KR101344719B1 - The method for differentiating stem cells into smooth muscle cells with strain and tissue engineering complex containing the smooth muscle cells - Google Patents

Abstract

The present invention provides a method for differentiating stem cells into smooth muscle cells while applying stress to the elastic scaffold inoculated with stem cells, and smooth muscle for tissue engineering for the treatment of vascular diseases or wounds containing smooth muscle cells differentiated by the method. Cell patch. The present invention demonstrates the synergistic effect of mechanical stress and biochemical factors on smooth muscle cell differentiation of human adipose stem cells and provides a useful application of stem cells in mechanical-active tissue engineering.

Description

The method for differentiating stem cells into smooth muscle cells with strain and tissue engineering complex containing the smooth muscle cells}

The present invention relates to a method for differentiating stem cells into smooth muscle cells and a complex for tissue engineering containing smooth muscle cells differentiated by the above method.

Recently, it has been reported that human adipose tissue contains an undifferentiated cell population which is excellent in self-renewal and can be differentiated into various cells such as liver, bone, cartilage, fat, blood vessels, heart and nervous system (B. Cousin et al., BBRC 301: 1016, 2003; Miranville et al., Circulation 110: 349, 2004; S. Gronthos et al., J. Cell Physiol. 189: 54, 2001; MJ Seo et al., BBRC 328: 258, 2005). Adipose-derived stem cells (ASCs), which are derived from adipose tissue-derived multipotent stem cells, are easily and securely obtained in that they can be extracted from adipose tissue in large quantities compared with mesenchymal stem cells. It is attracting attention as a new source of multipotent stem cells because it has advantages in terms of tissue accessibility, stability, efficacy, and economics due to its unlimited limitations and easy in vitro culture.

Development of biological substitutes fundamentally requires stable cell sources that can improve or preserve tissue function, optimal biochemical environments, physiological conditions and biologically stable scaffolds.

The copolymer of lactic acid and caprolactone (PLCL) has a mechanical property range determined by these component ratios, and has a rubber-like elastic force by the physical crosslinked structure of these components. Electrospinning also enables the fabrication of nanometer-thick fibrous scaffolds and induces cell growth, proliferation and differentiation. Nanofibrous scaffolds are characterized by mechanical stability, structural induction, and extracellular matrix (ECM) protein recombination. It provides an anchor suitable for interaction of cells with surrounding tissues in order to respond smoothly to biological and physiological changes. Scaffold materials for tissue engineering materials should be suitable materials for the mechanical properties of the design, polymer, and excellent biocompatibility. In the present invention, the PLCL nanofiber scaffold made by the electrospinning method will be utilized for scaffold development for differentiating fat stem cells into smooth muscle cells.

For example, the development of biotechnology, such as the use of adult stem cells extracted from bone marrow or blood to treat coronary artery disease, regenerates tissues and organs using stem cells to restore their function. Methods for restoration are actively being attempted. Stem cells are cells that have the ability of self-replicating and differentiate into two or more types of cells. Totipotent stem cells, pluripotent stem cells, and multipotent stem cells cells). Most of the differentiation studies of smooth muscle cells include conversion growth factor-β-1 (TGF-β-1), platelet-derived growth factor-BB (PDGF-BB), ascorbic acid (AA), retinoic acid (RA), and angiotensin II ( Liquid biochemical factors were used to induce differentiation factors such as Ang II), and various smooth muscle cell differentiation induction levels were shown.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors earnestly researched for the development of a method for differentiating stem cells into smooth muscle cells with high efficiency. As a result, the present invention has been completed by devising a method for differentiating stem cells by applying stress with biochemical factors.

It is therefore an object of the present invention to provide a method for differentiating stem cells into smooth muscle cells.

Another object of the present invention to provide a smooth muscle cell patch for tissue engineering for the treatment of vascular diseases or wounds containing smooth muscle cells differentiated by the method of differentiating stem cells of the present invention.

Still other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

The present invention provides a method for differentiating stem cells into smooth muscle cells while applying stress to the stem cells inoculated with the elastic scaffold.

The present inventors have made intensive studies to develop a method for differentiating stem cells into smooth muscle cells with high efficiency. As a result, biochemical factors such as growth factors and mechanical stimuli that can be expressed by stresses are applied to smooth muscle cells from stem cells. By providing a method for differentiation with high efficiency, the present invention has been completed.

Stem cells are those that remain undifferentiated into specific cells and then differentiate into each cell, if necessary, with the ability to differentiate into all kinds of cells that make up the body, including nerves, blood, and cartilage. Undifferentiated cells of all stages are collectively, and differentiation proceeds to specific cells by specific differentiation stimulus (environment). Stem cells, unlike differentiated cells that have ceased cell division, have the property of proliferation (expansion) by being able to self-renewal their own cells by cell division. It is characterized by having plasticity in differentiation because it can be differentiated into other cells by different environment or differentiation stimulus.

Stem cells can be classified into pluripotent stem cells, pluripotent stem cells, and multipotent stem cells according to their differentiation ability.

On the other hand, such stem cells may be classified according to their origin. The first is derived from embryos from embryos (embryonic stem cells) and the second is stem cells (adult stem cells) that appear during the development of each organ in the embryo or during adult development. Although functionally different, embryonic stem cells and adult stem cells are characterized by differentiation into various cell types.

As an exemplary embodiment of the present invention, the stem cells that can be differentiated by the present invention is not limited, and the cells having the characteristics of stem cells, that is, undifferentiated, infinite proliferation and differentiation into specific cells are cells to which the present invention can be applied. to be. Stem cells include embryonic stem cells, adult stem cells, induced pluripotent stem cells, embryonic germ cells and embryonic tumor cells, preferably adipose stem cells, mesenchymal stem cells, bone marrow stem cells, cord blood stem cells, neural stem cells and Induced pluripotent stem cells, more preferably fat stem cells.

 The term "fat tissue-derived stem cell" used in the present invention is an undifferentiated adult stem cell isolated from adipose tissue, and may be abbreviated herein as "fat stem cell". This can be recovered by culturing suspension containing fats containing saline derived from liposuction followed by trypsin treatment of the stem cell layer attached to the culture vessel, such as a flask, or by directly scraping off a small amount of saline by scraping with a scraper. It is obtained through the method or the like.

In one embodiment of the present invention, preferably human adipose stem cells can be used.

On the other hand, in order to increase the usefulness of stem cells as a cell therapeutic agent, a technique for efficiently differentiating stem cells into specific cells such as smooth muscle cells is required. In order to achieve the above object, the present invention provides an elastic nanofiber (scaffold) capable of applying mechanical stress.

Many cardiovascular diseases are treated with synthetic grafts that bypass blocked blood vessels; However, in many cases successful treatment has been limited due to the poor functioning of the synthetic materials used for tissue replacement. An alternative approach is to design tissue engineered constructs using a combination of cells and scaffolds.

In the design of scaffold materials for the fabrication of engineered tissue, candidate polymers must have adequate mechanical properties, which are suitable for target applications and should be biodegradable polymers whose degradation products are nontoxic during the implantation process. The biodegradable polymer is spontaneously degraded after a certain period of time in a living body, and refers to a polymer having at least one of biocompatibility, blood affinity, anti-calcification properties, cellular nutrients, and intercellular matrix formation. . Such biodegradable polymers are not particularly limited in the present invention, but typically, fibrin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid, polyglycolic acid (poly (glycolic acid), PGA), Poly (lactic-co-glycolic acid), PLGA, poly-ε- (caprolactone), poly (lactic acid-co-ε-caprolactone) (poly (L-lactide- co-ε-caprolactone), PLCL), polyanhydride, polyorthoester, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly-N-isopropylacrylamide, poly (ethylene oxide) -poly (propylene Oxide) -poly (ethylene oxide) copolymers, copolymers thereof, mixtures thereof and the like can be used. Preferably a copolymer of lactic acid and caprolactone (PLCL) can be used. PLCL exhibits a range of mechanical properties and rubber-like elasticity on its physical crosslinked structure, depending on their relative composition.

At this time, the content of the biodegradable polymer in the scaffold is preferably 5 to 100% by weight.

The composite scaffold, such as a scaffold, may be formed by a known method, for example, a salt-casting and particle-leaching technique, a gas forming technique, a polymer fiber into a nonwoven fabric, and a polymer mesh ( fiber extrusion and fabric forming process, thermally induced phase separation technique, emulsion freeze drying method, high pressure gas expansion, electrospinning The biodegradable polymer may be manufactured by molding). Preferably, the electrospinning method is good.

Electrospinning allows the production of nanofiber scaffolds with high pores for cell growth, proliferation and differentiation, with high surface area to volume ratios and fiber diameters below the nanometer range. By differentiating stem cells on the nanofiber scaffold, the present invention can differentiate stem cells into smooth muscle cells more efficiently than the floating culture and attachment culture methods used in the conventional stem cell differentiation. There is an advantage. Biodegradable nanofiber scaffolds manufactured by electrospinning have been spotlighted as materials for tissue engineering, and high-efficiency differentiation of fat stem cells into smooth muscle cells from nanofiber scaffolds, which is not a general culture vessel, is a biological nano-analysis of cardiovascular diseases. It can have great potential for substance-cell transplantation treatment.

The average fiber diameter of the nanofibers constituting the nanofiber scaffold may be in the range of 500 ~ 1000 nm.

In one embodiment of the present invention, the nanofiber scaffold may be further coated with an extra cellular matrix (ECM) protein to facilitate the attachment of stem cells. Extracellular matrix proteins include collagen, elastin, proteoglycans, glycosaminoglycans, fibronectin, laminin, vitronectin and the like. Among these, proteins coated on the nanofiber scaffold are preferably type 1 collagen, type 4 collagen, fibronectin, laminin. More preferably, type 1 collagen may be used as a coating for stem cell adhesion.

The term stem cell “differentiation” as used in the present invention includes not only the case of fully differentiated stem cells from specific cells, but also an intermediate step before the complete differentiation of stem cells from specific cells.

In another aspect, the present invention to inoculate the fat stem cells to the nanofibers, and incubating the fat stem cells in the state adhered to the surface of the nanofibers, including the step of applying a mechanical stress in combination with the differentiation factors, smooth muscle cells of the fat stem cells It provides a differentiation method.

As a medium used in the method for differentiating adipose stem cells, a conventional medium known in the art to be suitable for culturing stem cells may be used. Preferably, DMEM (Dulbecco modified Eagle medium) or Keratinocyte-SFM (Keratinocyte serum free medium) Can be used.

 In addition, the medium generally contains neutral buffers (such as phosphates and / or high concentration bicarbonates) and protein nutrients (such as serum, such as FBS, serum substitutes, albumin, or essential and non-essential amino acids, such as glutamine) in isotonic solutions. can do. Furthermore, lipids (fatty acids, cholesterol, HDL or LDL extracts of serum) and other components found in most preservative media of this kind (such as insulin or transferrin, nucleosides or nucleotides, pyruvate salts, any ionized form or salt) Sugar sources such as glucose, selenium, glucocorticoids such as hydrocortisone and / or reducing agents such as β-mercaptoethanol.

It may also be advantageous to include anti-clumping agents, such as those sold by Invitrogen (Cat # 0010057AE), for the purpose of preventing the cells from adhering to each other or forming too large a bundle. .

In vitro experiments, human-derived adipocytes have been reported to differentiate into smooth muscle cells in response to various biochemical factors. The biochemical factors include growth factors such as acidic fibroblast growth factor (acidic FGF), basic fibroblast growth factor (basic FGF), insulin-like growth factor-1 (IGF-1), insulin-like growth factor- 2 (IGF-2), keratinocyte growth factor (KGF), platelet derived growth factor (PDGF), human transformation growth factor-alpha (TGF-α), human transformation growth factor-beta (TGF-β), Vascular endothelial growth factor (VEGF), epidermal cell growth factor (EGF), neuronal growth factor (NGF) and the like. In addition, the biochemical factors may include ascorbic acid (AA), retinoic acid (RA), angiotensin II (Ang II) and the like. However, their low differentiation efficiency limits the application of adipose stem cells to clinical treatment.

The present invention provides the optimal stress by applying a mechanical stress device for the proliferation of adipose stem cells and efficient differentiation into smooth muscle cells on the elastic nanofiber implants and examining the synergistic effects with various differentiation factors under such stress. To develop culture methods and smooth muscle cell / scaffold complex.

In the present invention, the tension value consisting of the electronic portion and the mechanical portion is designed and customized to give a stress, it was possible to apply a stress within the general physiological conditions in the incubator. It is preferable to apply the tension in the range of 0.5 to 10 Hz, the stress is in the range of 1 to 10%, more preferably 5% stress, 1 Hz frequency.

In the present invention, the inventors have demonstrated in the elastic scaffold that the synergistic effect produced by the combined stimulation of biochemical factors and stress is more effective in the process of differentiating fat stem cells into smooth muscle cells than when using one stimulus. .

Smooth muscle cell markers such as smooth muscle cell alpha-actin (α-SMA) and myosin heavy chain (MHC) were used to demonstrate differentiation into smooth muscle cells.

α-SMA is an early marker of smooth muscle cell development, but its expression alone does not provide clear evidence that it is a smooth muscle line. However, MHC is not recognized in any other cell line and is expressed only in contractile smooth muscle cells. Therefore, in order to characterize the differentiation of smooth muscle cells, not only the expression of α-SMA but also the expression of MHC was evaluated in the present invention.

Differentiation factors used in the present invention did not affect the expression of markers of smooth muscle cells, such as smooth muscle cells α-actin (SMA) and myosin heavy chain (MHC) in political culture. However, it was influenced by the differentiation factor in the stress culture environment. In particular, adipose stem cells cultured under stress conditions in medium supplemented with RA or AA significantly increased the expression of SMA (3-fold) and MHC (2-fold) compared with stress-only or differentiation factor. . Differentiation efficiency through the present invention was the best when the stress at the same time in the medium containing RA or AA.

Particularly preferably, the addition of the biochemical factor RA significantly increased the proliferation and differentiation of adipose stem cells.

Therefore, the present invention provides a method for highly efficient differentiation of adipose stem cells into smooth muscle cells on a biodegradable nanofiber scaffold having elasticity, and provides a complex consisting of a scaffold and differentiated smooth muscle cells.

In another aspect, the present invention provides a tissue engineering complex for regeneration of blood vessels containing smooth muscle cells differentiated from stem cells by the above method as an active ingredient.

The tissue engineering complex according to the present invention is characterized in that smooth muscle cells differentiated from stem cells are seeded in a scaffold made by molding a biodegradable polymer.

The scaffold manufactured as described above serves to deliver the seeded smooth muscle cells into the transplanted tissue, and the cells adhere to and grow on the scaffold surface to form new tissue. On the other hand, it is preferable that the average thickness of a scaffold is 50-500 micrometers, tensile strength is 2-10 Mpa, elastic modulus is 5-15 Kpa, and elongation at break is 100-1000%.

In the scaffold for tissue engineering according to the present invention, it is preferable that smooth muscle cells differentiated from stem cells as an active ingredient are seeded at 1 × 10 ⁴ to 1 × 10 ⁶ cells / cm 2 with respect to the scaffold. If the smooth muscle cell concentration is less than the above range, the effect of stimulating blood vessel regeneration of the smooth muscle cell is insignificant, whereas if the concentration exceeds the above range, the cells inoculated due to the lack of nutrients and oxygen may occur.

Meanwhile, the smooth muscle cell-scaffold complex according to the present invention may include the smooth muscle cell-scaffold complex and a pharmaceutically acceptable carrier and / or additive as an active ingredient. Examples include sterile water, physiological saline, conventional buffers (phosphate, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives. can do. For topical administration, it is also preferable to combine organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. .

Smooth muscle cell-scaffold complex according to the present invention, if necessary according to the formulation, suspensions, solubilizers, stabilizers, tonicity agents, preservatives, adsorption agents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers , Sulfur-containing reducing agents, antioxidants and the like may be appropriately included. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.

The smooth muscle cell-scaffold complex of the present invention may be formulated with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art. It may be made in the form or prepared by incorporation into a multi-dose container.

The smooth muscle cell-scaffold complex of the present invention containing smooth muscle cells differentiated from stem cells as an active ingredient can be very useful for the treatment of wound treatment, cardiovascular disease, cerebrovascular disease, ischemic disease and the like.

Vascular diseases in the present invention include cardiovascular disease, cerebrovascular disease, ischemic disease and the like, for example, atherosclerosis, stable and unstable angina, peripheral cardiovascular disease, hypertension, heart failure, peripheral circulation disorders, myocardial infarction, stroke, Transient and ischemic attacks, subarachnoid hemorrhage, and the like.

As such, stem cells inoculated into the scaffold may be differentiated into smooth muscle cells to effectively induce tissue regeneration in a tissue in which the smooth muscle cell-scaffold complex for tissue engineering according to the present invention is transplanted.

In addition, the scaffold / smooth muscle cell complex prepared by the method of the present invention can be utilized as a patch for the treatment of myocardial infarction or smooth muscle cell sheet for tissue engineering vessels.

Adipose tissue-derived stem cells have been of primary interest in many tissue engineering studies due to their various potentials. We focused on the potential for smooth muscle cell differentiation of human adipose stem cells in cardiovascular tissue engineering. In the present invention, the combined effects of stress and differentiation factors on human adipose stem cell proliferation on elastic nanofiber scaffolds and resulting differentiation into smooth muscle cells were investigated. Cell proliferation was increased in mechanically stressed cultures, and it was found that the effect of medium composition was greater than that in stationary culture. Differentiation factor did not affect smooth muscle cell α-SMA and MHC expression in stationary culture. However, the expression of α-SMA and MHC was influenced by differentiation factors in stress cultures, especially the treatment of α-SMA (3 times) and MHC (2 times) in the treatment of retinoic acid as compared to the mechanical stimulation alone. It showed a significant increase in expression. The present invention demonstrates the synergistic effect of mechanical stress and biochemical factors on smooth muscle cell differentiation of human adipose stem cells and provides a useful application of stem cells in mechanical-active tissue engineering.

According to the present invention, adipose stem cells use a single stimulus of smooth muscle cell markers such as α-SMA and MHC due to synergistic effect when a combination of mechanical stress is applied in combination with RA or AA on the nanofiber scaffold, preferably RA. Proliferation was possible in a significantly increased state.

1 is a result of immunofluorescence staining of α-SMA of adipose stem cells after culturing multipotent adipose stem cells isolated from liver subcutaneous adipose tissue for 2 weeks in a smooth muscle differentiation medium or a general culture medium in a tissue culture plate,
FIG . 2A is a result of immunofluorescence staining confirming adhesion of adipose stem cells to an electrospun PLCL patch surface coated with various proteins (type 1 collagen, type 4 collagen, fibronectin, laminin) by DAPI staining . B is a graph quantified by measuring the cell adhesion rate by WST (Water-soluble Tetrazolium Salts) method,
3 is a photograph taken from the top and side of the culture apparatus for transferring stress,
Figure 4 is a photograph of the surface of the electrospun PLCL scaffold having a nanofiber form with an electron microscope (SEM),
Figure 5 is fixed with an electrospinning PLCL patch inoculated with adipose stem cells in a stress transfer culture apparatus, and then containing biochemical factors for two weeks to examine the effects of biochemical factors and mechanical stress on the proliferation of adipose stem cells It is a graph quantified by measuring the cell proliferation by WST assay after incubation in a stationary culture or stress with a medium,
FIG. 6 is a photograph taken after SEM inoculation of adipose stem cells into an electrospinning PLCL scaffold coated with type 1 collagen, followed by incubation for 2 weeks under static culture or stress in a medium containing biochemical factors,
A and C of Figure 7 immunological for α-SMA (A) and MHC (B) After culture, or stationary culture under stress (5% strain, 1 Hz) with a medium containing the biological factor fat stem cells The results of fluorescence staining, B and D are the results of quantification of the immunological fluorescence staining using Image J software.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

Example

Materials and Methods

1. Cell isolation and culture

Human subcutaneous adipose tissue samples were obtained from the abdomen of three different female donors ages between 42 and 55 years old. D. Hanson, ME Wall, B. Pourdeyhimi and EG Loboa, J. Biomater. Sci . Polymer As described in Edn 18, 1387 (2007), human adipose stem cells were isolated and characterized. Briefly, the adipose tissue was stored at 37 ° C. for 6 hours in DMEM / F12 medium (Welgene) containing 0.3% collagenase type I solution (Wako), 10 mg / ml bovine serum albumin (BSA, Sigma) and 1% penicillin. It was disassembled by shaking it intermittently. The resulting suspension (fat) was then filtered through 250 mm Nitex filters (Sefar America Inc) and centrifuged at 400 g for 8 minutes to remove impurities. Cells collected by centrifugation were suspended in maintenance medium (DMEM / F12 medium supplemented with 10% FBS). These primary cells were plated in tissue culture flasks under humid conditions containing 37 ° C. 5% carbon dioxide and 95% air for 48 hours. The cultures were washed away to remove unattached cells. The medium was changed once every two days and the cells were kept at sub-confluent level. Cells were passaged with 0.05% trypsin / 0.53 mM EDTA solution and inoculated at a density of 5 × 10 ³ cells / cm ² and cultured for cell proliferation under the same conditions (passage 1). The morphology of the cultured cells was observed under a phase change microscope (Nikon TE 2000-U). Cells of passages 4 and 5 were used in this study.

2. Fabrication of Electrospun PLCL Scaffolds

PLCL (50:50) is known as L-lactide (100 mmol), ε-caprolactone (100 mmol) and 1,6-hexanediol (0.5) using Stanus octoate (1 mmol) as catalyst polymerized in 100 ml glass ampoule containing mmol). simply. The ampoule was purged three times with nitrogen at 90 ° C., then sealed under vacuum and heated to 150 ° C. for 24 hours with stirring in an oil bath. After the reaction, the obtained polymer was dissolved in chloroform and filtered through a 4.5 μm pore membrane filter. The polymer was precipitated in excess methanol, filtered and dried under vacuum. The composition of the PLCL was confirmed in CDCl ₃ by 1 H NMR spectra (400 MHz; Varian). The number average molecular weight (Mn) and polydispersity (Mw / Mn) (Mw: molecular weight mass mean) of the copolymers measured by GPC were approximately 1.5 to 1.6 X 10 ⁵ and 2.7 to 2.9, respectively. The intrinsic viscosity of the said copolymer was 3.0-3.2. The total yield of PLCL was 81%. Nanofiber PLCL scaffolds were prepared by the electrospinning method. Briefly, the polymer solution (7% w / v) was prepared in hexafluoroisopropanol (HFIP) for the electrospinning process. First, fill the syringe with the solution and apply a voltage of 20 kV at a normal flow rate (1 ml / h) of mandrel (15 cm in diameter, 27 cm in length; nano NC) and 15 cm from the 22 gauge needle. And spun. After preparation, the scaffold fibers were dried overnight at room temperature. The scaffold was then sterilized by soaking in 70% ethanol for 20 minutes and washed three times for 5 minutes with sterile water and PBS.

3. Attachment of Human Adipose Stem Cells to PLCL Scaffold

Fibronectin (Sigma Chemical, St. Louis, Mo) and Laminin (Sigma Chemical) (20 μg / ml) were dissolved in PBS before coating the electrospun PLCL scaffold, and Type 1 and Type 4 collagen (Nitta Gelatin) ( 20 μg / ml) was dissolved in acetic acid (pH 3). After 4 hours at 37 ° C., the unabsorbed protein solution was removed from the plate and washed three times with PBS. Human adipose stem cells were attached to the PLCL scaffold at a density of 2 × 10 ⁴ cells / cm ² . Human adipocyte-inoculated scaffolds were then incubated in growth medium (DMEM / F12 medium supplemented with 10% FBS) for 3 days. Unattached cells were removed from the wells by washing with PBS. Cell viability was measured by Dojindo laboratories (WST) experiments, which depend on the ability of living cells to reduce tetrazolium salts to convert them into water-soluble colored formazan products. After 3 days the culture medium was removed and 20 μl of WST solution was added to each well containing 180 μl medium. Cells were then cultured for 4 hours at 37 ° C. 50 μl of each sample solution was transferred to a well of a 96 well plate and the absorption of the formazan product was measured by optical density (OD) at 450 nm. The blank value of the medium exposed to the cell-free scaffold is background and subtracted from each experimental value. The absorbance of each well was measured using a UV-microplate reader (model VERSA max, Molecular Devices).

 4. Biochemical Stimulation and Stress

Human adipose stem cells were inoculated at 2 × 10 ⁴ cells / cm ² in a type 1 collagen-coated electrospun PLCL scaffold and incubated for 3 days before stimulation in DMEM / F12 medium supplemented with 10% FBS (Welgene). . To study the combined effects of stress and biochemical stimulation on cell growth and differentiation, supplemented with one of the following chemicals or growth factors and reduced serum (DMEM / F12, 5% FBS, 1% penicillin) Human adipose stem cells were exposed for 2 weeks: (1) 1 ng / ml TGF β-1 (R & D system); (2) 50 μΜ β-mercaptoethanol (Amersham Pharmacia Biotech) and 0.3 mM ascorbic acid (AA, Sigma); (3) 1 μM all-trans retinoic acid (RA, Sigma); (4) 10 ng / ml PDGF-BB (Sigma) and smooth muscle induction medium; (5) DMEM-20% FBS; (6) 1 μM Angiotensin II (Sigma), 10% FBS, DMEM (Welgene); (7) SMIM supplemented with 1% FBS and 100 units / ml heparin (Sigma), consisting of MCDB 131 medium (Sigma). The control group was human adipose stem cells cultured in DMEM / F12 medium containing 10% FBS. A custom-made device was designed and manufactured to operate with a stress in the range of 5% and a frequency in the range of 1 Hz to apply tension. The tensioning device consisted of electrical and mechanical parts.

5. Immunofluorescence Staining

At the end of the experiment, cell-polymer constructs were fixed at 4% paraformaldehyde for 60 minutes at room temperature and immunostained according to standard immunocytochemistry manuals. Briefly, two washes with PBS and 0.1% Triton X-100 were applied to the cells for 15 minutes, after which nonspecific binding was blocked with 4% BSA. Monoclonal antibodies specific for α-SMA (mouse monoclonal, 1: 200; DAKO) and monoclonal antibodies specific for smooth muscle myosin heavy chain (mouse monoclonal, 1: 100; MHC, DAKO) Treated at room temperature for minutes. Primary antibodies were detected with Alexa Fluor 488-conjugated anti mouse goat IgG (1: 700; Invitrogen), Alexa Fluor 594-conjugated anti-rabbit goat IgG (1: 250; Invitrogen). Cells were reversed stained with DAPI (4,6-diamino-2-phenylindole dihydrochloride; Vector Laboratories). The primary antibody was omitted for the negative control. After washing with PBS, the stained cells were observed by fluorescence microscopy (DXM 1200F, Nikon). To determine the fraction of stained cells, images of the stained cells were taken with a Nikon TE 2000-U microscope. The processed images were fractionally analyzed using Image J software (NIH).

6. Electron Microscope Scanning

Morphology and surface morphology of scaffolds and tissue engineering grafts were studied by electron microscopy (SEM, Hitachi) operated at 15 kV. For SEM testing, the scaffolds were fixed in 4% v / v formaldehyde for 24 hours and dried by dehydration using a graded ethanol series. A small amount of scaffold or tissue engineering graft sample was attached to the SEM stub with carbon tabs and the stub surface was then coated with gold using a sputter coater (Eiko IB3).

7. Statistical Analysis

Statistical analysis was performed using an unpaired student's t test with a combination of various variables. Data are shown as mean ± SD. Statistical significance was defined as p <0.05.

Experiment result

1.Specification of Nanofiber PLCL Scaffolds

SEM micrographs of electrospun PLCL scaffolds revealed that the PLCL scaffolds formed under the optimal spinning conditions used in this study were beadless, porous, and nanoscale fiber scaffolds (FIG. 4). The nanofibers of the PLCL were obtained having a fiber diameter in the range of 750 ± 250 nm. The average thickness of the scaffolds was 100-150 μM as measured by gauge. The tensile properties of the electrospun PLCL scaffolds were specified as already known. The electrospun PLCL had a tensile strength and modulus of 4.7 Mpa and 8.4 Kpa, respectively. The scaffold had an elongation at break of 960%. This tensile property means that the electrospun PLCL scaffold is very elastic and physically strong enough to fit into a vascular graft.

2. Attachment of Human Adipose Stem Cells to Nanofiber PLCL Scaffolds

Electrospinning PLCL scaffold (FIG. 4) served as a substrate for measuring the differentiation capacity of human adipose stem cells into smooth muscle cells in the bioreactor system. Extracellular matrix protein coatings were used as a scaffold modification method to enhance cell adhesion to nanofiber PLCL scaffolds. Human adipose stem cells were attached to PLCL scaffolds coated or uncoated with extracellular matrix proteins (type 1 collagen, type 4 collagen, fibronectin, laminin) and cell adhesion was measured by WST experiments and DAPI staining. Compared to other scaffolds. It has been demonstrated that human adipose stem cells adhere with greater affinity to PLCL scaffolds coated with type 1 collagen. As can be seen in FIG. 2B, DAPI (shown as light blue dots) staining is a two-dimensional cell-substrate with human fat stem cells densely attached to the surface of a nanofiber PLCL scaffold coated with type 1 collagen. It was demonstrated that the network was formed and that more nuclei were observed in the PLCL scaffold coated with type 1 collagen than any other scaffold (FIG. 2A).

3. Effect of Medium on Stress Stimulated Human Adipose Stem Cell Proliferation

To demonstrate the combined effect of stress stimulation and biochemical factors on functions such as cell proliferation and differentiation of human adipose stem cells attached to the nanofiber scaffold, the attached cells were subjected to 5% stress within a range of normal physiological conditions, or In the absence of conditions, cultures were carried out in various media supplemented with biochemical factors that induce differentiation into smooth muscle cells in the light of what was previously reported. As shown in FIG. 5A, cell proliferation was not significantly affected by medium difference in stationary culture. Compared to the stationary culture, the application of stress resulted in an increase in human adipose stem cell differentiation (up to 150% of the political culture control, p <0.05) and the cell proliferation rate varied with medium (FIG. 5B). Under stressed conditions, when cultured with TGF-β1, AA, and RA-supplemented medium, human adipose stem cell growth was increased (p <0.05) compared to that with stress alone, but cultured with SIMM medium. Cell proliferation was decreased (p <0.001). SEM images show increased cell proliferation of human adipose stem cells cultured under stress stimulation conditions in nanofiber PLCL scaffolds (FIG. 6). After two weeks of treatment with TGF-β1, AA, and RA under stress, the cells increased enough to completely cover the nanofiber scaffold. However, it was observed that the cells grew sparsely in stress-stimulated scaffolds in stationary culture scaffolds and SIMM media, and the pores of the scaffolds were empty. These results indicate that biochemical factor matches should be considered even in stress stimulated cell proliferation.

4. Synergistic Effects of Stress and Differentiation Factors on the Differentiation of Human Adipose Stem Cells into Smooth Muscle Cells

To test the combined effect of differentiation factors and stress on differentiation of human adipose stem cells attached to nanofiber PLCL scaffolds into smooth muscle cells, the attached cells were cultured in the medium under or without stress. As shown in FIG. 6, fluorescence staining analysis was performed to detect early smooth muscle cell marker (α-SMA) and late smooth muscle cell marker (MHC). In stationary cultures, differentiation factors elicited α-SMA expression of human adipose stem cells cultured on the plate (FIG. 1), whereas α-SMA and MHC expression of human adipose stem cells attached to the nanofiber PLCL scaffold were differentiation factors. It was not significantly affected by the difference (FIG. 6). On the other hand, even in the control medium, the stress alone induced α-SMA expression of human adipose stem cells (176% of the political culture control, p <0.05) (FIGS. 4A and B). Interestingly, MHC expression was not affected by stress in control medium (FIGS. 4C and D). Cells cultured under stress in medium supplemented with differentiation factors demonstrated increased expression of α-SMA as well as MHC. In particular, the expression of α-SMA and MHC increased significantly (˜80%) compared to cells exposed only to stress when treated with RA while stressing the cells (FIGS. 6B and D). In FIG. 6, in the culture of stress-stimulated human adipose stem cells, the proliferative effects of TGF-β-1, AA, and RA were almost the same, but when combined with stress stimulation, RA showed a difference in differentiation into smooth muscle cells. It was.

Further discussion

Multipotent human adipose stem cells represent a potential source of mesenchymal cells such as smooth muscle cells in the blood vessel wall. In vitro experiments showed that cognitive stem cells differentiate into smooth muscle cells in response to biochemical factors. However, its low differentiation rate limits the further use of cognitive stem cells in clinical treatment. In the present invention, the inventors report that the combined stimulation of biochemical factors and stress produces a synergistic effect on the mechanism of human adipose stem cell differentiation than when they are used alone. In the present invention, it has been demonstrated that the smooth muscle cell markers such as α-SMA and MHC are significantly increased when human adipose stem cells are combined with RA and stress. Smooth muscle cell differentiation is very often described using smooth muscle cell markers. These smooth muscle cell markers include α-SMA and MHC. Although α-SMA is an early marker of smooth muscle cell development, its expression alone does not provide clear evidence that it is a smooth muscle cell line. MHC was not recognized in any other cell line and was expressed only in contractile smooth muscle cells. Therefore, in order to specify the differentiation of smooth muscle cells, not only the expression of α-SMA but also the expression of MHC was evaluated in the present invention. This finding is consistent with the discovery that early expression of smooth muscle cell markers in stationary cultures and stress culture systems and the need for RA to reach the phenotype of mature smooth muscle cells.

The basic requirements in the development of biological substitutes that can restore, maintain or improve the function of tissues are scaffolds of appropriate cell sources, optimal biochemical environment, physiological environment, and biocompatibility.

The present invention hypothesized the concept of 'smooth muscle cell-elastic nanofiber implants' as an approach for transplantation into the vascular system and heart disease.

This approach involves electrospun polymer nanofiber scaffolds containing smooth muscle cells differentiated from adipose tissue. Nanofiber scaffolds provide an anchorage for interacting with cells and surrounding tissues to respond smoothly to biological and physiological changes such as mechanical stability, structural induction, and extracellular matrix protein recombination.

It is well known that cell function is determined by the substrate to which the cell is attached. Although many reports have reported that stem cells containing human adipose stem cells differentiate into smooth muscle cells on tissue culture plates in differentiation medium, the present inventors need to identify whether human adipose stem cells differentiate into smooth muscle cells on elastic nanofiber scaffolds. there was. Unlike conventionally reported, the present invention shows that differentiation medium is not sufficient for differentiation of human adipose stem cells attached to nanofiber PLCL scaffolds into smooth muscle cells and stress is required for differentiation into smooth muscle cells. Some studies suggest that stress is a potential regulator of smooth muscle cell proliferation and differentiation. The present invention provides evidence that stress is a positive factor for the differentiation and proliferation of human adipose stem cells into smooth muscle cells on nanofiber PLCL scaffolds.

RA, AA, and TGF-β-1 have the most positive effect on human adipose stem cell proliferation, and their role has been reported in human adipose stem cell proliferation. In the present invention, the rate of differentiation of human adipose stem cells into smooth muscle cells was the highest in medium supplemented with RA under stress-stimulated culture conditions. RA is an active form of retinoids involved in embryonic development as well as cell differentiation and proliferation via cell signaling pathways. RA regulates transcription through several of the nuclear receptor superfamily. However, the retinoic acid signaling pathway of mesenchymal stem cells in smooth muscle cell differentiation is still unknown. The present invention provides for a useful tissue engineering approach in the differentiation of human adipose stem cells exposed to pretrans RA in stress-stimulated cultures with elastic biodegradable scaffolds, although the mechanism is not clearly identified.

In summary, the present invention shows that stress affects the growth and phenotype of human adipose stem cells. The application of stress significantly increased the proliferation of adipose stem cells compared to stationary cultures during the addition of the biochemical factor RA, and RA had a positive effect on the differentiation of adipose stem cells compared to the case of stress only.

Biodegradable nanofiber scaffolds manufactured by electrospinning are gaining attention as tissue engineering materials, and high-efficiency differentiation of fat stem cells into smooth muscle cells from nanofibrous scaffolds, which is not a general culture vessel, is a biological nano It can have great potential for substance-cell transplantation treatment.

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Claims (11)

A method of differentiating stem cells into smooth muscle cells while applying strain to the elastic nanofiber scaffold inoculated with the stem cells.
The method of claim 1, wherein the method comprises converting growth factor-β-1 (TGF-β-1), platelet derived growth factor-BB (PDGF-BB), ascorbic acid (AA), retinoic acid (RA) and angiotensin II. A method comprising the medium containing at least one biochemical differentiation factor selected from the group consisting of (Ang II).
The method of claim 2, wherein the biochemical differentiation factor is retinoic acid.
The method of claim 1 wherein said elastic scaffold is comprised of a biodegradable polymer.
The method of claim 4, wherein the biodegradable polymer is a copolymer of lactinic acid and caprolactone (PLCL).
The method of claim 1, wherein the scaffold is coated with at least one surface selected from the group consisting of type 1 collagen, type 4 collagen, fibronectin, and laminin.
7. The method of claim 6, wherein the scaffold is coated on its surface with type 1 collagen.
The method of claim 1, wherein the stem cells are selected from the group consisting of adipose stem cells, mesenchymal stem cells, bone marrow stem cells, umbilical cord blood stem cells, neural stem cells and induced pluripotent stem cells.
A smooth muscle cell-scaffold complex for tissue engineering containing smooth muscle cells differentiated from stem cells by the method of any one of claims 1 to 8 for the treatment of vascular diseases or wounds.
10. The smooth muscle cell-scaffold complex of claim 9 provided in the form of a patch.
The smooth muscle cell-scaffold complex according to claim 10, wherein the vascular disease is a cardiovascular disease or an ischemic disease.

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