Cell differntiation inhibiting agent, cell culture method using the same, culture medium, and cultured cell line

a cell differentiation and inhibiting agent technology, applied in the field of stem cell differentiation inhibiting agents, can solve the problems of complex preparation of mouse primary fibroblasts, insufficient donor numbers, and poor quality of primary fibroblasts

Inactive Publication Date: 2005-07-14
ASAHI KASEI KK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016] The object of the present invention is to provide a differentiation inhibiting agent which allows culture of a stem cell or an embryonic stem cell in an undifferentiated state without use of any feeder cell or any component derived from the feeder cell. In addition, the object of the present invention is to provide a method for culturing the stem cell or the embryonic stem cell in an undifferentiated state by using such the differentiation inhibiting agent without use of any feeder cell or any component derived from the feeder cell, and to provide a cell culture liquid which comprises such the differentiation inhibiting agent, and to provide a cell line prepared by culturing using such the differentiation inhibiting agent. Further another object of the present invention is to provide a novel bicyclic compound enabling culture of the stem cell or the embryonic stem cell in the undifferentiated state without use of any feeder cell or any component derived from the feeder cell.

Problems solved by technology

Particularly, a substantial organ such as heart, liver, kidney, or pancreas is essential for maintaining life, and thus hypofunction and a function thereof causes a death.
However, the number of donor is generally insufficient, and thus a new approach to solve the problem is necessary.
However, preparation of the mouse primary fibroblast is complicated as follows.
Since this cell is primary cell, quality management is complicated, management on a GMP-matched level is difficult, and the ability of maintaining undifferentiation may be different depending on the embryo used.
Therefore, the above culture method for maintaining undifferentiation of the ES cell by using cells derived from the mouse is not suitable for culturing the ES cell aiming to use for medicine.
However, also in this case, the ES cell under culturing is exposed to an unidentified factor secreted from the mouse cell, and therefore the ES cell cultured in such an environment is not suitable for the use in medicine.
Moreover, a danger of infection of the endogenous virus remains.
Consequently, the defect caused by coculture with the mouse primary fibroblast is not solved at all.
LIF is a cytokine and therefore, has problems of high cost and a bad preservation performance, resulting in unsuitableness for mass culture.
Especially, for the primate ES cell, it has been known that addition of LIF to the culture medium alone does not allow keeping the undiffentiation status (for example, Thomson et al., Proc. Natl. Acad. Sci.
However, the action of PD98059 depends on LIF and does not express independently the effect (Burdon et al., Dev. Biol. 210: p 30, 1999) and, hence, the aforementioned problem is not solved.
Consequently, so far, there was no differentiation inhibiting agent enabling safe mass culture of the totipotent stem cell at a low cost, and no method of safe mass culture of the totipotent stem cell at the low cost has been known.

Method used

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  • Cell differntiation inhibiting agent, cell culture method using the same, culture medium, and cultured cell line
  • Cell differntiation inhibiting agent, cell culture method using the same, culture medium, and cultured cell line
  • Cell differntiation inhibiting agent, cell culture method using the same, culture medium, and cultured cell line

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examples as implementation

of the present invention will be presented below.

[0264] These examples are provided to help those skilled in the art for practice of the present invention. These examples do never restrict the scope of the present invention in any modes. In addition, modification is allowed in a range not going out of the scope of the present invention. The “room temperature” described in the following examples represents a range from 0 to 30° C. “%” means a percent by weight unless otherwise stated.

example 1

(1) Preparation of a Mouse ES Cell Culture Medium

[0265] With a purpose to proliferate the ES cell, the factor was added to Dulbecco's modified Eagle medium (hereafter DMEM) (Invitrogen Corp. made, 11995) with a final concentration shown below to prepare an ES cell culture medium. 15% bovine fetus serum (Invitrogen Corp. made) or 15% knockout serum replacement: KSR (Invitrogen Corp. made), 0.1 mM β-mercaptoethanol (Sigma Corp. made), 1×nonessential amino acid stock (Invitrogen Corp. made, 11140-050), 2 mM L-Glutamine (Invitrogen Corp. made, 25030-081), 103 unit / mL ESGRO(CHEMICON International Inc., made).

[0266] ESGRO contains a mouse LIF as the active ingredient. As the culture medium for ES cell differentiation inhibition assay, an assay medium was prepared by excluding ESGRO from the ES cell culture medium as described above.

(2) Culture of the Mouse ES Cell

[0267] 5 mL of a sterilized 0.1% gelatin (SIGMA Corp. made, Type A: from porcinESkin, G2500) aqueous solution was added i...

example 2

[0279] (1) ES cell Differentiation Inhibition Assay 2

[0280] For the D3ES cells prepared by the method described in (3) preparation of a mouse ES cell of Example 1, 8×104 cells were seeded in a 10 cm-diameter cell culture dish coated previously with a 0.1% aqueous gelatin solution to make 10 mL of the ES cell assay medium. 1 mL of the differentiation inhibiting agent A to F, which were dissolved in dimethylsulfoxide (DMSO) or culture medium or their mixture to make 40 μg / mL per each dish, or ESGRO, which was adjusted to make 104 unit / mL, was added, and culturing was carried out at 37° C. in 5% CO2 incubator for 7 days. To the culture medium was added DMSO to make the final concentration 0.1% or lower.

(2) Isolation of RNA 1

[0281] A total RNA was extracted from the ES cells cultured by the method described in (1) ES cell differentiation inhibition assay 2 as described above, by using ISOGEN (K.K. Nippon Gene, Japan made) by the attached method. First, the medium was removed from th...

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Abstract

The object of the present invention is to provide a differentiation inhibiting agent which allows culture of a stem cell or an embryonic stem cell in an undifferentiated state without use of any feeder cell, a method for culturing using the same, a cell culture liquid using the same, and a cell prepared by culturing using this differentiation inhibiting agent. The present invention provides a differentiation inhibiting agent which comprises a low molecular weight compound, especially a tetrahydroisoquinoline derivative, as an active ingredient; a method for safely culturing a stem cell in large scale in undifferentiated state in the absence of feeder cell which comprises culturing a stem cell by using a tetrahydroisoquinoline derivative; a culture liquid for stem cells comprising a tetrahydroisoquinoline derivative; and a cell which is obtained by culture using a tetrahydroisoquinoline derivative as a differentiation inhibiting agent.

Description

TECHNICAL FIELD [0001] The present invention relates to a stem cell differentiation inhibiting agent comprising a low molecular weight compound, especially a tetrahydroisoquinoline derivative, as an active ingredient, a stem cell culture method by using the same, a culture medium, and a cultured stem cell line prepared by using the same. The present invention relates further to new bicyclic compounds having an action of maintaining an undifferentiated state of the stem cell. BACKGROUND ART [0002] An organ and a tissue damaged by an injury, a disease or aging require promotion of regeneration to recover its function. Particularly, a substantial organ such as heart, liver, kidney, or pancreas is essential for maintaining life, and thus hypofunction and a function thereof causes a death. Therefore transplantation treatment such as organ transplantation is popularly conducted intending life saving. However, the number of donor is generally insufficient, and thus a new approach to solve ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/404A61K31/47A61K31/472A61K31/655A61P1/02A61P1/16A61P3/10A61P9/10A61P9/12A61P17/02A61P19/02A61P19/10A61P25/00A61P25/02A61P25/16A61P25/28A61P27/02A61P29/00C07D217/16C12N5/00
CPCA61K31/404A61K31/47A61K31/472C12N2501/999C07D217/16C12N5/0018A61K31/655A61P1/02A61P1/16A61P1/18A61P3/10A61P9/10A61P9/12A61P17/00A61P17/02A61P17/14A61P19/00A61P19/02A61P19/08A61P19/10A61P25/00A61P25/02A61P25/14A61P25/16A61P25/28A61P27/02A61P29/00A61P37/02A61P43/00C07D217/00C12N5/00C12N5/06
Inventor MIYABAYASHI, TOMOYUKIYAMAMOTO, MASASHI
Owner ASAHI KASEI KK
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