Method for inducing cell death in pluripotent stem cells and differentiated cells other than cardiac myocytes

一种多能性干细胞、心肌细胞的技术，应用在对多能性干细胞和心肌细胞以外的分化细胞诱导细胞死亡领域

Inactive Publication Date: 2012-05-09
DAIICHI SANKYO CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, among the known methods for inducing cell death, there is still room for improvement in the purification method of cardiomyocytes that can be used in the treatment of myocardial diseases, and a new method for inducing cell death with higher efficiency is expected

Method used

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  • Method for inducing cell death in pluripotent stem cells and differentiated cells other than cardiac myocytes
  • Method for inducing cell death in pluripotent stem cells and differentiated cells other than cardiac myocytes
  • Method for inducing cell death in pluripotent stem cells and differentiated cells other than cardiac myocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Embryonic stem cell-derived cardiomyocytes and remaining undifferentiated embryos Immunostaining of Stem Cells

[0075] The purpose of this example is to confirm the mixed state of cardiomyocytes and undifferentiated cells in the cell mass (embryoid body) when the cell mass (embryoid body) containing cardiomyocytes was formed from stem cells.

[0076] Mouse embryonic stem cells (cell line name EB3, Nat Genet 2000; 24: 372-376) were kindly donated by Dr. Hitoshi Niwa of RIKEN. For the mouse embryonic stem cells, use the existing method (Bader A, et al., Differentiation 2001, 68, p31-43) (that is, use culture medium [α-MEM (Minimum Essential Medium, minimum essential medium) ( SIGMA), 10% FBS (EQUITEC BIO), 100 units / ml penicillin, 50 μg / ml streptomycin (GIBCO)] By the hanging drop method, 75 embryonic stem cells were cultured in the form of cell block for each EB for 7 days. Method) After differentiating the cell mass containing cardiomyocytes, the embryo...

Embodiment 2

[0079] Example 2: After mixing cardiomyocytes derived from embryonic stem cells and remaining embryos Sugar (sugar alcohols) treatment in the culture system of fetal stem cells

[0080] The purpose of this example is to confirm the culture state of cardiomyocytes and the culture state of other cells after treating a cell mass (embryoid body) containing cardiomyocytes formed from embryonic stem cells with sugar (sugar alcohols).

[0081] Mouse-derived embryonic stem cells were formed into embryoid bodies by the method described in Example 1 and differentiated to a stage containing programmed cardiomyocytes (precardiac mesoderm). The resulting embryoid bodies were carefully treated with digestive enzymes (trypsin, collagenase) to prevent cell damage, thereby partially dispersing them, and culturing them again for adherence. When continuing to culture for 5 days, if figure 2 As shown, a group of cardiomyocytes beating autonomously (the cell group surrounded by the red line)...

Embodiment 3

[0084] Embodiment 3: Glucose (sugar alcohols) induces cell death of mouse embryonic stem cells Guidance

[0085] The purpose of this example is to confirm the survival state of stem cells after treating mouse embryonic stem cells with sugars (sugar alcohols).

[0086] As for the mitochondrial membrane potential, which is lost in dead cells, when the membrane potential as an indicator of survival is detected and stained with the fluorescent mitochondrial membrane potential sensitive reagent TMRM (Molecular Probes), the reagent-derived The fluorescent signal is higher in live cells and becomes lower in dead cells.

[0087] Utilizing this property, the embryonic stem cells cultured by the method described in Example 1 were cultured in α-MEM medium containing mannitol 0.45M (equivalent to about 720mOsm / kg) and 1 μM TMRM for 0h (initial), 3h , 6h, and 20h later, the collected embryonic stem cells were washed, and then analyzed by FACS. Based on the fluorescence intensity of th...

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Abstract

Disclosed is a method which can induce cell death in pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells and differentiated cells other than cardiac myocytes derived from pluripotent stem cells and does not induce cell death in cardiac myocytes without employing any genetic modification. By adding a substance that is not recognized to have physical toxicity or a cell death-inducing activity to the culture conditions for a pluripotent stem cell or a non-cardiac myocyte, it becomes possible to establish a method for inducing cell death in cells other than cardiac myocytes with extremely high efficiency without employing any genetic modification.

Description

technical field [0001] The present invention relates to a method for inducing cell death to differentiated cells other than cardiomyocytes derived from pluripotent stem cells and pluripotent stem cells. Cells are relevant means for the purification of cardiomyocytes. Background technique [0002] Cardiomyocytes in adults have lost their proliferative activity, and they have to rely on heart transplantation in order to treat severe myocardial infarction, cardiomyopathy and other diseases. However, the current situation is that the problem of insufficient heart donors cannot be overcome, and finding a treatment method other than heart transplantation has become a top priority. In this regard, it is expected that the supplementation of cardiomyocytes produced and purified in vitro as a disease treatment is the most promising method for helping heart disease patients who have to rely on heart transplantation. [0003] Known methods for obtaining cardiomyocytes include methods ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00C12N5/07
CPCC12N5/0657C12N2506/02C12N5/0696C12N5/0081C12N2501/90C12N5/00C12N5/0606C12N5/0607
Inventor 服部文幸福田惠一
Owner DAIICHI SANKYO CO LTD
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