Stem cell differentiation promoting components

Abstract

The present invention relates to a composition for promoting the osteogenic differentiation efficiency of adipose-derived stem cells. The present invention has revealed that treating Adipose-Derived Stem Cells (ADSCs) from older donors with reduced differentiation efficiency with FGF2 and / or HGF during the early stages of differentiation can restore damaged paracrine function and maximize differentiation efficiency. Therefore, it can be utilized as an in vitro process technology for promoting stem cell differentiation efficiency before or during adipose-derived stem cell transplantation.

Description

Technical Field

[0001] The present invention relates to a composition for promoting stem cell differentiation, and more particularly to a composition for promoting the osteogenic differentiation efficiency of adipose-derived stem cells.

Background Art

[0002] A cell therapy product is defined as a pharmaceutical product used for therapeutic, diagnostic, and prophylactic purposes through a series of actions such as growing or sorting live autologous, allogeneic, or xenogeneic cells in vitro or otherwise changing the biological properties of cells (more-than-minimal manipulation). Among these, a stem cell therapy product specifically refers to the case of using stem cells, and the current typical application fields are actively being developed in cases where the recovery and regeneration of lost cells, such as in neurological diseases, heart diseases, lung diseases, liver diseases, cancers, etc., are essential but do not occur well naturally.

[0003] Stem cells are cells that can differentiate into various cell types that make up biological tissues. They are a general term for undifferentiated cells in the pre-differentiation stage obtained from the tissues of embryos, fetuses, and adults. Unlike cells that have stopped dividing after differentiation is complete and the differentiation process has progressed in response to differentiation stimuli (environment), stem cells have the characteristic of being able to self-renew by producing cells identical to themselves through cell division and proliferating (expansion). They can also differentiate into other cells in different environments or with different differentiation stimuli, and are characterized by their flexibility in differentiation (plasticity). Stem cells are broadly classified into embryonic stem cells (ES cells), which are totipotent and pluripotent, obtained from embryos and can differentiate into all cell types, and adult stem cells, which are multipotent, obtained from various tissues. The inner cell mass of the blastocyte, an early stage of embryonic development, is the part that will eventually form the fetus. Embryonic stem cells formed from this inner cell mass are, theoretically, pluripotent stem cells capable of differentiating into cells of all tissues that make up an individual. In other words, embryonic stem cells are undifferentiated cells that can proliferate indefinitely and differentiate into all cell types, while adult stem cells are cells that have the ability to differentiate into a wide variety of cells. Adult stem cells can be obtained from various sources such as bone marrow, dental tissue, and peripheral blood, but adipose tissue, in particular, is known as a rich source of stem cells with diverse potential. ADSCs (adipose-derived stem cells), like other adult stem cells, are cells derived from mesenchymal tissue and can differentiate into various types of cells such as adipocytes, fibroblasts, smooth muscle cells, endothelial cells, and adipose progenitor cells, as well as epithelial, cartilage, nerve, fat, and muscle cells. Furthermore, they have a rapid cell proliferation rate, are easily obtained by incidentally extracting large quantities of adipose tissue during liposuction, are easily separated by enzymes, and have been reported to have a low disease incidence after transplantation.

[0004] On the other hand, bone is maintained by a balance between osteoblast formation and osteoclast formation (Alliston T. et al. Interfering with bone remodelling. Nature. 2002;416:686-687.). Maintaining a balance between osteoblasts and osteoclasts is essential for maintaining bone homeostasis. The disruption of the balance between osteoclasts and osteoblasts due to aging occurs when the bone-destroying capacity of osteoclasts exceeds the bone-forming capacity of osteoblasts, leading to a breakdown of homeostasis. This means that suppressing bone resorption by osteoclasts is important for preventing and treating age-related bone diseases (Tanaka, Y., et al., 2005). Excessive osteoclast activity exceeding osteoblast activity causes various bone diseases, characterized by bone mass loss and structural deterioration of the skeleton (Kim N. et al. Osteoclast differentiation independent of the TRANCE-RANK-TRAF6 axis. J Exp Med. 2005;202:589-595.). Such bone diseases include intractable bone diseases such as osteoporosis, non-union fractures, osteonecrosis, osteomalacia, and bone defects. Osteoporosis, also known as osteoporosis or osteoporosis, is a metabolic bone disease characterized by a significant decrease in bone mass compared to a normal person, with the main lesion being a quantitative decrease in bone components. Generally, the pathology of osteoporosis itself is often asymptomatic or mild, but once a fracture occurs, it is generally difficult to treat, and even with osteosynthesis, complete recovery is difficult.

[0005] Conventional treatments for bone diseases include autografting, allografting, and artificial bone grafting. However, these methods have drawbacks, such as the induction of complications like infection and hematoma at the bone harvesting site (autografting), the possibility of disease transmission from the donor (allografting), and the failure to achieve fundamental bone formation (artificial bone grafting). [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide a composition for promoting stem cell differentiation.

[0007] Another object of the present invention is to provide a composition for inducing osteogenic differentiation.

[0008] Furthermore, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone diseases.

[0009] Another objective of the present invention is to provide a stem cell transplantation aid.

[0010] Another objective of the present invention is to provide a method for improving the osteogenic differentiation ability of stem cells.

[0011] Furthermore, an object of the present invention is to provide uses for FGF2 or HGF for use in stem cell differentiation.

[0012] Furthermore, an objective of the present invention is to provide uses for FGF2 or HGF for inducing osteogenic differentiation.

[0013] Furthermore, an object of the present invention is to provide stem cells treated with FGF2 or HGF for the prevention or treatment of bone diseases.

[0014] Furthermore, an object of the present invention is to provide a method for treating bone diseases. [Means for solving the problem]

[0015] To achieve the above objective, the present invention provides a stem cell differentiation promoting composition comprising FGF (fibroblast growth factor) 2 or HGF (hepatocyte growth factor) as an active ingredient.

[0016] Furthermore, the present invention provides a composition for inducing osteogenic differentiation, comprising FGF2 or HGF as an active ingredient.

[0017] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases, comprising FGF2 or HGF as an active ingredient.

[0018] Furthermore, the present invention provides a stem cell transplantation adjuvant comprising FGF2 or HGF.

[0019] Furthermore, the present invention provides a method for improving the osteogenic differentiation ability of stem cells.

[0020] Furthermore, the present invention provides uses for FGF2 or HGF for use in stem cell differentiation.

[0021] Furthermore, the present invention provides applications for FGF2 or HGF for use in inducing osteogenic differentiation.

[0022] Furthermore, the present invention provides stem cells treated with FGF2 or HGF for the prevention or treatment of bone diseases.

[0023] Furthermore, the present invention provides a method for treating bone disease, which includes the step of transplanting FGF2 or HGF into an individual suffering from bone disease.

[0024] Furthermore, the present invention provides a method for treating bone disease, comprising the step of transplanting stem cells treated with FGF2 or HGF into an individual suffering from bone disease. [Effects of the Invention]

[0025] According to the present invention, it has been confirmed that ADSCs derived from elderly donors have a reduced differentiation efficiency due to damage to their growth factor secretion ability, are unable to form / produce bone despite bone formation-inducing stimuli, and exhibit a damaged paracrine function. Thus, it has been clarified that by treating stem cells with FGF2 and / or HGF at the initial stage of differentiation, the activity of the stem cells can be promoted and the differentiation efficiency can be maximized. Therefore, it can be utilized as an in vitro engineering technology for promoting the differentiation efficiency of stem cells before or at the time of adipose stem cell transplantation.

Brief Description of the Drawings

[0026] [Figure 1] It is a figure for confirming the bone formation / paracrine function (potential) according to the age of the individual (donor) from which ADSCs were isolated: Figure 1a: Cell morphology of ADSC-Y and ADSC-E; Figure 1b: Cell doubling time; Figure 1c: Schematic diagram of the experiment for comparative analysis of bone formation of ADSC-Y and ADSC-E; Figure 1d: Alizarin Red S staining images of ADSC-Y and ADSC-E after 20 days of bone formation induction; Figure 1e: Quantitative graph of the Alizarin Red S staining images; Figure 1f: Results of Western blot analysis of bone formation markers 0, 1, 3, and 6 days (D0, D1, D3, and D6) after bone formation induction; Figure 1g: Quantitative graph of the results of Western blot analysis of Runx-1 0, 1, 3, and six days after bone formation induction; Figure 1h: Quantitative graph of the results of Western blot analysis of ALP 0, 1, 3, and 6 days after bone formation induction; Figure 1i: BMP-2 secretion levels of ADSC-Y and ADSC-E analyzed by ELISA; Figure 1j: VEGF secretion levels of ADSC-Y and ADSC-E analyzed by ELISA; Figure 1k: TGF-β1 secretion levels of ADSC-Y and ADSC-E analyzed by ELISA; Figure 1l: HGF secretion levels of ADSC-Y and ADSC-E analyzed by ELISA; and Figure 1m: Results of Western blot analysis of the protein level of FGF-2 in ADSC-Y and ADSC-E and its quantification graph. [Figure 2]This is a figure analyzing the expression patterns of bone formation factors in ADSC-Y and ADSC-E during osteogenic induction: Figure 2a: BMP-2 secretion levels of ADSC-Y and ADSC-E analyzed by ELISA 0, 1, 3, and 6 days after osteogenic induction; Figure 2b: TGF-β1 secretion levels of ADSC-Y and ADSC-E analyzed by ELISA 0, 1, 3, and 6 days after osteogenic induction; Figure 2c: VEGF secretion levels of ADSC-Y and ADSC-E analyzed by ELISA 0, 1, 3, and 6 days after osteogenic induction; Figure 2d: HGF secretion levels of ADSC-Y and ADSC-E analyzed by ELISA 0, 1, 3, and 6 days after osteogenic induction; Figures 2e - 2g: Western blot analysis results and quantification graphs of P-Met and c-Met in ADSC-Y and ADSC-E 0, 1, 3, and 6 days after osteogenic induction; and Figures 2h - 2j: Western blot analysis results and quantification graphs of FGFR2 and FGF2 in ADSC-Y and ADSC-E 0, 1, 3, and 6 days after osteogenic induction; [Figure 3] This is a figure confirming the effect of improving the osteogenic function of ADSC-E by FGF2 and / or HGF during osteogenic induction: Figure 3a: Schematic diagram of the osteogenic induction and FGF2 and / or HGF treatment experiment; and Figures 3b and 3c: Alizarin Red S staining images of ADSC-E under each condition and its quantification graph. [Figure 4] This is a figure confirming the regulatory effect of FGF2 and / or HGF on the expression of early osteogenic markers in ADSC-E during osteogenic induction: Figure 4a: Schematic diagram of the experimental process of treating ADSC-E with FGF2 and / or HGF during osteogenic induction and performing Western blot and ELISA analyses on days 1, 3, and 6; Figures 4b - 4f: Protein expression levels of FGFR2, Runx-2, Osterix, and ALP confirmed by Western blot analysis in ADSC-E; Figure 4g: BMP-2 secretion level confirmed by ELISA; and Figure 4h: VEGF secretion level confirmed by ELISA; and [Figure 5]The figures show the in vivo effect of FGF2 and / or HGF on improving the bone formation ability of ADSCs: Figure 5a: Schematic diagram of an experiment in which ADSC-E primed with FGF2 and / or HGF is transplanted into mice; Figures 5b and 5c: H&E staining results and quantification graphs for transplanted cells and bone complexes; and Figures 5d and 5e: Immunohistochemically stained human osteocalcin and its quantification graphs. [Best Mode for Carrying Out the Invention]

[0027] The present invention will be described in detail below with reference to the attached drawings and examples of its implementation. However, the following examples are presented as illustrations of the present invention, and if it is determined that a specific description of a well-known technology or configuration familiar to those skilled in the art would unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention will not be limited in this respect. The present invention can be modified and applied in various ways within the scope of equivalents described in the claims below and interpreted therefrom.

[0028] Furthermore, the terminology used herein is intended to adequately describe preferred embodiments of the invention and may vary depending on the intent of the user, operator, or the conventions of the art to which the invention pertains. Therefore, the definitions of these terms should be interpreted in light of the context of the entire specification. When, throughout the specification, a part of a specification is said to "include" a component, this means, unless otherwise stated, that it may include other components rather than excluding them.

[0029] All technical terms used in this invention are used in the sense that would be generally understood by a person skilled in the art in the relevant field, unless otherwise defined. While preferred methods or samples are described herein, similar or equivalent methods are also included within the scope of this invention. The contents of all publications cited herein as references are incorporated into this invention.

[0030] Throughout this specification, the percentages used to indicate the concentration of a particular substance refer to (w / w)%, (w / v)%, and (v / v)%, respectively, for solid / solid, unless otherwise specified.

[0031] In one aspect, the present invention relates to a stem cell differentiation promoting composition comprising FGF (fibroblast growth factor) 2 or HGF (hepatocyte growth factor) as an active ingredient.

[0032] In one embodiment, the composition of the present invention may contain both FGF2 and HGF.

[0033] In one embodiment, the composition of the present invention may contain FGF2 at a concentration of 0.5 to 10 ng / mL, HGF at a concentration of 5 to 100 ng / mL, and most preferably contains FGF2 at a concentration of 5 ng / mL and HGF at a concentration of 50 ng / mL.

[0034] In one embodiment, the stem cells may be adult stem cells, which may be derived from at least one of the following: bone marrow, blood, brain, skin, fat, umbilical cord blood, and Wharton's jelly of the umbilical cord, with adipose-derived stem cells (ADSCs) being most preferred.

[0035] In one embodiment, the composition of the present invention can promote the differentiation of stem cells into osteocytes.

[0036] In one embodiment, the composition of the present invention can enhance / improve / promote differentiation into osteoblasts under osteogenic conditions (osteogenesis differentiation medium), and the osteogenic conditions may be obtained by changing from a general culture medium to an osteogenic differentiation medium.

[0037] In one embodiment, the composition of the present invention can promote the differentiation of adipose-derived stem cells (ADSCs) into osteoblasts.

[0038] In one embodiment, the stem cells may be adipose-derived stem cells (ADSC-E) from an elderly donor aged 50-80 years, and adipose-derived stem cells from elderly donors may possess damaged paracrine potential and bone formation (production) function.

[0039] In one embodiment, the composition of the present invention can increase the bone formation function of adipose-derived stem cells (ADSC-E) from an elderly donor.

[0040] In one embodiment, the composition of the present invention can increase the bone formation / production function of adipose-derived stem cells from elderly donors and promote their differentiation into osteoblasts.

[0041] In one specific example, adipose-derived stem cells from older donors showed decreased expression of the growth factors Runx-2 and ALP, decreased secretion of paracrine factors BMP-2, VEGF, TGF-Beta1 and HGF, and decreased expression of C-Met phosphorylation, FGF2, and FGF2R compared to adipose-derived stem cells from younger donors aged 20-29 years (ADSC-Y).

[0042] In one embodiment, the composition of the present invention can increase the expression of an early bone formation marker, which may be FGFR2, Runx-2, Osterix, or ALP.

[0043] In one embodiment, the composition of the present invention can promote the secretion of vascular regeneration and bone formation-related factors, the factors of which may be BMP-2 or VEGF.

[0044] In one embodiment, the composition of the present invention can increase the expression of osteocalcin, a bone formation / production marker.

[0045] In one embodiment, the composition of the present invention may be a culture medium composition.

[0046] In one aspect, the present invention relates to a composition for inducing osteogenic differentiation, comprising FGF2 or HGF as an active ingredient.

[0047] In one embodiment, the composition of the present invention can increase the differentiation-inducing effect during osteocyte differentiation induction.

[0048] In one embodiment, the osteocytes may be osteoblasts.

[0049] In the present invention, the composition containing FGF2 or HGF as an active ingredient can not only enhance the in vivo effects of a cell therapy agent by mixing it with the cell therapy agent for treatment and injecting it into the body, but can also be used as a method for transplanting a cell therapy agent with increased function into the body after treating the stem cells themselves with the composition.

[0050] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of bone disease, comprising FGF2 or HGF as an active ingredient.

[0051] In one embodiment, the bone disease may be one or more selected from the group consisting of arthritis, bone defect disease, osteoporosis, osteopenia, osteolytic metastasis, senile kyphosis, and Paget's disease, and the arthritis may be synovitis, rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, pseudogout, spondyloarthritis (SpA), psoriatic arthritis, ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement, or Still's disease.

[0052] In one embodiment, the composition of the present invention may contain both FGF2 or HGF and stem cells.

[0053] The pharmaceutical composition of the present invention may further contain known bone disease treatment agents in addition to FGF2 and / or HGF as active ingredients, and can be used in combination with other known treatments for the treatment of these diseases.

[0054] In the present invention, the term "prevention" means all actions that suppress or delay the onset, spread, and recurrence of bone disease by administering the pharmaceutical composition according to the present invention, and "treatment" means all actions that improve or beneficially alter the symptoms of bone disease by administering the composition according to the present invention. In the art to which the present invention belongs, a person with ordinary skill will be able to grasp the precise criteria for diseases in which the composition of this application is effective and to determine the degree of improvement, enhancement, and treatment by referring to materials presented by the Korean Medical Association and other sources.

[0055] In this invention, the term "therapeutably effective amount" used in combination with the active ingredient means an amount effective in preventing or treating bone disease, and the therapeutically effective amount of the composition of this invention may vary depending on several factors, such as the method of administration, the site of target, and the patient's condition. Therefore, the dosage for use in humans should be determined to be appropriate, taking both safety and efficiency into consideration. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal experiments. These considerations when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0056] The pharmaceutical compositions of the present invention are administered in pharmaceutically effective amounts. As used in this invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, without causing side effects. The effective dose level can be determined by factors including the patient's health status, the cause and severity of the bone disease, the activity and sensitivity of the drug, the method of administration, the timing of administration, the route of administration and elimination ratio, the duration of treatment, the drugs used in combination or concurrently, and other factors well known in the medical field. The compositions of the present invention can be administered as individual therapeutic agents, in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and in single or multiple doses. Considering all the aforementioned factors, it is important to administer an amount that provides the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0057] The pharmaceutical compositions of the present invention may include carriers, diluents, excipients, or combinations thereof of two or more commonly used in biological formulations. As used in the present invention, "pharmaceutically acceptable" means exhibiting properties that are non-toxic to cells or humans to which the composition is exposed. The carrier is not particularly limited as long as it is suitable for intracellular delivery of the composition, and can be, for example, a mixture of one or more of the compounds listed in Merck Index, 13th ed., Merck & Co. Inc., physiological saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Diluents, dispersants, surfactants, binders, and lubricants may also be added, and the compositions may be formulated in commonly used dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Furthermore, the formulations can be developed in a desirable form according to each disease or component, using appropriate methods in the field or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0058] In one embodiment, the pharmaceutical composition may be one or more dosage forms selected from the group including oral preparations, topical preparations, suppositories, sterile injection solutions, and sprays, with oral or injection dosage forms being more preferred.

[0059] As used in this invention, the term "administration" means providing a given substance to an individual or patient by any suitable method, and can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically as an injectable dosage form) or orally, depending on the desired method. The dosage ranges widely depending on the patient's weight, age, sex, health status, diet, administration time, method of administration, excretion rate, and disease severity. Liquid formulations for oral administration of the compositions of this invention include suspensions, liquid formulations, emulsions, and syrups, and may contain various excipients in addition to commonly used simple diluents such as water and liquid paraffin, such as wetting agents, sweeteners, fragrances, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. The pharmaceutical compositions of this invention can also be administered by any device that allows the active substance to move to target cells. Preferred administration methods and formulations include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and intravenous drip injection. Injectable preparations can be manufactured using aqueous solvents such as physiological saline and Ringer's solution, non-aqueous solvents such as vegetable oil, higher fatty acid esters (e.g., ethyl oleate), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin), and may contain pharmaceutically acceptable carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA), emulsifiers, buffers for pH adjustment, and preservatives to inhibit microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol).

[0060] As used in this invention, the term "individual" refers to all animals, including humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quail, cattle, dogs, mice, rats, rabbits, or guinea pigs, that have developed or are at risk of developing the aforementioned bone disease. By administering the pharmaceutical composition of this invention to an individual, the aforementioned disease can be effectively prevented or treated. The pharmaceutical composition of this invention can be administered in combination with existing therapeutic agents.

[0061] The pharmaceutical composition of the present invention may further contain pharmaceutically acceptable additives, in which case the pharmaceutically acceptable additives can be starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicone dioxide, calcium hydrogen phosphate, lactose, mannitol, candy, acacia gum, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opa-dry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

[0062] In one aspect, the present invention relates to a stem cell transplantation adjuvant comprising FGF2 or HGF.

[0063] In one embodiment, the transplantation adjuvant can be administered simultaneously with or at different times to stem cell transplantation, but it is more preferable to administer them simultaneously or together, as this can enhance / improve or promote the ossification of transplanted stem cells.

[0064] In one aspect, the present invention relates to a method for improving the osteogenic differentiation ability of stem cells, comprising the steps of (a) isolating adult adipose-derived stem cells extracted from adipocytes isolated from an individual; and (b) treating the stem cells with FGF2 or HGF.

[0065] In one embodiment, step (b) may be priming by replacing the differentiation medium with one containing FGF2 or HGF.

[0066] In one example, FGF2 or HGF can be administered within 7 days after differentiation induction, and can be administered for the initial 3 or 6 days.

[0067] In one embodiment, the method described above may be a method for improving the differentiation ability of adipose-derived stem cells into osteoblasts.

[0068] As used herein, the term "priming" refers to the phenomenon in which the reactivity (activity) of stem cells is enhanced in order to promote their therapeutic effect.

[0069] In one aspect, the present invention relates to a method for producing a cell therapy agent with improved bone formation ability, comprising the step of priming adult adipose-derived stem cells isolated from an individual by treating them with FGF2 and / or HGF.

[0070] In one embodiment, the cell therapy agent may be an autologous cell therapy agent.

[0071] In one example, the individual may be an elderly person.

[0072] In one embodiment, the cell therapy agent may be injected back into the patient after priming stem cells isolated from the patient by apheresis outside the body.

[0073] In one aspect, the present invention relates to the use of FGF2 or HGF for use in stem cell differentiation.

[0074] In one aspect, the present invention relates to the use of FGF2 or HGF for inducing osteogenic differentiation.

[0075] In one aspect, the present invention relates to the use of stem cells treated with FGF2 or HGF for the prevention or treatment of bone diseases.

[0076] In one aspect, the present invention relates to a method for treating bone disease, comprising the step of transplanting FGF2 or HGF into an individual suffering from bone disease.

[0077] In one aspect, the present invention relates to a method for treating bone disease, comprising the step of transplanting stem cells treated with FGF2 or HGF into an individual suffering from bone disease. [Modes for carrying out the invention]

[0078] The present invention will be described in more detail by the following examples. However, the following examples are provided to illustrate the content of the present invention and do not limit it thereto.

[0079] Example 1. Isolation and culture of young and old ADSCs.

[0080] To comparatively analyze the cellular activity of young ADSCs (ADSC-Y) and elderly ADSCs (ADSC-E), ADSCs were isolated and cultured from young and elderly donors, respectively. Specifically, elderly adipose tissue collected from donors aged 50-70 years who gave written consent at Kyung Hee University Hospital [Seoul, Korea; (IRB#2016-12-022, donor:8, 2021-01-011, donor:20)] was washed with PBS containing 5% penicillin / streptomycin (Welgene, Daegu, Korea), and the tissue was enzymatically digested with 1% collagenase I at 37°C for 1 hour. The enzymatic reaction was stopped by adding the same volume of FBS, and after centrifugation, the stromal vascular fraction was filtered through a cell strainer (70 μm, Corning, NY, USA) to remove debris. ADSC pellets were then obtained by centrifugation at 1500 rpm for 5 minutes at 4°C. The ADSCs (Adipose-Derived Stem Cells) were resuspended in α-MEM containing 10% FBS, 1% penicillin, and streptomycin. ADSCs isolated from healthy 20-29 year old donors were purchased from ScienCell Research Laboratories (Carlsbad, CA). All ADSCs were cultured in a 5% CO2 incubator at 37°C, with the culture medium changed every other day. Subsequent experiments used ADSCs from passages 3-5. When comparing the cell morphology and doubling time of the isolated young ADSCs (ADSC-Y) and older ADSCs (ADSC-E), little difference in cell morphology was observed (Figure 1a), but a difference was shown in that ADSC-Y proliferated every 30 hours, while ADSC-E proliferated every 50 hours (Figure 1b).

[0081] Example 2. Analysis of osteogenic differentiation by induction of bone formation

[0082] To evaluate the bone formation function of young and older ADSCs, calcium deposition was confirmed by alizarin Red S staining analysis after culturing in bone formation-inducing culture medium for 20 days. Specifically, ADSC-Y and ADSC-E were each placed in 6-well plates at a rate of 5 × 10⁶ 4 After dispensing cells / well, when the cell density reached 80-90%, the culture medium was changed to Stempro osteogenesis differentiation media (Gibco, Grand Island, NY, USA), and the cells were cultured for 20 days to induce osteogenicity. On day 20 of osteogenicity induction, the cells were fixed with 3.7% formaldehyde (Sigma-Aldrich, ST. Louis, MO, USA) and stained with 2% Alizarin red S solution (Sigma-Aldrich, ST. Louis, MO, USA) for 10 minutes to visualize calcium deposition. Subsequently, Alizarin red S was eluted with 10% cetylpyridinium chloride solution (Sigma-Aldrich, ST. Louis, MO, USA), and calcium deposition was quantified by absorbance at 560 nm (Molecular Devices, Sunnyvale, CA, USA).

[0083] The results showed that ADSC-Y could differentiate into osteoblasts under conditions of high calcium deposition, while ADSC-E hardly differentiated into osteoblasts even under the same bone formation induction conditions (Figures 1c-e).

[0084] Example 3. Analysis of bone formation-related growth factor production induced by bone formation.

[0085] The expression levels of the transcription factor modulators of ADSC-Y and ADSC-E were confirmed by Western blot analysis under osteogenic conditions. Specifically, ADSC-Y and ADSC-E samples from Example 2 at 0, 1, 3, and 6 days of osteogenic induction were washed with PBS, then disrupted with 1X lysis buffer (Cell Signaling Technology, Danvers, MA, USA), and centrifuged at 12,000 rpm for 20 minutes at 4°C to collect the supernatant. Protein concentrations were determined from the supernatant by BCA (bicinchoninic acid) analysis (Thermo Fisher, Rockford, IL, USA), and electrophoresis was performed using SDS-PAGE. Subsequently, the samples were transferred to a nitrocellulose membrane, blocked with 5% skim milk, incubated with primary antibodies against Runx-2 (Cell Signaling Technology, Danvers, MA, USA), ALP (alkaline phosphatase) (Abcam, Cambridge, UK), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) (Abcam, Cambridge, UK), and reacted with a secondary antibody (Bio-rad, Hercules, CA, USA) conjugated with anti-IgG HRP (horseradish peroxidase). The blot was developed by adding ECL (Dogen Bio, Seoul, Korea), and chemiluminescence was visualized using an Amersham imager 600 (GE Healthcare, Buckinghamshire, UK). The expression levels of each protein were quantified using the ImageJ program (Version 1.53e, National Institutes of Health, Bethesda, Maryland, USA).

[0086] The results showed that activation of Runx-2, one of the major transcription factor modulators of early osteogenesis, promotes osteogenic cell differentiation, but Runx-2 expression was lower in ADSC-E than in ADSC-Y during the early stages of osteogenesis induction (Figure 1f and g). Furthermore, ALP expression was also higher in ADSC-Y (Figure 1f and h). This confirmed a correlation between the loss of osteogenesis function in ADSC-E and the decreased expression of the aforementioned growth factors.

[0087] Example 4. Comparison of paracrine factor production in young and older ADSCs.

[0088] Since Runx-2 activation is associated with signaling molecules such as TGF-Beta, FGF, and BMP-2, we evaluated the secretion of bone-forming growth factors, including BMP-2, VEGF, TGF-Beta1, and HGF, in ADSC-Y and ADSC-E using ELISA to confirm their paracrine potential. The results showed that BMP-2 and VEGF levels were significantly higher in ADSC-Y than in ADSC-E (Figure 1i and j), and contrary to expectations, TGF-Beta1 production was not affected by age and osteogenic capacity in ADSCs (Figure 1k). Furthermore, there was a significant difference in HGF (hepatocyte growth factor) secretion between ADSC-Y and ADSC-E, suggesting that HGF secretion is deeply related to the bone-forming function of ADSCs. Furthermore, when we examined the levels of FGF-2 (fibroblast growth factor-2), a representative factor that promotes bone formation, we found that it was significantly lower in ADSC-E compared to ADSC-Y (Figure 1m).

[0089] The results above indicate that aging reduces the rate of cell repopulation and differentiation function, which is due to a decrease in paracrine factors.

[0090] Example 5. Analysis of paracrine factor production induced by bone formation

[0091] As mentioned above, ADSCs with impaired osteogenic function showed impaired paracrine activity in response to osteogenic stimuli. Therefore, to confirm the correlation between osteogenic function and paracrine factors, the kinetics of osteogenic paracrine factors were investigated in ADSC-Y and ADSC-E, respectively, during osteogenic induction. Specifically, ADSC-Y and ADSC-E were cultured in osteogenic medium, and conditioned medium was collected at 0, 1, 3, and 6 days after osteogenic induction. The levels of BMP-2, TGF-β1, VEGF, and HGF were analyzed by ELISA.

[0092] As a result, bone formation induction / stimulation increased BMP-2 concentrations in ADSC-E to levels similar to those in ADSC-Y (Figure 2a). Furthermore, TGF-Beta1 levels were maintained in a similar pattern in both ADSC-Y and ADSC-E under bone formation conditions (Figure 2b), and VEGF secretion increased gradually in ADSC-Y, while showing little change for 6 days in ADSC-E (Figure 2c). In addition, HGF levels increased continuously in ADSC-Y after bone formation induction, while they were undetectable in ADSC-E (Figure 2d).

[0093] Example 6. Analysis of the expression pattern of bone morphogenetic factors by induction of bone formation

[0094] Since HGF binds to the receptor c-Met and induces various signaling pathways through autophosphorylation, Western blot analysis was performed using the method described in Example 3 to confirm the expression patterns of the osteogenic factors P-Met and C-Met induced by bone formation, using primary antibodies against C-Met, P-Met, FGF2 (Cell Signaling Technology, Danvers, MA, USA), FGFR2 (fibroblast growth factor receptor 2), and GAPDH (Abcam, Cambridge, UK) in ADSC-Y and ADSC-E, respectively.

[0095] As a result, ADSC-Y, which actively secretes HGF, showed significantly higher levels of C-Met phosphorylation compared to ADSC-E (Figures 2e-g). Furthermore, the expression of FGF2 and FGF2R was shown to be maintained at significantly higher levels in ADSC-Y compared to ADSC-E (Figures 2h-j).

[0096] Considering the paracrine function differences between ADSC-Y and ADSC-E in the early stages of bone formation induction confirmed in the above examples, BMP-2 or TFG-Beta is not expected to be directly associated with the loss of bone formation function in ADSC-E, because the difference between ADSC-E and ADSC-Y is not significant. Therefore, it can be inferred that the lack of HGF, FGF2, or VEGF directly affects the bone formation function of damaged ADSC-E.

[0097] Example 7. Confirmation of FGF2 and HGF's promotion of osteogenic differentiation of ADSCs.

[0098] To confirm whether supplementing ADSC-E with FGF2 and / or HGF can restore osteogenic function, ADSC-E was treated (primed) with FGF2 and / or HGF using the same process as in Figure 3A, and its osteogenic function was confirmed by Alizarin Red S staining. Specifically, to determine the optimal time for stimulating ADSC-E differentiation with FGF2 and / or HGF, the culture medium of ADSC-E was replaced with differentiation medium containing FGF2 (R&D Systems, Minneapolis, MN, USA) (1 or 5 ng / mL), HGF (R&D Systems, Minneapolis, MN, USA) (10 or 50 ng / mL), or FGF2 + HGF (Stempro osteogenesis differentiation media), and osteogenic function was confirmed on days 3 and 6 while osteogenicity was induced for 20 days (Figure 3a). During this 20-day differentiation period, HGF or FGF treatment was administered for the first 3 or 6 days, while the remaining 17 or 14 days were treated with basic osteogenic differentiation medium without FGF / HGF.

[0099] The results showed that 6 days of FGF2 or HGF treatment significantly improved the bone formation function of ADSC-E (Figure 3b). In particular, the effect of FGF-2 was hardly observed in ADSC-Y, but was significantly observed in ADSC-E, indicating the importance of FGF-2 supplementation for bone formation function. Furthermore, when ADSC-E was treated with a combination of FGF2 and HGF, its bone formation function was significantly restored to the same level as that of ADSC-Y (Figure 3). To determine the precise effect of each condition, Alizarin Red S was quantified, and a particularly significant improvement was observed when FGF2 and HGF were treated together compared to when FGF2 or HGF was treated alone (Figure 3c). Based on this, the optimal treatment concentrations of FGF2 and HGF were determined to be 5 ng / mL for FGF2 and 50 ng / mL for HGF, and these were used in subsequent experiments.

[0100] The results described above confirm that supplementation with FGF2 and / or HGF during the first six days of differentiation can promote the osteogenic capacity of ADSC-E.

[0101] Example 8. Confirmation of the mechanism by which FGF2 and HGF improve bone formation.

[0102] 8-1. Confirmation of changes in the expression of early bone formation markers

[0103] To determine which proteins are altered by FGF2 and HGF treatment and which enhance the osteogenic differentiation of ADSC-E cells, ADSC-E cells were treated with both FGF2 and / or HGF during osteogenic induction (untreated group: control group). Changes in the expression of FGFR2, Runx-2, Osterix, and ALP, which are early osteogenic markers in ADSC-E cells, were then examined by Western blot analysis on days 1, 3, and 6 (Figure 4a).

[0104] As a result, under osteogenic conditions, FGFR2 expression increased in a time-dependent manner, and this was particularly pronounced under FGF2 or FGF2+HGF conditions (Figures 4b and c). Runx-2 also showed a time-dependent increase in the untreated group, but FGF2 and / or HGF treatment promoted Runx-2 expression, shifting its peak to day 3, and Runx-2 expression levels were highest with the combined FGF2 and HGF treatment (Figures 4b and d). Furthermore, Osterix and ALP expression were shown to change slightly with FGF2 and / or HGF treatment compared to the control group (Figures 4b, e, and f).

[0105] Based on the aforementioned protein expression profiles, it was predicted that FGF2 and / or HGF would induce ADSC-E cells to enter the immature pre-osteoblast phase when compared to an untreated ADSC-E control group.

[0106] 8-2. Confirmation of changes in secretion of secretory factors

[0107] To determine which proteins are altered by FGF2 and HGF treatment to improve the osteogenic differentiation of ADSC-E cells, ADSC-E cells were treated with both FGF2 and / or HGF during osteogenic induction (untreated group: control group). On days 1, 3, and 6, the secretion levels of BMP-2 and VEGF, which are secretory factors with different mechanisms in ADSC-E and ADSC-Y cells, were evaluated by ELISA.

[0108] As a result, when FGF2 or HGF was treated alone, BMP-2 secretion levels were not significantly different between groups. However, when FGF2 and HGF were treated together, the highest concentration was observed from day 1 and was maintained at a significantly higher level compared to the control group (Figure 4g). Furthermore, VEGF secretion was shown to increase with bone induction, and its concentration was not affected at all by FGF2 or HGF. However, when FGF2 and HGF were combined, VEGF secretion was shown to increase significantly in ADSC-E (Figure 4h).

[0109] Example 9. Confirmation of enhanced ADSC bone formation ability by FGF2 and HGF in vivo.

[0110] To evaluate the osteogenesis capacity of FGF2 and / or HGF-treated ADSCs in vivo, ADSCs were treated with FGF2 and / or HGF under osteogenesis induction, and then stained for osteocalcin, an important bone production marker synthesized by osteoblasts, to confirm their osteogenesis capacity. Specifically, ADSCs were treated with FGF2, HGF, and FGF2+HGF for 3 and 6 days, respectively, under osteogenesis induction, and then stained for 2 × 10⁻⁶ days. 6Each ADSC was mixed with 40 mg of HA / β-TCP (hydroxyapatite / beta-tricalcium phosphate) ceramic powder (Biomatlante, Vigneux-de-Bretagne, France). The ADSC-HA / β-TCP mixture was incubated at 37°C for 2 hours and then subcutaneously implanted into the dorsal region of 6-week-old male Balb / c nude mice (20-22 g) (Figure 5a). After 12 weeks, the implants were collected and fixed with 3.7% formaldehyde. The samples were decalcified with 0.2 M EDTA (pH 7.2-7.4) for 2 weeks and then embedded in paraffin. The paraffin-embedded samples were sectioned to a thickness of 5 μm, deparaffinized and hydrated, and then stained with H&E (hematoxylin and eosin). To detect transplanted human ADSCs, samples were treated with an antibody against human osteocalcin and incubated with a biotin-conjugated secondary antibody. The enzyme-substrate reaction was performed with ABC reagent solution. Stained areas were visualized with Nova RED (Vector Laboratories, Burlingame, CA, USA), and control staining was completed with hematoxylin.

[0111] H&E staining revealed osteoid and neovascularization, demonstrating that the FGF2 and / or HGF-treated group improved bone formation compared to the control group (untreated group) (Figures 5b and c). In particular, cells treated with a combination of FGF2 and HGF showed significantly improved bone formation on both day 3 and day 6 of bone formation induction compared to cells treated with either FGF2 or HGF alone, with the highest bone formation capacity observed in ADSCs treated with a combination of FGF2 and HGF for 6 days (Figures 5b and c). Furthermore, immunohistochemical analysis of human-specific osteocalcin expression revealed a small area of ​​osteocalcin positivity in the control group, which was significantly increased by FGF2 and / or HGF priming (Figures 5d and e). In particular, the group treated with a combination of FGF2 and HGF for 6 days showed the most significant increase in the osteocalcin-stained area (Figures 5d and e), confirming that treatment with a combination of FGF2 and HGF most significantly improves the osteogenic function of ADSC-E and promotes differentiation into osteoblasts in vivo.

[0112] The above examples confirmed that initial priming with a combination of FGF2 and HGF is necessary for improving the bone formation function of ADSC-E both in vitro and in vivo.

Claims

1. A stem cell differentiation promoting composition comprising FGF (fibroblast growth factor) 2 or HGF (hepatocyte growth factor) as an active ingredient.

2. The stem cell differentiation promoting composition according to claim 1, comprising FGF2 and HGF.

3. The stem cell differentiation promoting composition according to claim 1, comprising FGF2 at a concentration of 0.5 to 10 ng / mL.

4. The stem cell differentiation promoting composition according to claim 1, comprising HGF at a concentration of 5 to 100 ng / mL.

5. The stem cell differentiation promoting composition according to claim 1, wherein the stem cells are adult stem cells.

6. The stem cell differentiation promoting composition according to claim 5, wherein the adult stem cells are derived from at least one of bone marrow, blood, brain, skin, fat, umbilical cord blood, and Wharton's jelly of the umbilical cord.

7. The stem cell differentiation promoting composition according to claim 1, which promotes the differentiation of stem cells into osteocytes.

8. The stem cell differentiation promoting composition according to claim 1, which promotes the differentiation of adipose-derived stem cells (ADSCs) into osteoblasts.

9. The stem cell differentiation promoting composition according to claim 1, wherein the stem cells are adipose-derived stem cells from an elderly donor aged 50 to 80 years.

10. A composition for inducing osteogenic differentiation, comprising FGF2 or HGF as an active ingredient.

11. The osteogenic differentiation induction composition according to claim 10, which increases the differentiation induction effect during osteogenic differentiation induction.

12. The osteocyte is an osteoblast, as described in claim 11.

13. A pharmaceutical composition for the prevention or treatment of bone disease, comprising FGF2 or HGF as an active ingredient.

14. The pharmaceutically active composition for the prevention or treatment of a bone disease according to claim 13, wherein the bone disease is at least one selected from the group consisting of arthritis, bone defect disease, osteoporosis, osteopenia, osteolytic metastasis, senile kyphosis, and Paget's disease.

15. A stem cell transplantation adjuvant containing FGF2 or HGF.

16. (a) the step of isolating adult adipose-derived stem cells extracted from adipocytes isolated from an individual; and (b) A method for improving the osteogenic differentiation ability of stem cells, comprising the step of treating stem cells with FGF2 or HGF.

17. The method for improving the osteogenic differentiation ability of stem cells according to claim 16, wherein step (b) is to replace the differentiation medium with one containing FGF2 or HGF.

18. A method for improving the osteogenic differentiation ability of stem cells according to claim 16, which improves the differentiation ability of adipose-derived stem cells into osteoblasts.

19. Uses of FGF2 or HGF for stem cell differentiation.

20. Uses of FGF2 or HGF for inducing osteogenic differentiation.

21. Uses of stem cells treated with FGF2 or HGF for the prevention or treatment of bone diseases.

22. A method for treating bone disease, comprising the step of transplanting FGF2 or HGF into an individual suffering from bone disease.

23. A method for treating bone disease, comprising the step of transplanting stem cells treated with FGF2 or HGF into an individual suffering from bone disease.

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