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Method for directionally differentiating human iPSCs (induced pluripotent stem cells)-derived 3D (three-dimensional) retinas by means of induction to obtain retinal ganglion cells

A retinal ganglion, induced differentiation technology, applied in the fields of developmental biology and biomedical engineering, can solve the problem of low induction function of the source of seed cells

Active Publication Date: 2016-01-27
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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Problems solved by technology

[0005] In order to overcome the problem of low induction function of existing seed cell sources, the present invention provides a method for inducing human iPSc-derived 3D retinal directed differentiation into retinal ganglion cells (RGCs) in vitro

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  • Method for directionally differentiating human iPSCs (induced pluripotent stem cells)-derived 3D (three-dimensional) retinas by means of induction to obtain retinal ganglion cells
  • Method for directionally differentiating human iPSCs (induced pluripotent stem cells)-derived 3D (three-dimensional) retinas by means of induction to obtain retinal ganglion cells
  • Method for directionally differentiating human iPSCs (induced pluripotent stem cells)-derived 3D (three-dimensional) retinas by means of induction to obtain retinal ganglion cells

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Embodiment 1

[0020] 1. Human iPSc-derived 3D retinal isolation and digestion:

[0021] Using the method of mechanical separation, the nerve fiber layer of human iPSc-derived 3D retina cultured for 50-55 days (specific preparation method reference: XiufengZhong, etal. -In Hanks solution, use a 0.45mm needle to separate the tissue under a 50-fold upright microscope, discard the pigmented part (pigment layer) in the retinal tissue, and keep the golden yellow tissue part (nerve fiber layer); separate the nerve fiber layer tissue Use a pipette to transfer to a 3.5cm culture dish, rinse with PBS for 10 minutes; absorb the PBS, digest the cells with accutase cell digestion solution, and place them in a 37°C incubator for 30 minutes; at the end of the process, the cells can be sucked into the centrifuge tube and blown at 10 Under a magnified upright microscope, it was seen that the cell mass was digested into a single cell, and then 2ml of induction differentiation medium was added to terminate th...

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Abstract

The invention discloses a method for directionally differentiating human iPSCs (induced pluripotent stem cells)-derived 3D (three-dimensional) retinas by means of induction to obtain retinal ganglion cells. The method includes digesting nerve fiber layers of the human iPSc-derived 3D retinas to obtain single cells; inoculating seed cells, namely the single cells, in induction differentiation culture media and cultivating the seed cells in the induction differentiation culture media; differentiating the seed cells to obtain the retinal ganglion cells. The induction differentiation culture media are culture media with neurotrophic factors and concentration-maintained retinal differentiation nutrient liquid. The method has the advantages that a novel functional RGCs (retinal ganglion cells) research approach is obtained from an angle of stem cell induction differentiation, a novel research idea is provided for directionally differentiating human iPSc-RGCs, a novel research clue is provided for investigating human iPSc-RGCs regulation mechanisms, a novel seed cell is provided for research on nerve tissue engineering, and theoretical and laboratory bases are laid for treating visual impairment by means of stem cell transplantation.

Description

Technical field: [0001] The invention belongs to the fields of developmental biology and biomedical engineering, and specifically relates to a method for inducing directional differentiation of human iPSc-derived 3D retina into retinal ganglion cells. Background technique: [0002] The damage and repair of retinal neurons is a common scientific problem in the occurrence, development and outcome of neurological visual damage. In recent years, with the rapid development of stem cell research, induced pluripotent stem cells (iPSCs) have brought new hope for the repair and treatment of optic nerve damage. Due to the self-replication and multi-differentiation potential of iPSCs, as a donor for repairing nervous system damage, it can not only meet the cell quantity required for transplantation, but also iPSCs can be induced to differentiate into different neural cell lines including neurons under certain conditions. Therefore, transplanting human-derived iPSCs to treat optic nerv...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 葛坚钟秀风李康寯
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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