Method and kit for in-vitro induction of cornea-like stem cells

A technology of limbal stem cells and inducers, which is applied in the field of a method and kit for inducing limbal stem cells in vitro, can solve many problems, and achieve high efficiency and good stability

Pending Publication Date: 2021-12-03
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the method of using small molecular substances to induce has made some progress, there are not many reports so far. How to use small molecular compounds to obtain corneal limbal stem cells in vitro for corneal regeneration and repair needs further improvement

Method used

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  • Method and kit for in-vitro induction of cornea-like stem cells
  • Method and kit for in-vitro induction of cornea-like stem cells
  • Method and kit for in-vitro induction of cornea-like stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Induction of H9 embryonic stem cells into limbal stem cells

[0074] (1) Culture of H9 cells

[0075] The H9 cell line was cultured in a petri dish covered with 10 mg / mL Matrigel matrigel (such as figure 1 ), the medium is mTeSR medium. Among them, Matrigel was purchased from BD Company (product number: 354234), and mTeSR medium was purchased from Stemcell Technologies (product number: 05850).

[0076] (2) Induction of surface ectoderm cells

[0077] When H9 is cultivated to a confluence of about 70%, the replacement medium is the first surface ectoderm induction medium, and the composition of the first surface ectoderm induction medium is DMEM / F12 medium containing 10% fetal bovine serum (purchased (from ThermoFisherScientic, product number: 10565-018), add 10 μ M SB431542 and 20 ng / mL bFGF, at 37 ° C, 5% CO 2 cultured in a carbon dioxide incubator for 1 day.

[0078] Subsequent replacement medium was the second surface ectoderm induction medium, the com...

Embodiment 2

[0090] Example 2 Induction of different pluripotent stem cell lines to limbal stem cells

[0091] Example 2 studied the induction of different pluripotent stem cells to limbal stem cells, wherein the pluripotent stem cells used were embryonic stem cell lines H1, H7 and induced pluripotent cell line 201B7 (purchased from ATCC, product number is ACS-1023), The culture method was slightly adjusted according to Example 1, wherein when culturing pluripotent stem cells, the medium used was changed from mTeSR medium to E8 medium (purchased from STEMCELL, product number 05940), and Vitronectin (Bolian Protein, purchased from ThermoFisher Scientific, product number: A14700) was used to coat the petri dish. In addition, whether it is the first surface ectoderm induction medium, the second surface ectoderm induction medium, the first corneal limbal stem cell induction medium or the second corneal limbal single cell medium, the basal medium components are changed to E6 culture base (purc...

Embodiment 3

[0094] Example 3 Comparison of Induction of Different Matrigels and Combinations to Limbal Stem Cells

[0095] Example 3 studied the effect of the type of matrigel in the culture dish on the induction of limbal stem cells before the induction from surface ectoderm cells to limbal stem cells. Cell line used is H9, and concrete implementation method is identical with embodiment 1, sets up three groups of different Matrigel experiments, is respectively:

[0096] Type IV collagen (Col IV), the use concentration is respectively type IV collagen 5μg / mL,

[0097] Laminin (LN521), the concentration of laminin is 2 μg / mL,

[0098] Collagen type IV (Col IV) and laminin (LN521) were combined, and the concentrations of collagen type IV (Col IV) and laminin (LN521) were 2.5 μg / mL and 1 μg / mL, respectively.

[0099] The p63 cells obtained from three different matrigel experiments were as follows: Figure 8 shown. The experimental results showed that whether type IV collagen, laminin or ...

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Abstract

The invention provides a method and kit for in-vitro induction of cornea-like stem cells. The provided method comprises: (1) performing first culture on pluripotent stem cells by using a first inducer to obtain surface ectoderm cells, wherein the first inducer comprises a first small molecule inhibitor and a first protein, the first small molecule inhibitor comprises a TGF-beta / Smad inhibitor, and the first protein comprising bFGF or BMP4; and (2) performing second culture on the surface ectoderm cells by using a second inducer so as to obtain the cornea-like stem cells, wherein the second inducer comprises a second protein, and the second protein comprises BMP4. The kit provided by the invention comprises the small molecule inhibitor, protein and a culture medium, wherein the small molecule inhibitor comprises SB505124 or SB431542, and the protein comprises bFGF or BMP4. By using the method or the kit, the cornea-like stem cells can be rapidly obtained, and the stability is good.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and kit for inducing limbal stem cells in vitro. Background technique [0002] Limbal stem cells are located in the limbal tissue at the junction of the cornea and sclera. They have a strong ability to proliferate and can effectively regenerate and repair the cornea. There are an estimated 400 million people with visual impairment worldwide, of whom more than 40 million are blind. Among these people, the number of blindness caused by corneal (cornea) dysfunction is about 10 million, and more than 1 million new blind patients are added every year. In addition, due to genetic, chemical, burn, infection and other reasons, the function of the corneal limbus cannot be performed normally, resulting in the occurrence of limbal stem cell deficiency (LSCD), which destroys the normal corneal epithelial homeostasis. One of the important causes of poor vision and blindness. At presen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12N5/079
CPCC12N5/0623C12N5/0621C12N2501/15C12N2501/115C12N2501/155C12N2506/02C12N2506/45C12N2533/90C12N2533/54C12N2533/52
Inventor 孙长斌张曦
Owner SHENZHEN HUADA GENE INST
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