KR20160142587A

KR20160142587A - Method for cardiomyogenic differntiation of stem cells by applying electric and mechanical signals

Info

Publication number: KR20160142587A
Application number: KR1020150078511A
Authority: KR
Inventors: 김병수; 윤정기; 방석호
Original assignee: 서울대학교산학협력단
Priority date: 2015-06-03
Filing date: 2015-06-03
Publication date: 2016-12-13
Also published as: WO2016195247A3; WO2016195247A2

Abstract

Stem cell differentiation efficiency can be further enhanced by simulating the microenvironment of in vivo myocardial cells that differentiate stem cells by simultaneously applying electrical stimulation and mechanical stimulation to stem cells inoculated on a support having both piezoelectricity and elasticity. Accordingly, the stem cell differentiation method according to the present invention can be applied not only to various studies, but also to the treatment of myocardial cell-related diseases including myocardial infarction.

Description

METHOD FOR CARDIOMYOGENIC DIFFERENTIATION OF STEM CELLS BY APPLICATION ELECTRIC AND MECHANICAL SIGNALS BACKGROUND OF THE INVENTION Field of the Invention [0001]

The present invention relates to a method for differentiating stem cells into myocardial cells by applying electromechanical stimulation.

Stem cells are differentiated into individual cells, which are maintained as they do not undergo differentiation into specific cells, and are capable of differentiating into all kinds of cells, such as nerves, blood, cartilage, and muscles, And the undifferentiated cells at the pre-stage. Unlike the differentiated cells in which the cell division is stopped, the stem cells are self-renewing and proliferate by the cell division, and when the differentiation stimulus (micro environment) is applied, It is characterized by plasticity in differentiation because it can be differentiated into other cells by different environment or differentiation stimulation.

In recent years, a method of differentiating mesenchymal stem cells from myocardial cells through microenvironmental simulation of the heart using an electric field or a cyclic strain has been widely used by utilizing the above-described features of stem cells. However, there are methods of applying only electrical stimulation using an electric field or current, and studies of applying only mechanical stimulation, but there is a limit to the efficiency of differentiation and there is room for improvement.

On the other hand, heart disease is the leading cause of death in developed countries and the prevalence rate is increasing rapidly in Korea. Currently, various procedures are performed to treat myocardial infarction and abnormal cardiac development, but there are many difficulties in curing due to the low regeneration ability of myocardial cells.

Currently, the most commonly performed procedures for treating heart disease are transplantation and replacement of part of the heart tissue with animal-derived tissue. However, these procedures have problems such as securing organ donors and rejecting immune rejection.

To solve these problems, many research groups are conducting research on how to restore heart function through cardiac cell transplantation. As a method for acquiring cells to be used for cardiomyocyte transplantation, it is most commonly proposed to obtain myocardial cells by differentiating adult stem cells, embryonic stem cells and induced pluripotent stem cells (iPSC) Method. However, embryonic stem cells and inducible pluripotent stem cells (ESCs) commonly suffer from the risk of teratoma formation, making them difficult to apply in clinical applications.

Disclosure of Invention Technical Problem [8] To overcome the limitations of the prior art as described above, it is an object of the present invention to provide a method for efficiently inducing differentiation of stem cells into cardiac myocytes.

In order to achieve the above object,

Inoculating stem cells onto a support having both piezoelectricity and elasticity, and

And a step of differentiating the stem cells into myocardial cells by applying a motility to the support on which the stem cells have been inoculated.

The present invention also provides a myocardial cell-support complex containing myocardial cells differentiated from stem cells by the above method.

Other details of the embodiments of the present invention are included in the following detailed description.

The method of the present invention for differentiating stem cells into myocardial cells by inoculating stem cells into a piezoelectric and elastic substrate (PES) having both piezoelectricity and elasticity, followed by generating electrical stimulation and mechanical stimulation at the same time, It is possible to further enhance the differentiation efficiency by simulating in vivo microenvironment. Therefore, the present invention can be applied to various studies such as treatment of cardiovascular diseases including myocardial infarction.

1 is a schematic diagram showing two microenvironments of the heart, an electrical signal and a mechanical signal.
FIG. 2 is a schematic diagram illustrating a process of differentiating mesenchymal stem cells into myocardial cells using PES according to the present invention.
3 is a schematic diagram of a cell culture apparatus capable of bending and stretching (stretching and contracting) a PES according to an embodiment of the present invention.
4 is a schematic diagram showing a manufacturing process of PES.
5 is a SEM image of (a) zinc oxide (ZnO) nano-rods used in the examples, and (b) SEM images of single zinc oxide nanorods used in PES.
Figs. 6 to 9 show electrical characteristics at the time of PES bending (bending motion) in Test Example 1. Fig.
10 shows the arrangement of ZnO nanorods in the PES for mechanical stimulation in Test Example 1. FIG.
Fig. 11 shows the permanent strain profile when subjected to 10 days of PES and PDMS bending and mechanical stimulation in Test Example 1. Fig.
FIGS. 12-14 relate to cytotoxicity when subjected to PES, electrical stimulation and mechanical stimulation in Test Example 1. FIG.
15 shows the arrangement of hMSCs by mechanical stimulation in Test Example 2 with F-action staining (blue = cell nucleus, scale bar = 30 μm).
Figures 16 to 18 show the improvement of myocardial differentiation of hMSCs by electrical stimulation and mechanical stimulation according to Test Examples 3 to 5.
19 is a schematic diagram of a mechanism of myocardial differentiation of hMSC according to electrical stimulation and mechanical stimulation.
20 shows the results of evaluation of expression of BMP-4, IGF, VEGF and TGF-? By the RT-PCR method of Test Example 6. Fig.
21 shows the expression of p38, SMAD, FAK and ERK1 / 2 by the Western blot method of Test Example 3. Fig.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

It will be understood that when a layer, film, film, substrate, or the like is referred to herein as being "on" or "on" another part, it also includes the case where there is another part in the middle, do. On the contrary, when a portion such as a layer, a film, a film, a substrate, or the like is referred to as being 'under' another portion, it includes not only a case where the other portion is 'directly below' but also a case where there is another portion in between.

Hereinafter, the present invention will be described in detail.

The stem cell differentiation method according to the present invention comprises

Inoculating stem cells onto a support (PES) having both piezoelectricity and elasticity, and

And applying a mobility to the support on which the stem cells are inoculated to differentiate the stem cells into myocardial cells.

In the present specification, 'elasticity' refers to a property that deforms when a force is applied, but returns to its original shape when a force is removed.

In the present specification, 'motility force' or 'kinetic energy' refers to a stimulus that may cause physical changes such as bending, stretching, and contraction, and does not discriminate the means or the method.

According to a preferred embodiment, the movement force may be either one of a bending and stretching motion and a stretching motion, or both. Exercise means bending, stretching, and stretching movements, which move back to their original state or contract. This movement is possible because the PES has elasticity.

In addition, the method of differentiating stem cells according to the present invention promotes differentiation by simultaneously applying electrical energy and mechanical energy to the stem cells, and the stretching motion or the stretching motion applied to the supporter can be carried out by using the piezoelectricity of the supporter, It may be to generate energy.

The electrical energy may be one that generates a voltage of 0.1 to 10 V by bending and rolling the support at a frequency of 0.1 to 10 Hz. A voltage of 0.5 to 5 V can be generated at a frequency of preferably 0.1 to 5 Hz.

The mechanical stimulation may be generated by stretching the support by a length of 1 to 20% of the original length at a frequency of 0.1 to 10 Hz and contracting the support. The voltage can be generated by increasing the length of 1 to 10% of the original length of the support by a frequency of preferably 0.1 to 5 Hz and shrinking it.

According to a preferred embodiment, the inoculated stem cells can be arranged in a direction perpendicular to the direction of the motive force applied to the support.

The stem cells may be selected from the group consisting of adipose stem cells, mesenchymal stem cells, bone marrow stem cells, cord blood stem cells, neural stem cells and induced pluripotent stem cells.

For example, mesenchymal stem cells (MSCs) are pluripotent undifferentiated cells derived from mesenchymal cells of the fetus and are morphologically stromal cells containing spongy fibers and spongy aggregates of nonspecific cells. Thus, it can be differentiated into organs such as connective tissue, bone, cartilage, lymphatic vessels, and blood vessels.

1 is a schematic diagram showing two microenvironments of the heart, an electrical signal and a mechanical signal. In other words, the cardiomyocyte differentiation is promoted by the application of electric field and mechanical stimulus (cyclic strain). The differentiation process of myocardial cells will be specifically described in the examples.

In the present invention, as a culture supporter for differentiating mesenchymal stem cells into myocardial cells, a PES capable of simultaneously generating or applying an electrical signal and a mechanical signal is used as shown in FIG.

The PES can be repeatedly stretched or bent using, for example, the device shown in FIG. 3, to deliver electrical stimulation and mechanical stimulation to the stem cells inoculated to the support.

Specifically, the motion of the motor of the apparatus of FIG. 3 is transmitted to the PES located in the cell culture section, thereby causing the PES to bend and bend, stimulating the piezoelectric material of the PES to generate an electrical signal, Stretching, stretching) motion of the elastic material.

According to a preferred embodiment, as shown in FIG. 4, the PES may have a structure in which the elastic layer and the piezoelectric material layer are alternately laminated one or more times, preferably two or more times. At this time, it is preferable that the outermost layer is an elastic layer.

According to one embodiment, the support comprises

And a piezoelectric material layer arranged between the first elastic layer and the second elastic layer. The first elastic layer, the second elastic layer, and the piezoelectric material layer are disposed between the first elastic layer and the second elastic layer.

The piezoelectric material may be at least one of piezoelectric materials including ZnO, BaTiO ₃ and NaNBO ₃ , but is not limited thereto. That is, ZnO is an example of a typical piezoelectric material, and the present invention is not limited thereto, and can be applied to the present invention as long as it is a piezoelectric material capable of converting an external mechanical force into electric potential. For example, ZnO, ZnSnO _3, GaN, it is also possible to use a piezoelectric material such as _{Te, CdTe, CdSe, KNbO 3} , NaNbO 3, InN, PVDF, PVDF-TrFE.

The piezoelectric material layer may include a plurality of piezoelectric material nanorods. The piezoelectric material nanorod may be a biaxially grown piezoelectric material nano-rod.

In addition, the piezoelectric material nano-rods may be arranged in one direction to form a piezoelectric material layer. In particular, the piezoelectric material nano-rods may be arranged in a unidirectional single layer to form a piezoelectric material layer.

As used herein, the term nanorod is a term commonly used by those skilled in the art, and may generally mean a rod with an aspect ratio of 10 or less, and, in some cases, And may be referred to as about 100 nm or less with respect to the size thereof. As such, it should be noted that the nanorod is interpreted as a material having the technical meaning commonly used by those skilled in the art. The ZnO nanorods used in one embodiment of the present invention were about 2.5 to 3 mu m in length and about 200 to 250 nm in diameter. These small-sized one-dimensional nanomaterials are characterized in that lattice strain can be easily induced by small mechanical energy (bending or shaking) from the outside.

In the embodiments described hereinafter, the nano-rods, specifically, biaxially-grown nano-rods are used to generate the piezoelectric potential, but the present invention is not limited thereto. That is, uniaxially grown nano-rods can also be employed. As the material having piezoelectric properties, a nano-sized film shape, a nanowire shape, or the like may be employed. In addition, the biaxially grown nano-rods employed in the examples were produced by the hydrothermal synthesis method, but the present invention is not limited thereto. That is, hydrothermal synthesis is used in the case of mass synthesis, but CVD may be used to form a nanorod having a small amount of high crystallinity.

The elastic layer is preferably made of a material capable of transmitting mechanical energy applied from the outside to the piezoelectric material layer.

In addition, it is preferable that the elastic layer is made of a material capable of transmitting the piezoelectric potential generated from the piezoelectric material layer to the inoculated stem cells.

According to a preferred embodiment, the elastic layer may be made of a material containing PDMS (polydimethylsiloxane), but is not limited thereto and any elastic material having cell suitability or dielectric constant can be used without limitation. That is, a dielectric material having a dielectric constant and easy to transmit a piezoelectric potential generated from the piezoelectric material can be applied to the present invention, and it is preferable that mechanical energy applied from the outside can be transmitted to the piezoelectric material without loss.

According to a preferred embodiment of the present invention, when the electrical stimulation and the mechanical stimulation are simultaneously given to the mesenchymal stem cells, the efficiency of differentiation into myocardial cells is increased.

The myocardial cells differentiated from the stem cells by the above-described method can be used for various uses as myocardial cell-support complexes.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, the description of technical constructions already known in the art will be omitted. For example, the uniaxial nano-rod, the method of synthesizing / manufacturing the biaxially grown nano-rods, and the process of aligning the nano-rods through the rubbing process are well-known processes related to the liquid crystal alignment, so detailed description thereof is omitted. Even if these explanations are omitted, those skilled in the art will readily understand the constitution of the cell support, its function, and the like, which are presented according to the present invention through the following description.

PREPARATION EXAMPLE 1 Preparation of zinc oxide nano-rods (ZnO NRs) powder

The zinc oxide nanorods powders were prepared by a wet chemical method.

0.42 g of zinc nitrate hexahydrate (Zn (NO ₃ ) ₂ .6H ₂ O, ≥99.0%) (Sigma Aldrich) was dissolved in 100 mL of deionized water and 0.24 g of HMTA (hexamethylene Tetramine, Sigma Aldrich) were separately dissolved in 100 mL of deionized water (all at room temperature) to prepare two precursor solutions. With stirring at 85 DEG C, the zinc precursor solution was continuously injected into the HMTA solution via a syringe pump at an injection rate of 2 mL / h for 25 minutes, and the process was terminated after 5 minutes. After centrifugation, the flocculated nanorods were separated from the suspension and washed three times with deionized water to remove unreacted Zn ²⁺ and other ions. The final precipitate was dried at 80 占 폚 and annealed at 400 占 폚 for 2 hours in vacuum to improve the crystallinity and ultimately to prepare a biaxially grown ZnO nano-rod powder.

PREPARATION EXAMPLE 2 Preparation of Piezoelectric and Elastic Substrate (PES) Having Piezoelectricity and Elasticity

The piezoelectric and elastic material (PES) disclosed in Fig. 4 has a structure in which a plurality of zinc oxide nano-rods (ZnO NRs) having piezoelectricity are arranged between elasticity polydimethyl-siloxane (PDMS) And the manufacturing process thereof can be referred to Korean Patent Registration No. 10-1497338.

The morphological characteristics of the zinc oxide nanorods were analyzed using a JEOL JSM-7000F field emission scanning microscope (FESEM, Jeol Ltd., Akishima, Tokyo, Japan;

The PDMS block and the substrate for the rubbing process were prepared with a curing agent in a weight ratio of 10: 1 (Sylgard 184, Dow Corning Chemicals). A single layer of zinc oxide nanorods was formed on the PDMS substrate with a PDMS block through a unidirectional rubbing process (see Fig. 5 (a)), and the untreated PDMS was coated onto a single layer of zinc oxide nano- At a weight ratio of 1: 1.

The diluted PDMS was applied to a PDMS substrate having a single layer of zinc oxide nanorods, spin-coated at 3,000 rpm for 30 seconds, and treated on a hot plate at 85 ° C for 30 minutes.

The above process was repeated 5 times to fabricate a PES (Piezoelectric and Elastic Substrate) having a 5-fold zinc oxide nano-rod layer (piezoelectric material layer) which gives an electrical signal of 3V.

Test Example 1 Characteristic Analysis of PES (Piezoelectric and Elastic Substrate)

In order to evaluate the electrical properties of the PES prepared in Preparation Example 1, a silver electrode (200 nm) was deposited on the upper and lower sides of the PES by thermal evaporation, and the resulting device was mounted on a 3 mm thick PDMS substrate After that, voltage and current signals were measured.

FIGS. 6 to 9 show electrical characteristics when the PES having a length of 5 cm was bent at a radius of curvature of 20 mm (flexing motion). The current and the voltage were measured using a picoammeter (Keithley 6485, Keithley Instruments, Cleveland, ) And an electrometer / high resistance meter (Keithley 6517, Keithley Instruments).

Specifically, FIG. 6 shows the open circuit voltage generated in the PES when connected in the forward direction, FIG. 7 shows the open circuit voltage generated in the PES when connected in reverse, FIG. 8 shows the short circuit current density generated in the PES 9 represents the short circuit current density generated at the PES when reverse connected.

Alignment of zinc oxide nanorods of PES was performed by photographing and evaluating photographs of 0 random and 10 day post-stimulation at 5 random sites using an optical microscope (BX41, Olympus, Tokyo, Japan) National Institutes of Health, Bethesda, Md., USA). Figure 10 shows the arrangement of ZnO NRs in PES for 0 day and 10 day flexion and mechanical stimulation.

To determine the permanent deformation of PES and PDMS, lengths before and after stimulation were compared and measured. Figure 11 shows the permanent strain profile when subjected to 10 days of PES and PDMS bending and mechanical stimulation.

As a result of Test Example 1, it was confirmed that ZnO NRs were arranged on the PDMS in a unidirectional, monolayer arrangement. When the 5 cm PES was moved with a radius of curvature of 20 mm, an electrical signal of 3 V was obtained there was.

Example 1 Induction of Myocardial Differentiation of Mesenchymal Stem Cells Using PES

Human mesenchymal stem cells (hMSCs) (Lonza, Walkerxville, MD, USA) were inoculated with 10% v / v FBS (Gibco BRL) and 1% v / v penicillin / (v / v) CO ₂ incubator at 37 ° C in a medium consisting of DMEM low glucose (Gibco BRL, Gaithersburg, MD, USA) containing streptomycin (Gibco BRL) , And only hMSCs cells with no more than 6 passage passages were used.

The mesenchymal stem cells were seeded at a cell density of about 5 x 10 ⁴ cells / cm ^{2 in} the cell attachment area of PES, treated with 5-azacytidine (5-azaC) (6 μmol / L) Lt; / RTI > In the apparatus shown in FIG. 3, after placing the PES inoculated with the stem cells, electrical signals are generated through the stimulation for about 10 days, and mechanical signals are generated through the stimulation that stretches and contracts, Stimulation of lobular stem cells. At this time, the electrical stimulation generates an electrical signal of about 3V through a radius of curvature of 20 mm at a frequency of 1 Hz, and the mechanical stimulation increases the length of 3% of the conventional length by 1 Hz and shrinks the mechanical stimulus And the cell culture medium of the cell culture was changed every 3 days.

On the other hand, the biocompatibility and stability of zinc oxide were not cytotoxic at concentrations of zinc oxide nanowires below 100 μg / ml.

To determine the degree of cell differentiation for various stimuli, the stimulation was controlled as shown in Table 1 below.

5-acacytidine
(6 μmol)
5-azaC A stimulus
(Bending and stretching stimulation)
Bending Stretching stimulus
(Stretching stimulation)
Cyclic Strain PES
(PZ) PDMS
(PD) TCP
(Cell culture dishes) Comparative Example 1 - - - - - o Comparative Example 2 o - - - - o Comparative Example 3 o - - - o - Comparative Example 4 o - - o - - Comparative Example 5 o o - - o - Comparative Example 6 o o - o - - Comparative Example 7 o o o - o - Example 1 o o o o - -

12 to 14 show cytotoxicity when bending, electrical stimulation and mechanical stimulation are applied to PES. Specifically, FIG. 12 shows the results of fluorescence immunoassay of caspase-3 (red dot indicated by an arrow) Fig. 14 shows the results of RT-PCR for caspase-3, Fig. 14 shows the results of BCL-2 and p53 (Fig. The RT-PCR results are shown in Fig. These results show that PES itself, CES induced by electric stimulation, bending and stretching motion (CS) generated by PES evolving does not appear.

Test Example 2 Induction of Myocardial Differentiation by Paloidine Staining F-Actin Immunostaining of Mesenchymal Stem Cells

F-Actin (filamentous actin) is one of the fibers that constitute the cytoskeleton that maintains the shape of the cell, changes the morphology or transfers the substance in the cell. It forms not only the structure of the myofibril but also the interaction with myosin A series of cells were confirmed through F-actin staining of mesenchymal stem cells of Comparative Examples and Examples in Table 1 using phalloidin staining directly involved in muscle contraction.

Actin cytoskeleton and Focal adhesion staining kit (FAK100, Millipore) were used to stain Paloidin of F-actin of mesenchymal stem cells. Staining of F-actin of stained F-Actin was performed using Actin Cytoskeleton and Focal adhesion staining kit Arrangements were confirmed using Image J software (National Institute of Health).

15 shows the F-actin arrangement of the mesenchymal stem cells of the above Examples and Comparative Examples stained with the Paloidine staining method. According to the results shown in Fig. 15, the cells had a tendency to be arranged in a direction perpendicular to the direction of increasing and contracting the PES Respectively. This is due to the tendency of the cells to be "oriented" to minimize their stimuli when they receive a specific mechanical signal, and this array of cells is a very important factor in myocardial differentiation. This is because cells arranged in a line are more likely to contain and distribute cell gap-binding proteins (for example, connexin 43), which play an important role in intercellular signaling, compared to randomly arranged cells. Particularly in the case of myocardium, this gap junction protein is a key factor for the delivery and pulsation of calcium ions.

These results suggest that mesenchymal stem cells differentiated by mechanical signals have better conditions to function as myocardial cells.

Test Example 3 Induction of myocardial differentiation by Western blotting Comparison of expression amounts of myocardial differentiation- related proteins of mesenchymal stem cells

Using Western blotting, cardiomyogenic differentiation in mesenchymal stem cells and molecular signaling related to myocardial differentiation were analyzed through a related protein marker.

Cells were washed three times with PBS (phosphate buffered saline, Gibco-BRL) and resuspended in SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% Bromophenol Blue). Proteins in the buffer were electrophoresed on 4-10% SDS-PAGE (SDS-polyacrylamide gel) and transferred to membranes (Millipore, Bedford, Mass., USA) and primary antibodies against Cx43, NKX2.5 , MEF-2, GATA4, sarcomeric alpha-actinin, beta-MHC, p38, pp38, SMAD, pSMAD, FAK, pFAK, ERK1 / 2, pERK1 / 2, and beta-actin (Abcam, Cambridge, After treatment, the cells were reacted overnight at 4 ° C, washed, treated with secondary antibody conjugated with horseradish peroxidase (HRP), and reacted at room temperature for 50 minutes. The blots were developed using ECL (enhanced chemiluminescence) (LumiGLO, KPL Europe, Guildford, UK).

FIG. 16 shows the expression of NKX 2.5, MEF-2, GATA4, β-MHC, sarcomeric α-actinin and Cx43 by the Western blot method and FIG. 21 shows the expression of p38, SMAD, FAK and ERK1 / 2 Lt; / RTI >

As can be seen from FIG. 16, the comparison of the early differentiation factors NKX 2.5, MEF-2, GATA 4 and late differentiation factors β-MHC, sarcomeric α-actinin and connexin 43 (Cx 43) As a result, in Comparative Example 1 in which 5-azacytidine (5-azaC) was cultured in TCP without electrical stimulation and mechanical stimulation, protein expression was hardly observed, and Comparative Example 2 in which only 5-azacytidine treatment was performed without electrical stimulation and mechanical stimulation And the protein expression in Comparative Example 5 was very slight. On the other hand, in Comparative Example 6 in which only electric stimulation was applied and in Comparative Example 7 in which only mechanical stimulation was applied, more protein expression was observed than in the non-electrical stimulation, and in Example 1 in which electrical stimulation and mechanical stimulation were simultaneously applied, It was observed that a large number of cardiac differentiation protein markers were expressed.

It can be seen that this shows a tendency similar to that of the protein markers expressed by the molecular signal related to myocardial differentiation shown in FIG.

These results indicate that cardiac differentiation is further promoted when electrical stimulation and mechanical stimulation are applied at the same time as when one stimulus is applied to mesenchymal stem cells.

Test Example 4 Induction of Myocardial Differentiation by Fluorescence Immunostaining The expression level of myocardial differentiation -related proteins in mesenchymal stem cells

The late differentiation factors sarcomeric alpha-actinin and connexin 43 of the myocardial differentiation-related proteins identified in Test Example 3 were analyzed by fluorescence immuno-staining (Immunocytochemistry).

The cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes and washed with PBS. The reaction was carried out using Cx43 and α-actinin as primary antibodies and then reacted with a secondary antibody (Jackson-Immunoreseaarch, West Grove, PA, USA) coupled with TRITC (tetramethyl rhodamine isothiocyanate) or FITC (fluorescein isothiocyanate) For 1 hour. All samples were mounted using a mounting solution containing DAPI (4,6-diamidino-2-phenylindole, Vector Laboratories, Burlingame, Calif., USA) and analyzed using a fluorescence microscope (Olympus, Tokyo, Japan) Respectively.

FIGS. 17A and 17B show the improvement of expression of Cx43 (green) and the expression of sarcomeric alpha-actin (red) by fluorescent immunoassay (blue = cell nucleus, scale bar = 50 μm).

17A and 17B, it was observed that much more myocardial differentiation protein markers were expressed in Example 1 in which electrical stimulation and mechanical stimulation were simultaneously performed, as shown in Test Example 3 above.

&Lt; Test Example 5 > Quantitative analysis Reverse transcription polymerase chain reaction (qRT-PCR)

Relative gene expression levels of cyclic nucleotide-gated potassium channel 2 (HCN2) and calcium channel, voltage-dependent, L-type and alpha 1C subunit (CACNA1C) were quantified using qRT-PCR.

Total RNA was extracted from the sample using 1 mL of the trizol reagent (Invitrogen) and 200 μL of chloroform. The lysed samples were centrifuged at 4,000C for 10 minutes at a rate of 12,000 rpm. The RNA pellet was washed with 75% (v / v) ethanol, dried and dissolved in RNase-free water. For qRT-PCR, iQ ™ SYBR Green supermix kit (Bio-Rad) and MyiQ ™ single color Real-Time PCR Detection System (Bio-Rad) were used. β-actin was used as an internal control.

Fig. 18 shows the results of evaluation of expression of HCN2 and CACNA1C by qRT-PCR.

In the above results, it can be confirmed that the cardiac differentiation is further promoted when the electrical stimulation and the mechanical stimulation are simultaneously applied, as compared with the case where only one stimulation is applied to the mesenchymal stem cells.

Test Example 6 Comparison of Expression Levels of Factors Related to Myocardial Differentiation Mechanism of Mesenchymal Stem Cells - RT-PCR (Reverse Transcription Polymerase Chain Reaction)

19 is a schematic diagram of a mechanism of myocardial differentiation of hMSC according to electrical stimulation and mechanical stimulation. As shown in FIG. 19, when electrical signals including mechanical fields or mechanical stimuli are applied to the myocardial differentiation mechanism, autocrine or near secretory proteins (BMP-4, TGF-beta, vascular endothelial growth factor , And IGF) can be increased.

Specifically, phosphorylation of SMAD-1, 4, 5, 8 occurs by BMP-4 expressed by electrical stimulation, and the expression of NKX 2.5, MEF-2 and β-MHC is increased by this phosphorylation reaction , VEGF and TGF-β are known to increase the expression of connexin43, a type of gap junction protein. IGF increases myocardial differentiation by increasing the phosphorylation of p38 and increasing the expression of MEF-2.

Expression of BMP-4, TGF-β, VEGF, and IGF, which are related to myocardial differentiation by electrical stimulation, was compared using reverse transcription polymerase chain reaction.

Samples were lysed by treatment with TRIzol reagent (Invitrogen Carlsbad, CA, USA) and the total RNA was extracted with chloroform (Sigma) and eluted with 80% (v / v) isopropanol After precipitation, the supernatant was removed, and the precipitated RNA pellet was washed with 75% (v / v) ethanol and dried, and then washed with 0.1% (v / v) DEPC treated diethyl pyrocarbonate- The pure total RNA was obtained by dissolving the pellet.

The total RNA extracted with the method of the cDNA was synthesized using ^{SuperScript TM II reverse transcriptase (Invitrogen)} .

The synthesized cDNA was subjected to denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 45 seconds and extension at 72 ° C for 45 seconds, followed by 35 cycles of incubation at 72 ° C for 10 minutes (Gel Doc 100, Bio-Rad, Inc.) after staining with Et-Br (ethidium bromide) and electrophoresed on a 2% (w / v) agarose gel. Hercules, CA, USA) and quantitated using an imaging densitometer (Bio-Rad). In this test, β-actin was used as an internal control.

20 shows the results of evaluation of expression of BMP-4, IGF, VEGF and TGF-? By the RT-PCR method. 20, the expression levels of BMP-4, IFG, VEGF and TGF-beta, which are self-secretion factors related to myocardial differentiation, were remarkably high in Example 1.

FIG. 21 shows the expression of p38, SMAD, FAK and ERK1 / 2 by the Western blotting method described in Test Example 3. As shown in FIG. 21, since the mesenchymal stem cells are stimulated by the autocrine factors, it can be seen that the electrical and mechanical stimulation further enhanced the protein expression of intercellular signaling molecules (pp38 and pSMAD) for myocardial differentiation. In addition, phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinases 1/2 (ERK1 / 2) was enhanced by mechanical / electrical stimulation. Phosphorylation of ERK1 / 2 causes myocardial differentiation by enhancing GATA4 expression. P38, pSMAD, pFAK, pERK1 / 2 were further enhanced when compared with p38, SMAD, FAK, and ERK1 / 2, when both electrical and mechanical signals were applied at the same time.

These results suggest that cardiomyocyte differentiation is promoted when electrical stimulation and mechanical stimulation are applied at the same time as when one stimulus is applied to mesenchymal stem cells.

Claims

Inoculating stem cells onto a support having both piezoelectricity and elasticity, and
And dividing the stem cells into myocardial cells by applying a mobility to the support on which the stem cells have been inoculated.

The method according to claim 1,
Wherein the exercise force is either one of a bending and stretching motion and a stretching motion, or both.

3. The method of claim 2,
Wherein the stretching or stretching motion applied to the support generates electrical energy and mechanical energy using the piezoelectricity of the support.

The method of claim 3,
Wherein the electrical energy generates a voltage of 0.1 to 10 V by bending and rolling the support at a frequency of 0.1 to 10 Hz.

The method of claim 3,
Wherein the mechanical stimulation is generated by a movement of increasing the length of the support by 1 to 20% relative to the original length at a frequency of 0.1 to 10 Hz and contracting the support.

The method according to claim 1,
Wherein the stem cells are arranged in a direction perpendicular to the direction of the kinetic force applied to the support.

The method according to claim 1,
Wherein the stem cells are selected from the group consisting of adipose stem cells, mesenchymal stem cells, bone marrow stem cells, cord blood stem cells, neural stem cells and induced pluripotent stem cells.

The method according to claim 1,
Wherein the supporter has a structure in which an elastic layer and a piezoelectric material layer are alternately laminated one or more times, and the elastic layer forms an outermost layer.

9. The method of claim 8,
Wherein the piezoelectric material layer comprises a plurality of piezoelectric substance nanorods.

10. The method of claim 9,
Wherein the piezoelectric material nanorod is a biaxial growth piezoelectric material nanorod.

10. The method of claim 9,
Wherein the piezoelectric material nanorods are arranged in one direction between the piezoelectric material nanorods to form a piezoelectric material layer.

10. The method of claim 9,
Wherein the piezoelectric material nanorods are arranged in a unidirectional monolayer.

9. The method of claim 8,
Wherein the elastic layer is made of a material capable of transmitting mechanical energy applied from the outside to the piezoelectric material layer.

9. The method of claim 8,
Wherein the elastic layer is made of a material capable of transferring the piezoelectric potential generated from the piezoelectric material layer to the inoculated stem cells.

9. The method of claim 8,
Wherein the elastic layer is made of a material including a dielectric material having a dielectric constant.

15. A myocardial cell-scaffold complex containing myocardial cells differentiated from stem cells by the method of any one of claims 1 to 15.