Method for producing microalbumin-positive nerve cells, cells, and differentiation-inducing agent

A differentiation inducing agent and technology of manufacturing method, which can be applied to nervous system cells, cell culture active agents, animal cells, etc., can solve the problem of low induction efficiency of parvalbumin-positive nerve cells, and achieve the advantages of less differentiation induction process, high efficiency, and high efficiency. high-manufacturing effect

Pending Publication Date: 2022-05-17
KEIO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, although a method of specifically inducing differentiation of inhibitory neurons including some parvalbumin-positive neurons and the like has also been reported, the induction efficiency of parvalbumin-positive neurons themselves is extremely low (Non-Patent Document 3)
In addition, Non-Patent Document 4 reports a method with the highest rate of emergence of parvalbumin-positive neurons among existing induction methods. However, this method requires multiple differentiation induction steps and produces parvalbumin-positive nerve cells. It takes about 80 days for nerve cells, and then the positive rate of parvalbumin-positive nerve cells produced is about 20-30%

Method used

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  • Method for producing microalbumin-positive nerve cells, cells, and differentiation-inducing agent
  • Method for producing microalbumin-positive nerve cells, cells, and differentiation-inducing agent
  • Method for producing microalbumin-positive nerve cells, cells, and differentiation-inducing agent

Examples

Experimental program
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Effect test

experiment example 1

[0092] (Maintenance culture of iPS cells)

[0093] For iPS cells (strain 1210B2), the seeding density of iPS cells at the time of cell passage was set to 1.5×10 in a 6-well plate 4 1.5 ml / well of undifferentiated cell medium Stem Fit (AK02N, manufactured by Ajinomoto Co., Ltd.) was injected into a 6-well plate immediately before cell seeding without the need for pre-plating at the time of cell passage. ), the medium contained 1.5 mL of 10 μg / ml ROCK inhibitor Y27632 (manufactured by Fuji Film Wako Pure Chemical Co., Ltd.) and 1.5 μg / ml iMatrix-511 (manufactured by Nippi Co., Ltd.), and the cells were directly Inoculation, other than the above, was cultured according to a known cell culture method disclosed by the iPS Research Institute of Kyoto University.

experiment example 2

[0095] (Introduction of Ascl1 and Dlx2 genes into iPS cells)

[0096] The protocol for introducing Ascl1 and Dlx2 genes into iPS cells is shown in Figure 1A . Specifically, the Ascl1 gene and the Dlx2 gene were introduced into iPS cells in the following manner.

[0097] (1) Preparation of medium and preparation for coating of plate for cell seeding after gene transfer operation

[0098] To Stem Fit (AK02N, manufactured by Ajinomoto Co., Ltd.), 20 μg / ml of Y27632 (manufactured by Fuji Film WakoPure Chemical Co., Ltd.) and 2.5 μg / ml of iMatrix-511 (manufactured by Nippi Co., Ltd.) were added at 6 Put it into 6 wells (1 plate) at 2ml / well in a well plate, and heat it at 37°C, 5% CO. 2 Incubation.

[0099] (2) Monocellularization of iPS cells

[0100] will be approximately 1.5 x 10 in a 6-well plate 4 The iPS cells seeded in 1 cell / well were cultured with Stem Fit (AK02N, manufactured by Ajinomoto Co., Ltd.) for about 1 week. After removing the medium with an aspirator, the ...

experiment example 3

[0113] (Introduction of MEF2C gene, miRNA-9 / 9 to iPS cells *, miRNA-124 and BclxL genes)

[0114] MEF2C gene, miRNA-9 / 9 will be introduced into iPS cells * , miRNA-124 and BclxL gene protocols are shown in Figure 1B . Specifically, the MEF2C gene and miRNA-9 / 9 were introduced into iPS cells in the following manner. * , miRNA-124 and BclxL genes.

[0115] (1) MEF2C gene, miRNA-9 / 9 were introduced * , miRNA-124 and BclxL gene purification of lentivirus

[0116] According to conventional methods, purification such as Figure 1C MEF2C gene, miRNA-9 / 9 were introduced as shown in the lower paragraph * , miRNA-124 and BclxL genes lentivirus. Specifically, the MEF2C gene and miRNA-9 / 9 were purified and introduced in the following manner. * , miRNA-124 and BclxL genes lentivirus.

[0117] A 0.01% poly-L-lysine solution (0.01%, #P4832, manufactured by Sigma) was mixed with PBS(-) to 1:100 to obtain a solution, which was used for coating cells and tissue culture After washing...

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Abstract

The present invention provides a method for producing microalbumin-positive nerve cells, which comprises an expression induction step for inducing the expression of an Ascl1 gene, a Dlx2 gene and an MEF2C gene in cells, and a differentiation step for culturing the cells after the expression induction to differentiate microalbumin-positive nerve cells from the cells. A cell into which an Ascl1 gene, a Dlx2 gene, and an MEF2C gene are introduced in an expressible manner. And a differentiation-inducing agent for inducing cell differentiation into microalbumin-positive nerve cells, the differentiation-inducing agent containing, as an active ingredient, an Ascl1 gene, a Dlx2 gene, and an MEF2C gene, or a gene product of the Ascl1 gene, the Dlx2 gene, and the MEF2C gene.

Description

technical field [0001] The present invention relates to a method for producing parvalbumin-positive neural cells, a cell and a differentiation-inducing agent. [0002] This application claims priority based on Japanese Patent Application No. 2019-157194 for which it applied in Japan on August 29, 2019, and the content is incorporated herein by reference. Background technique [0003] Parvalbumin-positive nerve cells are one of the important nerve cells associated with diseases of the cerebral nervous system. The presence and function of parvalbumin-positive nerve cells are thought to cause various neuropsychiatric diseases and neurodevelopmental disorders. (Non-Patent Document 1, Non-Patent Document 2). Therefore, the functional analysis thereof has attracted attention for a long time, and in particular, in recent years, a technique for producing parvalbumin-positive neural cells from pluripotent stem cells such as human iPS cells has been strongly desired. [0004] Howeve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793C12N5/10C12N15/113C12N15/12C12N15/85C12N15/867
CPCC12N5/0619C12N15/85C12N15/86C07K14/47C12N15/113C12N2510/00C12N2310/141C12N2740/15043C12N2800/107C12N2506/45C12N2501/65C07K14/4705
Inventor 石川充冈野荣之
Owner KEIO UNIV
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