Method for preparing retinal progenitor cells derived from human pluripotent stem cells

A technology of human pluripotent stem cells and precursor cells, applied in cell dissociation methods, biochemical equipment and methods, animal cells, etc., can solve problems such as degeneration and damage of retinal functional neuron cells, and achieve single cell types and strong proliferation effect of ability

Active Publication Date: 2020-05-01
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, RPE is only a support cell in the retina. In addition to RPE damage, retinal degenerative diseases also have degeneration and damage of retinal functional neurons, which require supplementation and replacement.

Method used

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  • Method for preparing retinal progenitor cells derived from human pluripotent stem cells
  • Method for preparing retinal progenitor cells derived from human pluripotent stem cells
  • Method for preparing retinal progenitor cells derived from human pluripotent stem cells

Examples

Experimental program
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Embodiment 1

[0030] 1. Maintenance of BC1-GFP cells and construction of embryoid bodies

[0031] Materials and Instruments:

[0032] ① Cells: BC1-GFP cells, bone marrow-derived induced pluripotent stem cells.

[0033] ②Reagents and consumables:

[0034] 1) mTeSR1 medium: STEM CELL, #05851, 4°C;

[0035] 2) EDTA: Invitrogen, 15575-038, normal temperature;

[0036] 3) PBS (1×): Gino Biomedical Technology Co., Ltd., 14111202, room temperature;

[0037] 4) Matrigel: Corning, 354277, -20°C;

[0038] 5) (-)-Blebbistatin: Sigma, B0560, -20°C;

[0039] 6) Six-hole plate: FALCON, 353046;

[0040] 7) Centrifuge tube: BD FALCON, 352096.

[0041] ③Instrument:

[0042] 1) CO 2 Incubator: SANYO, MCO-20A1C;

[0043] 2) Inverted microscope: Nikon, TS100.

[0044] Specific steps are as follows:

[0045] BC1-GFP cells were maintained in mTeSR1 medium, and in about 4-5 days the clones could grow to occupy about 80%-90% of the well bottom area. Differentiated cells were scraped off under a light ...

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Abstract

The invention discloses a method for preparing retinal progenitor cells derived from human pluripotent stem cells. The method comprises the following steps: selecting a 3d retinal cup which is subjected to induced differentiation for 50-60 days and derived from the human pluripotent stem cell, removing RPE and other non-3d retinal cup tissue, putting the retinal cup into a container with a liquidmedium, performing mechanical separation, separating and discarding a pigmentary layer of a dark and brown black part in the 3d retinal cup, retaining a nerve layer of a golden yellow part, further digesting the nerve layer into single cells, and putting the single cells into a culture plate which contains a RDM medium and is coated by Matrigel, so as to obtain the retinal progenitor cells. By adopting the method disclosed by the invention, 3d retinal tissue with stable quality can be obtained, retinal progenitor cells of which the number is controllable and the quality is controllable can beobtained, the time that the retinal progenitor cells are obtained can be shortened, obtaining procedures can be simplified, and the obtained retinal progenitor cells have good value increase performance and a capability of re-differentiated to other types of cells of retina.

Description

Technical field: [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method of retinal precursor cells derived from human pluripotent stem cells. Background technique: [0002] At present, the degeneration, damage and necrosis of retinal cells have become one of the important causes of blindness, and the replacement therapy of stem cells has become a new hope for the treatment of this type of retinal disease. With the widespread application of induced pluripotent stem cells (iPS), the ethical and immune rejection issues that originally limited the development of stem cells and regenerative medicine have been well resolved. As early as the 1980s, scientists had induced the stem cells of mice, frogs and other animals to differentiate into retinal ganglion cells and photoreceptors. In the time since then, retinal cells have been induced to differentiate, and many research teams have tried to transplant specific retinal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079
CPCC12N5/0621C12N2501/91C12N2509/00
Inventor 钟秀风谢冰冰彭福华
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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