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Method for producing cell aggregate comprising glial precursor cells

A technology of precursor cells and aggregates, applied in the field of cell aggregates

Pending Publication Date: 2022-04-19
KEIO UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the cell aggregates containing oligodendrocyte precursor cells reported so far (Non-Patent Documents 4 and 5 and Patent Document 1) were prepared in the presence of feeder cells such as mouse cells, and therefore may contain feeder cells or Xenogeneic cell-derived components derived from feeder cells

Method used

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  • Method for producing cell aggregate comprising glial precursor cells
  • Method for producing cell aggregate comprising glial precursor cells
  • Method for producing cell aggregate comprising glial precursor cells

Examples

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preparation example Construction

[0186] [Method for preparing cell aggregates including glial precursor cells]

[0187] As one embodiment of the method for producing a cell aggregate containing glial precursor cells of the present invention, a production method including the following steps can be mentioned:

[0188] In step (1), in the embryoid body formation medium containing more than one SMAD signal transduction inhibitor and more than one Wnt signal transduction activator, in the absence of feeder cells, the pluripotent stem cells are cultured in suspension 5 days to 10 days, thus forming cell aggregates;

[0189] In step (2), the cell aggregates obtained in step (1) are suspended and cultured in an embryoid body formation medium containing retinoic acid;

[0190] In step (3), the cell aggregates obtained in step (2) are suspended and cultured in an embryoid body formation medium or a neuroglial proliferation medium containing retinoic acid and more than one SHH signal transduction activator; as well a...

Embodiment 1

[0416]

[0417] As the iPS cell line, the QHJI01s04 strain, which is a clinical-grade peripheral blood-derived feeder-free iPS cell line established by the Kyoto University iPS Cell Research Institute, was used. Before differentiation, culture was maintained in StemFit (registered trademark) AK03N medium (manufactured by Ajinomoto Co., Inc.) supplemented with laminin 511E8 fragment (iMatrix-511E8; manufactured by Nippi. Inc.) (0.5 mg / ml) .

[0418] To induce the differentiation of iPS cells into neural progenitor cells, a rapid aggregate serum-free suspension culture method (SFEBq method) was used to form embryoid bodies. Specifically, the maintained cultured QHJI01s04 strain was isolated into individual strains using 0.5×TrypLE Select (manufactured by Thermo Fisher Scientific) / 0.25 mM EDTA (ethylenediaminetetraacetic acid) / PBS (phosphate-buffered saline, manufactured by Thermo Fisher Scientific). Cells were inoculated on a 96-well low-adsorption culture plate (trade name: ...

Embodiment 2

[0434]

[0435] To investigate gene expression changes during differentiation induction, RNA-sequencing-based gene expression analysis was performed on differentiation-induced intermediates. Using the QHJI01s04 strain, induction of differentiation was started by the method of Condition 3 in Example 1, and using the RNeasy Plus Mini Kit (manufactured by QIAGEN) from before differentiation (iPS cell line), on the 7th day of differentiation, on the 14th day, on the 21st day, on the day Total RNA was extracted from the 35-day-old cells, and a library was prepared using Illumina's TruSeq Stranded mRNA Library Prepkit, and HiSeq 2500 was used for 80 cycles of sequencing. Gene expression analysis based on data of more than 30 million reads per cell ( Figure 3A-3D ).

[0436] Comparing the gene expression profiles before induction of differentiation (iPS cells) and the 7th day of differentiation, the expression levels of NANOG and POU5F1 (OCT3 / 4), which are markers of pluripotency...

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Abstract

The method for producing a cell aggregate containing colloidal precursor cells according to the present invention comprises the steps of: (1) culturing pluripotent stem cells in suspension for 5-10 days in the absence of feeder cells in an embryoid formation medium containing one or more SMAD signaling inhibitors and one or more Wnt signaling activators, and then culturing the pluripotent stem cells in suspension in the culture medium for 5-10 days in the absence of feeder cells; thereby forming cell aggregates; (2) carrying out suspension culture on the cell aggregate obtained in the step (1) in an embryoid forming culture medium containing retinoic acid; a step (3) in which the cell aggregate obtained in the step (2) is subjected to suspension culture in an embryoid formation medium or a medium for neuroglial proliferation, said medium containing retinoic acid and one or more SHH signaling activators; and a step (4) in which the cell aggregate obtained in the step (3) is suspended and cultured in a culture medium for neuroglial proliferation, said culture medium not containing retinoic acid and containing one or more SHH signaling activators.

Description

technical field [0001] The present invention relates to a cell aggregate including glial precursor cells derived from pluripotent stem cells, a preparation method thereof, and the like. Background technique [0002] Demyelinating diseases including spinal cord injuries and glial cell-based diseases rarely have natural cures, and most cases not only shed myelin-forming glial cells, but also cause axonal degeneration and cell body degeneration And worse. Especially for spinal cord injury, although the effect of rehabilitation can be observed in some patients, there is no radical therapy to regenerate the lost nerve axons and glial cells, and the disease has unmet medical needs (unmet medical needs) higher. Cell replenishment by transplantation is considered promising as a therapeutic strategy based on nerve regeneration and remyelination. [0003] A large number of research results aimed at realizing transplantation of neural progenitor cells have been reported, and in rece...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/02C12N5/079C12N5/071C12Q1/02A61K35/30A61P25/00
CPCC12N5/0622C12N5/0618G01N33/5058G01N33/5014A61K35/30A61P25/00C12N2501/15C12N2501/415C12N2500/38C12N2501/155C12N2506/45C12N2501/13C12N2501/235C12N2501/395C12N2503/02G01N2500/10A61K35/545G01N33/56966C12N5/0623C12N2513/00C12N2533/52C12N2501/385C12N2501/41C12N2501/135C12N2501/105C12N2501/115C12N2501/11C12Q1/04A61P25/28C12N2500/32C12N2501/998
Inventor 神山淳镰田泰裕中村雅也冈野荣之齐藤美帆井上满博
Owner KEIO UNIV
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