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Method for obtaining analogous retinal tissues rich in cone and rod cells by using human induced pluripotent stem cells

A technology of pluripotent stem cells and rod cells, applied in the field of stem cell regenerative biology, can solve problems such as difficult to meet the needs of clinical treatment, and achieve the effect of promoting clinical transformation application, increasing the risk of cell contamination, and reducing the risk of contamination

Active Publication Date: 2018-11-13
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the current retinal induction scheme is difficult to meet the needs of clinical treatment

Method used

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  • Method for obtaining analogous retinal tissues rich in cone and rod cells by using human induced pluripotent stem cells
  • Method for obtaining analogous retinal tissues rich in cone and rod cells by using human induced pluripotent stem cells
  • Method for obtaining analogous retinal tissues rich in cone and rod cells by using human induced pluripotent stem cells

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Embodiment 1

[0025] 1. Maintenance culture of hiPSCs

[0026] 1. hiPSCs cells: human induced pluripotent stem cells derived from urine, cell line UE017

[0027] 2. Reagents and consumables:

[0028] 1) mTeSR1 medium: STEM CELL, #05851, 4°C

[0029] 2) EDTA: Invitrogen, 15575-038, room temperature

[0030] 3) dispase, sigma, D4693, normal temperature

[0031] 4) PBS (1X): Gino Biomedical Technology Co., Ltd., 14111202, room temperature

[0032] 5) Matrigel: Corning, 354277, -20°C

[0033] 6) Six-hole plate: FALCON, 353046

[0034] 7) Centrifuge tube: BD FALCON, 352096

[0035] 3. Instrument

[0036] 1) CO 2 Incubator: SANYO, MCO-20A1C

[0037] 2) Inverted microscope: Nikon, TS100

[0038] 4. Steps:

[0039] hiPSCs were maintained in mTeSR1 medium, and passaged when the density reached about 80%-90% of the bottom area (about 4-5 days), digested with 0.5mM EDTA or 1mg / mL dispase, the digested cells Seed on Matrigel-coated culture plates.

[0040] 2. The effect of the digestion mo...

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Abstract

The invention discloses a mmethod for obtaining analogous retinal tissues rich in cone and rod cells by using human induced pluripotent stem cells. The method comprises the following steps: digestinghiPSCs to obtain cell pellets, and then carrying out suspension cultivation on the cell pellets to obtain an embryoid body; reinoculating the embryoid body into a culture dish enveloped by Matrigel, and carrying out induced differentiation inside an induced cultivation solution to obtain nerve retina (NR) and RPE; stirring up the NR and the RPE, carrying out suspension cultivation to obtain 3D analogous retina comprising NR tissues, then carrying out continuous suspension cultivation inside an optimized culture solution without retinoic acid, carrying out NR differentiation on all retinal cells, comprising highly matured photoreceptor cells, Rhodopsin positive rod cells, L / M OPSIN positive red and green cone cells and S-OPSIN positive blue cone cells, and thus particularly obtaining the analogous retinal tissues rich in red and green cone and rod cells.

Description

Technical field: [0001] The invention belongs to the field of stem cell regeneration biology, in particular to a method for regenerating retinal-like organs from pluripotent stem cells and obtaining retinal seed cells. Background technique [0002] Degenerative retinal disease is a major eye disease leading to irreversible blindness, mainly due to irreversible damage to photoreceptor cells, RPE cells or ganglion cells. However, there is currently no effective treatment for this disease. Stem cell therapy has brought new hope for vision recovery for this type of disease. Obtaining seed cells with similar characteristics to human retinal cells is the key to vision recovery treatment and disease mechanism research for retinal degenerative diseases. [0003] In the past, human retinal cells were mostly isolated from aborted embryonic retinas, but the quantity was very limited, and the batch quality was difficult to control, which could not meet the needs of clinical treatment, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079
CPCC12N5/0621C12N2506/45
Inventor 钟秀风李桂兰谢冰冰彭福华
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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