Induction factor for inducing differentiation of iPS (induced pluripotent stem) cells into cardiac muscle cells and application thereof

A technology for inducing differentiation and cardiomyocytes, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of inapplicable cardiomyocytes, low efficiency, and complicated production process of "hanging drops", and achieve the efficiency of inducing differentiation high effect

Inactive Publication Date: 2011-06-15
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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Problems solved by technology

[0003] The reported differentiation method of iPS cells to cardiomyocytes is a method of differentiating mouse iPS cells into Flk+ cells, sorting Flk+ cells by flow cytometry, and co-cultivating them with OP9 cells to induce the differentiation of cardiomyocytes (Genta Narazaki et al. Circulation 2008; 11 8; 498-506), this method is cumbersome and ineffective
The other is to use the "hanging drop method" to form mouse iPS cells into EBs, and then use IMDM-based differentiation medium to induce the differentiation of iPS-EBs into cardiomyocytes (Christina Mauritz et al. Circulation 2008; 11 8; 507-5 1 7 ), the effect of this method is better (the differentiation rate is about 50%), but the production process of "hanging drop" is complicated, and it is not suitable for the preparation of a large number of cardiomyocytes
Another is to use low-adherence bacterial culture dishes to form human iPS cells into embryoid bodies (Embryoid bodies, EBs), and then use DMEM / F12-based differentiation medium to induce iPS-EBs to differentiate into cardiomyocytes (Jianhua Zhang et al. Circ .Res.2009; 1 04; e30-e41), this method is suitable for forming a large number of EBs, but the efficiency of EBs differentiation to myocardium is low (1-10%)
It can be seen from the above that at present, there is no simple and efficient method for inducing the differentiation of iPS cells into cardiomyocytes.

Method used

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  • Induction factor for inducing differentiation of iPS (induced pluripotent stem) cells into cardiac muscle cells and application thereof
  • Induction factor for inducing differentiation of iPS (induced pluripotent stem) cells into cardiac muscle cells and application thereof
  • Induction factor for inducing differentiation of iPS (induced pluripotent stem) cells into cardiac muscle cells and application thereof

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Experimental program
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Embodiment 1

[0046] The preparation of embodiment 1 induction differentiation medium

[0047] (1) Preparation of growth medium for embryoid bodies (EBs): Add 100ml serum (Gibco), 0.055mM β-mercaptoethanol, 2mM L-glutamine and 0.1mM non-essential to 400ml Knockout DMEM medium (Gibco) Amino acid (Gibco) obtained 500ml embryoid body growth medium;

[0048] (2) 10 ml of DMEM medium in step (1) is taken, and 0.01 molar amount of ascorbic acid powder is dissolved therein to obtain 1M ascorbic acid stock solution;

[0049] (3) The 1M ascorbic acid stock solution in step (2) is diluted 10 with the culture medium of step (1) respectively 7 , 10 6 , 10 5 , 10 4 , 10 3 times, respectively to obtain 10 -7 , 10 -6 , 10 -5 , 10 -4 , 10 -3 M Ascorbic acid-induced differentiation medium.

Embodiment 2

[0050] Example 2 Inducing the differentiation test of iPS-tet-B3 cells into cardiomyocytes using induction differentiation medium was carried out according to the following method:

[0051] (1) Preparation of feeder layer cells

[0052] Mouse embryonic fibroblasts were isolated from the fetuses of 14-day-gestational Kunming white mice (purchased from the Experimental Animal Center of the Academy of Military Medical Sciences), cultured and expanded in H-DMEM containing 10% serum (volume percentage); with 10 μg / ml Mitomycin solution was used to treat fibroblasts of the 2nd to 4th passages in good growth state for 3.5 hours, with 4-5×10 4 cells / cm 2 Inoculated in tissue culture dishes, that is, made into feeder cells, which can be used for inoculation of iPS cells the next day;

[0053] (2) Culture expansion of undifferentiated mouse iPS cells

[0054] Cultivate the iPS-tet-B3 cell line under the feeder layer system in step (1) (gifted by the Shanghai Institute of Biology, Chi...

Embodiment 3

[0061] Example 3 Comparison Test of Induction and Differentiation of EBs to Cardiomyocytes in Different Growth Days

[0062] Coat 96 petri dishes with 0.1% gelatin. The coating method is to add gelatin solution to the petri dish, cover the bottom of the dish, let it stand for 15 minutes, absorb the excess gelatin liquid, dry it and place it at 37°C, 5% CO 2 The incubator is ready for use; collect the EBs grown in suspension for 3 to 8 days in step (3) of Example 2, set aside to settle in a centrifuge tube, remove the embryoid body growth medium, and use ascorbic acid concentration of 10 -4 EBs were resuspended in the induction medium of M, seeded in a gelatin-coated 96-well culture dish at a density of 1 EB / well, and placed at 37°C, 5% CO 2 Culture in the incubator, replace the fresh induction differentiation medium on the third day, and replace it every other day; the ratio of EBs to cardiomyocytes induced differentiation is calculated as: the number of spontaneously beating ...

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Abstract

The invention belongs to the field of regenerative medicine of cardiac muscle tissues, and discloses application of ascorbic acid as an induction factor in inducting differentiation of iPS (induced pluripotent stem) cells into cardiac muscle cells and a differentiation-inducing culture medium containing ascorbic acid. The invention also discloses a method for inducing differentiation of iPS cells into cardiac muscle cells by using the differentiation-inducing culture medium, which comprises the following step: inducing an iPS cell derived embryoid body, which has grown in a suspension mode for 5-7 days, to differentiate by utilizing the differentiation-inducing culture medium, thereby obtaining cardiac muscle cells. By using ascorbic acid as the induction factor, the invention obviously enhances the differentiation efficiency of the iPS cells into the cardiac muscle cells, and the differentiation percentage can reach 50-60%; the differentiation-inducing culture medium does not have toxic or side effect on the cells; and in addition, the method provided by the invention is simple, has the advantage of low cost, can be operated in common laboratories, and is suitable for large-scale proceeding.

Description

technical field [0001] The invention belongs to the field of myocardial tissue regeneration engineering. In particular, it relates to an inducing factor for inducing iPS cells to differentiate into cardiomyocytes, an inducing differentiation medium containing the inducing factor, and a method for using the inducing differentiation medium to induce iPS cells to differentiate into cardiomyocytes. Background technique [0002] Pluripotent stem cells (pluripotent stem cells) have the potential to differentiate into a variety of cell tissues, and are of great significance to the research of regenerative medicine, drug screening, disease models and developmental biology. In 2006, Japanese scholars (Kazutoshi Takahashi et al. Cell 2006, 126: 1-14) transformed normal somatic animal fibroblasts into Pluripotent stem cells with highly similar biological properties to embryonic stem cells are called induced pluripotent stem cells (Induced poluroipotent stem cells, iPS). In addition t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 王常勇刘志强王海滨段翠密林秋霞
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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