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Method of preparing cell for transplantation

A cell and original cell technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problem of not being able to induce differentiation significantly, and achieve the effect of high cell survival rate and high cell incorporation rate

Inactive Publication Date: 2007-01-03
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, demethylating drugs such as 5-azacytidine, although proven safe in humans, do not significantly induce differentiation into one cell type, cardiomyocytes

Method used

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  • Method of preparing cell for transplantation
  • Method of preparing cell for transplantation
  • Method of preparing cell for transplantation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Method of inducing cardiomyocytes from bone marrow stem cells by treatment with biological growth factors

[0068] More than four weeks after myocardial infarction, 15-20 ml of bone marrow stem cells were obtained from dog ilium using a heparin-soaked syringe. These bone marrow stem cells were treated with 2-20% fetal bovine serum and 1-1,000 μM L-ascorbic acid-2-PO 4 , 5-15 ng / ml LIF (leukemia inhibitory factor) and 1-200 nM dexamethasone culture medium. All culture devices and slides were coated with 5 ng / ml collagen for 15 min at room temperature prior to receiving bone marrow stem cells. This in vitro condition allows bone marrow stem cells to maintain their self-renewal characteristics and expand through passage without loss of response to differentiation drugs such as growth factors. Moreover, this condition also allows bone marrow stem cells to maintain mesenchymal morphology and karyotype through subculture. figure 1 is a phase-contrast photomicrog...

Embodiment 2

[0074] Example 2: A method for inducing bone marrow stem cells to become cardiomyocytes with high cell incorporation rate and cell survival rate for a period of time

[0075] Bone marrow stem cells can be treated with biological growth factors in cardiac-specific medium for different periods of time, from 1 hour to 21 days, and during this period, about 40-90% of the cells express the nuclear protein MEF2. According to the different treatment time in heart-specific medium, the ratio of induced cardiomyocytes can be increased to 60-90%. The optimal treatment time can make 80-90% of the cells express MEF2 protein. Transplantation of 80-90% of these MEF2-expressing cells can optimally regenerate infarcted hearts.

[0076] Bone marrow stem cells isolated from adult dogs were cultured in cardiac-specific medium for 6 days, showing the optimal amount of MEF2 protein present in the nucleus under fluorescence microscopy. Transplantation of these cells into infarcted dogs showed inco...

Embodiment 3

[0079] Example 3: Transplantation of bone marrow stem cells and evaluation of cell incorporation rate and survival rate

[0080] Bone marrow stem cells differentiated in vitro into cardiomyogenic cell lines were transplanted into myocardial tissue of infarcted dogs. Myocardial infarction in dogs was produced by persistent occlusion of the left coronary artery. The infarction remained stable for at least 2 months prior to transplantation of bone marrow stem cells. To avoid immune rejection of the graft, bone marrow was collected from individual transplant recipient dogs and bone marrow stem cells were prepared as follows:

[0081] Approximately 4 weeks after ligation, after myocardial infarction was confirmed by echocardiography, bone marrow was aspirated after perforation on the ilium. Quickly mix the bone marrow and heparin, freeze and transfer to the tissue culture room with dry ice, where the bone marrow is thawed at 37°C, shaken, washed once with conventional DMEM, place...

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Abstract

The invention describs a method of producing cells for transplantation into myocardial tissue of a mammal. The method comprises the steps: (a) providing bone marrow stem cells that have not been immortalized; (b) passage culturing two times said bone marrow stem cells under conditions that maintain the characteristics of bone marrow stem cells; (c) culturing said bone marrow stem cells in cardiac specific media to differentiate into cardiomyogenic cells; and (d) collecting the cells of step (c) when about 80 to 90% of said cells are cardiomyogenic cells. The cardiomyogenic cells induced according to the method can be clinically applied more suitably than those induced using demethylating agent such as 5-azacytidine, and therefore, they provide induced bone marrow stem cells of a mammal into cardiomyocytes having high rates of cell incorporation and cell survival for treating a mammal (e.g., a human) diagnosed as having a disorder characterized by insufficient cardiac function.

Description

technical field [0001] The present invention relates to methods of producing cells for transplantation of cardiac tissue in mammals. More particularly, it relates to a method in which mammalian bone marrow stem cells are treated with biological growth factors to differentiate into cardiac cell lineages and the differentiated bone marrow stem cells are treated in vitro for a period of time to achieve efficient cell incorporation rates and cell viability, and then transplant induced bone marrow stem cells into mammals with myocardial infarction and evaluate their changes. technical background [0002] Mesenchymal stem cells obtained from the bone marrow of adults have been shown to have the ability to differentiate into various cells including adipocytes, bone cells, chondrocytes, hepatocytes and cardiomyocytes. Based on this ability, bone marrow stem cells have been proposed as a source of cell transplantation. [0003] Myocardial infarction is one of the leading causes of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/00C12N5/02C12N5/077
CPCC12N2501/155C12N2501/115C12N2501/105C12N5/0657C12N5/0662
Inventor 杰弗里·D·克鲁伊森特金泽承柳喜媛
Owner ANTEROGEN
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