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Method for increasing activity in human stem cell

A high-activity, high-quality stem cell technology, applied in the field of preparation of high-activity human mesenchymal stem cell clumps, can solve the problems of not being able to meet the requirements of use, changes, cell aging, etc.

Inactive Publication Date: 2012-12-12
SEOUL NAT UNIV HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this case, the cells age and change, making them unsuitable for use in therapy

Method used

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  • Method for increasing activity in human stem cell
  • Method for increasing activity in human stem cell
  • Method for increasing activity in human stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Inducing sphere formation of human mesenchymal stem cells

[0071] (1) Medium for inducing spheroid formation

[0072] Using low-adsorption culture dishes for non-adherent culture, the above-mentioned mesenchymal stem cells were cultured in the traditional medium of mesenchymal stem cells, that is, α-MEM medium (Invitrogen) supplemented with serum substitute (SR). However, induction of non-adherence was unsuccessful (see figure 1 A).

[0073] Next, use low-adhesion culture dishes for non-adherent culture, and culture mesenchymal stem cells in embryonic stem cell medium (ESM) with basic fibroblast growth factor (bFGF) removed (hereinafter referred to as bFGF-free ESM) . Successfully induced non-adherence (see figure 1 B). The medium did not contain any fetal bovine serum (FBS), but contained DMEM / F-12 (Invitrogen), 20% KnockOut SR (Invitrogen), 0.1 mmol / L β-mercaptoethanol (Sigma), 1% non-essential amino acids (Invitrogen ), 50 IU / mL penicillin and 50 m...

Embodiment 2

[0078] Example 2 Effect of spheroid formation - in vivo activity

[0079] Evaluation of in vivo activity of mesenchymal stem cells in a rat model of ischemic heart disease. Rat models of ischemic heart disease were prepared by inducing ischemia by coronary artery ligation.

[0080] The rat model of ischemic heart disease was divided into three groups for evaluation: one group was injected with spherical cell aggregates (spheroids) prepared in (2) in Example 1; one group was injected with single cells prepared from spherical cell aggregates ( Dissociate) (Dissociate); a group of cells whose injection did not induce the formation of any spherical cell clumps (blank control group) ( ). At least 7 rats per group.

[0081] (1) ECG measurement

[0082] The baseline electrocardiogram was measured 4 days after the rat model was prepared, and the rats were injected with stem cells 7 days after the rat model was prepared. Specifically, stem cells or cell clumps were injected int...

Embodiment 3

[0101] Example 3: Analysis of spheroid formation mechanism (in vivo activity)

[0102] Experiments were conducted to analyze the mechanism of spheroid formation, which can lead to different in vivo activities as shown in Example 2.

[0103] First, EDTA was added to stem cells that sequestered calcium ions, which play an important role as a cell adhesion factor, by inducing spheroid formation in bFGF-free ESM using a low-adsorption culture dish. Such as Figure 11 As shown, in the presence of EDTA, no spheroids were formed. In other words, since as Ca 2+ The addition of chelating agent EDTA, the spheroid formation of mesenchymal stem cells was blocked, thus it is considered that spheroid formation is through Ca 2+ dependent on cell adhesion molecules.

[0104] From this, detection of two distinct Ca in spheroid formation by Western blot analysis 2+ Expression of dependent cell adhesion molecules, namely neural cadherin and epithelial cadherin. Such as Figure 12 showed t...

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Abstract

Provided are a method for preparing a highly active human mesenchymal stem cell, which includes forming a spherical cell aggregate by cultivating human mesenchymal stem cells against gravity; a highly active stem cell prepared thereby; a cell therapeutic agent including the stem cell aggregate; and a method for forming a spherical cell aggregate by cultivating human mesenchymal stem cells, wherein the amount of E-cadherin in the mesenchymal stem cell is increased during the cultivation.

Description

technical field [0001] The invention relates to a method for preparing highly active human mesenchymal stem cell aggregates, the highly active stem cell aggregates obtained by the method, and a cell therapy agent containing the stem cell aggregates. Background technique [0002] Stem cells are capable of differentiating into various cells constituting tissues of a living body, and generally refer to undifferentiated cells obtained from various tissues of embryos, fetuses, and adults. Stem cells differentiate into specialized cells by differentiation stimuli (environment); unlike differentiated cells in which cell division has ceased, they proliferate (expand) by cell division (self-renewal) to produce identical cells; characterized by the plasticity of their differentiation, It can differentiate into another type of cell under different circumstances or through differentiation stimuli. [0003] According to their differentiation ability, stem cells can be divided into pluri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/02A61K35/12A61P9/00A61P37/00A61P19/00A61K35/28
CPCA61K2035/124A61K35/28C12N2501/58C12N5/00C12N2525/00C12N5/0665A61P19/00A61P29/00A61P37/00A61P9/00
Inventor 金孝洙姜炫在李银珠
Owner SEOUL NAT UNIV HOSPITAL
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