KR20180015312A - Method for differentiating cardiomyocytes from hair follicle cells and use thereof - Google Patents

Abstract

The present invention relates to a method for differentiating hair follicle cells into cardiomyocytes, cardiomyocytes differentiated by the method, and uses thereof. According to the present invention, the method for differentiating hair follicle cells into cardiomyocytes is a specific method capable of differentiating hair follicle cells into only cardiomyocytes without differentiation into neurons, neurogliocytes, keratinocytes, and the like, wherein the hair follicle cells are cultured in accordance the method to continuously maintain pulsatile ability, and to enable differentiation into cardiomyocytes which strongly express cardiomyocyte-specific differentiation markers. The cardiomyocytes differentiated according to the method can be used for screening a library of therapeutic substances for developing a therapeutic agent of cardiovascular diseases, and for studying a mechanism of cardiovascular diseases, and are expected to be widely used in various fields, such as a cell therapeutic agent or the like, by being directly applied to cells, proliferated, and re-injected into a patient.

Description

Methods for differentiating cardiomyocytes from cardiomyocytes into cardiomyocytes and uses thereof

The present invention relates to a method of differentiating follicular cells into myocardial cells, differentiated myocardial cells, and uses thereof.

Cardiovascular disease is a major cause of death worldwide. Myocardial infarction is a common symptom of ischemic heart disease that results in irreversible necrosis of myocardial cells due to ventricular arrhythmia, resulting in gradual heart failure. Such heart failure can generally be caused by limited dividing capacity of the final differentiated myocardial cells or sudden interruption of blood supply by coronary artery thrombosis. Approximately 450,000-750,000 patients with heart failure are currently waiting for a heart transplant, but only 1,000 patients are donating a heart for a year. So far, various studies have been conducted for the treatment of heart failure in pharmacological, medical, and surgical fields. However, until now, the treatment method is limited, so it is necessary to develop an alternative treatment method for treating many heart failure patients.

Stem cell-based therapy is considered as one of the alternative therapies, and has attracted attention as a promising therapy for filling up lost myocardial cells and regenerating myocardium. Conventionally, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), multipotent germline stem cells (mGSCs), and mesenchymal stem cells MSCs) have been performed. In particular, pluripotent stem cells are known to be mainly used for myocardial regeneration and differentiation. However, there is a limit to the possibility that teratomas can be produced by pluripotent stem cells not differentiated after transplantation when these cells are applied to clinical practice.

On the other hand, hair follicle (HF) is a skin organ that makes hair, a tissue with special regenerative capacity to circulate throughout the life of anagen, catagen, and rest period in mammals. The follicular stem / progenitor cells are located in the upper region of the hair follicle called the bulge area, and these follicular stem cells differentiate into various cells of the hair follicle and various epidermal cells and regenerate the hair follicle structure during the growing period. In addition, follicular stem cells have been reported to be capable of differentiating into outer-root sheath hair matrix cells, inner-root sheath, sebaceous-gland basal cells, and epidermis . Recently, it has been reported that the skin-derived precursor cells isolated from mammalian skin epidermis, which are pluripotent adult stem cells, can be differentiated into neurons, glia cells, smooth muscle cells and adipocytes (Toma, JG et al. 2001, Nat Cell Biol 3: 778-784), and that neural crest stem cells are present in the skin nipple of the hair follicles. In addition, nestin, a neural stem / progenitor cell marker, is selectively expressed in the cells of the expansion region of the hair follicle, and the cells expressing this nestin are expressed in vitro in neovasculature, neurons, glia cells, smooth muscle cells, Cells, and melanocytes.

In addition, there are hair-follicle-associated pluripotent (HAP) stem cells in all three regions (upper, middle, and lower) of the mouse hair follicle, which are not only pulsatile myocardial cells but also neurons, glial cells, keratinocytes, , And the ability to differentiate into different types of cells. However, these protocols have important drawbacks to be used in regenerative drugs and clinical applications because different types of cells are co-produced when hair-follicle-associated pluripotent (HAP) stem cells are differentiated into different cells, However, there is no established method for differentiating them.

In order to solve the above conventional problems, the present inventors have made intensive studies on a culturing method capable of specifically differentiating only follicular cells from myocardial cells. As a result, they have established a differentiation method capable of differentiating into only myocardial cells, Thus, the present invention was completed by culturing the hair follicle cells to obtain cardiomyocyte-like cells expressing myocardial cell shape and pulsatile ability and expressing myocardial cell-specific marker protein.

Accordingly, it is an object of the present invention to provide a method for differentiating follicular cells into cardiomyocytes, comprising culturing hair follicle cells on support cells using a myocardial cell differentiation medium containing growth factors.

It is another object of the present invention to provide a cardiomyocyte differentiated by the differentiation method and a cell therapy agent for treating cardiovascular diseases comprising the same.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the object of the present invention, the present invention provides a method for differentiating hair follicle cells into cardiomyocytes, comprising culturing hair follicle cells on supporting cells using a myocardial cell differentiation medium containing growth factors to provide.

In one embodiment of the present invention, the growth factor may be vascular endothelial growth factor (VEGF).

In another embodiment of the present invention, the myocardial cell differentiation medium may be mouse serum free medium (MSFM) or KO-DMEM / B27 (knock-out DMEM).

In another embodiment of the present invention, the supporting cell may be an OP9 cell.

In another embodiment of the present invention, the hair follicle cell may be cultured for 5 to 20 days.

The present invention also provides a myocardial cell differentiated by the differentiation method.

In addition, the present invention provides a cell therapy agent for treating cardiovascular diseases, comprising the above myocardial cell as an active ingredient.

In one embodiment of the present invention, the cardiovascular disease may be a disease selected from the group consisting of angina pectoris, myocardial infarction, ischemic heart disease, hyperlipidemia, stroke, arteriosclerosis, hypertension, arrhythmia, cerebrovascular disease, and coronary artery disease .

The method of differentiating follicular cells into myocardial cells according to the present invention is a specific differentiation method capable of differentiating follicular cells into only myocardial cells without differentiating hair follicular cells into different kinds of cells. The hair follicular cells are cultured according to the above- And can differentiate cardiomyocyte-specific differentiation markers into strongly expressing cardiomyocytes. Therefore, the differentiated myocardial cells according to the above method can be used for library screening of a therapeutic substance for the development of a therapeutic agent for cardiovascular diseases and for studying the mechanism of cardiovascular diseases, and also directly manipulating the cells, And it is expected to be widely used in various fields such as the use as a cell therapy agent.

FIG. 1 shows the effect of the culture medium on the differentiation of hair follicle cells into myocardial cells. After the hair follicle cells were cultured in the respective medium, the expression level of mRNA of Tnnt2, a myocardial cell-specific gene, was determined by qRT-PCR The result is confirmed.
FIG. 2 shows the results of culturing hair follicle cells in MSFM or KO-DMEM / B27 medium supplemented with GSI, BMP-4, activin A, Noggin, or VEGF to select growth factors effective for differentiation of hair follicle cells into cardiomyocytes And the expression level of mRNA of Tnnt2, a myocardial cell-specific gene, was confirmed by qRT-PCR.
FIGS. 3A and 3B show that the hair follicle cells were cultured on each support cell (OP9, STO, MEF, and C166) using MSFM medium supplemented with VEGF to select growth factors effective for differentiation of hair follicle cells into cardiomyocytes (Fig. 3A) and the expression level of mRNA of Tnnt2 were confirmed by qRT-PCR (Fig. 3B).
FIGS. 4A and 4B show that hair follicle cells were cultured in KO-DMEM / B27 medium supplemented with VEGF (OP9, STO, MEF, and MEF) in order to select growth factors effective for differentiation of hair follicle cells into cardiomyocytes C166), and the results of microscopic observation (FIG. 4A) and the expression level of mRNA of Tnnt2 were confirmed by qRT-PCR (FIG. 4B).
FIG. 5 shows the results of qRT-PCR for the expression level of mRNA of Tnnt2 in OP9 supporting cells cultured in MSFM or KO-DMEM / B27 medium and in hair follicle cells cultured on the supporting cells.
FIG. 6 is a graph comparing the expression level of Tnnt2 mRNA on OP9-supporting cells using MSFM or KO-DMEM / B27 medium supplemented with VEGF, This is a result.
FIG. 7 shows the result of immunocytochemical staining for confirming expression of various marker proteins (Pan-cadherin, cTnT, Nestin, CK15, CK19 and PLZF) in OP9 supporting cells.
FIG. 8 shows the result of immunocytochemistry to determine whether various marker proteins (Pan-cadherin, cTnT, Nestin, CK15, CK19, and PLZF) are expressed in hair follicular cells.
FIG. 9 shows the expression of various marker proteins (Pan-cadherin, cTnT, Nestin, CK15, CK19, and PLZF) after immunoprecipitation of OP9 supporting cells using KO-DMEM / B27 medium supplemented with VEGF The results were confirmed by cytochemical staining.
FIG. 10 shows the results of qRT-PCR analysis of mRNA expression levels of various cardiac cell-specific genes Tnnt2, Mesp1, Isl1, Mef2c, Nkx2.5 and Tbx5 in the cells cultured by the method of FIG.
Fig. 11 shows the result of observing the morphology of the cells cultured by the method of Fig. 9 with a microscope.

The present invention relates to a method of differentiating follicular cells into myocardial cells, differentiated myocardial cells, and uses thereof.

The present inventors have made intensive studies on the method of differentiating hair follicle cells into myocardial cells. As a result, we have established a differentiation method capable of specifically differentiating into only myocardial cells and cultured hair follicles according to the above method to obtain myocardial cells , Thereby completing the present invention.

Accordingly, the present invention provides a method for differentiating hair follicle cells into cardiomyocytes, comprising culturing hair follicle cells on supporting cells using a myocardial cell differentiation medium containing growth factors.

In the present invention, the growth factor may be, but is not limited to, vascular endothelial growth factor (VEGF).

The myocardial cell differentiation medium may be mouse serum free medium (MSFM) or KO-DMEM / B27 (knock-out DMEM), more preferably KO-DMEM / B27 and may be cultured as commonly used in the art Media additive.

The term " feeder cells " used in the present invention is known to be associated with undifferentiated maintenance mechanisms that secrete nutritional factors that contribute to maintaining the undifferentiated state of embryonic stem cells and mediate cell contact. The signaling substances secreted by the supporting cells contribute to regulate the undifferentiated maintenance or differentiation initiation of embryonic stem cells. The substances secreted from the supporting cells include Wnt (Wingless-type MMTV integration site family), BMPs (Bone Morphogenetic Proteins), TGF-β (Transforming Growth Factor-beta) and extracellular matrix. There have been reports of differentiation into embryonic stem cells or induced pluripotent stem cells using various supporting cells derived from mouse or human. In the present invention, the supporting cells may be OP9, STO, MEF, and C166, and more preferably, OP9 cells may be used, but the present invention is not limited thereto.

In the present invention, the hair follicle cells may be cultured for 5 days to 20 days depending on the culture conditions to induce differentiation into myocardial cells.

As used herein, the term " Cardiomyocyte " refers to a muscle cell constituting the heart muscle. Each cardiomyocyte is composed of a myofibrils (myofibrils), a specialized organs constituting a long chain of excretion, ). Myocardial cells have a flexible fiber shape for proper shape changes during beating of the heart. In the present invention, myocardial cells are preferably myocardial cells expressing myocardial cells, morphology, pulsatility, and myocardial cell specific genes (Pan-cadherin, cTnT, Tnnt2, Mesp1, Isl1, Mef2c, Nkx2.5 and Tbx5) Cells.

In the examples of the present invention, it was confirmed that the hair follicle cells were cultured according to the differentiation method and differentiated into myocardial cell-like cells expressing cardiomyocyte-like morphology, pulsatility, and cardiac cell-specific genes.

In one embodiment of the present invention, in order to establish a differentiation condition capable of specifically differentiating into follicular cells from a hair follicle cell, a culture medium, a growth factor, and a kind of supporting cells capable of effectively improving the differentiation ability are determined, Whereby an optimal differentiation method was established (see Examples 2 to 4).

In another embodiment of the present invention, the amount of mRNA expression of various cardiac specific genes is increased after culturing hair follicle cells according to the differentiation method according to the present invention, and the cells of other series, that is, neurons, glial cells, hair follicles, And that the expression of specific genes was not increased in the hair follicle cells. In addition, microscopic observation revealed that the differentiated cells showed a similar shape to the myocardial cells and showed sustained pulsatility (see Example 5).

From the above, the differentiation method according to the present invention is expected to be very useful for differentiating hair follicle cells into myocardial cells.

Accordingly, the present invention provides myocardial cells differentiated by the differentiation method.

In addition, the present invention provides a cell therapy agent for treating cardiovascular diseases comprising the myocardial cell as an active ingredient.

The term " cell therapy " used in the present invention means that a living individual, allogeneic, or heterogeneous cells are expanded, selected, or otherwise altered biological characteristics of cells to restore the functions of cells and tissues And to treat, diagnose, and prevent a specific disease through a series of actions, including, for example,

In the present invention, the cardiovascular disease may be a disease selected from the group consisting of angina pectoris, myocardial infarction, ischemic heart disease, hyperlipidemia, stroke, arteriosclerosis, hypertension, arrhythmia, cerebrovascular disease, and coronary artery disease.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental Preparation and Experimental Method

1-1. Experimental animal

The animal test of the present invention was carried out in accordance with the guidelines of the National Laboratory for Laboratory Animal Usage, approved by the Experimental Animal Management Committee of Chung Ang University. The mice were bred under conditions of 21 ± 2 ° C, 55 ± 10% humidity, and 12 hours of day / night cycles. The B6CBA-Tg (Pou5f1-EGFP) 2Mnn (OG2) mice used in the experiments were purchased from Harlan Laboratories (Indianapolis, IN, USA) and hair follicle cells were isolated from the mice.

1-2. Isolation and Collection of Hair Cells

To separate hair follicle cells from OG2 mice, mice were sacrificed and dorsal skin incisions were made to obtain a dorsal skin patch of 1 x 2 cm, and all surgical procedures proceeded in an aseptic environment. The dorsal skin obtained from the mice was treated with 2.5 mg / ml of dispase diluted with Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F12, 1: 1 mixture; Thermo Fisher Scientific, Waltham, Mass., USA) And incubated overnight at 4 ° C. The next day, the whole hair follicle was pulled off from the skin with elaborate tweezers, and a solution of 0.25% trypsin-EDTA (Thermo Fisher Scientific) and 7 mg / ml DNase I (Roche, Basel, Switzerland) The cells were treated with DPBS (Dulbecco's Phosphate Buffered Saline) for 5 min at 37 ° C and treated with 10% FBS to inhibit trypsin activity. The cell suspension was then passed through a 40-μm diameter nylon mesh (BD Biosciences, San Jose, CA, USA) and cell viability was measured using a trypan blue reagent.

1-3. Follicular cell culture and differentiation

The hair follicles isolated from the dorsal skin of the mice were divided into 6-well plates at a density of 1.0 × 10 ⁶ per well and cultured in DMEM supplemented with 10% FBS, 20 μl / ml of B27 supplement, 20 ng / ml of basic fibroblast growth Factor (bFGF), 10 ng / ml of glial cell line-derived neurotrophic factor (GDNF), 75 ng / ml GFRα1 (GDNF family receptor α1) and 1% penicillin / streptomycin And cultured in DMEM / F12 (3: 1) medium at 37 ° C and 5% CO ₂ .

In order to differentiate the hair follicle cells in myocardial cells, the hair follicle cells, respectively, 10% FBS and 1% penicillin / streptomycin is added to the MEMα (minimum essential medium alpha) medium, 1% B27, 0.5% N _2, 100 μM of β- (N2 / B27 (DMEM-F12) medium supplemented with 1% penicillin / streptomycin, 2% B27 and 1% penicillin / DMEM / B27 medium (knock-out DMEM) medium, or MSFM (mouse serum free medium) medium. In order to examine the induction of differentiation by growth factors, 15 μM of GSI (gamma-secretase inhibitor), 25 ng / ml of BMP-4 (bone morphogenetic protein-4), 2 ng / / Ml of noggin, or 5 ng / ml of vascular endothelial growth factor (VEGF) was added to each medium. In addition, to examine the effect of supporting cells, hair follicle cells were cultured on each OP9, STO, MEF, or C166 feeder cell in which mitosis was inactivated.

1-4. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNA was isolated from each sample using the PureLink RNA Mini Kit and cDNA was synthesized by reverse transcription polymerase chain reaction according to the manufacturer's protocol using Superscript III Reverse Transcriptase. qRT-PCR was performed using TaqMan Gene Expression Master Mix (Applied Biosystems, G Grand Island, NY, USA). Expression levels of all genes were determined by calibrating the qRT-PCR results to the expression level of GAPDH. The TaqMan probes used were Tnnt2, Mesp1, Isl1, Mef2c, Nkx2.5 and Tbx5, synthesized by Applied Biosystems.

1-5. Immunocytochemical Analysis

Immunocytochemical staining was performed to observe the expression of specific marker proteins in cells at the cell level. To this end, the cells were fixed with 4% paraformaldehyde for 30 minutes in hair follicular cells, and 0.1% Triton X-100 diluted in DPBS was treated at room temperature for 10 minutes to increase the permeability of the cells. After the above procedure, cells were treated with 5% (w / v) BSA (bovine serum albumin) (Sigma-Aldrich) solution at room temperature for 30 minutes to inhibit nonspecific binding. Next, the primary antibodies specific for the protein to be identified in the cell are Pan-cadherin (Santa Cruz Biotechnology), cardiac troponin T (cTnT; Thermo Fisher Scientific), nestin (Santa Cruz Biotechnology), cytokeratin 15 , Cambridge, Cambridgeshire, UK), cytokeratin 19 (CK19; Abcam), and PLZF (EMD Millipore) were diluted 1: 200 with 5% BSA solution and reacted overnight at 4 ° C. The next day, cells were washed twice with DPBS and incubated with Alexa flour 568 goat anti-mouse (Thermo Fisher Scientific), Alexa flour 568 donkey anti-goat (Thermo Fisher Scientific) or Rhodamine (TRITC ) goat anti-rabbit (Jackson ImmunoResearch, West Grove, PA, USA) secondary antibody was reacted at room temperature for 1 hour while blocking light. After the reaction, the cells were rinsed twice with DPBS and then counterstained with Vectashield medium diluted with DAPI (4 ', 6-diamidino-2-phenylindole, Vector Laboratories, Burlingame, CA). Then, the stained cells were observed with a Nikon TE2000 fluorescence microscope (Nikon, Chiyoda-ku, Tokyo, Japan) and the degree of protein expression was evaluated.

1-6. Statistical analysis

All statistical analyzes were performed using SPSS (SPSS Inc., Mechanicsburg, PA, USA) program. ANOVA and Tukey's honestly significant difference (HSD) were performed to assess differences between groups. Statistical significance was judged based on P <0.05, and all experiments were repeated three times.

Example 2. Effect of culture medium on the differentiation of hair follicular cells into myocardial cells

To evaluate the influence of the culture medium on the differentiation of hair follicle cells into myocardial cells, hair follicles were cultured in different media without addition of other growth factors, namely, HF medium, MEMα / FBS, N2 / B27, MSFM, or KO-DMEM / B27 medium for 6 days. After induction of differentiation into cardiomyocytes through the above culture, qRT-PCR was performed on each hair follicle cell according to the method of Example 1-4 to compare mRNA expression levels of Tnnt2, a myocardial cell-specific gene.

As a result, as shown in FIG. 1, the hair follicle cells cultured in the MSFM and KO-DMEM / B27 medium showed remarkable differences in the amount of expression compared to the culture in the HF medium, whereas the MEMα / FBS and N2 / B27 medium, it was confirmed that the mRNA expression level of Tnnt2 was similar to that of HF medium. Since the expression levels of mRNA of intracellular Tnnt2 cultured in the MSFM and KO-DMEM / B27 medium were similar, experiments for determining the differentiation factors were performed using both media in the future.

Example 3: Confirmation of the increase in the induction of differentiation of hair follicle cells into cardiomyocytes by VEGF

To investigate the effect of GSI, BMP-4, activin A, Noggin, and VEGF on the differentiation of hair follicular cells into myocardial cells in vitro , various growth factors known to enhance the differentiation into cardiomyocytes were studied. To determine the most efficient growth factors for inducing differentiation, hair follicle cells were cultured in MSFM or KO-DMEM / B27 medium supplemented with each growth factor for 6 days, and the expression level of Tnnt2 mRNA in each cell was confirmed.

As a result, as shown in Fig. 2, when the hair follicle cells were cultured using the MSFM medium, the expression of Tnnt2 remarkably increased only when VEGF was added to the medium. On the other hand, when GSI, BMP-4, activin A, and Noggin were added to KO-DMEM / B27 medium, the expression levels of Tnnt2 and Control (control) , And when VEGF was added, the expression level was increased.

These results indicate that VEGF is a useful growth factor for improving the ability of hair follicle cells to differentiate into cardiomyocytes.

Example 4: Determination of support cells and culture medium effective to induce differentiation from hair follicle cells into cardiomyocytes

In order to examine the effect of supporting cells on the induction of differentiation from hair follicle cells to cardiomyocytes, hair follicles were cultured in various support cells inactivated mitotic capacity using MSFM or KO-DMEM / B27 medium supplemented with VEGF, that is, OP9 , STO, MEF, and C166, and the cells were observed under an optical microscope during the incubation period.

As a result, when hair follicle cells were cultured using MSFM supplemented with VEGF and KO-DMEM / B27 medium as shown in FIG. 3A and FIG. 4A, Similar cells were identified. In addition, when mRNA expression level of Tnnt2, a cardiac myocyte-specific differentiation marker, was measured, as shown in Figs. 3B and 4B, when cultured in MSFM and KO-DMEM / B27 medium, And the expression of mRNA of Tnnt2 was significantly increased when cultured on the above.

These results indicate that OP9-supporting cells are most effective in differentiating follicular cells into myocardial cells compared to other cells.

Further, in order to determine the more effective medium for the differentiation from hair follicle cells into cardiomyocytes in MSFM and KO-DMEM / B27 medium using the OP9 supporting cells, the hair follicle cells were cultured in MSFM or KO-DMEM / B27 medium supplemented with VEGF , And the expression level of Tnnt2 mRNA was measured after incubation for OP9 support cells. At this time, OP9 support cells were cultured only in MSFM or KO-DMEM / B27 medium as a control group to exclude the influence of OP9 support cells.

As shown in FIG. 5, the expression of Tnnt2 mRNA was markedly increased when the hair follicle cells were cultured with the OP9 support cells using the above-mentioned media, compared with the case where only the OP9 support cells were cultured using the respective media Respectively. As a result of comparing the effect of MSFM and KO-DMEM / B27 medium on the differentiation of hair follicle cells into myocardial cells, as shown in FIG. 6, it was found that when KO-DMEM / B27 medium was used, Induced differentiation into myocardial - like cells was more efficiently induced.

From the above results, it was confirmed that KO-DMEM / B27 medium was more effective in inducing differentiation from hair follicle cells into myocardial cells. Experiments were conducted using KO-DMEM / B27 medium .

Example 5. Characterization of myocardial-like cells differentiated from hair follicle cells

In order to analyze the characteristics of myocardial-like cells differentiated from hair follicle cells, hair follicle cells were cultured on OP9 support cells for 2 weeks using KO-DMEM / B27 medium supplemented with VEGF, Immunocytochemical staining analysis was performed according to the method of 5. At this time, in order to exclude the case that various marker proteins were expressed in support cells before the experiment, it was first verified that the markers were expressed by OP9 cells. As a result, it was confirmed that the marker proteins of pan-cadherin, cTnT, Nestin, CK15, CK19 and PLZF to be confirmed were not expressed in OP9-bearing cells as shown in Fig. Furthermore, as shown in FIG. 8, it was confirmed that the cTnT protein was weakly expressed in the hair follicle cells. On the contrary, in the case of cells obtained by culturing hair follicle cells for 2 weeks according to the above method, it was confirmed that cTnT, a cardiac cell-specific marker, and pan-cadherin protein as an adhesion marker for cardiac cells are strongly expressed. On the other hand, we observed that the nestin / progenitor cell marker nestin, the keratinocyte markers CK15 and CK19, and the undifferentiated sperm cell marker, PLZF proteins, were not expressed at all. The expression results of the marker proteins are summarized in Table 1 below.

OP9 feeder cells HF cells Cardiomyocyte-
like cells Pan-cadherin (Intercellular adherence junction in cardiac muscle) X X O Troponin (cTnT, cardiomyocyte) X O
(very few) O Nestin (Neural stem / progenitor marker) X X X CK15 (Keratinocyte marker) X X X CK19 (Keratinocyte marker) X X X PLZF (undifferentiated spermatogonia marker) X X X

In conclusion, when the hair follicle cells are cultured on OP9 support cells using KO-DMEM / B27 medium supplemented with VEGF, the expression levels of cardiac cell specific genes are increased and neurons, neuroglial cells, hair follicles, and gonadal cells are included And that the differentiation is induced only in the myocardial cells when the hair follicle cells are cultured under the above conditions through the absence of the genes specific to the other lines of cells.

In addition to the above results, mRNA expression levels of Tnnt2, Mesp1, Isl1, Mef2c, Nkx2.5 and Tbx5, which are additional various cardiac cell specific genes, were analyzed in cells obtained by culturing hair follicle cells for 2 weeks according to the above method. As a result, as shown in FIG. 10, it was confirmed that the expression of the genes was significantly increased in the differentiated myocardial cells compared to the unspecialized hair follicle cells, and thus, the culture conditions significantly differentiated the hair follicle cells from the hair follicle cells And the differentiation into myocardial - like cells was observed.

Further, as shown in FIG. 11, the cells were spiral-like and thin-cell-like, similar to myocardial cells, and showed spontaneous pulsation even in the absence of any stimulus. It was observed to last more than 3 months.

These results indicate that spontaneous differentiation from hair follicle cells into myocardial cells with pulsatile function is induced by culturing hair follicle cells on OP9 supporting cells using KO-DMEM / B27 medium supplemented with VEGF.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (8)

A method for differentiating hair follicle cells into myocardial cells, comprising culturing hair follicle cells on supporting cells using a myocardial cell differentiation medium containing growth factors.
The method according to claim 1,
Wherein the growth factor is a vascular endothelial growth factor (VEGF).
The method according to claim 1,
Wherein the myocardial cell differentiation medium is mouse serum free medium (MSFM) or KO-DMEM / B27 (knock-out DMEM).
The method according to claim 1,
Wherein the supporting cell is an OP9 cell.
The method according to claim 1,
Wherein the hair follicle cells are cultured for 5 to 20 days.
A myocardial cell differentiated by the differentiation method of claim 1.
A cell therapy agent for treating cardiovascular diseases, comprising the myocardial cell of claim 6 as an active ingredient.
8. The method of claim 7,
Wherein the cardiovascular disease is a disease selected from the group consisting of angina pectoris, myocardial infarction, ischemic heart disease, hyperlipidemia, stroke, arteriosclerosis, hypertension, arrhythmia, cerebrovascular disease, and coronary artery disease.

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