WO2024053957A1 - Chemically-induced osteogenic cells and use thereof in disease modeling, and scaffold for bone grafting - Google Patents

Abstract

The present invention provides a medium composition for direct differentiation of osteogenic cells and a method for direct differentiation of osteogenic cells, which induce osteogenic cells from somatic cells, the composition comprising, as active ingredients, chemicals, that is, a TGF-β receptor inhibitor (RepSox), a cAMP signaling activator (Forskolin) and an epithelial Na⁺ channel inhibitor (Phenamil). It has been identified that a composition comprising osteogenic cells prepared through the method is effective in treating or preventing osteogenesis imperfecta, which is a prototypical bone disease, and Proteus syndrome, and thus can be effectively used as a composition for preventing or treating bone diseases.

Description

Chemically induced osteogenic cells and their use in disease modeling and scaffolds for bone transplantation

The present invention relates to chemically induced osteogenic cells, a composition for preventing or treating bone diseases containing the osteogenic cells, and a scaffold for bone transplantation with a PCL-based nanofiber structure.

Bone repair following injury or disease requires bone regenerative factors such as osteogenic molecules and progenitor/stem cells. Osteogenic progenitor cells include bone marrow-derived mesenchymal stem cells (MSCs), which are mainly recruited to the site of injury. These cells have an innate regenerative capacity and secrete osteoinductive molecules such as growth factors and hormones to generate bone extracellular matrix (ECM) and promote matrix mineralization. However, in cases of severe bone damage that exceeds a critical size and is accompanied by infection, bone regeneration may be difficult due to severe inflammation and damaged vasculature.

In these cases, transplantation of exogenous bone progenitor cells may improve bone regeneration. MSCs aspirated from patients are a promising source of osteogenic progenitor cells that play excellent roles in osteogenic differentiation, immunomodulation, and angiogenesis activation. However, the use of MSCs for bone treatment has limitations such as tissue morbidity at the cell collection site and limited number of cells. To solve these problems, improvements in cell separation technology and cell reprogramming are needed.

Cells such as fibroblasts reprogrammed from widely available tissues are promising candidates for obtaining large quantities of osteogenic progenitor cells. The use of fibroblasts in cell reprogramming allows fibroblasts to acquire pluripotency through forced expression of transcription factors (becoming induced pluripotent stem cells (iPSCs)) and to further differentiate into a variety of lineage-specific cells, including osteoblasts. It started with the groundbreaking discovery that it was possible. At the same time, fibroblasts can also be directly converted into specific target cell types, such as cardiomyocytes, neurons or neural stem cells, chondrocytes, hepatocytes, hematopoietic cells, etc., without going through the pluripotency stage, by introducing a series of transcription factors. Small molecule compounds approved for clinical use have been used to induce the formation of a variety of cell types, such as iPSCs, neurons, neural stem cells, hepatocytes, cardiomyocytes, and myogenic stem cells.

Osteoblasts were derived from mouse and human fibroblasts using viral vectors. However, due to safety concerns regarding the use of viral vectors, recently, chemicals such as TGF-β receptor and vitamin D3 inhibitors have been used, or the DNA methylation inhibitor 5-aza-2'-deoxycytidine has been used to treat bone morphogenetic proteins. ; BMP) activation or a virus-free medium to generate osteoblasts, such as using the protein insulin-like growth factor binding protein 7 (IGFBP7) combined with osteoblast conditioned medium. approach was used. Osteoblasts generated by this method express an osteoblastic phenotype, mineralize in vitro and promote bone formation in vivo.

Recently, as the fields of biomedical engineering, materials engineering, and surgery have developed greatly, tissue engineering for the purpose of replacing and regenerating lost body tissue has been developed. Tissue engineering is a fusion of life science, engineering, and medicine to understand the correlation between the structure and function of biological tissues and, based on this, to replace or regenerate damaged tissues or organs with normal tissues. The purpose is to maintain, improve or restore body functions through tissue.

The purpose of bone tissue engineering is to induce bone formation and develop actual bone tissue by transplanting engineered cultured osteogenic cells to areas where bone is needed. For such tissue engineering, osteogenic cells, a scaffold on which these cells can attach and survive, and growth factors that promote induced differentiation for bone regeneration are required. If each component affects at the same time and maintains the appropriate time and environment, bone tissue can be formed.

In particular, osteoconductive materials such as scaffolds are important for osteogenic cells to live and form bone. The osteoconductive materials must be similar to the mineralization stage of bone, be biocompatible, and have surface activity and physical support that are closely linked to the surrounding bone. will be provided. These bone conductive materials are diverse, including ceramics, collagen, and biodegradable polymers.

After the scaffold is implanted in a subject, it induces the engraftment of cells necessary for tissue regeneration and initiates the formation of new tissue. It disappears over time and the newly formed tissue must fill the space. Accordingly, the scaffold is preferably biodegradable, does not require surgical removal, does not cause immune rejection, inflammatory response, or long-term fibrous encapsulation, does not undergo shrinkage of the graft volume, and is free from serious complications like prosthetic implants. do.

Therefore, in order to efficiently induce tissue remodeling while naturally disappearing after serving as a physical support for an appropriate period of time, the scaffold must have a certain level of mechanical strength and elasticity along with biodegradability. For this purpose, the most suitable natural or synthetic polymer and Selecting the most appropriate structure is very important.

Against this background, we used a virus-free approach to reprogram fibroblasts to generate osteogenic cells that can further differentiate into osteoblasts. A two-step culture process of induction and maturation was introduced to activate fibroblasts to the osteogenic cell state in the first step and generate more differentiated osteoblasts in the second step. We developed this process by screening potential chemical inducers and culture conditions, such as serum concentration and oxygen concentration, at each step. Additionally, we characterized chemically-induced osteogenic cells (ciOG) in terms of the expression of osteogenic phenotypes and in vivo osteogenesis, and regulated the production of ciOG to take advantage of the cells' in vivo regenerative capacity ( An engineered substrate was used to modulate. Finally, we confirmed whether the ciOG platform can be an effective model of bone disease for testing candidate drugs using patient-derived fibroblasts, and fabricated a scaffold for bone transplantation using the osteogenic cells and PCL-based nanofiber structure to achieve tissue regeneration. , the present invention was completed by confirming that it provides improved physical properties while maintaining the original functions such as wound healing and providing in vivo binding force.

The purpose of the present invention is to provide a medium composition for direct differentiation of osteogenic cells that induces osteogenic cells from somatic cells, comprising a TGF β receptor inhibitor, a cAMP signaling activator, and an epithelial Na ⁺ channel inhibitor as active ingredients.

Another object of the present invention is to provide a direct differentiation method for inducing osteogenic cells from somatic cells by culturing them in a medium containing a TGF β receptor inhibitor, a cAMP signaling activator, and an epithelial Na ⁺ channel inhibitor as active ingredients.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating bone diseases containing osteogenic cells produced by the above method as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving bone disease containing osteogenic cells produced by the above method as an active ingredient.

Another object of the present invention is to use osteogenic cells derived from somatic cells and PCL-based nanofiber structure for bone transplantation by culturing them in a medium containing a TGF ß receptor inhibitor, cAMP signaling activator, and epithelial Na ⁺ channel inhibitor as active ingredients. It provides a scaffold.

The advantages and features of the present invention and methods for achieving them will become clear by referring to the embodiments described in detail below along with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms. The present embodiments are merely provided to ensure that the disclosure of the present invention is complete and to provide a general understanding of the technical field to which the present invention pertains. It is provided to fully inform the skilled person of the scope of the present invention, and the present invention is only defined by the scope of the claims.

The terminology used herein is for describing embodiments and is not intended to limit the invention. As used herein, singular forms also include plural forms, unless specifically stated otherwise in the context. As used in the specification, “comprises” and/or “comprising” does not exclude the presence or addition of one or more other elements in addition to the mentioned elements. Like reference numerals refer to like elements throughout the specification, and “and/or” includes each and every combination of one or more of the referenced elements. Although “first”, “second”, etc. are used to describe various components, these components are of course not limited by these terms. These terms are merely used to distinguish one component from another. Therefore, it goes without saying that the first component mentioned below may also be a second component within the technical spirit of the present invention.

Unless otherwise defined, all terms (including technical and scientific terms) used in this specification may be used with meanings commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not interpreted ideally or excessively unless clearly specifically defined.

The present invention provides a medium composition for direct differentiation of osteogenic cells that induces osteogenic cells from somatic cells, comprising a TGF β receptor inhibitor, a cAMP signaling activator, and an epithelial Na ⁺ channel inhibitor as active ingredients.

The TGF β receptor inhibitor of the present invention is RepSox (1,5-Naphthyridine, 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]), SB431542 (4-[4-(1) ,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), an inhibitor of TGF-β RI, may be selected from the group consisting of ALK4 and ALK7, preferably may be RepSox (1,5-Naphthyridine, 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]), but is not limited thereto.

The cAMP signaling activator of the present invention may be selected from the group consisting of Forskolin, isoproterenol, isopreterenol, NKH 477, isoprotereno (Chemical based), PACAP 1-27, and PACAP 1-38 (peptide based). Not limited.

The epithelial Na ⁺ channel inhibitor of the present invention may be Phenamil.

The type of somatic cell of the present invention is not particularly limited, and any somatic cell can be used. The cells may be cells constituting an adult with limited differentiation and self-renewal abilities. Specifically, the somatic cells may be somatic cells constituting the skin, hair, blood, etc. of an individual. For example, in addition to fetal somatic cells, mature somatic cells or adult somatic cells may be used. Preferably, the somatic cells are fibroblasts, and in the present invention, fibroblasts may include all fibroblasts derived from animals such as humans, mice, horses, sheep, pigs, goats, camels, dogs, and rabbits.

Osteogenic cells of the present invention can be induced under conditions in which FBS (fetal bovine serum) is not added.

In a specific example of the present invention, it was confirmed that the osteogenic cells had significantly higher expression of osteogenic genes than non-induced fibroblasts. The osteogenic gene may be one or more selected from the group consisting of RUNX2 (Runt-related transcription factor 2), BSP (bone sialoprotein), ALP (alkaline phosphatase), and OCN (osteocalcin), but is not limited thereto.

Osteogenic cells of the present invention can exhibit nuclear translocation of RUNX2. In a specific example of the present invention, it was confirmed that the nuclear intensity of RUNX2 and the ratio of nuclear translocation cells in the osteogenic cells were higher than those in fibroblasts.

BMP signaling genes may be upregulated in the osteogenic cells of the present invention. Upregulation of the BMP signaling gene means that the BMP agonist gene is activated and the BMP antagonist gene is suppressed and regulated. The BMP agonist gene may be BMP1-11 or ID1, and the BMP antagonist gene may be GREM1 or GREM2, but is not limited thereto.

IGF signaling genes may be upregulated in the osteogenic cells. The IGF signaling gene may be IGF1, IGF2, IGF1R, IGF2R, or IGFBP2-7, but is not limited thereto.

Additionally, the present invention provides a direct differentiation method for inducing osteogenic cells from somatic cells by culturing them in a medium containing a TGF β receptor inhibitor, a cAMP signaling activator, and an epithelial Na ⁺ channel inhibitor as active ingredients.

In the present invention, culture may be performed for 4 to 12 days, preferably 5 to 10 days, but is not limited thereto.

The somatic cells may be, but are not limited to, fibroblasts.

In addition, the present invention provides a pharmaceutical composition for preventing or treating bone diseases containing osteogenic cells prepared by the above method as an active ingredient.

The bone disease of the present invention may be one or more selected from the group consisting of fracture, osteoporosis, rheumatoid arthritis, periodontitis, Paget's disease, osteomalacia, osteopenia, bone atrophy, osteoarthritis, osteogenesis imperfecta, Proteus syndrome, and avascular femoral necrosis. It is not limited to this.

Prevention in the present invention refers to all actions that suppress or delay bone disease.

Treatment of the present invention means suppressing the occurrence or recurrence of a disease, alleviating symptoms, reducing direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving, alleviating, or improving the prognosis of the disease state.

In the present invention, the content of the composition is not greatly limited depending on the purpose or aspect of use, for example, 0.01 to 99% by weight, preferably 0.5 to 50% by weight, more preferably 1 to 30% by weight, based on the total weight of the composition. It may be %. In addition, the pharmaceutical composition according to the present invention may further include additives such as pharmaceutically acceptable carriers, excipients, or diluents in addition to the active ingredients.

The pharmaceutically acceptable carrier is commonly used in preparation and includes, but is limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc. If necessary, other common additives such as antioxidants and buffers may be added. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. The pharmaceutical composition of the present invention is not particularly limited in formulation, but can be formulated as an injection, inhalation agent, topical skin agent, or oral ingestion agent.

When administered parenterally, the pharmaceutical composition of the present invention may be formulated with a suitable parenteral carrier according to methods known in the art. Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, through the skin, nasal cavity, respiratory tract, intramuscular injection, or intrathoracic injection) according to the desired method. The dosage varies depending on the patient's condition and weight, degree of disease, drug type, administration route and time, but can be appropriately selected by a person skilled in the art.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, a pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and drug dependence of the patient's disease. It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

Additionally, the present invention provides a food composition for preventing or improving bone disease containing osteogenic cells produced by the above method as an active ingredient.

Improvement in the present invention means any action that results in at least a reduction in the severity of the parameters associated with the condition being treated, such as symptoms.

The food composition of the present invention may include a health functional food composition.

In the health functional food composition of the present invention, the active ingredients can be added directly to food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement). Generally, when producing food or beverages, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

Additionally, the present invention provides a method for preventing or treating bone disease, comprising treating damaged hard tissue of a subject other than humans with a composition containing osteogenic cells produced by the above method as an active ingredient.

A subject of the present invention refers to a subject in need of treatment of a disease to which the composition of the present invention can be administered, and more specifically, a human or non-human primate, mouse, dog, cat, horse, and mammals such as cows, and may be mammals other than humans.

Bone diseases that can be prevented or treated by the composition may be caused by an imbalance in the activity of osteoblasts and osteoclasts. Because bone is a living tissue, old bone is constantly destroyed and goes through a remodeling process to create new bone. During this process, osteoclasts destroy old and unnecessary bone tissue to release calcium into the bloodstream to help maintain body functions, and osteoblasts play a role in regenerating destroyed bones. This process continues 24 hours a day, and approximately 10 to 30% of an adult's bones are rebuilt in this way each year. Therefore, the balance between osteoclasts and osteoblasts is very important, and this balance is regulated by various hormones and other body chemical components.

The bone disease may be one or more selected from the group consisting of fracture, osteoporosis, rheumatoid arthritis, periodontitis, Paget's disease, osteomalacia, osteopenia, bone atrophy, osteoarthritis, osteogenesis imperfecta, Proteus syndrome, and avascular femoral osteonecrosis, but is limited thereto. It doesn't work.

In addition, the present invention provides a scaffold for bone transplantation made of osteogenic cells derived from somatic cells and a PCL-based nanofiber structure by culturing in a medium containing a TGF ß receptor inhibitor, a cAMP signaling activator, and an epithelial Na ⁺ channel inhibitor as active ingredients. provides.

PCL (polycaprolactone) of the present invention is a biodegradable polymer and is a useful fibrous matrix due to its relatively low toxicity, controllable degradability, and ease of fabrication into engineering scaffolds due to its porous form.

In the PCL nanofibers of the present invention, Rho signaling genes, actin formation genes, myosin-related genes, and integrin-related genes can be upregulated. The Rho signaling gene may be one or more selected from the group consisting of ARHGDIB, CIT, ARHGAP27, and ARHGEF38, but is not limited thereto. The actin-forming gene may be ACTR3C, the myosin-related gene may be MYO1F or MYH3, and the integrin-related gene may be ITGB2, but are not limited thereto.

The PCL nanofiber topology of the present invention increases the induction of actomyosin contraction, and the actomyosin contraction increases RUNX2 and TAZ nuclear migration, and the RUNX2 and TAZ complex can increase reprogramming and maturation of osteogenic cells.

In one specific embodiment of the present invention, the mechanism by which nanofibers accelerate ciOG reprogramming is as shown in FIG. 6J. That is, i) the nanofiber topology induces more actomyosin contraction due to its unique and longer structure, ii) actomyosin contraction causes more RUNX2 and TAZ nuclear translocation, and iii) the RUNX2/TAZ complex leads to ciOG reprogramming. It was confirmed that it improves efficiency and further maturation.

The scaffold for bone transplantation containing osteogenic cells and PCL nanofibers of the present invention can increase bone induction in vivo .

The scaffold of the present invention is an artificial scaffold or cell carrier, and is one of the important basic elements in the field of tissue regeneration engineering. It may be a structure that serves to provide an environment suitable for cell adhesion, differentiation, and proliferation and differentiation of cells moving from the tissue periphery.

The culture of the present invention can be performed for 5 to 30 days, preferably 7 to 28 days, but is not limited thereto.

The present invention directly induces osteogenic cells from somatic cells containing chemicals, that is, TGF-β receptor inhibitor (Repsox), cAMP signaling activator (Forskolin), and epithelial cell Na ⁺ channel inhibitor (Phenamil) as active ingredients. A differentiation medium composition and a method for direct differentiation of osteogenic cells are provided, and it is confirmed that a composition containing osteogenic cells produced by the above method is effective in treating or preventing osteogenesis imperfecta and Proteus syndrome, which are representative bone diseases, It can be usefully used as a composition for preventing or treating bone diseases, and a scaffold for bone transplantation with a PCL-based nanofiber structure can provide excellent bone tissue regeneration, bone induction, wound healing, and in vivo cohesion.

Figure 1 is a diagram showing the chemical induction of fetal-derived human fibroblasts into osteogenic cells and maturation into osteoblasts, and Figure 1a shows the chemical induction of fetal-derived human dermal fibroblasts (fHDF) into osteogenic cells (ciOG). Figure 1b shows the culture timetable and conditions for 7 after treatment with RepSox (R), CHIR99021 (C), forskolin (F), and tranylcypromine (T) individually or in combination with a total of 16 chemical sets. Figure 1c shows the expression level of RUNX2 on day 7 using RF but replacing the protein BMP2 with other osteogenic chemicals individually or in combination, Figure 1d shows the expression level of RUNX2 on day 7 during chemical induction. Optical images showing changes in cell morphology, Figure 1e is a schematic diagram showing the optimized chemical combination and culture conditions used to produce ciOG, and Figure 1f shows the expression of osteogenic genes in ciOG at day 7 under optimized culture conditions. Figure 1g is a diagram showing an immunostaining image showing the RUNX2 signal in cells, Figure 1h is a diagram showing that induction conditions inhibit cell proliferation in cell numbers on day 7, and Figure 1i shows the culture schedule and conditions. It is a schematic diagram, and Figure 1j is a diagram showing an image of mature ciOG stained with ALP on day 14, and Figure 1k is a diagram showing an image of mature ciOG stained with ARS.

Figure 2 is a diagram showing the production of ciOG from adult-derived human fibroblasts, Figure 2a is a schematic diagram showing culture schedule and conditions, Figure 2b is an optical image showing changes in cell morphology during chemical induction for up to 7 days, and Figure 2c is A diagram showing the activation of the RUNX2 gene by chemical induction for 7 days, Figure 2D is a diagram showing an immunostaining image of RUNX2 on day 7, Figure 2E is a diagram showing the expression of osteogenic genes on day 7, and Figure 2F is a diagram showing Western blot analysis of RUNX2 and OCN in cells that were induced (ciOG) for 7 days and then matured (ciOG(M7p)), Figure 2g is a diagram showing OCN immunostaining images during 7 days of maturation, Figure 2h is a diagram showing ALP and ARS staining images of ciOG (M7p and M14p, respectively), and Figures 2i and 2j are images from different adult human fibroblast sources (oral gingival tissue from a 28-year-old Caucasian and arm skin tissue from a 26-year-old East Asian), respectively. This is a diagram showing the creation of ciOG using .

Figure 3 is a diagram showing bulk RNA and single cell RNA sequencing to confirm ciOG characteristics during reprogramming, and Figure 3a is a global sequence of cell samples (aHDF, ciOG, ciOG (M7p) and human osteoblast (hOB)). A diagram showing distance-based clustering analysis in transcriptional changes, Figure 3b is a diagram showing expression heat maps of fibroblast, osteogenesis, osteoblast/cell, and anti-osteogenesis-specific genes in the global transcriptome profile, and Figure 3c is a diagram showing the expression of ciOG Figure 3D is a diagram showing single cell RNA sequencing, and Figure 3D is a diagram showing the distribution of cells in each cluster of representative markers of cell identification (ID) related to metabolism (Jun), osteogenesis (ALP), and mesenchyme (VIM). Figure 3e shows that only active cells were taken and ALP(+) and VIM(High+) (~29%) were confirmed to be osteogenic, while ALP(-) and VIM(Low+)(~13%) were confirmed to be fibroblasts. It's a degree.

Figure 4 is a diagram showing that the production of ciOG depends on BMP signals, and Figure 4a compares the global transcriptome (24,426 genes) of ciOG and adult human dermal fibroblasts (aHDF), showing that ciOG is differentially expressed by more than 2-fold. It is a diagram showing 1,089 genes, Figure 4b is a diagram showing genes setting expression related to BMP and IGF signaling, and Figure 4c is a diagram showing chemical inhibitors of BMP and IGF signaling (LDN-193189 and BMS-536924, respectively). is a diagram showing the use across various capacities with RFP during ciOG creation.

Figure 5 is a diagram showing that ciOG implanted in vivo promotes bone repair and ectopic mineralization, Figure 5A is a schematic diagram showing ciOG implantation into critical-sized bone defects in immunosuppressed NOD/SCID mice, and Figure 5B is a diagram showing 12 A diagram showing micro-CT analysis of a tissue sample at week 5, Figure 5C is a diagram showing H&E and MT staining images showing new bone formation, Figure 5D is a diagram showing an immunohistochemical staining image for human OCN, and Figure 5E is a schematic diagram showing transplantation of mature ciOG (M7p) into subcutaneous tissue to test ectopic mineralization, Figure 5f is a diagram showing Micro-CT images and quantitative analysis results, and Figure 5g is a diagram showing H&E and MT staining of bone and It showed the same mineralized tissue ('B' area), and immunohistochemical staining for human OCN confirmed that it showed high OCN+ cells mainly at the front of bone formation (area in contact with fibrous tissue).

Figure 6 is a diagram showing that culture matrix nanotopography regulates the production and osteogenic ability of ciOG, Figure 6a is a schematic diagram showing induction and maturation of ciOG in a biomaterial matrix, and Figure 6b is a diagram showing ciOG on day 7 of culture. Figure 6c shows RUNX2 gene expression, Figure 6c shows RUNX2 staining of cells, and Figure 6d shows a strong increase in the expression of osteogenic genes (BSP, ALP and OCN) in ciOG when cells were cultured on nanofiber substrates. Figure 6e is a diagram showing biomineralization of mature ciOG (M14p) in a nanofiber matrix by ARS staining, and Figure 6f is a diagram showing cell morphology on various matrices analyzed by F-actin staining. Figure 6g shows the inhibition test to determine whether actin-related mechanotransduction is involved in ciOG generation in nanofibers, and Figure 6h shows that inhibitor treatment reduces the biomineralization ability of ciOG(M14p) as revealed by ARS staining. Figure 6i is a diagram showing the expression of the main mechanotransduction markers (pMLC and TAZ) of aHDF in nanofibers with and without mechanotransduction inhibitors (Blebbistatin and Y-27632), and Figure 6j shows the nanofibers containing ciOG material. This diagram summarizes the mechanisms that accelerate programming.

Figure 7 is a diagram showing in vivo bone tissue engineering using a PCL nanofiber-ciOG construct, and Figure 7a shows the timeline used for induction and maturation of ciOG in the engineered nanofiber matrix and for repairing critical size defects in the mouse calvarium. Schematic diagram showing the bone tissue engineering approach, Figure 7b shows a histological image (H&E and MT staining) of a sample 12 weeks after implantation, Figure 7c shows a high magnification image showing new bone formation, and Figure 7d Immunohistochemical staining of human OCN, a key late-stage bone matrix protein, showed an intense OCN signal in the newly formed matrix, indicating osteoinduction by nanofiber-supported ciOG.

Figure 8 is a diagram showing the feasibility of the ciOG platform for bone disease modeling and drug testing, Figure 8A is a schematic diagram showing the use of ciOG produced from patient-derived fibroblasts for bone disease modeling and drug discovery, and Figure 8B is A diagram showing the expression of osteogenic genes (RUXN2, OCN, and BSP) and mineralization of mature cells (ciOG(M28p)) by ciOG, and Figures 8C and 8D are diagrams showing the results of drug testing using the ciOG platform. Figure 8e is a diagram confirming the inhibitory role of ARQ092 in AKT signaling confirmed by Western blot analysis.

Hereinafter, the contents of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, and includes modifications of the technical idea equivalent thereto.

Example 1. Experimental materials and methods

1-1. General cell culture

All types of fibroblasts [fHDF (foetal human dermal fibroblasts; PH1060F, Genlantis), aHDFs (adult human fibroblasts; PH10605A, Genlantis) (GM00726, Corielle institute for medical research), HGF (human gingival fibroblasts; PCS-201-018, ATCC) and aHDFs derived from patients with Proteus syndrome (GM12260, Corielle) and osteogenesis imperfecta (GM17602, Corielle)] were grown in growth medium [GM: DMEM-HG, 10% FBS (Corning), 1% Penicillin-Streptomycin (Gibco). ), 1% MEM non-essential amino acids solution (Gibco), 2 mM Glutamax (Gibco), and 0.1 mM 2-mercaptoethanol (Gibco)], and the medium was changed every 2-3 days.

iPSC (WiCell) was maintained without a feeder. That is, DMEM/F-12 (Invitrogen) was mixed with Matrigel (reduced growth factor; corning) at a ratio of 11:1 on ice, applied to a 35 mm dish, and incubated at room temperature for 1 hour. After washing twice with PBS, iPSCs were seeded and cultured.

hMSC (human mesenchymal stem cells; PCS-500-012, ATCC) and hOB (human osteoblasts; C-12720, PromoCell) were grown in MSC basal medium (ATCC®PCS-500-030™) supplemented with MSC growth kit. Each was cultured in osteoblast growth medium (C-27001) supplemented with medium SupplementMix (C-39615).All cells were cultured at 37°C under humidified conditions with 5% CO ₂ .

1-2. Chemical bond screening and derivation of ciOG

HDFs were seeded on day -1 in 24-well plates at a density of 60,000 cells/well and cultured in GM. The medium was incubated with the four chemicals (R, F, C, T) individually or in different combinations in the presence of BMP2 on days 0, 2, and 4. Osteogenic medium containing a low concentration of FBS (DMEM-HG, 2% FBS) , 1% P/S, 50 μg/ml ascorbic acid, 10 mM β-glycerolphosphate, and 10 nM dexamethasone).

On day 7, RUNX2 gene expression was assessed by qRT-PCR, and the RF combination showing the highest level was selected. Next, various small molecules known as osteogenic activators were evaluated to replace BMP2 by determining RUNX2 gene levels in the presence of RF and BMP2. Four chemical (P, Sim, FK, CSA) treatment groups expressed higher RUNX2 gene levels than BMP2, and different combinations of these were RF tested in the absence of BMP2. In conclusion, the RFP combination was selected, and the full names and working concentrations of the chemicals used are listed in Table 1.

[Table 1]

1-3. Induction medium composition and cycle optimization

such as serum concentration, oxygen concentration (2/10% FBS, 2/10% serum replacer, 10% FBS under hypoxic conditions - (2%)) and induction period (0, 2, 4, 7, 14 and 21 days). Culture conditions were screened with defined chemical factors (RFP) to optimize them.

1-4. Quantitative RT-PCR

Total RNA was isolated using Ribospin ^TM (304-150, Geneall), and cDNA was isolated using Accupower RT premix (K-2043, Bioneer) or iScript cDNA Synthesis Kit (1708891, Bio-Rad) according to the manufacturer's protocol. synthesized. qRT-PCR was performed with cDNA mixed with 2X SensiMix SYBR Hi-ROX Mastermix (QT-605-05, Bioline) by the StepOnePlus real-time PCR system (Applied Biosystems).

Gene expression levels were analyzed and normalized to GADPH for each sample. 2- The fold change in gene expression was calculated using ^{the △△} CT method. Primers were purchased from Bioneer or Integrated DNA Technologies, Inc., and each primer sequence is listed in Table 2.

[Table 2]

1-5. Osteogenic maturation of ciOG

ciOG was matured in conventional osteogenic medium (with 10% FBS) with or without 10 μM Phenamil. The medium was changed every 3 days.

1-6. Alizarin red S and alkaline phosphatase staining.

To confirm biomineralization, cultured cells at each time point were washed with PBS and fixed with 4% PFA for 10 minutes at room temperature. The fixed cells were washed with deionized water and then stained with 40 mM Alizarin red S (A5533, Sigma Aldrich) solution at pH 4.2 for 30 minutes. Afterwards, the samples were washed three times with deionized water and stained images were obtained using an Epson Perfection V300 photo scanner.

For quantification, the dye was dissolved in 10% cetylpyridinium chlofide (Sigma-Aldrich), and the absorbance was measured at 562 nm using a spectrophotometer (Varioskan LUX, Thermo Fisher Scientific, MA, USA) (n=3). For alkaline phosphatase staining, FAST BCIP/NBT (B5655, Sigma) dissolved in 10 ml deionized water was applied to fixed cells and incubated at 37°C for 1 hour. Stained images were obtained using a fluorescence microscope (IX71, Olympus Australia, Mt. Waverley, Australia).

1-7. Colony forming unit assay (CFU assay)

1,000 cells of aHDF and ciOG were each seeded in a 100 mm culture dish and cultured in DMEM supplemented with 10% FBS for 2 weeks. On day 14, the medium was changed every 3 days, and the cells were fixed with a 1:1 acetone/methanol fixative, stained with 0.5% crystal violet solution in methanol for 30 minutes, and then washed with DIW. Plates were scanned with an Epson Perfection V300 photo scanner and colonies were counted (n=3).

1-8. Cartilage formation and alcian blue staining

Normal 2D monolayer cultures for chondrogenesis, aHDF and ciOG were seeded at a density of 3.16 x 10 ⁴ /cm ² in 24-well plates. The next day, the medium was replaced with chondrogenic medium (DMEM-HG containing 1% ITS, 1% PS, 50 μg/ml ascorbic acid, and 10 ng/ml TGF-β3), and cultured for 14 days, followed by 3 days. Recharged every time. Micromass culture method was used for 3D culture. That is, 10 μl of ciOG was seeded at a density of 8x10 ⁶ cells/ml and cultured in GM (DMEM-LG containing 10% FBS and 1% PS) in a 24-well plate.

After culturing in GM for 4 days, the medium was replaced with chondrogenic medium and renewed every 3 days. After 14 days, the 2D monolayer and micromass pellet were washed with PBS, fixed with 4% PFA for 10 minutes, and then washed with DIW. A 1% alcian blue solution prepared in 3% acetic acid at pH 2.5 was applied for 30 minutes. After rinsing with DIW, the plate was scanned and images were taken under a microscope.

1-9. Lipogenesis and oil red O staining

aHDF and ciOG were seeded at a density ^{of 2.1} were exposed to 10% StemXVivo Adipogenic Supplement (CCM011, R&D Systems Inc.) and 1% PS), and the medium was changed once every 3 days. On day 14, cells were fixed with 4% PFA for 10 min, rinsed with DIW, and maintained with 60% isopropanol for 5 min.

Subsequently, the Oil Red O working solution was newly prepared by diluting the stock solution (0.3% oil red O in isopropanol) with DIW at a ratio of 3:2 and then filtering. Plates were incubated with Oil Red O working solution for 10 min and rinsed with DW. Images of lipid droplets were taken under a microscope. For quantification, the stained plate was desalted by shaking with isopropanol, and then the supernatant was measured for absorbance at 500 nm.

1-10. Inhibition assay

For RUNX2, ICC was performed 7 days after treatment of Y-27632 and Blebbistatin in ciOG on nanofibers, respectively, when the medium was changed (every 2-3 days).

1-11. Immunostaining

Cultured cells were washed with PBS and fixed with 4% PFA for 10 minutes. PFA was removed by washing three times with PBS. Permeability was treated with 0.5% Triton Non-specific binding was blocked by adding 1% bovine serum albumin (BSA, Solmate) in PBS and removed after 1 hour. Primary antibodies [RUNX2 (1: 50, ab76956, abcam), OCT4 (1 μg/ml, ab19857, abcam), and OCN (osteocalcin; 20 μg/ml, MAB1419, R&D systems)] diluted with 1% BSA were applied. and maintained at 4°C overnight.

The next day, the primary antibody was removed, washed three times with PBS, and secondary antibody [Fluorescein (FITC) AffiniPure Donkey Anti-Mouse IgG (H+L) (715-095-150, Jackson ImmunoResearch Laboratories) or Anti-Rabbit IgG was added. (H+L) (711-095-152, Jackson)] was applied, washed with PBS, and incubated at room temperature for 1 hour.

After three washes, nuclei and actin filaments were visualized with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, D1306, ThermoFisher Scientific) and Alexa Fluor™ 546 Phalloidin (A22283, Invitrogen), respectively. Images were obtained by digital microscopy (CLS-01-00026, Logos Biosystems, Inc., Gyeonggi-do, Korea) or fluorescence microscopy. Quantification was performed using Image J.

Analysis of cell number per frame, cell circularity, RUNX2 nuclear intensity, and RUNX2 nuclear translocation cells were performed using Image J. The number of cells was counted in DAPI-stained images. Cell circularity was analyzed by cell boundaries identified in F-actin staining images. RUNX2 nuclear intensity was measured by accumulating RUNX2-stained images, which were normalized to the control level. The number of cells with RUNX2 nuclear translocation per frame was also normalized to the total number of cells in the frame.

1-12. Using the ciOG platform for drug testing in bone diseases

ARQ 092 (21388, Cayman chemical, Ann Arbor, MI, USA) was treated at a concentration of 125 or 250 nM after medium replacement during induction or post-induction maturation. Cells were fixed at day 14 (M7p) for ARS staining and harvested at days 7 (ciOG) and 14 (M7p) for Western blot analysis.

1-13. Western blot

Cells were lysed for 30 min on ice with RIPA buffer (EBA-1149, ElpisBio) containing phosphatase and protease inhibitors (78441, Thermo) and removed by centrifugation at 12,000 g for 30 min at 4°C. .

Total protein concentration of cell lysates was measured by Pierce™ BCA Protein Assay Kit (23225, Thermo). 10 μg of each sample was mixed with 5X SDS loading buffer and denatured at 95°C for 5 minutes. The reduced samples were loaded and run on 10% or 20% SDS-PAGE, separated, and transferred to NC membrane (Bio Rad).

Afterwards, the membrane was blocked with 5% BSA in TBS-T (blocking buffer) for 1 hour at room temperature, and 1:2000 diluted pan-AKT (#2920, Signaling Technologies), pAKT (S473) (# 4060, Signaling Technologies), pPRAS40 (#2997, Signaling Technologies) diluted 1:1000, RUNX2 (ab-76956) diluted 1:250, OCN (PA5-11849, Invitrogen) diluted 1:2000 and diluted 1:1000. The cells were incubated in blocking buffer overnight at 4°C with an antibody specific for beta-actin (SC-47778, Santa Cruz Biotechnology, Santa Cruz, CA).

The membrane was then rinsed with TBS-T, and then the secondary antibody in TBS-T was applied for 1 hour. The secondary antibody mouse-HRP (Santa Cruz Biotech.) was used at a dilution of 1:10,000 and incubated at room temperature for 1 hour. After washing with TBST, protein signals were visualized using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (34579, Thermo Fisher scientific) and captured by the iBright 1500 imaging system (Thermo Fisher Scientific). Densitometric analysis of Western blots was performed using Image J.

1-14. RNA sequencing

For total mRNA bulk sequencing, total RNA was isolated from cells of three groups [aHDF, ciOG, ciOG(M7p), and hOB] using Ribospin ^™ according to the manufacturer's instructions. Total mRNA sequencing was commissioned commercially (E-biogen. Inc., Seoul, South Korea). The obtained raw data were analyzed using ExDEGA software (v1.6.7, Ebiogen), and significantly expressed genes with fold change greater than 2 were selected.

62 genes were selected and plotted in a heatmap using ExDEGA GraphicPlus (v2.0, E-biogen, Seoul, Korea). Gene classification was based on a search performed in DAVID (david.abcc.ncifcrf.gov (accessed December 16, 2020)). Library preparation, sequencing, and quality control analysis were performed.

Gene profiling was analyzed from the limited amount of RNA available from matrix cultured cells (i.e. RNA from ciOG and ciOG(M7p) cultured with PCL films and nanofibers). Library construction was performed using the QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria), and QuantSeq 3' mRNA-Seq reads were aligned using Bowtie2.

To determine transcriptome changes at the single-cell level, single-cell RNA sequencing was performed with ciOG (5 × 10 ⁵ /mL), 10x Loupe Browser (10X Genomics, Pleasanton, CA, USA) and ExSCEA (Ebiogen Inc. , Korea) was used to perform clustering analysis with UMAP for data mining and graphic visualization. That is, library construction was performed using 10X Chromium Single Cell 3' Reagent Kits v3.1 (10X Genomics), and samples were sequenced by Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) according to the protocol. (sequencing) was done.

1-15. laboratory animals

Immunodeficient mice (NOD-SCID mice, male, 8 weeks old) were obtained from KOATECH (Daegu, Korea). Animal care and experimental protocols were approved by the Animal Care and Use Committee of Dankook University, Korea (Approval #17-011). Animals were acclimatized for 5–8 days before surgery, and each mouse was housed in a cage in a temperature- and humidity-controlled environment and exposed to a 12-h light/dark cycle with ad libitum access to water and food. General anesthesia was performed by intramuscular injection of a mixture of xylazine (10 mg/kg) and ketamine (80 mg/kg) before transplantation.

1-16. Ectopic bone formation study

To evaluate the ability of ciOG on mineral formation, an ectopic bone formation assay was performed. ciOG(M7p) or aHDF were trypsinized and resuspended in growth factor-reduced Matrigel (354230, Corning) at a density of 2 x 10 ⁶ /100 μl.

After incubation for 1 hour for gelation, the encapsulated cells were transplanted into a subcutaneous pocket on the back of anesthetized mice (n=5). Nine weeks after transplantation, samples with surrounding tissue were collected under CO ₂ inhalation euthanasia conditions.

1-17. mouse bone marrow model

The following two sets of experiments were performed. First, ciOG was prepared in DMEM and mixed with growth factor-reduced Matrigel at a ratio of 1:2, and the density was 1 x 10 ⁶ /6 μl (n=5). Second, aHDF and ciOG(M7p) were cultured on PCL nanofibers with a diameter of 4 mm (thus, approximately 2.2 x 10 ⁴ cells per nanofiber sample) (n=5). For comparative study, ciOG (M7p) in DMEM of Matrigel with reduced growth factors was used at a ratio of 1:2 (2.2 x 10 ⁴ /6 μl) (n=5).

After shaving the skull lesion of an anesthetized mouse, the surgical area was rubbed with iodine and ethanol, and a linear skin incision was made. A full-thickness flap was retracted, and the calvarial bone was exposed. Two 4 mm diameter cranial bone defects were created on each side of the parietal bone under cooled conditions with sterile saline using a dental handpiece and a 4 mm diameter trephine drill (South Korea).

After implanting the sample, the periosteum was sutured with absorbable sutures (4-0 Vicryl®Ethicon, Germany), and the external wound was fixed with non-absorbable sutures (4-0 Prolene, Ethicon, Germany). Animals were monitored daily for possible clinical signs related to infection, inflammation and any adverse events. At 12 weeks after surgery, the animals were euthanized by CO ₂ inhalation, and tissue sections of the skull bone surrounding the defect were collected and fixed in 10% neutral buffered formalin for 24 hours at room temperature.

1-18. Histology and immunohistochemistry

All samples were fixed in 10% neutral buffered formalin and decalcified before paraffin embedding. After decalcification, routine paraffin embedding was selected to identify osteogenic proteins by immunohistochemistry with routine dye staining.

The tissue was dehydrated and embedded in paraffin. The paraffin-embedded sample was cut into 5 μm pieces, deparaffinized with xylene, and then rehydrated. PCL nanofibers were almost completely degraded during the xylene process. For histological analysis, tissue slides were stained with hematoxylin and eosin (H&E) and Masson's trichrome (MT) and visualized under a light microscope.

Immunohistochemical tests were performed to confirm whether transplanted ciOG or ciOG(M7p) is involved in bone regeneration. Blocking solution (5% BSA, 5% normal donkey serum, 0.2% Triton , MAB1419, R&D Systems) and cultured overnight at 4°C.

After washing with PBS, secondary antibody (goat anti-mouse, 1:200, Jackson) was applied at room temperature for 1 hour, and then DAPI solution was added. Tissue slides were covered with Aqueous Mounting Medium (F4680, Sigma Aldrich) containing Fluoromount and visualized under a digital microscope (CELENA® Logos Biosystems, Inc.).

1-19. statistical analysis

Statistical comparisons were performed with GraphPad Prism 8.0 software using the tests (Student t-test, one-way ANOVA, or two-way ANOVA) described in the figure captions. The sample number for each test is also listed in the figure caption. P values of less than 0.05, 0.01, 0.001, or 0.0001 were considered statistically significant.

Experimental Example 1. Chemical induction of human fibroblasts into osteogenic cells and maturation into osteoblasts

To generate ciOG, we started from fetal human dermal fibroblasts (fHDF). A series of compounds were screened to determine which chemical cocktail induces the formation of ciOG by reprogramming fibroblasts into pluripotent cells or other cell types: TGF-β receptor inhibitor RepSox (R); GSK3 inhibitor CHIR99021 (C), adenylyl cyclase activator forskolin (F), and lysine-specific demethylase 1 inhibitor tranylcypromine (T).

These chemicals are 50 μg/mL ascorbic acid, 10 ^-8 M dexamethasone, and 0.01 M β-, supplemented with 2% fetal bovine serum (FBS) and the osteogenic growth factor bone morphogenetic protein 2 (BMP2), which plays an important role in bone development. Conventional osteogenic medium (OM) containing glycerophosphate was used individually and in different combinations to treat fHDFs on days 0, 2, and 4 for 7 days (Figure 1a).

Only 2% FBS was used to inhibit proliferation of fibroblasts during chemical induction. After 7 days, qPCR was performed to evaluate the expression of Runt-related transcription factor 2 (RUNX2), an osteogenic lineage-specific marker essential for activating genes required for osteoblast differentiation. Among 15 combinations of R, C, F and T with BMP2, R + F and R + F + T showed the highest expression of RUNX2, comparable to the expression of human mesenchymal stem cells (hMSCs) (Figure 1b) .

Eighteen osteogenic chemical candidates were selected to chemically replace the protein BMP2 (Table 1). Each of these 18 chemicals was used in combination with R and F to treat fHDF, and RUNX2 expression was measured. Phenamil, simvastatin, FK 506, and cyclosporin A, which have been shown to promote osteogenesis through BMP/Smad and NFATc1/Fra-2 signaling, induced much higher levels of RUNX2 gene expression than BMP2 with R and F (Figure 1c). These four osteogenic chemicals were then tested individually and in combination with R and F in a total of 15 sets, and phenamil in combination with R and F (RFP) was identified as the most effective treatment. ‘RF’ has been identified as a chemical substitute for SOX2 and OCT4 and consequently enhances stemness in the early stages of reprogramming, whereas ‘P’ downregulates Smurf1, an antagonist of SMAD, thereby activating BMPs, enhancing BMP signaling. Jada. As a result, the SMAD signal, which is an upstream type of BMP signal, is increased.

Therefore, this set of three chemicals was selected as an optimized chemical cocktail to induce fHDFs to become osteogenic cells that highly express RUNX2. During osteogenic reprogramming, dramatic morphological changes, including reduced cell size (a well-known phenomenon in cell reprogramming) and a change in cell shape to cuboids or polygons, a phenomenon observed when fibroblasts differentiate from MSCs to osteoblasts, are observed in optical fibers. was observed as an image (Figure 1d). After phalloidin and DAPI staining, the cell morphology of ciOG maintained a fibroblast morphology, but actin was less developed (Figure 1g).

Next, culture conditions were optimized in the induction phase. As a result of comparing 2% FBS under the original conditions of normoxia (21% oxygen) with other combinations of 2% and 10% FBS and 2% and 21% oxygen, it was confirmed that the original conditions resulted in the highest RUNX2 expression. Based on RUNX2 and osteocalcin (OCN) gene expression, a 7-day RFP treatment period was also shown to be superior to other periods up to 21 days. ciOG obtained using early passage cells (passage #3) showed higher osteogenic gene expression than ciOG from late passage cells (passage #8), indicating that the efficiency of RFP-mediated cell reprogramming is dependent on cell passage number. suggests.

Osteogenic cells were induced from human fetal fibroblasts by treating the fibroblasts with a combination of RepSox, forskolin, and phenamil in osteogenic medium containing 2% FBS under normoxic conditions (Figure 1e). ciOG was further analyzed in terms of the expression of other osteogenic genes, including osterix, bone sialoprotein (BSP), alkaline phosphatase (ALP) and osteocalcin (OCN). Significantly greater expression of these genes was observed in ciOG than in uninduced fibroblasts (Figure 1f). To confirm whether RUNX2 ⁺ cells have osteogenic function, RUNX2 was immunostained and its location was analyzed. Nuclear translocation of RUNX2 is an osteogenic function. While 100% of ciOGs showed nuclear translocation of RUNX2, only 5–10% of fHDFs showed that the intensity of RUNX2 in the nucleus of ciOGs was approximately three times that of fHDFs (Figure 1g). Considering the nuclear translocation of RUNX2, which represents functional osteogenic cells, the induction efficiency was almost 100% ( Fig. 1H ). As observed in the change in cell number over 7 days, FBS at a level of 2% effectively inhibited the proliferation of uninduced fibroblasts.

Then, ciOGs were matured into osteoblasts in osteogenic medium containing 10% FBS in the presence or absence of phenamil (Figure 1i). After 14 and 28 days of maturation, alkaline phosphatase (ALP) activity and biomineralization (calcium deposit formation) were assessed by ALP and Alizarin Red S (ARS) staining, respectively (Figures 1j and 1k). Maturation of ciOG with phenamil for 14 and 28 days (coded as ‘ciOG(M14p)’ and ‘ciOG(M28p)’, respectively) human osteoblasts matured for 14 and 28 days in osteogenic medium without phenamil. (human osteoblast; hOB) ('hOB(M14)' and 'hOB(M28)') and human mesenchymal stem cells (hMSC) ('hMSC(M14)' and 'hMSC(M28)') showed significant levels of ALP activity and calcium deposition, much higher than those observed in .

However, control fHDF showed very weak ALP expression even in osteogenic culture using phenamil ('fHDF(M14p)'). Therefore, phenmamil appears to promote osteogenic differentiation of ciOG during maturation. The initial induction phase for 7 days with RFP in 2% FBS-OM is important to activate fibroblasts to acquire stemness and osteogenic potential, whereas the subsequent maturation phase for 7–28 days with P (without RF) only 10 % FBS-OM is required to obtain osteoblast lineage. Continuous induction with RFP for 28 days did not result in ciOG mineralization, suggesting that overinduction of cells may inhibit their maturational conversion into lineage-specific cells.

Next, we attempted to generate ciOG from adult human fibroblasts. A culture platform optimized for fetal human fibroblasts was applied to adult cells (Figure 2A). Adult human dermal fibroblasts (aHDF) were derived from facial skin tissue of a 58-year-old Caucasian person. During chemical induction for up to 7 days, cell morphology was significantly altered, similar to fetal cells (Figure 2b). Expression of the RUNX2 gene was significantly higher in the generated ciOG than in the original fibroblasts (Figure 2c).

RUNX2 immunostaining and semiquantification revealed a much higher nuclear signal in ciOG than in aHDF, and the RUNX2 nuclear intensity and proportion of nuclear translocation cells in ciOG were almost twice that of native fibroblasts (Figure 2D). Expression of other osteogenic genes, including ALP, BSP, and OCN, was also greater in ciOG than aHDF at day 7 (Figure 2e).

Additionally, marker expression was further investigated at the protein level. OCN was chosen because it is a marker of mature osteogenesis/osteoblasts. Western blot analysis showed a clear band indicating OCN expression in mature cells (ciOG(M7p)) (Figure 2f). Increased OCN expression decreased RUNX2 expression. OCN immunostaining showed mostly OCN ⁺ cells at day 7 of maturation; However, the fraction of OCN ⁺ cells decreased at longer maturation periods (14 days) when mineralization became prominent (Figure 2g). Mature cells (ciOG(14Mp)) secreted significant levels of ALP and minerals (Figure 2h). Production of ciOG from different adult human fibroblast sources in oral gingival tissue of a 28-year-old Caucasian and arm skin tissue of a 26-year-old East Asian was driven by significant upregulation of RUNX2 and osteogenic genes ALP, BSP, and OCN and levels of mineralization. confirmed (Figures 2i and 2j).

Experimental Example 2. Bulk and single cell RNA sequencing to identify ciOG characteristics and signaling pathways during reprogramming

Changes in overall transcription were confirmed during the generation and maturation of ciOG. Hierarchical clustering analysis showed that the transcriptome profile of ciOG was similar to that of human osteoblasts (hOB) during osteogenic maturation (Figure 3A). The expression levels of fibroblast, osteogenesis, osteoblast/cell, and anti-osteogenesis-related genes were classified. In particular, the expression pattern of ciOG after maturation (ciOG(M7p)) was similar to that of hOB (Figure 3b). Mature ciOG(M7p) showed a higher correlation with hOB (-0.04) than with aHDF (-0.49). Two-dimensional principal component analysis (PCA) of cell types according to differentially expressed genes showed that native fibroblasts (aHDF, ‘no.1’), chemically induced intermediates (ciOG, ‘no.2’) and hOBs ( 'no.3') was distinguished. Accordingly, ciOG(M7p) was similar to hOB during maturation.

Afterwards, single-cell RNA sequencing (Chromium single-cell RNA-seq, 10x Genomics) was performed. After rigorous quality control and normalization analysis, 5,901 single cells were isolated and analyzed. Using clustering analysis and the Uniform Manifold Approximation and Projection (UMAP) method, we identified three separate cell clusters in the ciOG while preserving global distances (Fig. 3c). To analyze the cell identity of each cluster, metabolic and cell type markers were used to classify cell activity and type (osteoblasts, fibroblasts, and hematopoietic stem cells).

Cellular reprogramming often leads to low gene expression clusters with a low number of unique molecular identifiers due to reduced metabolic activity. General cellular metabolic markers such as Jun, GAPDH and RPS8 and other cell type specific markers were less abundant in cluster 3, representing an inactive cell cluster (~39% of cells) during ciOG reprogramming (Figure 3C).

Based on the expression levels of osteogenic and mesenchymal genes (Figure 3D), osteogenic and fibroblasts were distinctly clustered in clusters 1 (~49%) and 2 (~12%), respectively. Both clusters were negative for the hematopoietic stem cell marker CD34, suggesting that the induction conditions adopted (RFP in OM with 2% FBS) may not allow aHDFs to cross the mesenchymal barrier.

Similar to human MSCs, ciOGs did not express the pluripotency marker Oct4, showing that aHDFs were partially converted to ciOGs, resolving the tumorigenicity associated with the presence of Oct4 ⁺ pluripotent cells. Single cell analysis of ciOG further revealed a set of highly expressed genes in cluster 1 (OLFM2, TMEM119, TNFRSF11B, TGF-β1, CDH11, SPARC, COL1A1) involved in osteogenic processes or osteoblast signaling and function. Considering only active cells (n=3,583), ~29% of cells were ALP ⁺ (an osteogenic marker) and VIM ^high+ (a marker abundant in pluripotent cells of mesenchymal origin), whereas ~13% of cells were ALP ^- and VIM ^low+ ) was (Figure 3e). Projecting single cell RNA expression into t-SNE space and trajectory analysis allows us to visualize cell fate transitions during osteogenic induction, with cells moving from cluster 2 (ALP ^- /VIM ^low+ ) to cluster 1 (ALP ⁺ /VIM ^high+). ) or Cluster 3 (inactive), indicating that it has passed a separate step.

Next, we sought to identify the signal transduction pathway involved in ciOG production. For this purpose, the global transcriptomes of ciOG and aHDF were compared. Among the 24,426 genes analyzed, 1,089 genes were differentially expressed more than two-fold between ciOG and aHDF, with 501 genes upregulated and 588 genes downregulated in ciOG (Figure 4A).

When differential expression of ciOG was functionally annotated with Gene Ontology (GO), the top 20 GO categories indicated osteogenic function with loss of fibroblast growth behavior. In GO analysis of genes upregulated in ciOG, development of the skeletal system was the category with the lowest P -value. Signaling proteins in this category included BMP, IGF, and TGF-β.

Therefore, this signaling pathway and the WNT signaling pathway, another osteogenic pathway, were analyzed. Most genes related to BMP and IGF signaling were highly upregulated in ciOG (Figure 4b). BMP agonist genes (BMP1-11 and ID1) were globally activated, whereas BMP antagonist genes GREM1 and 2 were repressed in ciOG. In IGF signaling, IGF1 and 2, their receptors (IGF1R and IGF2R) and their stabilizing proteins (IGFBP2-7) were highly expressed. In contrast, changes in TGF-β and WNT signaling genes were variable.

To confirm the role of BMP and IGF signaling pathways, chemical inhibitors of each signaling pathway (LDN-193189 and BMS-536924, respectively) were used together with RFP. Alleviation of RUNX2, ALP and OCN gene expression was detected only when LDN193189 was used in combination with RFP, and the expression of these osteogenic genes gradually decreased in a dose-dependent manner, indicating the involvement of the BMP signaling pathway in ciOG generation (Figure 4c). The use of the BMP activator phenamil suggests that BMP signaling is involved in the ciOG reprogramming process.

Experimental Example 3. In vivo implanted ciOG for bone repair and ectopic mineralization

The in vivo response of ciOG generated in an anisotropic bone repair model and ectopic mineralization was tested. First, ciOG with growth factor-reduced Matrigel was implanted into a critical-sized bone defect area (4 mm diameter) in immunosuppressed NOD/SCID mice (8 weeks old, male) (Figure 5a). At 12 weeks after transplantation, micro-CT 3D structural images were analyzed to measure bone volume, surface area, and surface density (Figure 5b). This osteogenic index was significantly higher in the ciOG implant group than in the Matrigel alone group.

Histological analysis using hematoxylin and eosin (H&E) and Masson's trichrome (MT) staining showed that new bone was formed in the defective area (black arrow) in the ciOG transplant group. This was in striking contrast to the Matrigel-only group, in which no bone formation was observed (Figure 5c). Immunohistochemical staining (red) of human OCN showed that bone matrix proteins were deposited on the osteogenic surface in the ciOG graft group (Figure 5D).

After confirming that ciOG implantation benefited bone growth in an orthotopic model, we investigated the potential for ectopic mineralization to evaluate the in vivo osteogenic potential of cells in a microenvironment relatively impaired for osteogenesis. To compensate for the impaired osteogenic microenvironment, mature ciOG(M7p), which showed the highest OCN expression without calcification, was transplanted into the dorsal skin subcutaneous tissue of immunosuppressed NOD/SCID mice (Figures 2g and 5e).

ciOG(M7p) is as shown in Figure 1g. To evaluate the bone regeneration potential of the transplanted cells, intermediately mature (not completely calcified) osteoblast-like cells were transplanted into mice. At 8 weeks after transplantation, micro-CT imaging showed the formation of mineralized tissue in the ciOG(M7p) group: hard tissue was formed in 4 out of 5 ciOG(M7p) transplant samples. The samples showed significant bone density, bone volume, surface area, and surface density in contrast to the aHDF graft group where no mineralization was observed (Figure 5f). H&E and MT staining revealed bone-like tissue with mineralization ('B' region), while immunohistochemical staining of human OCN showed predominantly in the osteogenic front, i.e. in the area bordering fibrous tissue (collagenous early bone matrix). showed high OCN ⁺ cells.

Experimental Example 4. Matrix-stimulated ciOG generation and bone tissue engineering

We sought to use engineered substrates in ciOG production to potentially regulate the induction and maturation of ciOG and enhance osteogenic functions such as bone ECM formation (Figure 6a). Titanium (milled “Ti” or sandblasted and acid-etched “Ti(SLA)” aligned nanofibers of polycaprolactone (flat film “PCL film” or “PCL nanofibers” with an average diameter of 700 nm) and calcium phosphate A variety of commercial culture substrates used for bone repair or bone cell culture were tested, including substrates based on (“bone assay plates”).

Ti-based materials have been widely used as bone implants (dental and orthopedic), and SLA with micro/submicron structure improves roughness, is most widely used in titanium implants with unique hydrophobic properties and excellent osteogenic effects. PCL nanofibers have great potential for bone formation due to their collagen-mimicking nanofiber structure. The osteoassay plate provided a calcium phosphate microcrystalline support (scaffold) for investigation of osteoclast and osteoblast function.

First, we measured RUNX2 gene expression in cells (ciOG) induced for 7 days on various substrates under optimized induction conditions (RFP in 2% FBS-OM). After induction, RUNX2 expression was significantly higher in Ti (SLA), PCL nanofibers, and bone analysis plates than in TCP (Figure 6b). In particular, PCL nanofibers induce higher RUNX2 gene levels than their bare PCL counterparts (PCL films), suggesting that the nanofiber structure, along with other biophysical factors, may play a role in increasing the efficiency of reprogramming to ciOG. do. Therefore, we first focus on the effect of the nanofiber structure while mimicking the nanofiber topology of collagen in regenerating the matrix (matrix) for calcification during the ciOG reprogramming process, and other matrix parameters (roughness, composition, hydrophilicity, etc.) It will be optimized for application. RUNX2 staining of cells after 7 days of induction revealed significant nuclear translocation of RUNX2 on PCL nanofiber substrates (Figure 6c). Other key osteogenic genes (BSP, ALP, and OCN) were also significantly upregulated in ciOG on the nanofiber matrix (Figure 6d). Maturation of ciOG for 14 days on nanofiber substrates further enhanced biomineralization as revealed by ARS staining (Figure 6e).

Next, we analyzed global transcriptional changes between ciOG and ciOG(M7p) derived from planar films of PCL versus PCL nanofibers. QuantSeq analysis enabled genetic profiling from limited amounts of RNA obtained from cultured cells. Hierarchical clustering analysis revealed significant differences in transfer between ciOG (unmatured) and planar films of PCL nanofibers. The overall transcriptional changes of ciOG produced on PCL nanofibers were similar to those of mature ciOG (M7p) on planar PCL films. Additionally, the expression pattern of osteogenic-, osteoblast/cell-, and distal stage-specific genes in ciOG (not mature) cultured on nanofibrous substrates was consistent with upregulated osteogenic and osteoblast/osteocyte genes and downregulated distal stage genes. Together, the stage genes resembled the expression pattern of mature ciOG (M7p) on planar PCL films, suggesting that the nanofibrous matrix provided a topographic signal for activation of osteogenic maturation.

Based on these results, we determined that nanofiber matrices can provide nanotopographic signals that alter adhesion-dependent mechanotransduction cell signaling during cell reprogramming, and demonstrated the global potential of ciOG cultured on PCL nanofiber substrates and planar PCL films. As a result of analyzing carcasses (25,7376 genes), it was confirmed that 1,706 genes were differentially expressed more than 4-fold. That is, 836 genes were confirmed to be up-regulated in ciOG, and 870 genes were down-regulated in nanofibers compared to film substrates. The top 20 hits from the GO term analysis involved involvement in mechanotransduction, intracellular signal transduction, spindle assembly and organization, calcium ion transmembrane transport, hemophilia cell adhesion, and transmembrane receptor protein tyrosine kinase signaling, all of which depend on cell-matrix interactions. indicated.

Among the top 30 GO categories of upregulated genes, those related to mechanotransduction include Rho protein signal transduction, phosphatidylinositol-3-kinase signaling, and cytosolic calcium ion concentration. Genes related to Rho signaling (ARHGDIB, CIT, ARHGAP27, ARHGEF38), actin formation (ACTR3C), myosin (MYO1F, MYH3), and integrin (ITGB2) were upregulated 5- to 24-fold. Because gene sets from different osteogenic pathways, such as BMP, WNT and TGF-β signaling, were inconsistently regulated, these mechanotransduction signals represent a unique mechanism that may explain the altered cell behavior in ciOG produced on nanofibers. .

As a result of analyzing cell morphology, it was confirmed that there was a clear difference between cells derived from nanofibers and flat film substrates. Cells on the nanofiber substrate elongated in the direction of the 700 nm diameter fiber within 4 hours after seeding, indicating inhibition of cell proliferation by the nanofiber (Figure 6f). Regardless of matrix type, this cell elongation or decrease in cell circularity during 7 days of induction is a result of the change from fibroblasts to osteogenic cells. Cell shape changes in nanofiber matrices are related to intracellular mechanotransduction signals. Accordingly, we examined whether mechanotransduction and mechanosensitive machinery are involved in ciOG production.

Inhibitors of mechanosensitive molecules such as Y27632 (Rho-kinase inhibitor) and blebbistatin (nonmuscle myosin II inhibitor) were used during the induction of ciOG on nanofiber substrates. At day 7, RUNX2 nuclear translocation and its positive cell number were reduced by inhibitor treatment, especially blebbistatin treatment (Figure 6g). Moreover, the inhibitor reduced biomineralization of mature ciOG(M14p) as shown by reduced ARS staining, thereby nullifying the effect of nanofiber matrix signal (Figure 6h).

To determine the underlying mechanism by which nanofibers accelerate ciOG reprogramming more than films, we investigated representative cell contractility markers (pMLC) and mechanotransduction transcriptional coactivators (TAZ, transcriptional coactivators with PDZ binding motifs), and nanofibers It was confirmed that the pMLC intensity increased and the proportion of cells in which TAZ was translocated to the nucleus increased, and the group treated with Y-27632 (rho kinase inhibitor) was confirmed to recover to a baseline level similar to the film. On the other hand, after treatment with Blebbistatin, an actomyosin contraction inhibitor, it was confirmed that Taz nuclear translocation was directly alleviated regardless of pMLC (Figure 6i). In other words, the mechanism by which nanofibers accelerate ciOG reprogramming is summarized as follows (Figure 6j). i) the nanofiber topology induces more actomyosin contraction due to its unique and longer structure, ii) actomyosin contraction causes more RUNX2 and TAZ nuclear migration, iii) the RUNX2/TAZ complex induces ciOG reprogramming efficiency and Further improved maturation.

Considering the role of the nanofiber matrix in ciOG induction and maturation, we tested whether nanofiber-generated ciOG is more effective than Matrigel-generated ciOG in promoting the regeneration of bone defects in vivo. After maturation for 7 days, ciOG-nanofibers and ciOG-Matrigel structures were implanted into critical-sized calvarium defects of NOD/SCID mice (Figure 7a). Tissue samples were analyzed 12 weeks after surgery. H&E and MT staining showed that the ciOG(M7p)-nanofiber group exhibited greater collagenous bone matrix formation and newly calcified bone (Figures 7b and 7c). Immunohistochemical staining for human OCN, a late bone matrix protein, showed a strong signal in the newly formed matrix, confirming greater osteoinduction by nanofiber-supported ciOG (Figure 7d). These results suggest that generating ciOG on nanofiber matrices should promote bone induction in vivo and that implantation of ciOG(M7p)-nanofiber structures could be an effective bone tissue engineering approach.

Experimental Example 5. Feasibility of the ciOG platform for bone disease modeling and drug testing

After confirming the efficacy of ciOG in repairing defective bone tissue in vivo, we tested whether ciOG would be useful in bone disease modeling and drug screening. Two genetic bone diseases, osteogenesis imperfecta (OI) and Proteus syndrome (PS), were selected (Figure 8a). OI and PS are caused by mutations in the collagen type-1 alpha 1 chain (COL1A1) and AKT serine/threonine kinase 1 (AKT1) genes, respectively, and are characterized by low and overgrowth of connective tissues such as bone.

Using OI and PS patient-derived fibroblasts, we applied the ciOG generation platform and determined whether ciOG could reconstruct the pathological features of these diseases. Expression of osteogenic genes (RUNX2, OCN and BSP) by ciOG and mineralization by mature cells (ciOG(M28p)) showed significant differences between genetic conditions: PS > Normal > OI (Figure 8b). This indicates that ciOG generated from patient-derived cells reproduced the genetic and phenotypic characteristics of bone defect (OI) and overgrowth (PS).

Next, PS-derived fibroblasts during the maturation stage were treated with the chemical ARQ092, which inhibits AKT signaling, an important signaling pathway in PS. OCN synthesis and mineralization were reduced in an ARQ092-dose dependent manner (Figures 8C and 8D). Moreover, ARQ092 treatment significantly reduced the phosphorylation of AKT signaling molecules (Figure 8e), suggesting that ARQ092 inhibits osteogenic differentiation through the AKT signaling pathway. To determine whether AKT inhibition is effective during ciOG induction, ARQ092 was used to treat fibroblasts for 7 days during the induction phase. In an ARQ092-dose dependent manner, AKT signaling molecules ( p AKT and p PRAS40) were significantly downregulated, and RUNX2 expression was also confirmed to be decreased.

Above, embodiments of the present invention have been described with reference to the attached drawings, but those skilled in the art will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. You will be able to understand it. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

Claims (22)

1. A medium composition for direct differentiation of osteogenic cells that induces osteogenic cells from somatic cells, comprising a TGF β receptor inhibitor, a cAMP signaling activator, and an epithelial Na ⁺ channel inhibitor as active ingredients.
2. According to paragraph 1,

   The TGF β receptor inhibitors include RepSox (1,5-Naphthyridine, 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]), SB431542 (4-[4-(1,3 -benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), an inhibitor of TGF-β RI, ALK4, and ALK7. A medium composition.
3. According to paragraph 1,

   The cAMP signaling activator is a medium composition characterized in that it is selected from the group consisting of Forskolin, isoproterenol, isopreterenol, NKH 477, isoprotereno (Chemical based), PACAP 1-27, and PACAP 1-38 (peptide based) .
4. According to paragraph 1,

   A medium composition, wherein the epithelial cell Na ⁺ channel inhibitor is Phenamil (3,5-diamino-6-chloro- N -( N '-phenylcarbamimidoyl)pyrazine-2-carboxamide).
5. According to paragraph 1,

   A medium composition, wherein the somatic cells are fibroblasts.
6. According to paragraph 1,

   A medium composition, characterized in that the osteogenic cells are induced under conditions in which FBS (fetal bovine serum) is not added.
7. According to paragraph 1,

   A medium composition, wherein the osteogenic cells have significantly higher expression of osteogenic genes than non-induced fibroblasts.
8. In clause 7,

   A medium composition, wherein the osteogenic gene is at least one selected from the group consisting of RUNX2 (Runt-related transcription factor 2), BSP (bone sialoprotein), ALP (alkaline phosphatase), and OCN (osteocalcin).
9. According to paragraph 1,

   A medium composition, characterized in that the osteogenic cells exhibit nuclear translocation of RUNX2.
10. According to clause 9,

    A medium composition, characterized in that the nuclear intensity of RUNX2 and the ratio of nuclear translocation cells of the osteogenic cells are higher than those of fibroblasts.
11. According to paragraph 1,

    A medium composition, characterized in that the BMP signaling gene is up-regulated in the osteogenic cells.
12. According to clause 11,

    A medium composition, wherein the up-regulation of the BMP signaling gene results in the activation of the BMP agonist gene and the inhibition and regulation of the BMP antagonist gene.
13. According to clause 12,

    The medium composition, characterized in that the BMP agonist gene is BMP1-11 or ID1, and the BMP antagonist gene is GREM1 or GREM2.
14. According to paragraph 1,

    A medium composition, characterized in that the IGF signaling gene is up-regulated in the osteogenic cells.
15. According to clause 14,

    A medium composition, wherein the IGF signaling gene is IGF1, IGF2, IGF1R, IGF2R, or IGFBP2-7.
16. A direct differentiation method for inducing osteogenic cells from somatic cells by culturing them in a medium containing a TGF β receptor inhibitor, cAMP signaling activator, and epithelial Na ⁺ channel inhibitor as active ingredients.
17. According to clause 16,

    Direct differentiation method, characterized in that the culture is performed for 4 to 12 days.
18. According to clause 16,

    Direct differentiation method, wherein the somatic cells are fibroblasts.
19. A pharmaceutical composition for preventing or treating bone disease, comprising osteogenic cells produced by the method of claim 16 as an active ingredient.
20. According to clause 19,

    The bone disease is one or more bone diseases selected from the group consisting of fracture, osteoporosis, rheumatoid arthritis, periodontitis, Paget's disease, osteomalacia, osteopenia, bone atrophy, osteoarthritis, osteogenesis imperfecta, Proteus syndrome, and avascular femoral osteonecrosis. Pharmaceutical composition.
21. A food composition for preventing or improving bone disease, comprising osteogenic cells produced by the method of claim 16 as an active ingredient.
22. A method for preventing or treating bone disease comprising treating damaged hard tissue of a subject other than humans with a composition containing osteogenic cells produced by the method of claim 16 as an active ingredient.

Images