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Method of inducing differentiation into myocardial cells using g-csf

A technique for cardiomyocytes and differentiation inducers, applied in the field of cardiomyocytes and differentiation inducers of cardiomyocytes, can solve the problems of low efficiency of cardiomyocyte induction and no reports pointing out G-CSF cardiomyocyte differentiation induction.

Inactive Publication Date: 2010-06-02
福田惠一
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report that G-CSF directly differentiates bone marrow stem cells into cardiomyocytes, and there is no report that G-CSF is expressed in embryonic cardiomyocytes or directly used for induction of cardiomyocyte differentiation.
Furthermore, there is no report at all that G-CSF acts directly on ES cells to induce differentiation into cardiomyocytes.
[0016] As mentioned above, the efficiency of cardiomyocyte induction by conventional methods alone is low, and a more efficient and selective method for inducing cardiomyocyte differentiation is required

Method used

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  • Method of inducing differentiation into myocardial cells using g-csf
  • Method of inducing differentiation into myocardial cells using g-csf
  • Method of inducing differentiation into myocardial cells using g-csf

Examples

Experimental program
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Effect test

Embodiment 1

[0117] Example 1: Expression of G-CSF receptor in myocardium derived from mouse embryonic heart

[0118] The expression of G-CSF receptor in the myocardium derived from mouse embryonic heart was investigated by the following method.

[0119] (1) Research by in situ hybridization

[0120]Pregnant ICR wild-type mice were purchased from CLEA, Japan. Embryos were removed at days 7.5, 8.5, 9.5, and 10.5 of embryonic development, and using a digoxigenin-labeled RNA probe, as described in the literature (Sasaki H et al., Development 118, 47-59 (1993) ) for whole-mount in situ hybridization (WISH) of the heart. The full-length cDNAs of mouse G-CSF receptor (csf3r) and cardiac-specific transcription factor Nkx2.5 (accession numbers NM_008711, NM_008700, respectively) were obtained by reverse transcription PCR (RT-PCR) and subcloned into pBluescript plasmid middle. Use 5'-CCC CTC AAA CCT ATC CTG CCT C-3' (SEQ ID NO: 2) as the sense primer of csf3r, and 5'-TCC AGG CAG AGA TCA GCG AAT...

Embodiment 2

[0125] Example 2: In vivo role of G-CSF in cardiogenesis (1)

[0126] The mice were opened on pregnancy day 9.0, and G-CSF (100 ng / kg) or PBS (phosphate-buffered saline) as a control was directly administered in utero. BrdU (bromodeoxyuridine) was then intraperitoneally administered to the mother on day 9.5 of pregnancy. BrdU is taken up during DNA synthesis and proliferation can be assessed by immunostaining. Embryos were removed on day 12.5 of gestation to make heart slices. Test with the same method as the immunostaining described in embodiment 1 (2), its result is shown in Figure 4 . It was confirmed that G-CSF promotes myocardial proliferation in embryos.

[0127] In addition, heart sections were stained with hematoxylin and eosin, and the results of microscopic observation are shown in Figure 5 a. In the hearts of G-CSF-administered embryos, elongation of the trabecular layer was observed.

[0128] Furthermore, G-CSFR knockout mice lacking the G-CSF receptor (he...

Embodiment 3

[0132] Example 3: In vivo role of G-CSF in cardiogenesis (2)

[0133] On the 9.0th day of gestation, 2 ng was directly administered into the uterus of pregnant mouse mothers. Embryos were removed on day 13.5 of pregnancy, heart sections were made, and immunostained with Phospho-Histon H3. Phospho-Histon H3 is a pigment that is specifically stained during cell proliferation. The results obtained are shown in Figure 7 . G-CS administration was observed to promote myocardial proliferation in embryos.

[0134] The labeling index was calculated by the following formula.

[0135] Labeling index = Phospho-Histon H3 positive nuclei / total nuclei × 100 (%)

[0136] The results obtained are shown in Figure 8 . G-CSF was observed to strongly enhance myocardial proliferation from embryonic day 8.5 to day 10.5.

[0137] In addition, in order to study the effect of G-CSF on the apoptosis of the embryonic heart, Tunel staining was performed on the heart on the 10.5th day of embryoni...

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Abstract

A method of inducing the differentiation of ES cells into myocardial cells which comprises bringing the ES cells into contact with a G-CSF receptor agonist.

Description

technical field [0001] The present invention provides a method for inducing differentiation of ES cells into cardiomyocytes, cardiomyocytes obtained by the method, a differentiation inducer for inducing differentiation into cardiomyocytes, and the like. Background technique [0002] Although cardiomyocytes actively undergo cell division while beating autonomously before birth, they lose their ability to divide after birth, and because they do not have undifferentiated precursor cells, if cardiomyocytes are under various stresses such as myocardial infarction or myocarditis, Down and die, the lost cardiomyocytes cannot be replenished. As a result, the remaining cardiomyocytes maintain cardiac function due to compensatory hypertrophy, but if various pressures continue beyond their permissible range, they will further induce fatigue and death of cardiomyocytes, thereby presenting a reduction in myocardial function (ie heart failure). ). [0003] Therefore, it can be considere...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N15/09A61L27/00C12N5/077
CPCC12N2501/155C12N2506/02C12N2501/22A61K35/12C12N5/0657
Inventor 福田惠一汤浅慎介下地显一郎
Owner 福田惠一
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