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Method for inducing human pluripotent stem cell to differentiate into RPE cell

A pluripotent stem cell and cell technology, applied in the field of inducing human pluripotent stem cells to differentiate into RPE cells, can solve the problems of low cell viability and poor treatment effect, and achieve the effect of improving the differentiation efficiency and shortening the differentiation cycle.

Active Publication Date: 2019-01-04
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in clinical research, it was found that the cell viability of RPE cell suspension transplantation was low, and the therapeutic effect was not good.

Method used

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  • Method for inducing human pluripotent stem cell to differentiate into RPE cell
  • Method for inducing human pluripotent stem cell to differentiate into RPE cell
  • Method for inducing human pluripotent stem cell to differentiate into RPE cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Component media induce human pluripotent stem cells to differentiate into RPE cells.

[0060] 1. Neuroepithelial induction: After passage of hPSCs to Matrigel-coated culture dishes, wait for 30 minutes to 1 hour for adherent growth, and then use the component medium supplemented with 2 μM / mL SB431542 and 2 μM / mL DMH1 to culture, and replace the culture every day base. After 6 days in culture, the cells grew to confluence. The fused neural precursor cells were digested and passaged with Accutase, cultured in a component medium supplemented with 50ng / mL Shh, 10ng / mL IGF1, and 20ng / mL EGF, and the medium was replaced every day. After 3-4 days the cells became confluent and were neuroepithelial.

[0061] 2. Optic neuroepithelial induction: After the neuroepithelium is formed, use the component culture medium supplemented with 50ng / mL Shh, 10ng / mL IGF1, 20ng / mL EGF, 10μg / mL Activin A and 2μM / mL Chir99021.

[0062] 3. Naive RPE differentiation: Induce the optic neuroepithe...

Embodiment 2

[0066] Detection of differentiation efficiency during induction of hPSC differentiation into RPE

[0067] 1. Cell fixation: After digesting the cells in the cell culture dish with Accutase enzyme, blow gently with 2mL PBS, transfer to a 15mL centrifuge tube, and centrifuge at 1500rpm for 3 minutes; remove the supernatant after centrifugation, add 1mL 4% paraformaldehyde to weight Hang and fix for 5 minutes.

[0068] 2. Specific antibody labeling: fixed cells were permeabilized with 0.1% TritonX-100 for 10 minutes, centrifuged at 1500rpm for 3 minutes; rinsed with PBS once for 5 minutes; continued centrifuged at 1500rpm for 3 minutes, added 3% BSA to resuspend gently by pipetting, room temperature Incubate for 1 hour; centrifuge at 1500rpm for 3 minutes, add primary antibody diluted with 3% BSA, incubate at room temperature for 1 hour, add IgG with the same antibody concentration as the negative control; centrifuge at 1500rpm for 3 minutes, add 1mL PBS to rinse 3 times, each ti...

Embodiment 3

[0072] Identification of neuroepithelium, optic neuroepithelium, and naive RPE during induction

[0073] 1. Cell sample collection: On the 0th day and the 6th day of differentiation, pass the differentiated cells to the cell slide, induce differentiation according to the steps in Example 1, and on the 3rd day, 6th day, 9th day, 15th day, 18th day Cells were collected, rinsed twice with PBS to remove dead cells, and fixed by adding 4% paraformaldehyde for 5 minutes.

[0074] 2. Permeabilization and sealing: The fixed cells were permeabilized with 0.1% Triton X-100 for 5 minutes, rinsed twice with PBS, 5 minutes each time; added 3% bovine serum albumin to seal at room temperature for 1 hour;

[0075] 3. Antibody staining: Remove the blocking solution, add the primary antibody diluted with 3% bovine serum albumin, incubate overnight at 4°C; remove the primary antibody, rinse with PBS for 3 times, 5 minutes each time; add diluted with 3% BSA fluorescent secondary antibody, incubate...

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Abstract

The invention relates to a method for inducing a human pluripotent stem cell to differentiate into an RPE cell. According to the method, an inhibitor is used for inducing the human pluripotent stem cell to differentiate into a neural precursor; after Shh, IGF1 and EGF growth factors are utilized to activate ERK 1 / 2, JNK, Sonic hedgehog signal routes, the cell is presented as the shape of neuroepithelium, Activen A and Chir99021 are added to activate TGFbeta and Wnt signal routes respectively to induce a neuro-epithelial cell into a retinal progenitor cell; adherent culture is conducted on theretinal progenitor cell to obtain an RPE cell at an underdeveloped state. The invention further studies the field of applying the underdeveloped RPE cell to treating retinal degenerative diseases. Compared with the prior art, the method firstly makes three stages of differentiating the human pluripotent stem cell to the underdeveloped RPE cell and related small molecular substances and growth factors used therein clear. Compared with a matured RPE, the underdeveloped RPE obtained through differentiation can treat the retinal degenerative diseases more effectively after being transplanted.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for inducing human pluripotent stem cells to differentiate into RPE cells. Background technique [0002] Retinal degeneration (RD) disease is a disease characterized by progressive visual function decline, which is hereditary and acquired. It is a common and intractable blinding eye disease in clinical practice. It mainly includes various types of retinitis pigmentosa (RP), Stargardt disease and age-related macular degeneration (age-related macular degeneration, AMD). The pathogenesis of RD is mostly related to the lesions and apoptosis of RPE cells in the eye. The RPE is a polarized monolayer of pigment epithelium between Bruch's membrane and the retina. RPE plays a very important role in the process of vision formation: RPE not only contributes to the formation of the blood-brain barrier, but also completes the exchange of nutrients and metabolites with photorece...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079A61K35/30A61P9/10A61P27/02A61P25/00A61P25/28
CPCA61K35/30A61P9/10A61P25/00A61P25/28A61P27/02C12N5/0621C12N2501/105C12N2501/11C12N2501/15C12N2501/155C12N2506/45
Inventor 徐国彤吕立夏金彩霞田海滨欧庆健
Owner TONGJI UNIV
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