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A method for obtaining retinal-like tissue rich in cones and rods 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 the problems that are difficult to meet the needs of clinical treatment, and achieve the effect of stable and controllable activity and short digestion time

Active Publication Date: 2021-08-24
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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  • A method for obtaining retinal-like tissue rich in cones and rods by using human induced pluripotent stem cells
  • A method for obtaining retinal-like tissue rich in cones and rods by using human induced pluripotent stem cells
  • A method for obtaining retinal-like tissue rich in cones and rods 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 method for obtaining retinal tissue rich in cone and rod cells by using human induced pluripotent stem cells. It includes digesting hiPSCs to obtain cell pellets, and then suspending the cell pellets to obtain embryoid bodies; the embryoid bodies are then inoculated into culture dishes pre-coated with Matrigel, and induced to differentiate in the induction medium to obtain neural retina (NR) and RPE; NR and RPE were re-provoked and cultured in suspension to obtain 3D retinal tissue including NR tissue, and then continued to be cultured in suspension in an optimized culture medium without retinoic acid. NR differentiated into all retinal cells, including highly mature photoreceptors cells, Rhodopsin-positive rod cells, L / M OPSIN-positive red-green cone cells and S-OPSIN-positive blue cone cells, especially retinotopic tissues rich in red-green cone and rod cells were obtained.

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 Patents(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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