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Method for inducing mesenchymal stem cells to directionally differentiate into cardiac cells

A cardiomyocyte and stem cell technology, which is applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., to achieve good clinical application prospects.

Inactive Publication Date: 2013-05-08
北京清美联创干细胞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the directed differentiation of human mesenchymal stem cells (hMSCs) into cardiomyocytes induced by MS-818.

Method used

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  • Method for inducing mesenchymal stem cells to directionally differentiate into cardiac cells
  • Method for inducing mesenchymal stem cells to directionally differentiate into cardiac cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1. Preparation of MS-818 (2-oxidine-6-methyl-5-oxo-5,6-dihydro(7H)-pyrrole[3,4-d]pyrimidinemaleic acid)

[0012] In 2.25 g (6 mmol) of ethylene-4-chloromethyl-2-(4-benzylpiperazine)pyrimidine-5-carboxylate in ethanol (10 ml), NH was added dropwise at 20°C 4 OH solution (10 ml, 59 mmol) and stirred for 12 hours. The mixture was poured into NaHCO 3 Aqueous solution with CHCl 3 Extract. The extract was rotary evaporated and recrystallized from toluene to give light brown crystals (0.70 g, yield: 38%). A suspension of the above crystals (0.7 g, 2.3 mmol) and 10% Pd-C (0.07 g) in AcOH (13 ml) was stirred under hydrogen for one hour. Crystals were obtained by filtration, and the filtrate was subjected to rotary evaporation. with NaHCO 3 The residue was basified with aqueous solution and extracted with toluene. MgSO for extract 4 Drying and evaporation yielded 0.3 g (yield: 66%) of a light brown oil.

Embodiment 2

[0013] Example 2. Isolation, purification and primary and subculture of bone marrow mesenchymal stem cells

[0014] Extract the bone marrow of healthy volunteers under sterile conditions, add DMEM / F12 (1:1) medium and mix thoroughly, centrifuge to discard the supernatant and fat layer, add complete medium and mix well, then gently overlay the bone marrow fluid On the Percoll separation solution with a density of 1.0739 / ml, centrifuge at 900g for 30 minutes (at room temperature), take the part above the white blood cell membrane layer, add DMEM / F12 medium containing 10% fetal bovine serum (pre-added penicillin-streptomycin 100u / ug.ml -1 ), mixed well, centrifuged and washed for later use, and the cells were separated by 2 x 10 5 / m 2 The concentration of the inoculated 50ml plastic culture flask, placed at 37 ° C, 5% CO 2 Culture in an incubator with saturated humidity, replace the culture medium after 48h, discard the non-adherent cells, and change the medium every 2-3 day...

Embodiment 3

[0015] Example 3. Directional induction of bone marrow mesenchymal stem cells to differentiate into cardiomyocytes

[0016] The human MSCs were isolated and cultured for 3 passages, digested with 0.25% trypsin, washed with PBS, and washed with 2 x 10 5 / cm 2 The density of the cells was inoculated into a 24-well plastic culture plate, and 30uM, 50uM, and 100uM MS-818 were added to the complete medium. After inducing differentiation for 24 hours, the culture medium was discarded, washed twice with PBS, and continued to be cultured in complete medium. The medium was changed every 2 to 3 days, and the cells were digested and passaged with 0.25% trypsin when the cells reached 80% confluence. 14 and 21 days after induction, the cell slides were fixed, and the expressions of desmin, cardiac early transcription factor GATA4, cardiac-specific cTnI and intercalated disc protein connexin43 were observed by immunofluorescence staining. 7.5 x 10 5 MSCs were obtained after 12 passages o...

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Abstract

The present invention provides a method for inducing amplified human bone marrow mesenchymal stem cells in vitro to directionally differentiate into cardiac cells. According to the present invention, by using the biological characteristics of MSCs, human MSCs are isolated and enriched by the density gradient centrifugation method, and stem cells are amplified to obtain a large number of stem cells by using stem cell self-renewal and proliferation characteristics. The isolated stem cells are induced at a specific cultivation generation (3rd-7th generation, and 10th generation) by specific small molecule pyrimidine compounds and a specific concentration (100[mu]M). The in vitro induction directional differentiation scheme of the isolated stem cells has another feature of promoting cell proliferation and phenotype expression of cardiac myocytes by the use of bFGF. The method provides a supplementary source for injured cardiac myocytes, establishes a foundation for replacing clinical treatment of myocardial infarction and later period heart failure by stem cell transplantation, and has good prospects for clinical application.

Description

technical field [0001] The invention relates to a method for using the biological characteristics of bone marrow mesenchymal stem cells to separate and expand adult bone marrow mesenchymal stem cells, add a specific concentration of small molecular organic compound MS-818, and induce differentiation into cardiomyocytes in vitro. Background technique [0002] Coronary heart disease, myocardial infarction and heart failure are one of the main diseases that seriously affect people's quality of life and longevity. Research data show that mature cardiomyocytes lack the ability to regenerate. After myocardial infarction (MI), the myocardium in the infarcted area can only be replaced by fibrous tissue proliferation and replaced by scar tissue without systolic function, resulting in decreased cardiac function. There is a lack of fundamental treatment methods for infarcted myocardium regeneration and reconstruction, and cell replacement therapy has emerged as an ideal treatment strat...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/077
Inventor 田杰
Owner 北京清美联创干细胞科技有限公司
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