelial cells or the differentiation efficiency is very low.

The small molecule induction technology is an important basis established by animal-origin-free in vitro induction technology, utilizes a small molecule inducer to regulate extracellular signal paths, simulates the development process of cells, efficiently induces the differentiation of pluripotent stem cells to myocardial cells, nerve cells and other cell types, and is an animal-origin-free in vitro induction technology. The inventor of the invention combines the existing basis of the Limbal Stem Cell (LSC) development and in vitro induction technology and the LSC development approach to establish a stable and efficient LSC induction technology, and in vitro proves that the limbal stem cell-like can be obtained by pluripotent stem cells. To this end, the present invention provides a method for inducing limbal-like stem cells in vitro.

Specifically, the invention provides the following technical scheme:

in a first aspect of the invention, the invention provides a method of inducing limbal-like stem cells in vitro comprising: (1) subjecting pluripotent stem cells to a first culture using a first inducing agent comprising a first small molecule inhibitor comprising a TGF-beta/Smad inhibitor and a first protein comprising at least one selected from bFGF or BMP4, to obtain surface ectodermal cells; (2) performing a second culture of the surface ectodermal cells using a second inducing agent to obtain limbal-like stem cells, the second inducing agent comprising a second protein comprising BMP 4.

The present invention can obtain a limbal-like stem cell with stable performance by two-step induction, i.e., induction of surface ectodermal cells and induction of limbal-like stem cells, for example, the obtained limbal-like stem cell has stable expression of p63 gene.

According to an embodiment of the present invention, the above method for inducing limbal-like stem cells in vitro may further comprise the following technical features:

according to the embodiment of the invention, under the action of the first small molecule inhibitor, TGF-beta signals of the pluripotent stem cells are inhibited, and the differentiation of the pluripotent stem cells to specific cell types, namely surface ectodermal cells, is promoted. According to a particular embodiment of the invention, the TGF-beta/Smad inhibitor comprises at least one selected from SB505124 or SB 431542.

According to an embodiment of the present invention, the step (1) further comprises:

(1-1) subjecting the pluripotent stem cells to a first induction culture using a first surface ectoderm induction medium comprising a first small molecule inhibitor and bFGF;

(1-2) replacing a second surface ectoderm induction medium containing a first small molecule inhibitor and BMP4 to perform a second induction culture on the product of step (1-1) to obtain the surface ectoderm cells.

In the first culture according to the embodiment of the invention, bFGF is adopted in the first induction culture stage to stimulate the activation of FGF signal pathways, BMP4 is adopted in the second induction culture stage to stimulate the activation of Bone Morphogenetic Protein (BMP) signal pathways, and specific proteins are further adopted in different development stages of the pluripotent stem cells to stimulate the activation of corresponding different signal pathways, so that the efficient obtaining of surface ectodermal cells is realized.

According to an embodiment of the present invention, the step (2) further comprises:

(2-1) subjecting the surface ectodermal cells to a third induction culture using a first limbal stem cell induction medium comprising BMP 4;

(2-2) replacement of the second limbal stem cell-inducing medium without BMP4 the product of step (2-1) is subjected to a fourth induction culture to obtain the limbal stem cell-like cells.

In the second culture according to the example of the present invention, BMP4 was used in the third induction culture stage and the culture medium containing no BMP4 was replaced in the fourth induction culture stage to attenuate the stimulation of BMP4 signaling pathway.

According to an embodiment of the invention, the first surface ectoderm-inducing medium comprises at least one selected from DMEM/F12 medium or E6 medium, further comprising at least one selected from fetal bovine serum, platelet lysate or surrogate. It should be noted that the DMEM/F12 medium described herein refers to DMEM and F12 medium in a ratio of 1: 1, which can be obtained by direct purchase, or by self-mixing the DMEM medium with the F12 medium.

According to an embodiment of the invention, the second surface ectoderm-inducing medium comprises at least one selected from DMEM/F12 medium or E6 medium, further comprising at least one selected from fetal bovine serum, platelet lysate or surrogate.

According to an embodiment of the present invention, the first limbal stem cell induction medium comprises at least one selected from DMEM/F12 medium or corneal epithelial medium, further comprises at least one selected from fetal bovine serum, platelet lysate or substitutes;

according to an embodiment of the present invention, the second limbal stem cell induction medium comprises at least one selected from DMEM/F12 medium or corneal epithelial medium, further comprising at least one selected from fetal bovine serum, platelet lysate or substitutes.

According to an embodiment of the present invention, the first small molecule inducer is used at a concentration of 5 to 15 μ M in the first induction culture, and the first small molecule inducer is used at a concentration of 0 to 15 μ M in the second induction culture.

According to the embodiment of the invention, the first protein is used at a concentration of 5-50 ng/mL. Further, the efficiency of induction of surface ectodermal cells was further improved.

According to the embodiment of the invention, the second protein is used at a concentration of 5-15 ng/mL. Further improving the induction efficiency of the limbal-like stem cells.

According to an embodiment of the invention, the method further comprises: culturing the pluripotent stem cells to a confluency of 60-80%, and performing first induction culture on the pluripotent stem cells by using a first surface ectoderm induction culture medium containing a first small molecule inhibitor and bFGF, wherein the culture time is 1-2 days.

According to the embodiment of the invention, the culture time of the second induction culture is 1-3 days.

According to an embodiment of the invention, the pluripotent stem cells are cultured using a feeder-free cell culture medium comprising at least one selected from mTeSR culture medium, E8 culture medium.

According to an embodiment of the present invention, before the first culturing of the pluripotent stem cells with the first inducing agent, the method further comprises: coating the culture dish with a first Matrigel including at least one selected from Matrigel and Vitronectin Matrigel. Thereby creating a microenvironment suitable for the differentiation of the pluripotent stem cells to specific cell-surface ectodermal cells.

According to the specific embodiment of the invention, the concentration of the Matrigel is 8-12 mg/mL.

According to the specific embodiment of the invention, the concentration of the Vitronectin matrigel is 0.35-0.75 mu g/mL.

According to an embodiment of the invention, before the second culturing of the surface ectodermal cells with the second inducer, further comprises: coating the culture dish with a second matrigel comprising at least one selected from the group consisting of type IV collagen, laminin. The inventor finds that the culture medium coated by the second matrigel can effectively promote the attachment and growth of surface ectodermal cells, provides a microenvironment for further inducing development of the surface ectodermal cells, and further improves the efficiency of inducing the surface ectodermal cells into the limbal stem cells.

In a second aspect of the present invention, the present invention provides a kit for inducing limbal stem cells in vitro, comprising:

a small molecule inhibitor comprising a TGF-beta/Smad inhibitor;

a protein comprising at least one member selected from the group consisting of bFGF, BMP 4; and

and (4) a culture medium.

According to an embodiment of the present invention, the kit described above may further comprise the following technical features:

according to an embodiment of the invention, the TGF-beta/Smad inhibitor comprises at least one selected from SB505124 or SB 431542.

According to an embodiment of the present invention, the medium comprises at least one selected from DMEM/F12 medium, E6 medium, corneal epithelial medium, further comprises at least one selected from fetal bovine serum, platelet lysate or substitutes.

According to an embodiment of the invention, the kit further comprises: a petri dish coated with Matrigel comprising at least one selected from Matrigel, Vitronectin Matrigel, type IV collagen, laminin.

The beneficial effects obtained by the invention are as follows: the method or the kit provided by the invention is used for in vitro induction of the limbal-like stem cells, and the limbal-like stem cells with excellent performance can be obtained among different pluripotent stem cell lines by a two-step induction method, and the method or the kit has the advantages of high efficiency and good stability.

Drawings

FIG. 1 is a microscopic observation of embryonic stem cell H9 provided according to an embodiment of the present invention.

FIG. 2 is a graph showing the expression profiles of TFAP2A, SOX1, and PAX6 genes in ectodermal cells, provided in accordance with an example of the present invention.

FIG. 3 is a microscopic observation of the differentiated limbal-like stem cells from pluripotent stem cells provided in accordance with an embodiment of the invention.

Fig. 4 shows the flow analysis result of the limbal-like stem cells p63 differentiated from H9 cells according to the present invention.

FIG. 5 shows the results of qRT-PCR of KRT3 and KRT12 genes provided by the present invention.

FIG. 6 is a graph showing the results of microscopic observation of different pluripotent stem cells provided according to an embodiment of the present invention.

FIG. 7 shows the results of flow analysis of limbal-like stem cells differentiated using different pluripotent stem cells, according to an embodiment of the invention.

FIG. 8 shows the flow analysis results of different matrigel and combined differentiation to obtain limbal-like stem cells p63 according to the embodiment of the present invention.

Detailed Description

The following detailed description of embodiments of the invention is intended to be illustrative, and is not to be construed as limiting the invention. While certain terms have been explained and illustrated herein to facilitate understanding by those skilled in the art, it is to be understood that such explanations and illustrations are provided for convenience and should not be construed as limiting the scope of the invention.

As used herein, reference to pluripotent stem cells refers to stem cells that have pluripotency and unlimited expansion capacity. Pluripotent stem cells include Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs).

As used herein, the term "limbal stem cell-like" refers to a cell that is characteristic of a limbal stem cell and is obtained in vitro by human intervention, such as a cell that expresses the p63 gene and functions to differentiate into corneal cells. The limbal stem cells obtained in the present application are distinguished from limbal stem cells produced by humans themselves without any intervention, and are termed limbal-like stem cells.

The invention establishes a method for inducing the limbal stem cells in vitro by utilizing the pluripotency of the pluripotent stem cells, and the method can be used as one of approaches for the source of the limbal stem cells, thereby providing a technical basis for the induced differentiation of clinical-grade limbal stem cells.

In one aspect of the invention, the invention provides a method of inducing limbal-like stem cells in vitro. According to an embodiment of the invention, there is provided a method comprising:

(1) subjecting pluripotent stem cells to a first culture using a first inducing agent comprising a first small molecule inhibitor comprising a TGF-beta/Smad inhibitor and a first protein comprising at least one selected from bFGF or BMP4, to obtain surface ectodermal cells;

(2) performing a second culture of the surface ectodermal cells using a second inducing agent to obtain limbal-like stem cells, the second inducing agent comprising a second protein comprising BMP 4.

The method for inducing the limbal-like stem cells in vitro provided by the invention obtains the limbal-like stem cells with excellent performance by two-step induction, wherein the two steps respectively comprise the induction of surface ectodermal cells and the induction of the limbal-like stem cells.

According to an embodiment of the invention, the TGF-beta/Smad inhibitor comprises at least one selected from SB505124 or SB 431542. And further selectively inhibit TGF-beta I type receptors, and activin receptor-like kinases (ALK)4, 5 and 7.

In at least some embodiments of the invention, step (1) further comprises: (1-1) subjecting the pluripotent stem cells to a first induction culture using a first surface ectoderm induction medium comprising a first small molecule inhibitor and bFGF; (1-2) replacing a second surface ectoderm induction medium containing a first small molecule inhibitor and BMP4 to perform a second induction culture on the product of step (1-1) to obtain the surface ectoderm cells.

The first surface ectoderm induction medium includes but is not limited to: the basic medium is DMEM (Dulbecco's Medium) containing 10% fetal bovine serum, platelet lysate or serum replacementF12 Medium (purchased from KnockOut)^TMSerum Replacement or purchased from thermofisherscientific) or well-defined E6 medium.

The first small molecule inhibitor can be adjusted to a suitable use concentration. In at least some embodiments of the invention, the SB505124 or SB431542 is used at a concentration of 5-15 μ M and the bFGF is used at a concentration of 5-50 ng/mL.

The second surface ectoderm induction medium includes but is not limited to: the basal medium was DMEM/F12 medium (purchased from KnockOut) containing 10% fetal bovine serum, platelet lysate or serum replacement^TMSerum Replacement or purchased from thermofisherscientific) or well-defined E6 medium.

The BMP4 is used at a concentration of 5-50 ng/mL, and the SB505124 or SB431542 is used at a concentration of 0-15 μ M.

In the process of inducing and obtaining surface ectodermal cells, the pluripotent stem cells are first cultured, for example, in a feeder-free medium. Feeder-free media that may be used include, but are not limited to: the basal medium is a commercially available mTeSR medium or a well-defined E8 medium. When the culture dish is used for culturing the pluripotent stem cells, the culture dish can be covered by Matrigel or Vitronectin Matrigel in advance, so that a microenvironment suitable for the differentiation of the pluripotent stem cells to specific cell-surface ectodermal cells is created. According to the embodiment of the invention, the culture dish can be covered with Matrigel with the concentration of 8-12 mg/mL or Vitronectin Matrigel with the concentration of 0.35-0.75 mu g/mL.

Then, the cultured pluripotent stem cells are used for induction to obtain surface ectodermal cells. According to the embodiment of the present invention, when pluripotent stem cells are cultured to a confluency of 60 to 80%, the first surface ectoderm-inducing medium is added and the cells are cultured for 1 to 2 days, and then the cells are further cultured for 1 to 3 days by replacing the cells with the second surface ectoderm-inducing medium.

The obtained surface ectodermal cells can be used for obtaining the limbal-like stem cells through induction. Prior to induction, a petri dish matrigel coating treatment may be performed. When the culture dish is coated with matrigel, the utility model can be usedMethods commonly used in the art. For example, a culture dish may be covered with matrigel and left at room temperature for 2 hours or more before inoculation. Useful matrigel include, but are not limited to: type IV collagen, laminin, or a combination of these matrigels. According to the embodiment of the invention, the IV type collagen is used at the concentration of 3-10 mu g/cm²The using concentration of laminin is 0.5-10 mu g/cm²。

The previously obtained surface ectodermal cells were then digested and seeded into matrigel coated petri dishes as described above. For example, commercially available Accutase (INVOVIVE CELL, AT104-500) or TrypLE can be used^TMThe induced surface ectoderm cells were digested with cell digest at 37 ℃ to form a single cell suspension, the cells were collected by centrifugation, plated on a matrigel-coated petri dish, and then cultured for 12 hours with the addition of a second surface ectoderm induction medium containing 10 μ M Y27632. The inventor finds that Y27632 can be used as a ROCK inhibitor to promote the survival rate of cells after passage.

Limbal-like stem cells are then induced to be obtained. According to the embodiment of the invention, the culture medium is replaced by the first limbal stem cell induction culture medium for 2-3 days, the culture medium is replaced by liquid every day, and then the culture medium is replaced by the second limbal stem cell induction culture medium for 2-3 days.

The first type of limbal stem cell induction culture Medium comprises a base culture Medium which is a DMEM/F12 culture Medium or a commercially available Corneal epithelial culture Medium (Corneal Epithelium Medium, purchased from CELLNTEC and having a product number of CnT-30) added with 0-10% of fetal bovine serum, platelet lysate or serum substitute, and also comprises an inducer BMP 4. The concentration of the BMP4 inducer is 5-15 ng/mL.

The basal medium of the second limbal stem cell induction medium is a corneal epithelium medium added with 0-10% of fetal bovine serum, platelet lysate or serum substitute.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1H 9 Induction of embryonic Stem cells into limbal Stem cells

(1) Culture of H9 cells

The H9 cell line was cultured in a petri dish covered with 10mg/mL Matrigel (see FIG. 1), and the medium was mTeSR medium. Among them, Matrigel was purchased from BD company (cat # 354234), and mTeSR medium was purchased from Stemcell Technologies (cat # 05850).

(2) Induction of surface ectodermal cells

H9 culture to about 70% confluency, the first surface ectoderm induction medium was replaced with DMEM/F12 medium (purchased from ThermoFisher scientific, Cat.: 10565-018) containing 10% fetal bovine serum, 10. mu.M SB431542 and 20ng/mL bFGF were added, and 5% CO was added at 37 ℃ and 5% CO₂The culture was carried out in a carbon dioxide incubator for 1 day.

Then, the medium was changed to a second surface ectoderm-inducing medium consisting of DMEM/F12 medium containing 10% fetal bovine serum, 5. mu.M SB431542 and 25ng/mL BMP4 were added, and 5% CO was added at 37 ℃ to the medium₂The culture was carried out in a carbon dioxide incubator for 2 days. SB431542 was purchased from ABCAM (cat # ab120163), bFGF was purchased from ThermoFisher (cat # 13256-&D (cargo number: 314-BP-050).

(3) Culture dish coating

Adding the IV type collagen dry powder into a DPBS solution (Dulbecco's Phosphate Buffered Saline) containing 0.25% glacial acetic acid to prepare a solution with the concentration of 1 mg/mL; dissolving at 2-8 ℃, and shaking occasionally; calculating the appropriate dilution concentration according to the basal area concentration to form 5 μ g/cm²The concentration of coverage. Type IV collagen was purchased from Sigma (cat # C5533-5 MG).

(4) Digestion and inoculation of surface ectodermal cells

Using TrypLE^TMDigesting the ectodermal cells induced in step (2), digesting at 37 ℃ for 10 minutes, gently blowing the cells to form a single cell suspension, collecting the cells by centrifugation, and resuspending the cells in a second surface ectodermal induction medium containing 10. mu. M Y27632 at 2X 10⁵Cells/cm²Was inoculated into the coated dish of (3), and a second surface ectoderm-inducing medium was added thereto to continue the culture for 12 hours. Wherein TrypLE^TMPurchased from ThermoFisher under item number 12604013, and Y27632 purchased from Sigma under item number Y0503-5 MG.

Meanwhile, single cell sequencing is used for detection, and the single cell sequencing result shows that most of cells do not express the human pluripotent stem cell core transcription factors POU5F1, SOX2 and NANOG, and simultaneously accord with the expression characteristics of surface ectodermal cells (TFAP2A + SOX1-PAX6-SOX10-) (as shown in figure 2, wherein the abscissa in figure 2 represents the identity of the gene, each point represents one cell, and the ordinate represents the expression amount (unit: UMI) of the gene in each cell).

(5) Induction of limbal stem cells

The culture medium was continuously changed to the first limbal stem cell induction medium, which was composed of DMEM/F12 medium supplemented with 10% fetal bovine serum and BMP4 supplemented with 10ng/mL inducer, and was cultured for 2 days with daily change of the medium. Then, the culture medium was changed to a second limbal stem cell induction medium, which was a corneal epithelial medium, and the culture was changed every 2 to 3 days, and the cells were cultured for 15 days, collected and subjected to flow analysis, and the cells formed a distinct polygonal shape (as shown in fig. 3). Wherein the corneal epithelial medium was purchased from Cellntec corporation.

(6) Flow analysis

The P63 protein serves as a marker of the limbal-like stem cells and can be used to indicate the limbal-like stem cells. Induced cells were flow analyzed using anti-p 63 antibody. The p63 antibody was purchased from abcam (cat # ab 124762). The results are shown in FIG. 4.

(7) Differentiation into corneal epithelial cells

Epithelial cell stratification experiments are used for determining the differentiation effect of the epithelial cells to the cornea, and qRT-PCR results shown in figure 5 show that the expression levels of the differentiated cells KRT3 and KRT12 are obviously improved.

Example 2 Induction of limbal Stem cells by different pluripotent Stem cell lines

Example 2 the induction of limbal stem cells by different pluripotent stem cells was studied, wherein embryonic stem cell lines H1, H7 and induced pluripotent stem cell line 201B7 (from ATCC under the ACS-1023) were used, and the culture method was modified slightly according to example 1, wherein the culture medium used was changed from mTeSR medium to E8 medium (STEMCELL under the No. 05940) and the culture dishes were coated with Vitronectin (Vitronectin under the ThermoFisher scientific under the No. A14700). In addition, the basal medium composition in the first surface ectoderm-inducing medium, the second surface ectoderm-inducing medium, the first limbal stem cell-inducing medium, or the second limbal single cell medium was changed to E6 medium (purchased from ThermoFisher scientific, cat # A1517001), the inducer was changed from SB431542 to SB505124 (purchased from SIGMA, cat # S4696-5MG), and the others were unchanged.

The results of microscopic observation of the culture of different pluripotent stem cells using the E8 medium are shown in FIG. 6.

Flow analysis shows that the several cell lines induce p63 cells, and compared with example 1, there is no obvious difference, and the cell yield rates all show better consistency as shown in figure (7).

Example 3 comparison of Induction into limbal Stem cells by different matrigels and combinations

Example 3 the effect of the matrigel class in the dishes on limbal stem cell induction prior to induction of limbal stem cells by surface ectodermal cells was studied. The cell line used was H9, the specific implementation method was the same as example 1, and three different matrigel experiments were set up, which were:

type IV collagen (Col IV), used at a concentration of 5. mu.g/mL of type IV collagen,

laminin (LN521) at a concentration of 2. mu.g/mL,

the combination of type IV collagen (Col IV) and laminin (LN521) at concentrations of 2.5 μ g/mL and 1 μ g/mL, respectively.

P63 cells obtained from three different matrigel experiments are shown in FIG. 8. The experimental results show that no matter the type IV collagen, the laminin or the mixture of the type IV collagen and the laminin are adopted as the matrigel, the P63 cell obtained has no obvious influence.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.