KR20190040957A - Maturation method of differentiated ventricular cardiomyocytes - Google Patents

Abstract

The present invention relates to a method for maturation of differentiated ventricular muscle cells, particularly ventricular muscle cells differentiated from human pluripotent stem cells. The method for maturation of differentiated ventricular muscle cells using a medium containing albumin, ascorbic acid, and lactate according to the present invention not only expresses a cardiac cell marker, cTnT protein, and simultaneously, a ventricular-like marker, MLC2V protein at a high ratio, but also promotes maturation to ventricular muscle cells, thereby being useful for testing efficacy or toxicity of heart disease-related drugs in vitro.

Description

[0001] MATURATION METHOD OF DIFFERENTIATED VENTRICULAR CARDIOMYOCYTES [0002]

The present invention relates to a method of maturation of differentiated ventricular muscle cells, particularly ventricular myocytes differentiated from human pluripotent stem cells.

Stem cells are capable of differentiating into various cells, and have autoproliferative capacity. They are embryonic stem cells isolated from early embryos, isolated from primordial germ cells of the embryo Three types of embryonic germ cells and multipotent adult progenitor cells isolated from adult bone marrow are most well known. Stem cells have become the subject of research as a cellular resource for functional recovery of various organs. There are a number of studies that try to find out the effects of cytokines, physiologically active proteins, and simple compounds to induce the differentiation of stem cells into specific cells. ought.

On the other hand, in order to develop and study a therapeutic agent for patients suffering from heart disease, studies using cardiac muscle cells isolated from atrial or ventricular tissue of various species including humans are underway. However, cardiac myocytes obtained by this method have a limited cell proliferation rate, so that the quantitative supply and demand is limited, the differentiation can proceed to other kinds of cells other than the desired cells in the cell differentiation process, And difficulty in securing a variety of human-derived tissues depending on the therapeutic purpose. Thus, it is difficult to develop related technologies.

Therefore, various studies are being conducted to establish the technique of differentiation of cardiac muscle cells. Recently, it has been known that human embryonic stem cells can induce differentiation into cardiac muscle cells (Kehat et al. , J. Clin . Invest. , 108: 407, 2001) The differentiation-induced cardiac myocytes have not only self-sustaining pulsatile function but also myocardial specific proteins such as myosin heavy / light chain, a-actinin, troponin and artificial natriuretic peptide (ANP) . In addition, Matrigel, which mimics the extracellular microenvironment, can be used to differentiate cardiac muscle cells by creating a human-like environment (Nature Methods, 2014), or to produce methacrylated And differentiation techniques such as differentiation of cardiac myocytes by using a cell supporter containing gelatin and the like (Korean Patent Laid-Open No. 10-2014-0165408).

However, the technique of differentiating cardiac myocytes from embryonic stem cells or embryonic germ cells by the conventional method is not limited to cardiomyocytes, but may also include other types of cells such as hematopoietic cells, vascular cells, neural cells, intestinal cells, There is a problem that cells are formed in a mixed form. The efficiency of differentiating cardiac muscle cells from stem cells is very low, and the proportion of cardiac muscle cells in these differentiated cells is only 5 to 20% of the total. In addition, mature functional ventricular myocytes, which are also important for cardiac contraction and relaxation functions and for drug efficacy testing and drug toxicity testing, are well known for their ability to successfully differentiate into cardiac myocytes. (Lian X et al. , Stem Cells. 2013). It is also well known that the efficiency of differentiation into ventricular-like type cells in nodal type, atrial like type or ventricular like type cells, which are heart-forming cells, is remarkably low (Zuhang J., et al., Cir Res., 2012).

Accordingly, the present inventors tried to establish a method of maturing ventricular myocytes differentiated from human pluripotent stem cells, and cultured differentiated ventricular muscle cells using a culture medium containing albumin, ascorbic acid and lactate The present inventors have completed the present invention by confirming that the rate of cells expressing the cTnT protein, which is a marker of cardiac cells, and the MLC2V protein, which is a ventricle-like type marker, are matured at a rapid rate.

It is an object of the present invention to provide a maturation method of differentiated ventricular myocytes and a mature ventricular muscle cell in this manner.

In order to achieve the above object, the present invention provides a method for maturing ventricular myocytes, comprising culturing the differentiated ventricular myocytes in a culture medium containing albumin, ascorbic acid and lactate, and matured.

The present invention also provides mature ventricular myocytes in the manner described above.

The method of matured differentiated ventricular myocytes using the medium containing albumin, ascorbic acid and lactate according to the present invention comprises the steps of: expressing a cardiac cell marker, cTnT protein, and simultaneously, a ventricular-like marker, MLC2V protein at a high ratio In addition, it can be useful for testing the efficacy or toxicity of heart-related drugs in vitro by promoting maturation into ventricular myocytes.

Fig. 1 shows the results of culturing cTnT, MLC2A And the expression of MLC2V marker protein by FACS.
Fig. 2 shows the results of culturing cTnT, MLC2A < RTI ID = 0.0 > And the expression of MLC2V marker protein by FACS.
Figure 4 shows the expression of cTnT, MLC2A < / RTI > expressed in ventricular myocytes from culture day 38 from the day of culture using M5, M6 or M7 medium And the amount of MLC2V marker protein expressed in the M5 medium was defined as 1, and the fold increase was obtained.
FIG. 3 is a graph showing percentage of cells expressing cTnT marker protein expressed in ventricular myocytes from culture day 38 from culture day, using M5, M6 or M7 medium, and simultaneously expressing MLC2A or MLC2V marker protein.
FIG. 5 is a photograph of the morphology of ventricular myocytes matured using M7 medium under a microscope.

Hereinafter, the present invention will be described in detail.

The present invention provides a method of maturing ventricular myocytes including culturing differentiated ventricular myocytes in a culture medium comprising albumin, ascorbic acid and lactate.

As used herein, the term " differentiated ventricular muscle cells " refers to cells that have induced differentiation from human pluripotent stem cells into ventricular muscle cells.

The differentiated ventricular myocytes may be obtained by a step comprising:

1) culturing human pluripotent stem cells in a medium containing matrigel;

* 2) culturing the cultured cells of step 1) in a medium containing wnt signaling promoter and insulin-free medium;

3) culturing the cultured cells of step 2) in a medium containing a wnt signal transduction inhibitor and no insulin.

The human pluripotent stem cells of step 1) may be human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

The wnt signal transduction promoter of step 1) above may be any wnt signal transduction promoter known in the art. The wnt signal transduction promoter may be added at a concentration of 5 to 20, specifically 6 to 15 [mu] M, more specifically 8 to 13 [mu] M. Specifically, the wnt signal transduction promoter may be a compound described in Table 1 below.

name The 2-yl) -2-pyrimidinyl] amino] ethyl] amino] -3-pyridinecarbonitrile

The wnt signal transduction inhibitor of step 2) may be any wnt signal transduction inhibitor known in the art. The wnt signal transduction inhibitor may be added at a concentration of 1 to 15 [mu] M, specifically 2 to 12 [mu] M, more specifically 3 to 8 [mu] M. Specifically, the wnt signal transduction inhibitor may be a compound described in Table 2 below.

name The 2 - [(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno [3,2-d] pyrimidin-2-yl) thio] -acetamide

2 - [[3- (4-fluorophenyl) -3,4,6,7-tetrahydro-4-oxothieno [3,2-d] pyrimidin- benzothiazolyl) -acetamide

3- (2-methoxyphenyl) -4-oxothieno [3,2-d] pyrimidin-2-yl] thio] -acetamide

4- (2-Methyl-4-pyridinyl) -N- [4- (3-pyridinyl) phenyl] benzeneacetamide

N- (6-chloro-2-benzothiazolyl) -3,4-dimethoxy-benzenepropanamide

The albumin may be added at a concentration of 100 to 1,000 μg / ml, specifically 200 to 800 μg / ml, more specifically 400 to 700 μg / ml. On the other hand, the ascorbic acid may be L-ascorbic acid 2-phosphate. The ascorbic acid may be added at a concentration of 10 to 500 μg / ml, specifically 50 to 400 μg / ml, more specifically 100 to 300 μg / ml. The lactate may also be sodium DL-lactate. The lactate may be added at a concentration of 0.1 to 20 mM, specifically 1 to 10 mM, more specifically 3 to 8 mM.

The method according to the present invention may further comprise the step of culturing the mature ventricular myocytes in a medium containing B27.

As used herein, the term " B27 " means a serum-free supplement. B27 is used for the cultivation of neurons, growth and survival of cardiac cells and embryonic cells, and it contains antioxidants, which reduce damage caused by active oxygen.

In a specific embodiment of the present invention, the present inventors differentiated human pluripotent stem cells to obtain ventricular myocytes and then cultured the ventricular myocytes with BSA, L-ascorbic acid 2-phosphate and sodium DL-lactate When cultured in RPMI-1640 medium without glucose, it was confirmed that the ratio of cells expressing the cTnT protein as a cardiac cell marker and the MLC2V protein as a ventricular-like type marker was high (see Figs. 1 to 4) .

In addition, it was confirmed that the cells cultured in the culture medium had a faster rate of maturation into ventricular-like cardiac cells elongated than those cultured in RPMI-1640 medium without glucose containing B27 and sodium DL-lactate (See FIG. 5).

Therefore, the method according to the present invention can be useful for quickly and efficiently matured differentiated ventricular myocytes.

The present invention also provides mature functional ventricular myocytes in this manner.

The ventricular myocytes may be prepared by culturing the differentiated ventricular myocytes as described above in a culture medium containing albumin, ascorbic acid, and lactate, and matured into mature functional ventricular myocytes.

For example, the albumin may be added at a concentration of 100 to 1,000 μg / ml, specifically 200 to 800 μg / ml, more specifically 400 to 700 μg / ml. On the other hand, the ascorbic acid may be L-ascorbic acid 2-phosphate. The ascorbic acid may be added at a concentration of 10 to 500 μg / ml, specifically 50 to 400 μg / ml, more specifically 100 to 300 μg / ml. The lactate may also be sodium DL-lactate. The lactate may be added at a concentration of 0.1 to 20 mM, specifically 1 to 10 mM, more specifically 3 to 8 mM.

The method according to the present invention may further comprise the step of culturing the mature ventricular myocytes in a medium containing B27.

In a specific embodiment of the present invention, the inventors of the present invention found that the ventricular myocytes obtained by differentiating human pluripotent stem cells were treated with BSA, L-ascorbic acid 2-phosphate and glucose-free When cultured in RPMI-1640 medium, the ratio of cells expressing the cTnT protein, which is a marker of cardiac cells, and the MLC2V protein, which is a ventricular-like marker, is high (see FIGS. 1 to 4) (See Fig. 5).

Therefore, the mature ventricular myocytes prepared by the above method can be usefully used for in vitro tests for the efficacy or toxicity of drugs related to heart diseases.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are intended to illustrate the present invention, but the present invention is not limited thereto.

Example 1. Culture of human pluripotent stem cells

First, 10 占 퐂 of Vitronectin XF (Vitronectin XF, Cat # .07180, Stem cell technologies) per well was added to the cell culture plate, and the wells of the plate were coated. Human pluripotent stem cells such as H9 cells to the coated plate by using the (Wicell, WA09-FD) TeSR ™ -E8 ™ medium (Cat # .05940, Stem cell technologies ) prepared by incubation under 37 ℃, 5% CO ₂ conditions Respectively. During the subculture period, the medium was changed daily.

Example 2. Pre-culture of human pluripotent stem cells for differentiation into ventricular myocytes

First, the Matrigel-hESC qualified Matrix (Corning, Cat # .354277) was diluted to a volume ratio of 200: 1 using DMEM / F12 medium. This was added to the volume of 200 ml per well of the 6-well plate, and the plate was allowed to stand for 1 hour under the condition of 37 ° C and 5% CO ₂ to coat the wells of the plate. The human pluripotent stem cells prepared in Example 1 were dispensed into the wells of the coated plates at a rate of 1.5 × 10 ^{5 per} well. The cells were treated with mTeSR ™ (10 μM), Y27632 (Stemgent, Cat # 04-0012-02) -1 medium (Cat # .5850, Stem cell technologies). This was cultured for 5 days while changing the medium every day, and the medium was mTeSR ™ -1 medium. Two days after the culture, the Matrigel-hESC qualified Matrix was replaced with mTeSR ™ -1 medium diluted to a volume ratio of 200: 1 and cultured for 24 hours.

Example 3. Differentiation into ventricular myocytes

First, the growth factor reduced form Matrigel (Corning, Cat # 354230) and B27 supplement without insulin (Invitrogen, Cat # .A1895601) were added to 50 ml of RPMI-1640 medium (Gibco, Cat # .11875) And then diluted to a volume ratio to prepare a differentiation medium.

On the other hand, the culture medium of human pluripotent stem cells pre-cultured in Example 2 was removed, and the prepared differentiation medium was added to 2 ml per well. 10 mu M of CHIR99021 (Selleckchem, Cat #. S2924) was added thereto and cultured. On day 1, 3, 5, 7 and 9, the medium was replaced with RPMI-1640 medium supplemented with B27 supplement without insulin, and 5 μM of IWP4 (Stemgent, Cat #. 04-0036) was added.

As a result, the ventricular myocyte was observed from 8 days after culture.

Example 4. Maturation of differentiated ventricular myocytes

First, a mature medium containing the composition shown in Table 3 below was prepared.

M5 DMEM (Gibco, Cat # .11966025), B27-Minus Insulin,
5 mM sodium DL-lactate (Sigma-Aldrich, Cat # .L4263) M6 RPMI-1640 (Gibco, Cat # .11879), B27-Minus Insulin, 5 mM sodium DL-lactate M7 RPMI-1640, BSA, 200 [mu] g / ml L-ascorbic acid 2-phosphate,
5 mM sodium DL-lactate

On the 10th day after culturing of ventricular myocytes differentiated in Example 3, the culture medium of the cells was removed, replaced with M5, M6 or M7 medium, and further cultured for 10 days. At this time, the mature medium was changed once every two days. After 10 days of culture, matured medium was removed and mature cardiac myocyte cells were further cultured for 70 days by adding RPMI-1640 medium containing B27 supplement 50x (Invitrogen, Cat # 17504-044) containing insulin .

Experimental Example 1. Confirmation of differentiation rate into ventricular muscle cells

Cells of 24 days and 38 days of culture cultured in Example 4 were sampled to express the cTnT protein as a marker of cardiac cells while simultaneously expressing atrial like type marker MLC2 A protein or ventricular like type marker, MLC2V protein, was analyzed by FACS method.

Specifically, trypsin-EDTA was added to the cells on the 38th day of culture, and the cells were separated into single cells, fixed with 4% paraformaldehyde for 20 minutes, and then subjected to Permeabilization Perm / Ferm solution (250 μl / tube) And cultured at 4 DEG C for 30 minutes. After the reaction, the cells were washed with PBS, and then primary antibody (Cat #: 564767, 5 μl, BD Bioscience) for cTnT and primary antibody (Cat # 130-106-141, Miltenyi Biotec, 10 μl ) And a primary antibody (Cat # 130-106-134, Miltenyi Biotec, 10 μl) for MLC2V-APC were added and reacted at 4 ° C for 30 minutes. Cells reacted with the antibody were washed with PBS, and the expression ratios of the respective protein markers were confirmed using a FACS instrument (BD FACSCalibur flow cytometer). The results are shown in FIGS. 1 to 4.

As shown in FIG. 1, in the cells cultured on the 24th day of culture using the M7 medium, the proportion of cells expressing the cTnT marker and expressing the ventricle-like marker MLC2V protein was high (FIG. 1). From this, it was confirmed that the differentiated and matured cells were significantly differentiated into ventricular-like ventricular myocytes by the above method.

On the other hand, as shown in Figs. 2 to 4, the ratio of cells expressing the cTnT marker and expressing the MLC2V protein at the 38th day of culture was the highest when the M7 medium was used (Fig. 2). On the other hand, when these results were cultured in the M5 medium, when the amount of the expressed protein was 1, it was increased to a multiple thereof. As a result, when cultured in M7 medium, cells expressing cTnT protein and expressing both MLC2A and MLC2V (MLC2A + / MLC2V +) expressing the cTnT protein while expressing the cTnT protein and the MLC2V-expressing cell (MLC2A + / MLC2V +) expressing the cTnT protein and the MLC2V-expressing cell (MLC2A + / MLC2V +) about 4 times (Fig. 4). Also, the proportion of cells expressing the cTnT protein and simultaneously expressing MLC2A or MLC2V protein was 7.4%, 13.03% and 65% in the M5, M6 and M7 medium, respectively (Fig. 3).

Experimental Example 2. Identification of differentiated ventricular myocyte morphology

The morphology of differentiated ventricular myocytes matured using the M5, M6 or M7 medium in Example 4 was confirmed microscopically. At this time, the morphology of cells on day 92 of culturing in M5 medium, on day 67 of culturing on M6 medium, and on day 30 after culturing on M7 medium were compared.

As a result, as shown in Fig. 5, matured ventricular myocytes differentiated using M7 medium were matured into elongated ventricular-like heart cells within 30 days of culture. On the other hand, when the M5 and M6 medium were used, they matched to elongated ventricular-like cardiac cells on days 92 and 62 of culture (FIG. 5).

From the above, it was found that the culture medium supplemented with BSA, ascorbic acid and lactate effectively maturated the differentiated ventricular muscle cells.

Claims (9)

1) culturing human pluripotent stem cells in a culture vessel coated with matrigel, with a medium containing no matrigel;
2) culturing the cultured cells of step 1) in a medium containing matrigel;
3) culturing the cultured cells of step 2) in a medium containing wnt signaling promoter and Matrigel and without insulin;
4) culturing the cultured cells of step 3) in a medium containing a wnt signal transduction inhibitor and insulin-free medium to differentiate into ventricular myocytes; And
5) culturing the differentiated ventricular myocytes of step 4) in a culture medium containing albumin, ascorbic acid and lactate to mature into mature functional ventricular myocytes.
The method according to claim 1, wherein said albumin is contained at a concentration of 100 to 1,000 占 퐂 / ml.
The method of claim 1, wherein the ascorbic acid is L-ascorbic acid 2-phosphate.
The method of claim 1, wherein the ascorbic acid is included at a concentration of 10 to 500 [mu] g / ml.
2. The method of claim 1, wherein the lactate is sodium DL-lactate.
The method of claim 1, wherein the lactate is included at a concentration of 0.1 to 20 mM.
The method of claim 1, further comprising culturing the mature ventricular myocytes in a medium containing B27.
The method according to claim 1, wherein the human pluripotent stem cells are embryonic stem cells, inducible pluripotent stem cells or a mixture thereof.
A mature functional ventricular myocyte according to the method of claim 1.

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