A method to induce the directed differentiation of human iPSC-derived 3D retina into 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: 2018-01-09
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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[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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Embodiment 1
[0020] 1. Human iPSc-derived 3D retinal isolation and digestion:
[0021] Using the method of mechanical separation, culture 50-55 days of human iPSc-derived 3D retina (specific preparation method reference: Xiufeng Zhong, et al. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs, Nat Commun.2014Jun 10;5:4047) into the D-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); transfer the isolated nerve fiber layer tissue to a 3.5cm culture dish with a pipette, rinse with PBS for 10 minutes; absorb the PBS, digest the cells with accutase cell digestion solution, and place in a 37°C incubator for 30min ; At the end of the process, the cells can be sucked into the centrifuge tube and pipetted. Under a 10-fold upright microscope, the cell clusters can be seen to be digested ...
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The invention discloses a method for inducing directional differentiation of human iPSc-derived 3D retina into retinal ganglion cells. It digests the nerve fiber layer of the human iPSc-derived 3D retina into single cells, and uses the single cells as seed cells to be cultured in induction differentiation medium, and the seed cells are differentiated into retinal ganglion cells. The induced differentiation The medium is composed of neurotrophic factors and retinal differentiation nutrient solution at a maintenance concentration. The present invention provides a new research approach for obtaining functional RGCs from the perspective of stem cell induced differentiation, provides a new research idea for the directed differentiation of human iPSc-RGCs, and provides a new method for exploring the regulation mechanism of human iPSc-RGCs The research clues provide a new seed cell for the research of neural tissue engineering, and lay a theoretical and experimental basis for stem cell transplantation to treat visual impairment.
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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IPC IPC(8): C12N5/071
Inventor 葛坚钟秀风李康寯
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV