Method for maturation of retinal tissue containing continuous epithelium

A technique for epithelial tissue and retina, used in tissue culture, biochemical equipment and methods, embryonic cells, etc.

Pending Publication Date: 2020-03-31
RIKEN +2
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, it is known that in retinal tissue derived from pluripotent stem cells including the CMZ, the range in which a continuous epithelial structure can be maintained is limited to the vicinity of the CMZ (Non-Patent Document 3)

Method used

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  • Method for maturation of retinal tissue containing continuous epithelium
  • Method for maturation of retinal tissue containing continuous epithelium
  • Method for maturation of retinal tissue containing continuous epithelium

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preparation example Construction

[0099] Preparation of targeting vectors for homologous recombination of target genes and effective screening of homologous recombinants can be carried out according to the methods described in: Gene Targeting, A Practical Approach (gene targeting, practical methods), IRL Press at Oxford University Press (1993); Biomanual Series 8, Gene Targeting, Making of Mutant Mouse using ES cell (gene targeting, using ES cells to produce mutant mice), YODOSHA CO., LTD. (1995); etc. As the targeting vector, either of a replacement type or an insertion type can be used. As the screening method, methods such as positive selection, promoter selection, negative selection, polyadenylic acid (polyA) selection and the like can be used.

[0100] Examples of methods for selecting desired homologous recombinants from selected cell lines include DNA hybridization method (Southern hybridization method) for genomic DNA, PCR method, and the like.

[0101] The term "suspension culture" or "suspension cul...

Embodiment approach

[0147] As a preferred embodiment of preparing retinal tissue at an early stage of development, a method comprising the following steps can be enumerated:

[0148] (1) The first step is to form cell aggregates by suspending and culturing pluripotent stem cells in a serum-free medium;

[0149] (2) In the second step, the aggregates formed in the first step are suspended in a serum-free medium or a serum-containing medium that does not contain the SHH signal transduction pathway action substance but contains the BMP signal transduction pathway action substance, Aggregates comprising retinal progenitor cells or neural retinal progenitor cells are obtained.

[0150] The aggregate comprising retinal progenitor cells or neural retinal progenitor cells obtained by this method can be used as a retinal tissue at an early stage of development as a starting material used in the method of the present invention.

[0151] [for the first step]

[0152] The first step can be performed accord...

Embodiment 1

[0630] (Preparation example of cell aggregates including retinal tissue using human ES cells and method of cutting retinal tissue)

[0631] Human ES cells (derived from KhES-1; Nakano, T. et al. CellStem Cell 2012, 10(6), 771-785) knocked into CRX::Venus were obtained according to "Ueno, M. et al. PNAS 2006, 103 (25), 9554-9559" and "Watanabe, K. et al. Nat Biotech 2007, 25, 681-686". For the medium used for culturing human ES cells, DMEM / F12 medium (Sigma) supplemented with 20% KSR (KnockOut TM Serum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1x non-essential amino acids, 7.5 ng / mL bFGF medium.

[0632] Cell aggregates including retinal tissue were prepared by partially modifying the method described in "Kuwahara et al. Nat Commun 2015, 19(6), 6286". That is, after the cultured ES cells were dispersed into single cells using TrypLEExpress (Invitrogen), the dispersed human ES cells were suspended in a cell-nonadherent 96-well culture plate (Sumilon...

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Abstract

The present invention provides a method for retaining the continuous epithelial structure of retinal tissue. The method includes culturing a retinal tissue in a culture medium containing: a methyl group donor or a substrate of a methyl group donor at a concentration that inhibits cellular differentiation of neuroretinal precursor cells; and a neurite outgrowth inhibitor at a concentration that inhibits neurite outgrowth.

Description

technical field [0001] The present invention relates to methods for culturing retinal tissue capable of maintaining a continuous epithelial structure. Background technique [0002] The retinal tissue is a neural tissue with a multilayered epithelial structure produced by the diencephalon, converts light stimulation into electrical signals, and transmits the signals to the brain. In recent years, methods for preparing multilayered retinal tissues from pluripotent stem cells have been reported (Patent Document 1 and Non-Patent Document 1). In addition, it is known that by forming uniform aggregates of pluripotent stem cells in a serum-free medium containing a Wnt signal transduction pathway inhibitory substance, and culturing them in suspension in the presence of a basement membrane preparation and then culturing them in serum Suspension culture in medium to obtain multi-layered retinal tissue (Patent Document 2 and Non-Patent Document 2), in which homogeneous aggregates of p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079
CPCC12N5/062C12N2500/32C12N2500/38C12N2500/99C12N2501/115C12N2501/155C12N2501/39C12N2501/392C12N2501/70C12N2501/999C12N2506/02C12N2500/00C12N2501/71C12N2501/998
Inventor 坂口秀哉笹井芳树永乐元次糠谷大树桑原笃
Owner RIKEN
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