KR101818762B1

KR101818762B1 - Methods to promote differentiation into smooth muscle cells and composition thereof

Info

Publication number: KR101818762B1
Application number: KR1020150100239A
Authority: KR
Inventors: 서원희
Original assignee: 중앙대학교 산학협력단
Priority date: 2015-07-15
Filing date: 2015-07-15
Publication date: 2018-01-16
Also published as: KR20170010135A

Abstract

The present invention relates to a method for promoting differentiation into smooth muscle cells and a composition using the same, and more particularly, to a method for promoting differentiation into smooth muscle cells, And inducing overexpression of FGF-12 in the starting cell to change the differentiation state of the starting cell; And confirming the phenotype of the smooth muscle cells in the starting cells. The method of the present invention includes a method of promoting differentiation of smooth muscle cells from a starting cell comprising a polynucleotide encoding FGF-12 as an active ingredient, The present invention relates to a composition for promoting the differentiation of a plant. INDUSTRIAL APPLICABILITY The present invention is very effective in promoting the differentiation of undifferentiated stem cells into smooth muscle cells using FGF-12 as a single factor or promoting the differentiation into smooth muscle cells from fibroblasts.
The present invention can easily and efficiently promote the differentiation of smooth muscle cells from various starter cells using FGF-12, and thus can be usefully used for developing a gene therapy agent or a cell therapy agent for smooth muscle cell degenerative diseases.

Description

[0001] The present invention relates to a method for promoting differentiation into smooth muscle cells,

The present invention relates to a method for promoting differentiation into smooth muscle cells and a composition using the same, and more particularly, to a method for promoting differentiation into smooth muscle cells, And inducing overexpression of FGF-12 in the starting cell to alter the differentiation state of the starting cell; And a step of identifying a phenotype of smooth muscle cells in the starting cells. The method of promoting the differentiation from the starting cells to the smooth muscle cells comprising the steps of: (a) culturing the starting cells comprising the polynucleotide encoding FGF-12 as an active ingredient And to a composition for promoting differentiation into smooth muscle cells.

Smooth muscle cells constitute most of the intestinal muscle walls except for the heart. They regulate hormones and the autonomic nervous system to shrink and relax, allowing these organs to perform their respective functions smoothly. In the blood vessels, smooth muscle cells form the inner wall of the medial layer and are very important for the development of a mature and stable vascular system. Structures support the blood vessels, regulate blood flow and blood pressure, synthesize and secrete extracellular matrix essential for blood vessel durability and elasticity. In addition, smooth muscle cells may exhibit adaptive changes in regeneration state in which shrinkage is decreased and cell division and mobility are increased in order to recover from damage of blood vessels. Thus, smooth muscle cells can not maintain their normal physiological functions if their function is impaired due to inherited or acquired reasons such as genetic damage, infection, or other diseases. Therefore, it is necessary to develop a method that can effectively promote differentiation into smooth muscle cells for the treatment of smooth muscle cell degenerative diseases.

The specific molecular mechanisms of the regulation of smooth muscle cell differentiation and smooth muscle cell specific gene expression are not well known. There have been attempts to differentiate human pluripotent stem cells or inducible pluripotent stem cells into smooth muscle cells by using retinoic acid, various growth factors or extracellular matrix. However, (Descamps et al., Vascul Pharmacol (2012) 56 (5-6): 267-79).

Accordingly, the present inventors have studied how to obtain smooth muscle cells effectively, and found that when FGF-12 is overexpressed, differentiation is induced from stem cells into smooth muscle cells and that the fibroblasts are induced to differentiate into smooth muscle cells Thereby completing the present invention.

It is therefore an object of the present invention

(a) providing a starter cell; And

(b) inducing overexpression of FGF-12 in the starting cell to change the differentiation state of the starting cell; And

(c) confirming the phenotype of smooth muscle cells in the starter cell of step (b). The present invention also provides a method for promoting differentiation of smooth muscle cells from a starter cell.

Another object of the present invention is

The present invention provides a composition for promoting differentiation of smooth muscle cells from a starting cell comprising a polynucleotide encoding FGF-12 as an effective ingredient.

To achieve these and other advantages and in accordance with the purpose of the present invention,

(a) providing a starter cell; And

(c) confirming the phenotype of smooth muscle cells in the starter cell of step (b). The present invention also provides a method for promoting differentiation from a starting cell to a smooth muscle cell.

In order to achieve another object of the present invention,

There is provided a composition for promoting differentiation from a starting cell to a smooth muscle cell comprising a polynucleotide encoding FGF-12 as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention

(a) providing a starter cell; And

Hereinafter, the method of the present invention will be described step by step.

The present invention provides (a) a method of promoting differentiation from a starting cell to a smooth muscle cell comprising the step of providing a starting cell.

In the present specification, 'differentiation' means a phenomenon in which the structure or function of a cell changes or is specialized.

&Quot; Smooth muscle cell (SMC) " means smooth muscle muscle cells. 'Smooth muscle' is a muscle that has no horizontal pattern and is also called as a fringe pattern. In vertebrates, muscles of the internal organs other than the heart, namely the gastrointestinal tract, the airways of the respiratory system, the blood vessels, the bladder, and the uterus are all smooth muscles. Smooth muscle can not move consciously, smooth muscle contraction is directly controlled by the autonomic nervous system, and is affected by hormones. In general, smooth muscle is formed through myogenesis process from the mesoderm in the developmental stage, but it is formed in the ectomesenchyme of the neural crest like the aorta or the pulmonary artery There is also something derived.

The smooth muscle cells of the present invention may preferably be vascular smooth muscle cells (VSMC). Vascular smooth muscle cells form the structure of the vasculature and regulate blood pressure and blood flow by contracting and relaxing through control of the nervous system and hormones. Differentiated smooth muscle cells express smooth muscle cell-specific contractile proteins such as SM MHC (SM MHC), SM22, calponin, smooth muscle α-actin and desmin at high levels And has shrinkage.

In the present invention, the 'starting cell' is a cell to be differentiated or transformed into a smooth muscle cell according to the method of the present invention, and may be undifferentiated from a desired smooth muscle cell, State, or may be terminally differentiated. The final differentiated cells do not proliferate or have unipotency, which allows them to divide only into one kind of cell even when proliferating. The starting cells that are undifferentiated from the smooth muscle cells in the present invention may be stem cells, and the stem cells may be pluripotent stem cells or adult stem cells. In the present invention, the pluripotent stem cells may preferably be induced pluripotent stem cells or embryonic stem cells, and the adult stem cells may preferably be mesenchymal stem cells. In the present invention, the specifically differentiated or terminally differentiated starting cells may be fibroblasts, and preferably they may be dermal fibroblasts. The starting cell may also be a naturally occurring cell or a genetically modified cell. The starter cell may be isolated or cultured according to a suitable method known in the art depending on the kind thereof.

The present invention provides a method comprising: (b) inducing overexpression of FGF-12 in a starter cell to alter the differentiation state of the starter cell.

The step of changing the differentiation state of the starting cell means to induce or accelerate the ability of the starting cell to differentiate, change or convert into another kind of cell. The method of the present invention is a method for inducing or promoting a change from a starter cell to a smooth muscle cell comprising the step of altering the differentiation state of the starter cell by overexpressing FGF-12 in the starter cell.

The step of changing the differentiation state of step (b) of the present invention may be a method characterized in that the differentiation of the starting cells proceeds.

The progression of the 'differentiation of the starting cells' means that the starting cells change from a less specific state to a more specific state. This may mean that the number of mature and fully differentiated cells that can be formed by the differentiation of the starter cell is reduced, that is, the cell potency is limited. Therefore, when the method of the present invention changes the differentiation state by progressing the differentiation of the starting cells, it means that the starting cells are undifferentiated cells rather than the smooth muscle cells. Cells that are undifferentiated from smooth muscle cells are cells that can be differentiated into other types of cells besides smooth muscle cells.

The starting cells that are undifferentiated from the smooth muscle cells may be stem cells.

"Stem cell" refers to a cell capable of self-renewal and differentiation to form unlimitedly specialized cells of tissues and organs. 'Self renewal' is the ability to repeat the cell division cycle while maintaining undifferentiated state. Stem cells can divide into two daughter stem cells, or one daughter stem cell and one derived (or transit) cell, and eventually differentiate into the mature and complete form of the tissue. The stem cells in the present invention may be derived from mammals, and may be derived from humans.

The stem cells in the present invention may be pluripotent stem cells.

'Pluripotent stem cells' refers to stem cells with pluripotency that can differentiate into three glands that make up the body, namely endoderm, mesoderm, and ectoderm. In general, embryonic stem cells, induced pluripotent stem cells (iPC or iPCS), embryonic germ cells, and embryonic carcinoma cells are allogenic stem cells . The pluripotent stem cells in the present invention may be derived from mammals, and may be derived from humans.

The pluripotent stem cells in the present invention may preferably be induced pluripotent stem cells or embryonic stem cells.

Inducible pluripotent stem cells (iPCs or iPCSs) are cells that do not undergo pluripotency, usually embryonic somatic or adult differentiation cells, Or overexpression of the pluripotent stem cells. Four reprogramming factors, Oct4, Sox2, Klf4, and cMyc, are the most commonly used reprogramming factors.

'Embryonic stem cell (ESC)' is a cell cultured in vitro from the inner cell mass of blastocyst, which is just before embryo implantation, The cells are pluripotent. Broadly, embryonic stem cells derived from embryonic stem cells are included. 'Embryonic body or embryoid body (EB)' refers to a mass of spherical stem cells produced in suspension culture and potentially has the ability to differentiate into endoderm, mesoderm, and ectoderm, so that tissue-specific differentiation It is used as a precursor in most induction of differentiation to secure cells.

In addition, the stem cells of the present invention may be adult stem cells.

"Adult stem cells" are stem cells found in adult tissues or organs such as brain, bone marrow, peripheral blood, blood vessels, muscles, skin, teeth, heart, stomach, liver, ovaries, , Stem cells that play a role in maintaining the tissue and repairing the damage. Adult stem cells exist only in a very small amount in each tissue of a body, and generally, the kinds of differentiable cells exhibit multipotency which is limited to the cells constituting the specific tissue. Adult stem cells include neural stem cells that can differentiate into neurons, hematopoietic stem cells that can differentiate into blood cells, mesenchymal stem that can differentiate into bone, cartilage, fat, muscle, But are not limited to, mesenchymal stem cells, hepatic stem cells capable of differentiating into hepatocytes. The adult stem cells of the present invention may be derived from mammals, and may be derived from humans.

The adult stem cells in the present invention may preferably be mesenchymal stem cells.

Mesenchymal stem cells (MSCs), also called mesenchymal stromal cells, are multipotent stem cells capable of differentiating into mesenchymal stem cells. It can be differentiated into osteocyte, chondrocyte, tendinocyte, adipocyte, myocyte, fibroblast, and the like. The mesenchymal stem cells of the present invention may be derived from tissues such as umbilical cord, umbilical cord blood, placenta, amniotic fluid, amniotic membrane, bone marrow, fat, muscle, skin, peripheral blood and the like. Preferably a mesenchymal stem cell derived from human bone marrow.

Meanwhile, the step of changing the differentiation state of the starting cells in the step (b) may be such that the starting cells are differentiated into smooth muscle cells.

Transdifferentiation is the process by which one type of differentiated cell is converted into another type of differentiated cell. One mature somatic cell has a pluripotency state or an intermediate stage such as a progenitor cell But rather to a different type of mature somatic cell line. "Somatic cells" refers to cells that are not eggs or sperm that do not directly transfer genetic material such as DNA to the next generation. In general, the probability of specific somatic cells to differentiate into somatic cells is very limited.

Therefore, when the method of the present invention is to change the differentiation state of the starter cell through the conversion differentiation, the starter cell may be a cell in a differentiated state in which the type and function of the cell is specified, or may be a terminally differentiated state. The final differentiated cells do not proliferate or have unipotency that can only divide into one kind of cell even when proliferating.

The starting cell may be a fibroblast when the method of the present invention is to alter the differentiation state of the starting cell through conversion differentiation.

'Fibroblast' is a cell that synthesizes and secretes extracellular matrix and collagen and elastin fiber proteins that form and support connective tissue structure. It is derived from primitive mesenchyme during development and is the most common cell in the connective tissue of animals and is also very important for wound healing. Fibroblasts can be seen in various forms depending on the tissue or activity of the fibroblasts. Fibroblasts may be referred to as activating states, and fibroblasts may be referred to as inactive states. The fibroblasts in the present invention may be derived from mammals, and may be derived from humans.

The fibroblasts in the present invention may preferably be dermal fibroblasts, and most preferably human dermal fibroblasts. Dermal fibroblasts are the major constituent cells of the dermis of the skin along with macrophages and adipocytes. Unlike fibroblasts of other tissues, dermal fibroblasts do not proliferate well, and once they are completed, it is known that they are unlikely to change into other types of cells without external stimuli such as scratches or damage.

The method of the present invention is a method comprising (b) inducing overexpression of FGF-12 in the starter cell to change the differentiation state of the starter cell.

In the present invention, 'FGF-12' refers to fibroblast growth factor 12 and may be referred to as FGF12, FGF12B, FHF1, or the like. FGF-12 is similar in structure to other fibroblast growth factors (FGFs) but has no N-terminal secretion signal sequence, And it is biochemically distinguishable in that it exists in the cytoplasm and does not activate the FGF receptor on the cell surface. Therefore, it may be classified as a fibroblast growth factor (FGF) homologous factor (FHF). The mRNA sequence or amino acid sequence of human FGF-12 is selected from the group consisting of NM_021032.4 (FGF 12 isoform 1, mRNA), NP_066360.1 (FGF 12 isoform 1, protein), NM_004113.5 (FGF 12 isoform 2, mRNA), NP_004104.3 (FGF 12 isoform 2, protein).

The FGF-12 of the present invention may preferably be human FGF-12, more preferably a protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

As used herein, the term 'protein' is used interchangeably with 'polypeptide' or 'peptide', for example, a polymer of amino acid residues as commonly found in proteins in nature.

FGF-12 of the present invention includes functional equivalents thereof. Refers to a polypeptide having at least 70%, preferably at least 80%, more preferably at least 90% sequence homology (i.e., identity) with the amino acid sequence of FGF-12 of the present invention. For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% Sequence homology to the sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, Refers to a polypeptide having substantially the same physiological activity as FGF-12 of the present invention. &Quot; Substantially homogenous physiological activity " means an activity capable of inducing and promoting differentiation or conversion to smooth muscle cells when expressed in a starter cell.

Such functional equivalents may result from the addition, substitution or deletion of some of the amino acid sequences of FGF-12 of the present invention. The substitution of the amino acid is preferably a conservative substitution. Examples of conservative substitutions of amino acids present in nature include: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids , Glu), basic amino acids (His, Lys, Arg, Gln, Asn) and sulfur containing amino acids (Cys, Met). Such functional equivalents also include modifications in which a part of the amino acid is deleted from the amino acid sequence of FGF-12 of the present invention. The amino acid deletion or substitution is preferably located in a region that is not directly related to the bioactivity of the protein of the present invention. In addition, deletion of the amino acid is preferably located in a portion not directly involved in the physiological activity of FGF-12 of the present invention. Also included are variants in which some amino acids are added at both ends or sequences of the amino acid sequence of FGF-12. Also included within the scope of the functional equivalents of the present invention are proteins or polypeptide derivatives in which some of the chemical structures of the proteins are modified while maintaining the basic skeleton of the protein according to the present invention and its physiological activity. This includes, for example, structural modifications to alter the stability, shelf stability, volatility, or solubility of the protein of the present invention.

In the step (b) of the present invention, induction of FGF-12 overexpression in the starter cell may be performed by transforming the starter cell with a polynucleotide encoding FGF-12.

The polynucleotide encoding FGF-12 of the present invention may preferably comprise the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.

As used herein, a "polynucleotide" or "nucleic acid" refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in the form of single- or double-stranded. Unless otherwise constrained, also includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.

Transforming the starter cell with a polynucleotide encoding FGF-12 can be carried out by selecting a suitable polynucleotide delivery method for transforming cells known in the art and selecting a suitable standard technique according to the starting cell Can be performed. Such methods include, but are not limited to, in vitro transfection, injection or microinjection, electroporation, heat shock, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, The use of silicon carbide whiskers, shaking with silicon carbide fibers, sonication, transfection with liposomes, receptor-mediated transfection, Agrobacteria mediated transformation, polyethylenglycol But are not limited to, precipitation, methods using polyethyleneimine or dextran sulfate, lipofectamine, microparticle impact, particle gun bombardment, and the like.

The transformation in the present invention may be mediated by a recombinant expression vector into which a polynucleotide encoding FGF-12 is inserted.

Expression refers to the generation of a protein or nucleic acid in a cell, and a recombinant expression vector is a vector capable of expressing a target protein or a target nucleic acid (RNA) in a suitable host cell. The expression vector may be a polynucleotide Gene) insert that is operably linked to the expression of the gene construct. The term 'operably linked' refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA to perform a general function, It means connected so that it can be expressed. An " expression control sequence " means a DNA sequence that regulates the expression of a polynucleotide sequence operably linked to a particular host cell. Such regulatory sequences include, but are not limited to, promoters for conducting transcription, any operator sequences for regulating transcription, sequences coding for suitable mRNA ribosome binding sites, sequences regulating termination of transcription and translation, initiation codons, termination codons, polyadenylation Signal and an enhancer. The recombinant expression vector of the present invention is not particularly limited as long as it is a vector ordinarily used in the field of cloning, and examples thereof include, but are not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector and a viral vector. Recombinant vectors can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage are performed using enzymes generally known in the art.

Therefore, the recombinant expression vector of the present invention is a gene construct comprising both a polynucleotide encoding FGF-12 and an essential expression control sequence for expressing FGF-12 in the starter cell, and these are operably and functionally linked. The recombinant expression vector of the present invention may contain a selection marker and / or a replication origin for selecting a host cell in the production of the recombinant expression vector, and the expression vector may further comprise an expression control sequence, Or secretory signal sequences, leader sequences, and the like, and may be prepared variously according to purposes, including a sequence of a reporter or marker gene for displaying and confirming the characteristics of a starting cell or smooth muscle cell. have. Sequences for the above expression control sequences and other additional functions may optionally be included as starting cells or specific for smooth muscle cells.

The recombinant expression vector of the present invention may be a recombinant viral vector.

In the present invention, the recombinant viral vector may be selected from the group consisting of an adenovirus vector, a retrovirus vector, a herpes virus vector, a lentivirus vector and an avipoxvirus vector. The recombinant viral vector of the present invention may preferably be an adenoviral vector. In one embodiment of the present invention, the adenovirus vector pAdEasy-1 vector was used.

When the method of the present invention is to transform a starting cell via a recombinant viral vector, overexpression of FGF-12 in the starting cell may be induced by a method comprising the following steps.

(i) preparing a polynucleotide encoding FGF-12, a recombinant viral vector operatively linked so that the expression control sequence can be expressed in the starting cell by FGF-12; And

(ii) transforming the recombinant viral vector into a virus producing cell line to prepare and isolate an FGF-12 expressing recombinant virus; And

(iii) infecting the starting cells with the FGF-12 expressing recombinant virus; And

(iv) further culturing the virus-infected starter cell to overexpress FGF-12.

The additional culturing step of (iv) may be carried out appropriately until the amount of FGF-12 sufficient to induce smooth muscle cells in the starting cell is expressed according to the starting cell, the recombinant viral vector used for transformation, the kind of virus, and the like . Preferably about 6 to 14 days. In the examples of the present invention, adenovirus containing pAdEasy-1 vector containing the base sequence of FGF-12 was used to infect starter cells. When the starting cells were mouse embryonic stem cells, And cultured for 10 days to 14 days. When the starting cells were human dermal fibroblasts or human bone marrow-derived mesenchymal stem cells, the cells were further cultured for 6 days after virus infection.

The present invention provides a method for promoting differentiation from a starting cell to a smooth muscle cell comprising the step of (c) confirming the phenotype of smooth muscle cells in the starter cell of step (b).

The smooth muscle cell phenotype is a smooth muscle cell-specific expression trait distinguished from other cells, and may be a smooth muscle cell specific structure or shape, expression pattern of a smooth muscle cell specific gene combination, a smooth muscle cell specific response to hormone or external stimulus , Such as a cellular contraction reaction by an acetylcholine receptor agonist, or the like. Any other known expression traits specific for smooth muscle cells can be used without limitation. Whether or not the starter cells have acquired smooth muscle cell specific expression traits can be determined by comparing and contrasting the starter cells, the positive control cells, and the negative control cells for a specific myocyte phenotype. As the positive control cells, smooth muscle cells can be used, and the negative control cells can be preferably the untransformed starting cells or the starting cells transformed with a polynucleotide that does not contain a base sequence encoding FGF-12 .

When measuring the level of expression of a smooth muscle cell specific gene, the mRNA or protein expression level of the gene can be measured. Examples of assay methods include reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR (competitive RT-PCR), and the like. ), Real-time RT-PCR, RNase protection assay (RPA), northern blotting, DNA microarray chip, RNA sequencing ), But the present invention is not limited thereto. Methods for measuring the expression level of a protein can be used without limitation in the methods known in the art, and examples thereof include western blotting, dot blotting, enzyme-linked immunosorbent assay Immunohistochemical staining, immunoprecipitation, complement fixation, flow cytometry (FACS), or protein chip method (RIA), radioimmunoassay (RIA), radioimmunoprecipitation, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, And the like, but are not limited thereto.

In the examples of the present invention, expression levels of myocardin, SRF,? -SMA, SM22?, Smoothelin, and SM-MHC in the starting cells infected with the adenovirus containing the base sequence of FGF-12 (Ad-FGF12) RT-PCR, Western blotting and immunohistochemical staining. Also, adenovirus-infected starting cells (Ad-LacZ) containing the nucleotide sequence of untreated, untreated viruses, LacZ (beta -galactosidase) were used as negative controls or human aortic smooth muscle cells (HASMC) as positive controls Respectively. For genes specifically expressed in smooth muscle cells, the following references can be found: Miano J. J Biomed Res (2010) 24 (3): 169-80; Mack P. Arterioscler Thromb Vasc Biol (2011), 31 (7): 1495-1505.

In another embodiment of the present invention, the contraction phenotype of the starting cells infected with Ad-FGF12 was measured using a collagen gel contraction assay. Cells were cultured with collagen to form a cell-collagen polymer, and the size of the cell-collagen mass before and after stimulation with carbacol, a cholinomimetic cholinomimetic, And the shrinkage was confirmed.

The above-described method of the present invention is specifically shown in the embodiment of the present invention.

In one embodiment of the present invention, the expression of smooth muscle cell-specific genes such as myocardin, α-SMA, SM22α, and smoothelin in the embryo formed from mouse embryonic stem cells infected with Ad-FGF12 was observed 10 days after viral infection, -PCR. In addition, the expression of α-SMA in the embryoid body on the 14th day after viral infection was significantly increased by immunofluorescence staining. That is, the over-expression of FGF-12 promotes the differentiation of embryonic stem cells into smooth muscle cells.

In another embodiment of the present invention, the expression of α-SMA was markedly increased at 6 days after viral infection in human bone marrow-derived mesenchymal stem cells infected with Ad-FGF12 by immunofluorescence staining. That is, the overexpression of FGF-12 promotes the differentiation of mesenchymal stem cells into smooth muscle cells.

In another embodiment of the present invention, the expression of α-SMA in the human dermal fibroblasts infected with Ad-FGF12 was observed at 6 days after viral infection by immunofluorescence staining, and SRF, α-SMA, SM22α, smoothelin, SM -MHC was significantly increased by RT-PCR. In addition, human dermal fibroblasts overexpressing FGF-12 showed shrinkage similar to that of human aortic smooth muscle cells in collagen gel shrinkage experiments. That is, the overexpression of FGF-12 promotes the differentiation of fibroblasts into smooth muscle cells.

Finally, the present invention provides a composition for promoting differentiation from a starting cell to a smooth muscle cell comprising a polynucleotide encoding FGF-12 as an active ingredient.

The polynucleotide encoding FGF-12 may preferably include the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. The polynucleotide encoding FGF-12 may also be contained in a recombinant expression vector operatively linked to an expression control sequence so as to be capable of expression in the starter cell. Preferably, the recombinant expression vector may be a recombinant expression vector.

Therefore, the present invention relates to a method of inducing overexpression of FGF-12 in a starting cell to promote differentiation or conversion of the cell into a smooth muscle cell, and a method of promoting differentiation into smooth muscle cells containing the polynucleotide encoding FGF-12 as an active ingredient &Lt; / RTI > The method of the present invention is effective for facilitating differentiation of stem cells into smooth muscle cells by using FGF-12 as a differentiation factor. In addition to fibroblasts as well as stem cells in undifferentiated state, it is effective to promote the differentiation into smooth muscle cells.

Figure 1 shows Western blotting results showing the expression of transcription factors involved in smooth muscle cell differentiation in adenovirus-infected human aortic smooth muscle cells (HASMC). β-actin is a loading control.
Figure 2 shows RT-PCR results showing the expression of FGF-12 in adenovirus-infected mouse embryonic stem cells (mESC). GAPDH is a loading control.
FIG. 3 shows immunofluorescence microscopic photographs showing the expression of smooth muscle actin in an embryonic body composed of adenovirus-infected mESCs. Red indicates smooth muscle actin and blue indicates DAPI. The size of the scale bar indicated by white diaphragm in the drawing is 200 μm.
Figure 4 shows RT-PCR results showing the expression of smooth muscle cell marker genes in an embryonic body composed of adenovirus-infected mESCs. * p < 0.05
FIG. 5 shows immunofluorescence microscopic photographs showing the expression of smooth muscle actin in adenovirus-infected human dermal fibroblast (HDF). Red indicates smooth muscle actin and blue indicates DAPI. The size of the scale bar indicated by white diaphragm in the drawing is 200 μm.
FIG. 6 shows RT-PCR results showing the expression of smooth muscle cell marker genes in adenovirus-infected HDF.
FIG. 7 shows collagen gel shrinkage experiment results to confirm shrinkage of adenovirus-infected HDF. * p < 0.05
FIG. 8 shows immunofluorescence microscopic photographs showing the expression of smooth muscle actin in adenovirus-infected human bone marrow-derived mesenchymal stem cells (BM-MSC). Red indicates smooth muscle actin and blue indicates DAPI. The size of the scale bar indicated by white diaphragm in the drawing is 200 μm.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<Experimental Method>

Cell culture

Human dermal fibroblast (HDF) and bone marrow-derived mesenchymal stem cells (BM-MSC; Lonza) were cultured in 10% fetal bovine serum (FBS; Gibco) supplemented with Dulbecco's modified Eagle's medium (DMEM; Invitrogen). Human Aortic Vascular Smooth Muscle Cells (HASMC; ScienCell Research Laboratories) were cultured in smooth muscle growth medium (SMGM; Scientific Research Laboratories) or basal medium. Murine embryonic stem cells (mESC; D3, ATCC) were cultured in mESC medium. For induction of differentiation, mESCs were infected with adenovirus encoding LacZ (Ad-LacZ) or FGF12 (Ad-FGF12) in a feeder-free condition for 7 hours. Infected mESCs were washed and replaced with medium supplemented with 15% FBS and 2 × mouse leukemia inhibitory factor (mLIF). The mESCs were cloned into embryonic bodies using standard hanging drop method and cultured in mESC medium without mLIF. The embryoid bodies were incubated in a hanging drop for 2 days and coated with gelatin - coated cell culture dish. And continued to be cultured until day 14. HDF and BM-MSC were infected with adenovirus encoding LacZ (Ad-LacZ) or FGF12 (Ad-FGF12) for 7 hours, then washed and further incubated in fresh medium for 72 hours. Cells were cultured in fibronectin coated cell culture dishes for 6 days in DMEM supplemented with 10% FBS.

Immunofluorescent staining

Cells were stained with anti-smooth muscle actin primary antibody (α-SMA; Sigma) and fluorescently labeled secondary antibody (Molecular Probes). The nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI). The stained cells were observed with a fluorescence microscope (Nikon, Melville). The immunofluorescent staining photographs shown represent the results of three separate, repeated experiments.

Reverse transcriptase Chain reaction

Total RNA of cells or tissues for reverse transcription polymerase chain reaction (RT-PCR) was isolated using Trizol reagent (Invitrogen). CDNA was synthesized from the whole RNE using the corresponding gene-specific primer and Superscript first-strand synthesis kit (Invitrogen), and amplified by PCR for 30-35 cycles. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Real-time PCR was performed using the SYBR-Green PCR master mix (Applied Biosystems) and the StepOnePlus ™ Real Time PCR System (Applied Biosystems). Data were analyzed according to the △ ΔCt method and standardized for GAPDH. The primers used in the PCR reaction are shown in Table 1 .

Adenovirus making

Human FGF-12 cDNA was subcloned into pShuttle-CMV vector and a recombinant adenovirus plasmid was constructed using pAdEasy-1 vector (Qbiogene). The recombinant adenovirus used in the experiment was purchased from ViraQuest Inc. .

Western Blat

The cell lysate was separated by SDS-PAGE, and the protein band was transferred to blot. Blood was incubated with appropriate primary antibodies such as myocardin (Sigma), serum response factor (SRF), MRTF-A (Santa Cruz Biotechnology), MRTF-B (Santa Cruz biotechnology), b- actin (Santa Cruz biotechnology) and horseradish peroxidase And then reacted with a secondary antibody. The protein band reacted with the antibody was confirmed by chemiluminescent reagent (Amersham Biosciences).

Collagen gel shrinkage experiment

Cells for collagen gel contraction assay were dispersed in 2 mg / ml collagen gel solution (BD biosciences) at a final density of 1 × 10 ⁵ cells / 100 ml of SMCM basal medium (ScienCell). The cell / collagen mixture was placed in a 12-well culture dish and incubated at 37 ° C. until polymerization occurred. SMCM basal medium (ScienCell) with or without carbachol (5 μM; Sigma) was added to induce the cell contraction reaction, and immediately thereafter the collagen gel was removed from the surface of the culture dish. The size of the collagen gel was measured by CCD camera and NIH Image software. The degree of shrinkage of the collagen gel containing cells was determined by dividing the size of the gel at 2 hours after the addition of carbachol by the size of the initial gel.

< Example 1>

FGF By -12 Smooth muscle cell Increased transcription factor expression

We investigated whether FGF-12 could induce the expression of important transcription factors in vascular smooth muscle cell differentiation.

Human Aortic Smooth Muscle Cells (HASMC) were infected with adenovirus (Ad-FGF) containing the FGF-12 gene or adenovirus (Ad-LacZ) containing the LacZ gene and the expression of the transcription factor protein important for the differentiation of the smooth muscle cell line Expression patterns were observed by Western blotting.

As shown in FIG. 1 , the expression of MYOCD, SRF and MRTF-A was significantly increased in human smooth muscle cells infected with Ad-FGF12 as compared to human smooth muscle cells infected with no virus or Ad-LacZ.

< Example 2>

FGF -12 Overexpression of mouse embryonic stem cells Into smooth muscle cells differentiation

In contrast to HASMC, we examined whether FGF-12 overexpression in embryonic stem cells originally not expressing FGF-12 could induce differentiation into smooth muscle cells by expressing an important transcription factor for smooth muscle cell differentiation.

Mouse embryonic stem cells (mESC) were not infected with the virus or infected with Ad-LacZ or Ad-FGF12 adenovirus and the expression of FGF-12 in each embryonic stem cell was confirmed by RT-PCR ( FIG. 2 ). Each embryonic stem cell was allowed to aggregate into an embryonic body and cultured for 10 days or 14 days in a medium not containing LIF so that spontaneous differentiation of the embryoid body occurred. ( Fig. 3 ) and RT-PCR ( Fig. 4 ) of the expression patterns of representative marker genes of smooth muscle cells in the differentiated embryoid bodies. The mRNA expression level of the RT-PCR of FIG. 4 is expressed as a relative value to the embryonic stem cells that had not been treated with virus (Day 0) before the average of the results of five repeated experiments for each gene.

As shown in the RT-PCR result of FIG. 2 , in the mouse embryonic stem cells not infected with the virus or infected with Ad-LacZ, mRNA of FGF-12 was not detected at all but in the mouse embryonic stem cells infected with Ad- 12 mRNA was overexpressed.

As shown in the immunochemical staining image of Fig. 3 , the embryos formed from embryonic stem cells infected with Ad-FGF12 expressed α-smooth muscle actin (α-SMA) at a high level on both 10th day and 14th day of culture , The level of smooth muscle cell actin was significantly higher than that of embryonic stem cell embryos not infected with virus or infected with Ad-LacZ.

The RT-PCR result of FIG. 4 shows that embryos formed from embryonic stem cells infected with Ad-FGF12 overexpress smooth muscle cell differentiation marker genes such as MYOCD, SM22? And Smoothelin including? -SMA observed in FIG. 3 . The smooth muscle cell differentiation marker gene is expressed at a low level on the 10th day of culture even in the embryo of a stem cell which is not infected with the virus or infected with Ad-LacZ. However, the expression level of smooth muscle cell marker gene in the embryos expressing FGF- Was significantly higher than that of the control group, showing a statistically significant difference.

These results show that FGF-12 overexpression in FGF-12-expressing embryonic stem cells can effectively promote differentiation into smooth muscle cells.

< Example 3>

FGF -12 Overexpression of human dermal fibroblast Into smooth muscle cells Transitional eruption

We examined whether FGF-12 overexpression in FGF-12-expressing fibroblast can induce transdifferentiation into smooth muscle cells.

Human dermal fibroblasts were cultured in DMEM containing 10% FBS for 6 days without infection with the virus or with Ad-LacZ or Ad-FGF12 adenovirus, Smooth muscle cell marker gene expression (Example 3-1) and degree of shrinkage of cells (Example 3-2).

< Example 3-1>

In human dermal fibroblasts Smooth muscle cell Marker Induction of gene expression

We examined whether FGF-12 overexpression in fibroblasts can induce the differentiation into smooth muscle cells through expression patterns of smooth muscle cell marker genes.

RT-PCR ( Fig. 6 ) for immunofluorescence staining for smooth muscle actin (α-SMA) ( FIG. 5 ) and smooth muscle marker genes (SM-MHC, SRF, Smoothelin, α-SMA and SM22α) were confirmed. In addition to fibroblasts, human aortic smooth muscle cells (HASMC) cultured in a basal medium for 3 days were included as a positive control in RT-PCR experiments.

As shown in the immunofluorescent staining image of FIG. 5 , the smooth muscle actin was hardly detected in the fibroblasts infected with the virus or with the Ad-LacZ, but the fibroblasts infected with the Ad-FGF-12 showed a very high level Expressing smooth muscle actin.

In the RT-PCR of FIG. 6 , it was confirmed that the fibroblasts infected with Ad-FGF12 overexpressed a variety of smooth muscle cell marker genes such as SM-MHC, SRF, Smoothelin, α-SMA and SM22α. Fibroblasts infected with non-virus-infected fibroblasts or Ad-LacZ express only smooth muscle cell differentiation marker genes or only at extremely low levels, whereas fibroblasts infected with Ad-FGF12 have similar levels to smooth muscle cells (HASMC) As a result.

< Example 3-2>

Induction of contractile phenotype expression in human dermal fibroblasts

The ability of fibroblast overexpressing FGF-12 to induce essential contractile phenotype in smooth muscle cells was confirmed by collagen gel shrinkage experiment ( Fig. 7 ) using carbacol which causes vasoconstriction.

The fibroblasts that did not infect or infected with the virus were included in the collagen gel, and cultured in a basal medium containing carbachol or PBS to evaluate the degree of shrinkage of the gel (the results corresponding to carbachol and PBS in Fig. 7 are white Indicated by bars and black bars). The degree of shrinkage of the collagen gel containing fibroblasts was expressed as the average of four replicates of the gel size divided by the initial gel size after 2 hours of incubation in the primary culture medium. In addition to fibroblasts, human aortic smooth muscle cells (HASMC) were included as positive controls

The results of the collagen gel shrinkage experiment of FIG. 7 show that fibroblasts overexpressing FGF-12 have shrinking properties similar to those of smooth muscle cells. Fibroblasts not infected with the virus or infected with Ad-LacZ showed little shrinkage due to the difference in size of collagen gel stimulated with PBS or carbacol. On the other hand, fibroblasts infected with Ad-FGF12 decreased the size of collagen gel by about 25% compared with before stimulation by carbachol stimulation, and there was a statistically significant difference compared to PBS-treated gel. In other words, it can be seen that fibroblasts acquire a contractile phenotype by carbachol stimulation due to overexpression of FGF-12.

The results of Example 3 above show that overexpression of FGF-12 effectively promotes the differentiation of fibroblasts into smooth muscle cells that are functionally shrinking as well as expressing marker genes of smooth muscle cells.

< Example 4>

FGF -12 human by overexpression Bone marrow origin Intermediate lobe Stem cell Into smooth muscle cells differentiation

We examined whether FGF-12 overexpression in human stem cells could induce differentiation into smooth muscle cells.

Human bone marrow-derived mesenchymal stem cells (BM-MSC) were cultured in DMEM containing 10% FBS for 6 days without infection with the virus or with Ad-LacZ or Ad-FGF12, and the α- The expression of SMA was confirmed by immunofluorescence staining ( Fig. 8 ).

As can be seen from Fig. 8 , In the mesenchymal stem cells not infected with the virus or infected with Ad-LacZ, α-SMA was almost not expressed, but it was confirmed that α-SMA was expressed at a very high level in the mesenchymal stem cells infected with Ad-FGF12 . In other words, it can be shown that FGF-12 overexpression in human mesenchymal stem cells can effectively defat smooth muscle cells.

As described above, the present invention provides a method and a composition capable of promoting differentiation or conversion of smooth muscle cells from various starter cells using FGF-12 as a single differentiation factor. By using the present invention, smooth muscle cells can be easily and efficiently produced and provided. Therefore, the present invention relates to a gene therapy agent or a cell therapy agent capable of effectively regenerating smooth muscle cells in a disease accompanied with dysfunction or degeneration of smooth muscle cells such as aortic aneurysm, Marfan syndrome, ) Can be used to develop.

<110> CHUNG ANG University industry Academic Cooperation Foundation <120> Methods to promote differentiation into smooth muscle cells and composition thereof <130> NP15-0072 <160> 28 <170> Kopatentin 2.0 <210> 1 <211> 243 <212> PRT <213> Homo sapiens FGF-12 isoform 1 <400> 1 Met Ala Ala Ala Ile Ala Ser Ser Leu Ile Arg Gln Lys Arg Gln Ala 1 5 10 15 Arg Glu Ser Asn Ser Asp Arg Val Ser Ala Ser Lys Arg Arg Ser Ser 20 25 30 Pro Ser Lys Asp Gly Arg Ser Leu Cys Glu Arg His Val Leu Gly Val 35 40 45 Phe Ser Lys Val Arg Phe Cys Ser Gly Arg Lys Arg Pro Val Arg Arg 50 55 60 Arg Pro Glu Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Phe Ser Gln 65 70 75 80 Gln Gly Tyr Phe Leu Gln Met His Pro Asp Gly Thr Ile Asp Gly Thr 85 90 95 Lys Asp Glu Asn Ser Asp Tyr Thr Leu Phe Asn Leu Ile Pro Val Gly 100 105 110 Leu Arg Val Val Ala Ile Gln Gly Val Lys Ala Ser Leu Tyr Val Ala 115 120 125 Met Asn Gly Glu Gly Tyr Leu Tyr Ser Ser Asp Val Phe Thr Pro Glu 130 135 140 Cys Lys Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr Ser 145 150 155 160 Ser Thr Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe Leu Gly 165 170 175 Leu Asn Lys Glu Gly Gln Ile Met Lys Gly Asn Arg Val Lys Lys Thr 180 185 190 Lys Pro Ser Ser His Phe Val Pro Lys Pro Ile Glu Val Cys Met Tyr 195 200 205 Arg Glu Pro Ser Leu His Glu Ile Gly Glu Lys Gln Gly Arg Ser Ser Arg 210 215 220 Lys Ser Ser Gly Thr Pro Thr Met Asn Gly Gly Lys Val Val Asn Gln 225 230 235 240 Asp Ser Thr <210> 2 <211> 732 <212> DNA <213> Homo sapiens FGF-12 isoform 1 (NM_021032: 827-1558) <400> 2 atggctgcgg cgatagccag ctccttgatc cggcagaagc ggcaggcgag ggagtccaac 60 agcgaccgag tgtcggcctc caagcgccgc tccagcccca gcaaagacgg gcgctccctg 120 tgcgagaggc acgtcctcgg ggtgttcagc aaagtgcgct tctgcagcgg ccgcaagagg 180 ccggtgaggc ggagaccaga accccagctc aaagggattg tgacaaggtt attcagccag 240 cagggatact tcctgcagat gcacccagat ggtaccattg atgggaccaa ggacgaaaac 300 agcgactaca ctctcttcaa tctaattccc gtgggcctgc gtgtagtggc catccaagga 360 gtgaaggcta gcctctatgt ggccatgaat ggtgaaggct atctctacag ttcagatgtt 420 ttcactccag aatgcaaatt caaggaatct gtgtttgaaa actactatgt gatctattct 480 tccacactgt accgccagca agaatcaggc cgagcttggt ttctgggact caataaagaa 540 ggtcaaatta tgaaggggaa cagagtgaag aaaaccaagc cctcatcaca ttttgtaccg 600 aaacctattg aagtgtgtat gtacagagaa ccatcgctac atgaaattgg agaaaaacaa 660 gggcgttcaa ggaaaagttc tggaacacca accatgaatg gaggcaaagt tgtgaatcaa 720 gattcaacat ag 732 <210> 3 <211> 181 <212> PRT <213> Homo sapiens FGF-12 isoform 2 <400> 3 Met Glu Ser Lys Glu Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Phe 1 5 10 15 Ser Gln Gln Gly Tyr Phe Leu Gln Met His Pro Asp Gly Thr Ile Asp 20 25 30 Gly Thr Lys Asp Glu Asn Ser Asp Tyr Thr Leu Phe Asn Leu Ile Pro 35 40 45 Val Gly Leu Arg Val Val Ala Ile Gln Gly Val Lys Ala Ser Leu Tyr 50 55 60 Val Ala Met Asn Gly Glu Gly Tyr Leu Tyr Ser Ser Asp Val Phe Thr 65 70 75 80 Pro Glu Cys Lys Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile 85 90 95 Tyr Ser Ser Thr Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe 100 105 110 Leu Gly Leu Asn Lys Glu Gly Gln Ile Met Lys Gly Asn Arg Val Lys 115 120 125 Lys Thr Lys Pro Ser Ser His Phe Val Pro Lys Pro Ile Glu Val Cys 130 135 140 Met Tyr Arg Glu Pro Ser Leu His Glu Ile Gly Glu Lys Gln Gly Arg 145 150 155 160 Ser Arg Lys Ser Ser Gly Thr Pro Thr Met Asn Gly Gly Lys Val Val 165 170 175 Asn Gln Asp Ser Thr 180 <210> 4 <211> 546 <212> DNA <213> Homo sapiens FGF-12 isoform 2 (NM_004113: 247-792) <400> 4 atggagagca aagaacccca gctcaaaggg attgtgacaa ggttattcag ccagcaggga 60 tacttcctgc agatgcaccc agatggtacc attgatggga ccaaggacga aaacagcgac 120 tacactctct tcaatctaat tcccgtgggc ctgcgtgtag tggccatcca aggagtgaag 180 gctagcctct atgtggccat gaatggtgaa ggctatctct acagttcaga tgttttcact 240 ccagaatgca aattcaagga atctgtgttt gaaaactact atgtgatcta ttcttccaca 300 ctgtaccgcc agcaagaatc aggccgagct tggtttctgg gactcaataa agaaggtcaa 360 attatgaagg ggaacagagt gaagaaaacc aagccctcat cacattttgt accgaaacct 420 attgaagtgt gtatgtacag agaaccatcg ctacatgaaa ttggagaaaa acaagggcgt 480 tcaaggaaaa gttctggaac accaaccatg aatggaggca aagttgtgaa tcaagattca 540 acatag 546 <210> 5 <211> 20 <212> DNA <213> Human FGF12 forward <400> 5 agcttggttt ctgggactca 20 <210> 6 <211> 24 <212> DNA <213> Human FGF12 reverse <400> 6 ctcttctgag atgagtttct gctc 24 <210> 7 <211> 20 <212> DNA <213> Human a-SMA forward <400> 7 agcgacccta aagcttccca 20 <210> 8 <211> 20 <212> DNA <213> Human a-SMA reverse <400> 8 catagagaga cagcaccgcc 20 <210> 9 <211> 20 <212> DNA <213> Human SM22 forward <400> 9 ggagcagtgg gtgcatttca 20 <210> 10 <211> 20 <212> DNA <213> Human SM22 reverse <400> 10 tgcactagcc aagtcatccg 20 <210> 11 <211> 22 <212> DNA <213> Human SM-MHC forward <400> 11 atgaggccac ggagagcaac ga 22 <210> 12 <211> 21 <212> DNA <213> Human SM-MHC reverse <400> 12 ccattgaagt ctgcgtctcg a 21 <210> 13 <211> 20 <212> DNA <213> Human SRF forward <400> 13 ccagtgtgtg ggggagattc 20 <210> 14 <211> 20 <212> DNA <213> Human SRF reverse <400> 14 aaccccgaca tttggtctcc 20 <210> 15 <211> 20 <212> DNA <213> Human smoothelin forward <400> 15 tgcaaaccaa gaccttctcc 20 <210> 16 <211> 20 <212> DNA <213> Human smoothelin reverse <400> 16 tccagcttct caatcatggc 20 <210> 17 <211> 19 <212> DNA <213> Human GAPDH forward <400> 17 gaaggtgaag gtcggagtc 19 <210> 18 <211> 19 <212> DNA <213> Human GAPDH reverse <400> 18 gaagatggtg atggatttc 19 <210> 19 <211> 20 <212> DNA <213> Mouse a-SMA forward <400> 19 gtgcctatct atgagggcta 20 <210> 20 <211> 20 <212> DNA <213> Mouse a-SMA reverse <400> 20 gccatctcat tttcaaagtc 20 <210> 21 <211> 20 <212> DNA <213> Mouse SM22 forward <400> 21 caagactgac atgttccaga 20 <210> 22 <211> 20 <212> DNA <213> Mouse SM22 reverse <400> 22 tgggctttct tcataaacca 20 <210> 23 <211> 20 <212> DNA <213> Mouse MYOCD forward <400> 23 agttgtcacc tccttctgtg 20 <210> 24 <211> 20 <212> DNA <213> Mouse MYOCD reverse <400> 24 atccataggg gaattcagat 20 <210> 25 <211> 20 <212> DNA <213> Mouse smoothelin forward <400> 25 gccatgattg agaagctaga 20 <210> 26 <211> 20 <212> DNA <213> Mouse smoothelin reverse <400> 26 gaggagaagt tctggatgtc 20 <210> 27 <211> 20 <212> DNA <213> Mouse GAPDH forward <400> 27 atgactccac tcacggcaaa 20 <210> 28 <211> 20 <212> DNA <213> Mouse GAPDH reverse <400> 28 atgatgaccc ttttggctcc 20

Claims

(a) providing starting cells from isolated or cultured cells; And
(b) inducing overexpression of FGF-12 in the starting cell to change the differentiation state of the starting cell; And
(c) identifying a phenotype of smooth muscle cells in the starter cell of step (b); and promoting differentiation from the starting cells to smooth muscle cells.

[2] The method according to claim 1, wherein the step of modifying the differentiation of step (b) is performed by progressing the differentiation of the starting cells.

3. The method of claim 2, wherein the starter cell is a stem cell.

4. The method according to claim 3, wherein the stem cells are pluripotent stem cells or adult stem cells.

5. The method according to claim 4, wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

5. The method according to claim 4, wherein the adult stem cells are mesenchymal stem cells.

2. The method according to claim 1, wherein the step (b) is performed by transdifferentiation of the starting cells.

8. The method of claim 7, wherein the starting cell is a fibroblast.

9. The method of claim 8, wherein the fibroblasts are dermal fibroblasts.

2. The method according to claim 1, wherein the FGF-12 comprises the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3.

The method according to claim 1, wherein inducing overexpression of FGF-12 in step (b) is carried out by transforming the starting cell with a polynucleotide encoding FGF-12.

12. The method according to claim 11, wherein the polynucleotide encoding FGF-12 comprises the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

13. The method of claim 12, wherein said transformation is mediated by a recombinant expression vector into which a polynucleotide encoding FGF-12 is inserted.

14. The method of claim 13, wherein the recombinant expression vector is a recombinant viral vector.

15. The method of claim 14, wherein the recombinant viral vector is selected from the group consisting of an adenovirus vector, a retrovirus vector, a herpes virus vector, a lentivirus vector, and an avipoxvirus vector.

A composition for promoting differentiation from a starting cell to a smooth muscle cell comprising a polynucleotide encoding FGF-12 as an active ingredient.