WO2025258968A1 - Composition pour diagnostiquer la nécrose aseptique de la tête fémorale ou prédire le stade de nécrose aseptique de la tête fémorale - Google Patents

Composition pour diagnostiquer la nécrose aseptique de la tête fémorale ou prédire le stade de nécrose aseptique de la tête fémorale

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WO2025258968A1
WO2025258968A1 PCT/KR2025/007902 KR2025007902W WO2025258968A1 WO 2025258968 A1 WO2025258968 A1 WO 2025258968A1 KR 2025007902 W KR2025007902 W KR 2025007902W WO 2025258968 A1 WO2025258968 A1 WO 2025258968A1
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disease
bone
bone disease
stage
paragraph
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Korean (ko)
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송정식
박관규
양헌무
이영한
이승환
이은주
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University Industry Foundation UIF of Yonsei University
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Definitions

  • the present invention relates to a composition for diagnosing or predicting the stage of osteonecrosis of the femoral head.
  • Osteonecrosis of the femoral head is a progressive degenerative disease that causes the destruction of subchondral bone and structural collapse of the hip joint. It can occur even in young adults and is a serious bone disease that may require early joint replacement.
  • the pathogenesis of ONFH has been primarily understood to be local ischemia due to vascular damage or structural stress due to increased mechanical pressure. Accordingly, previous studies have primarily analyzed the pattern of subchondral bone destruction, osteocyte death, and vascular structural changes at the imaging or histological level. Furthermore, studies have observed gene expression changes through bulk RNA sequencing at the tissue level, but these studies have been limited by their inability to sufficiently reflect intercellular heterogeneity or spatial context.
  • this study categorized the distribution and functional status of cell subtypes within ONFH lesions according to spatial anatomic regions and quantitatively analyzed these data, thereby comprehensively elucidating the spatial regulation of the WNT signaling network and the structure of cell-cell interactions.
  • This study demonstrated for the first time that disease progression is regulated by lineage reprogramming and local signaling networks, rather than simple damage. This represents a significant turning point, transcending existing pathological understanding and proposing the functional hierarchy among stromal cell subtypes and their regulatory signals as potential diagnostic and therapeutic targets.
  • the present invention aims to elucidate the distribution and cell fate changes among spatially compartmentalized stromal cell subtypes within a femoral head osteonecrosis lesion, and to interpret the interactions between them and the WNT signaling regulation mechanism.
  • the present invention aims to provide a composition for use in diagnosing and predicting the stage of osteonecrosis of the femoral head.
  • ischemic necrosis means ischemic necrosis of bone tissue caused by blockage of blood supply or local metabolic abnormality, and refers to a pathological condition including cell death within the tissue, microvascular damage, bone matrix collapse, and secondary bone remodeling failure.
  • osteonecrosis of the femoral head refers to a localized osteonecrosis that occurs in the femoral head forming the articular surface of the femur, causing collapse of the subchondral bone, collapse of the articular surface, and structural collapse of the hip joint, and is a progressive degenerative disease that may lead to pain, limitation of motion, and early artificial hip replacement if the disease progresses.
  • mesenchymal stromal cells refers to a multipotent precursor cell population that expresses genes such as COL1A1 and ACTA2 and can differentiate into mesenchymal lineages such as bone, cartilage, and fat.
  • mesenchymal lineages such as bone, cartilage, and fat.
  • core cell lineage that exists in various subtypes (FC, ALC, CLC, OLC, USC) within ONFH lesions, as cells that perform functions such as tissue remodeling, extracellular matrix composition, immune regulation, and damage repair.
  • FC, ALC, CLC, OLC, USC subtypes
  • endothelial cells refer to cells that express genes such as PECAM1 (CD31), EMCN, and A2M, form the inner walls of blood vessels, and participate in the regulation of blood flow, oxygen supply, angiogenesis, and inflammatory responses within tissues. Furthermore, in ONFH lesions, numerical and functional changes are observed in areas where blood flow is blocked and microvascular damage occurs.
  • lymphoid cell refers to an adaptive immune system or innate immune system cell that expresses genes such as CD3D, CD3E, TRBC2, and CCL5, and is composed of T cells, B cells, and NK cells. It also refers to a cell lineage that contributes to regulating immune responses and creating an inflammatory environment within tissues.
  • myeloid cell refers to an innate immune system cell that expresses genes such as LYZ, CXCL8, and SPARC1L, and is composed of macrophages, monocytes, dendritic cells, and neutrophils. It is also a group of cells that perform tissue damage response, phagocytic function, cytokine secretion, and antigen presentation functions.
  • Fibrochondrocyte refers to a subset of stromal cells that are characterized by the expression of genes such as SFRP4, SOX9, FOXO1, and CTHRC1 in single-cell transcriptome analysis and are present in an expanded state in areas of necrosis, mediating chondrogenesis and fibrous matrix formation, and involved in atypical calcification and WNT signaling activation.
  • fibrochondrocytes are major recipients in several pathways, including MK, PTN, and ANGPTL.
  • the genes PPP2R3A, DKK3, GREM1, FZD1, FERMT1, NXN, GRB10, CTHRC1, APCDD1L, SFRP4, ALPK2, WNT5A, BICC1, IGFBP6, VGLL4, TPBG, FOXO1, SOX9, PRICKLE1, LZTS2, and FZD6 were characterized by higher expression compared to the control group (see Fig. 4d).
  • ALC Addipogenic Lineage Cell
  • RSPO3, ANKRD6, PRICKLE2, and FZD5 genes that express genes such as RSPO3, ANKRD6, PRICKLE2, and FZD5
  • ALC has the highest sender/receiver signal ratio among all stromal subtypes and functions as a major upstream signal transduction cell.
  • ALC is a strong transmitter in many pathways such as MK, PTN, and ANGPTL.
  • the genes SFRP1, DAAM2, MAGI2, GPC3, ANKRD6, FZD3, FZD5, FRZB, FZD4, PRICKLE2, FZD8, RSPO3, and PRICKLE1 are characterized by higher expression compared to the control group (see Fig. 4h).
  • chondrogenic lineage cell means a cell that expresses genes such as ACAN, SOX9, and COL2A1, is a subtype of stromal cells with enhanced cartilage differentiation characteristics, exists together with FC in necrotic areas, and contributes to the accumulation of extracellular matrix components and the chondrogenesis program.
  • Osteogenic lineage cell means a differentiated cell that expresses genes such as SP7, BGLAP, and IBSP and is a stromal cell subtype located in the terminal differentiation stage according to pseudotime analysis, and can contribute to bone formation and bone regeneration.
  • uncommitted stromal cell means a progenitor cell that expresses genes such as TBX2 and A2M without expression of differentiation markers specific to a specific lineage, maintains an initial state of mesenchymal origin, and has the potential to differentiate into various lineages (cartilage, fat, bone) depending on external signals.
  • chondrogenic marker gene means
  • the chondrogenic differentiation marker genes may include type II and type IX collagen family genes (COL2A1, COL9A1), cartilage matrix regulatory factors (SOX9, ACAN, CNMD), and genes specifically expressed in an atypical mineralized or fibrocartilaginous environment (AMTN, etc.).
  • FCs fibrochondrocytes
  • CLCs chondrogenic cells
  • COL9A1 is a type IX collagen alpha 1 chain gene, which is one of the major collagen proteins that constitute the ECM (extracellular matrix) of cartilage, and is a gene involved in structural stability and cell-matrix interaction in hyaline cartilage, and is used as an indicator indicating a state of chondrocyte differentiation at the intermediate stage or higher.
  • AMTN is the amelotin gene, which is mainly known as a protein related to enamel formation, but in recent studies, it is expressed in cartilaginous tissue and fibrocartilaginous environments, and is a gene suggested to be associated with mineralization tendency of tissues and atypical calcification, and in this specification, it is used as a marker indicating the pathological characteristics of fibrochondrocytes.
  • CNMD refers to the chondromodulin gene, which is known as an antiangiogenic factor that regulates proliferation and differentiation of chondrocytes and inhibits angiogenesis, plays an important role in the avascular nature of cartilage and stabilization of chondrocytes, and is used as a marker reflecting functional chondrogenesis in the CLC or FC subtype.
  • SFRP4 Small Frizzled-Related Protein 4
  • SFRP4 refers to a secreted glycoprotein that inhibits Wnt signaling by binding to a Wnt ligand and thereby inhibiting the interaction with the Wnt receptor (Frizzled), and which can be encoded by the human SFRP4 gene.
  • SOX9 SRY-Box Transcription Factor 9
  • SRY-Box Transcription Factor 9 is a transcription factor that regulates chondrocyte differentiation and fibrochondrocyte characteristics, promotes ECM composition and chondrogenic gene expression, and can be encoded by the human SOX9 gene.
  • FOXO1 Formhead Box O1
  • a transcription factor involved in cell survival, metabolism, and stem cell maintenance and has the function of regulating WNT signal inhibition and fibrotic differentiation pathway. It can also be encoded by the human FOXO1 gene.
  • CTHRC1 Collagen Triple Helix Repeat Containing 1
  • ECM remodeling and tissue regeneration processes whose expression is increased in fibrochondrocytes and is associated with WNT signaling inhibition. It may also be encoded by the human CTHRC1 gene.
  • FZD1 (Frizzled Class Receptor 1) refers to a cell membrane receptor protein that binds to Wnt ligands in the Wnt signaling pathway and mediates signal transduction, and may be encoded by the human FZD1 gene. It also contains a cysteine-rich domain (CRD) and may be involved in the activation of various Wnt pathways, including the Wnt/ ⁇ -catenin pathway.
  • CCD cysteine-rich domain
  • NXN Nucleoredoxin
  • RSPO3 R-spondin 3
  • LGR receptor LGR receptor
  • WNT receptor WNT receptor
  • PRICKLE1 refers to a cell membrane-associated protein involved in the regulation of the Planar Cell Polarity (PCP) pathway, which may affect cell polarity and tissue morphogenesis, and may be encoded by the human PRICKLE1 gene.
  • PRICKLE1 may act as a key regulator in the Wnt/PCP pathway, a non-canonical sub-pathway of the Wnt signaling pathway.
  • WNT5A is a secreted signaling protein involved in the Wnt signaling pathway, inducing non-canonical Wnt signaling, and may be involved in cell motility, polarity formation, differentiation, and inflammation regulation. It may also be encoded by the human WNT5A gene.
  • BICC1 (Bic-C Family RNA Binding Protein 1) is an RNA-binding protein that can regulate the stability and translation of target mRNA, and may be involved in the regulation of Wnt signal transduction, cell differentiation, and developmental processes.
  • the protein may be encoded by the human BICC1 gene.
  • IGFBP6 Insulin-like Growth Factor Binding Protein 6
  • IGF-II insulin-like growth factor II
  • IGF-II insulin-like growth factor II
  • VGLL4 Vestigial-like family member 4 refers to a transcriptional coregulator that can suppress YAP (Yes-associated protein)-mediated transcriptional activity through interaction with TEAD transcription factors, and can perform cell proliferation inhibition, differentiation promotion, and tumor suppression functions. It can also be encoded by the human VGLL4 gene.
  • RSPO3 R-spondin 3
  • R-spondin 3 refers to a secreted protein that inhibits internalization of Wnt receptors and enhances Wnt/ ⁇ -catenin signaling through interaction with LGR receptors and ZNRF3/RNF43 proteins, and can be encoded by the human RSPO3 gene.
  • FZD8 (Frizzled Class Receptor 8) refers to a cell membrane receptor protein that binds to Wnt ligands and mediates the activation of the Wnt signaling pathway, and may be encoded by the human FZD8 gene.
  • the protein may be involved in both the canonical Wnt/ ⁇ -catenin signaling pathway and the non-canonical Wnt pathway.
  • SFRP1 Small Frizzled-Related Protein 1
  • SFRP1 Small Frizzled-Related Protein 1
  • This protein may particularly contribute to negative regulation of the canonical Wnt/ ⁇ -catenin pathway.
  • PPP2R3A Protein Phosphatase 2 Regulatory Subunit B'' Alpha
  • PPP2A protein phosphatase 2A
  • DKK3 Dickkopf-related protein 3
  • Wnt/ ⁇ -catenin pathway a secreted protein that regulates the Wnt/ ⁇ -catenin pathway, and may be involved in cell-cell interaction and differentiation regulation and cell growth inhibition within the tumor microenvironment. It may be encoded by the human DKK3 gene. Although this protein does not directly bind to the Wnt receptor, it may contribute to tumor suppression and tissue homeostasis maintenance by indirectly suppressing Wnt signaling.
  • GREM1 (Gremlin 1) refers to a secreted protein that can inhibit Bone Morphogenetic Protein (BMP) signaling by directly binding to it, thereby inhibiting its interaction with the BMP receptor, and can be encoded by the human GREM1 gene. It can also be involved in cell differentiation, tissue development, fibrosis regulation, and tumor suppression/promotion.
  • BMP Bone Morphogenetic Protein
  • FERMT1 (Fermitin family homolog 1) is a cell membrane adhesion-related protein that mediates integrin activation, regulates cell motility, signal transduction, and interaction with the extracellular matrix, and may be encoded by the human FERMT1 gene.
  • the protein may perform physiological and pathological functions related to Kindler syndrome, tissue regeneration, and cancer invasiveness.
  • GRB10 Crowth factor receptor-bound protein 10
  • IGF-1 receptor insulin receptor
  • IGF-1 receptor insulin receptor
  • tyrosine kinase receptors tyrosine kinase receptors
  • APCDD1L Addenomatosis Polyposis Coli Down-Regulated 1 Like
  • APCDD1L Adenomatosis Polyposis Coli Down-Regulated 1 Like
  • ALPK2 Alpha Kinase 2
  • ⁇ -kinase family is an atypical protein kinase belonging to the ⁇ -kinase family, which can contribute to cell signaling, cell cycle regulation, or tissue-specific functions by transferring a phosphate group to a specific serine or threonine residue, and can be encoded by the human ALPK2 gene.
  • BMP2 (Bone Morphogenetic Protein 2) is a secreted protein belonging to the TGF- ⁇ superfamily, which can be involved in differentiation of osteoprogenitor cells, bone formation, cartilage development, cell survival regulation, etc. by binding to BMP receptors on the cell membrane and inducing SMAD-mediated signaling pathways, and can be encoded by the human BMP2 gene.
  • LZTS2 Leucine Zipper Tumor Suppressor 2
  • LZTS2 Leucine Zipper Tumor Suppressor 2
  • FZD6 (Frizzled Class Receptor 6) is a G-protein coupled receptor present in the cell membrane, which mediates canonical or non-canonical Wnt signaling pathways through binding to Wnt ligands, and may be involved in maintaining cell polarity, tissue development, and tumor metastasis, and may be encoded by the human FZD6 gene.
  • ANKRD6 Alkyrin Repeat Domain 6
  • ANKRD6 is a protein involved in the regulation of non-canonical WNT signaling, and has functions related to cell polarity and cell fate regulation. It can also be encoded by the human ANKRD6 gene.
  • PRICKLE2 Primary Cell Polarity Protein 2
  • ALCs adipose tissue cells
  • FZD5 (Frizzled Class Receptor 5) is a receptor that binds to WNT ligands and induces non-canonical WNT signaling, and is activated at the border of osteonecrotic lesions. It may also be encoded by the human FZD5 gene.
  • GPC3 (Glypican-3) is a heparan sulfate proteoglycan bound to the cell membrane, which interacts with growth factors such as Wnt and Hedgehog to regulate signal transduction, and in particular, can contribute to cell growth, differentiation, and tumorigenesis by activating the Wnt/ ⁇ -catenin pathway, and can be encoded by the human GPC3 gene.
  • DAAM2 (Dishevelled Associated Activator of Morphogenesis 2) is a downstream protein of the Wnt signaling pathway, which interacts with Dishevelled (DVL) to mediate non-canonical Wnt/PCP or Wnt/RhoA pathway, and may be involved in cell migration, cytoskeletal reorganization, tissue morphogenesis, and cell differentiation, and may be encoded by the human DAAM2 gene.
  • FZD3 (Frizzled Class Receptor 3) is a G-protein coupled receptor that binds to Wnt ligands and mediates non-canonical Wnt/PCP signaling, may be involved in cell polarity regulation, neural development, and cell migration, and may be encoded by the human FZD3 gene.
  • the specific WNT signature score calculation process is as follows.
  • the calculated Wnt signature score is increased compared to low-stage disease, it is likely to be high-stage bone disease or corresponds to high-stage bone disease.
  • stage 3A the ALC WNT signature score is increased compared to low-stage (stage 3A), it is likely to be high-stage osteonecrosis or corresponds to high-stage (stage 3B, 4) osteonecrosis.
  • “pseudotime analysis” refers to an analysis method that predicts the differentiation progression path and temporal sequence between cells based on single-cell transcriptome data using Monocle3 or a similar algorithm.
  • CellChat analysis refers to a computational method for modeling the directionality and centrality (sender/receiver cells) of signaling networks between cell subtypes based on ligand-receptor interaction data between cells.
  • EMT Epithelial-Mesenchymal Transition pathway
  • EMT Epithelial-Mesenchymal Transition pathway
  • ECM extracellular matrix
  • MMPs matrix-degrading enzymes
  • fibrosis cancer metastasis
  • tissue remodeling a process in which epithelial cells lose cell-to-cell adhesion and polarity, acquire mesenchymal cell characteristics, and increase motility and invasiveness.
  • EMT plays a central role in physiological and pathological processes such as extracellular matrix (ECM) reorganization, increased expression of matrix-degrading enzymes (MMPs), fibrosis, cancer metastasis, and tissue remodeling.
  • ECM extracellular matrix
  • MMPs matrix-degrading enzymes
  • Activation of the EMT pathway is closely related to increased expression of genes such as COL1A1, PLOD1, LOX, FN1, TGFB1, MMP14, POSTN, and VEGFC, and in the present invention, activation of the EMT pathway was specifically observed in the necrotic area among osteonecrosis lesions of the femoral head.
  • adipogenesis refers to a biological process by which precursor cells (e.g., mesenchymal stromal cells) differentiate into adipocytes, which involves the acquisition of intracellular fat storage capacity, changes in insulin sensitivity, and the activation of transcriptional programs related to the regulation of fatty acid metabolism and energy homeostasis.
  • precursor cells e.g., mesenchymal stromal cells
  • the adipogenesis process is closely associated with increased expression of genes such as PPARG, CEBPA, FABP4, ADIPOQ, LPL, and PLIN1, and in the present invention, it was observed as a key pathway reflecting the differentiation characteristics of adipose lineage cells (ALCs), particularly in the marginal region.
  • ALCs adipose lineage cells
  • adipogenesis may also include the meaning of “adipocyte generation” or “lipogenesis.”
  • the term “marginal region (mar)” refers to a region of intermediate cell density corresponding to the histological boundary around a necrotic area. Histologically, it is observed to have an intermediate cell density, and at the transcriptomic level, it is characterized by a high concentration of adipose-like cells (ALCs), and it includes an early regulatory zone of stromal differentiation where selective expression of canonical WNT signaling inhibitory genes (SFRP1, RSPO3, etc.) occurs.
  • the term “marginal region” as used herein may include the meaning of "marginal region", “border tissue”, “marginal region”, “marginal area”, “marginal region”, or “marginal area”.
  • control region refers to structurally preserved femoral head tissue located outside of an avascular osteonecrosis of the femoral head lesion. Histologically, it refers to a non-lesion-related control area with normal trabecular bone structure and low cell density, transcriptomically, without specific expansion of stromal cell subtypes, and with balanced WNT-related gene expression.
  • normal region may also encompass the meanings of "normal area,” “normal tissue,” “control area,” and "control tissue.”
  • bone disease means a disease that causes structural, functional, or metabolic damage due to an abnormality in the homeostasis of bone tissue in the body, and may include all bone pathologies that may be caused by abnormal function of bone cells, destruction of bone matrix, abnormal blood flow, inflammation, or hormonal imbalance.
  • Bone diseases include, but are not limited to, osteoporosis, osteopenia, osteomalacia, rickets, Paget's disease of bone, osteonecrosis, osteonecrosis of the femoral head (ONFH), Legg-Calve-Perthes disease, Kienbock's disease, Preiser's disease, Scheuermann's disease, osteomyelitis, tuberculous osteomyelitis, Pott's disease, osteosarcoma, chondrosarcoma, Ewing's sarcoma, and metastatic bone cancer. These may include osteodysplasia, osteomalacia, osteoporosis, osteomyelitis, osteoarthritis, osteomyelitis metastasis, osteomalacia ...
  • the above bone disease may be a disease accompanied by the collapse or spatial reorganization of trabecular bone, which is similar to the morphological changes observed in the tissue of osteonecrosis (e.g., osteonecrosis of the femoral head) targeted in the present invention.
  • the Wnt-related gene-based diagnosis and analysis method according to the present invention is not limited to osteonecrosis, but can also be applied to various bone diseases characterized by collapse or spatial reorganization of bone columns as described above.
  • the “ARCO (Association Research Circulation Osseous) classification” refers to an internationally recognized imaging staging standard for evaluating and describing the pathological progression stages of bone disease, osteonecrosis, or osteonecrosis of the femoral head (ONFH). This classification is divided into several stages according to the severity of the lesion, including the extent of the osteonecrotic lesion, the presence and degree of subchondral depression, and the presence or absence of joint space and cartilage damage. This specification includes the following three staging stages as main criteria.
  • Stage 1 is the initial stage, meaning there are small lesions in the skull.
  • Stage 2 refers to cases where the lesion is slightly enlarged and the shape of the bone head is deformed.
  • Stage 3 refers to cases where the femoral head begins to collapse and a fracture may occur, and can be classified into 3A and 3B depending on the depth of the femoral head subsidence.
  • Stage 3A is a relatively early stage in which collapse of the femoral head articular surface is observed, but the depth of collapse is less than 2 mm. Subchondral collapse has begun, and the cartilage structure is partially preserved, but progression of the lesion is imminent. This can be classified as a low-stage disease.
  • Stage 3B is a stage where the joint surface depression exceeds 2 mm, and structural collapse is more evident. Pain worsens with weight-bearing, and a marked decline in joint function may be evident. Imaging reveals asymmetrical depression of the femoral head.
  • Stage 4 is an advanced stage with not only subchondral depression but also joint space narrowing, cartilage destruction, or secondary osteoarthritis, in which the joint function between the femoral head and the acetabulum is virtually lost, and total hip arthroplasty is generally considered. Stages 3B and 4 can be classified as advanced stages.
  • stage means a stage corresponding to a relatively early stage of bone disease or osteonecrosis according to the ARCO classification criteria, and may specifically include stages 1 to 3, more specifically stages 1 to 3A, and most specifically stage 3A.
  • advanced stage means a state in which the disease of bone disease or osteonecrosis is considerably advanced according to the ARCO classification criteria, and may specifically include stages 3 and 4, and more specifically stages 3B and 4.
  • spatial remodeling refers to a pathological process in which different cell subtypes within a tissue are rearranged according to specific spatial locations, and thus cell-cell interactions, signal transduction, and cell fate determination change in a spatially organized manner.
  • osteonecrosis specifically osteonecrosis of the femoral head, this refers to a phenomenon in which specific cell subtypes are distributed and function differently according to specific locations within the lesion, such as fibrochondrocytes (FCs) and chondroblasts (CLCs) expanding in the center of the necrotic area, and adipose-like cells (ALCs) being localized in the border area.
  • FCs fibrochondrocytes
  • CLCs chondroblasts
  • ALCs adipose-like cells
  • diagnosis refers to a series of analytical activities that confirm the presence of a bone disease or osteonecrosis, or evaluate the stage or degree of progression of the disease, based on the number of cells measured from a biological sample (e.g., tissue, cells, body fluid, etc.) or the expression level of genes or proteins.
  • a biological sample e.g., tissue, cells, body fluid, etc.
  • diagnosis includes not only the determination of the presence or absence of a disease, but also the act of deriving information such as the spatial characteristics of the disease, pathological severity, or prognosis prediction.
  • the diagnosis of bone disease herein includes a diagnosis of osteonecrosis or osteonecrosis of the femoral head.
  • FCs fibrochondrocytes
  • CLCs chondrocytes
  • “staging prediction” means the process of evaluating the degree of progression or severity of a disease or estimating which stage a disease belongs to based on specific biological indicators or sample analysis results.
  • ALCs adipose tissue cells
  • RSPO3, ANKRD6, PRICKLE2, FZD5 ALC-specific WNT genes
  • prevention may include, without limitation, any act of blocking, suppressing, or delaying disease symptoms using the pharmaceutical composition of the present invention.
  • treatment and “improvement” may include, without limitation, any action that improves or benefits disease symptoms by examining the pharmaceutical composition of the present invention.
  • the term "diagnostic composition” or “composition for staging prediction” refers to a composition used to determine the presence or absence, stage, prognosis, etc. of a specific disease from a biological sample, and includes one or more components such as an antibody, primer, probe, label, enzyme, etc. for detecting the presence or expression level of a target cell or a target marker (e.g., protein, nucleic acid, etc.).
  • the composition can be applied to various analysis platforms such as ELISA, RT-PCR, DNA chip, protein chip, and immunoassay, and can be used alone or as part of a diagnostic kit.
  • the composition may additionally include a binding moiety (e.g., an antibody or nucleic acid probe) specific for a particular gene or protein marker and a buffer, detection label, enzyme or other auxiliary reagent suitable for the assay conditions.
  • a binding moiety e.g., an antibody or nucleic acid probe
  • the term "agent for measuring the number of fibrochondrocytes (FC)” means a reagent or substance configured to quantitatively or qualitatively measure the presence and quantity of FC in a sample, and may include a nucleic acid probe, primer, antisense oligonucleotide, or a kit containing the same capable of detecting the expression of the FC-specific marker gene (e.g., SERP4, FOXO1, CTHRC1, SOX9), or an antibody, antibody-enzyme conjugate, antibody-fluorophore conjugate, or antibody-based immunostaining reagent that recognizes a protein expressed by the gene.
  • FC-specific marker gene e.g., SERP4, FOXO1, CTHRC1, SOX9
  • the agent may be used to indirectly estimate or directly detect the number of FC, for example, through RT-PCR, qPCR, RNA in situ hybridization, immunohistochemical staining (IHC), ELISA, FACS analysis, flow cytometry, or immunofluorescence analysis.
  • the “agent for measuring the number of adipogenic lineage cells (ALC)” refers to an agent for selectively detecting or quantifying ALC from a biological sample, and may include an antibody that binds to a protein specifically expressed in the ALC (e.g., RSPO3, ANKRD6, PRICKLE2, and FZD5, etc.), or a primer or probe that specifically binds to the mRNA of the gene.
  • a lipid staining composition including a dye such as Oil Red O, Nile Red, or BODIPY for visualizing the accumulation of neutral fat.
  • the agent may be used to directly or indirectly measure the number of ALC through, for example, RT-PCR, qPCR, RNA in situ hybridization, immunohistochemical staining (IHC), ELISA, FACS analysis, flow cytometry, or immunofluorescence analysis.
  • diagnostic kit means a diagnostic kit comprising a diagnostic composition including an antibody or nucleotide primer specific to a biomarker to be diagnosed, and further comprising a detection means for measuring the expression level.
  • kits may be, but is not limited to, an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.
  • the diagnostic kit may further include one or more other component compositions, solutions, or devices suitable for the analysis method.
  • the kit may further include essential elements required for performing a reverse transcription polymerase reaction.
  • the reverse transcription polymerase reaction kit includes a pair of primers specific for a gene encoding a marker protein.
  • the primers are nucleotides having a sequence specific for the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp.
  • the kit may include a primer specific for the nucleic acid sequence of a control gene.
  • Other reverse transcription polymerase reaction kits may include test tubes or other suitable containers, reaction buffers (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.
  • the diagnostic kit may include essential components required for performing a DNA chip.
  • the DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or a fragment thereof are attached, and reagents, agents, enzymes, etc. for producing a fluorescently labeled probe.
  • the substrate may also include cDNA or oligonucleotides corresponding to a control gene or a fragment thereof.
  • the diagnostic kit may include essential components required for performing an ELISA.
  • the ELISA kit includes an antibody specific for the protein. Antibodies are antibodies that have high specificity and affinity for a marker protein and little cross-reactivity with other proteins. They can be monoclonal, polyclonal, or recombinant.
  • ELISA kits may also include antibodies specific for a control protein.
  • ELISA kits may also include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies), and their substrates or other substances capable of binding to antibodies.
  • the “target subject” of the present invention refers to a subject whose onset of a disease is uncertain, and is a subject with a high possibility of onset of a disease.
  • the "biological sample” of the present invention means any material, biological fluid, tissue or cell obtained from or derived from an individual, for example, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, It may include, but is not limited to, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.
  • diagnostic device means a system that performs the function of measuring the expression level of a specific gene or the concentration of a protein from a biological sample (e.g., blood, tissue, body fluid, etc.) isolated from a target individual, and diagnosing or predicting the presence or absence, progression, subtype, etc. of a disease based on the measured level.
  • the diagnostic device may be composed of one or more hardware and/or software modules, and may include a measuring unit, a judgment unit, a detection unit, etc.
  • the term "measurement unit” refers to a means for quantitatively or qualitatively analyzing the number of cells, the level of gene expression, or the concentration of proteins present in a biological sample obtained from a target individual.
  • the measurement unit can detect the corresponding indicator using various biological or biochemical methods, such as flow cytometry, quantitative PCR, immunoassays (e.g., ELISA, Western blot), microarray, RNA-seq, FACS analysis, and mass spectrometry.
  • the “judgment unit” means a system that performs a logical operation or algorithmic judgment to determine whether the target individual has developed or is likely to develop osteonecrosis based on the number of cells, expression level of genes or proteins measured in the measurement unit, and a calculation algorithm or rule-based judgment system that determines whether the stage of the osteonecrosis is low stage or high stage.
  • the judgment unit compares input data from the measurement unit with a preset threshold to make a judgment, and the judgment criterion includes a step of judging osteonecrosis if the ratio of fibrochondrocytes (FC) and chondrogenic lineage cells (CLC) to uncommitted stromal cells (USC) measured by the measurement unit is higher than the preset threshold compared to the reference value of the normal control group, and a step of judging that the patient is likely to have a bone disease or osteonecrosis or corresponds thereto if the expression levels of SFRP4, SOX9, FOXO1, CTHRC1, FZD1, NXN, PRICKLE1, WNT5A, BICC1, IGFBP6, VGLL4, RSPO3, FZD8 and SFRP1 measured by the measurement unit are higher than the preset threshold compared to the reference value of the normal control group.
  • FC fibrochondrocytes
  • CLC chondrogenic lineage cells
  • USC uncommitted stromal
  • the judgment unit compares the input data from the measurement unit with a preset threshold to make a judgment
  • the judgment criterion includes a step of judging osteonecrosis if the ratio of adipogenic lineage cells (ALC) to uncommitted stromal cells (USC) measured by the measurement unit is equal to or greater than the preset threshold compared to the reference value of the normal control group, based on the number of undifferentiated stromal cells and the number of adipogenic lineage cells measured by the measurement unit, and a step of judging that the expression levels of RSPO3, ANKRD6, PRICKLE2, FZD5, GPC3, FZD8, FZD4, SFRP1, and MAGI2 measured by the measurement unit are equal to or greater than the preset threshold compared to the reference value of the normal control group, thereby highly proving to be a high-stage bone disease or osteonecrosis or corresponding thereto.
  • ALC adipogenic lineage cells
  • USC uncommitted stromal cells
  • the "detection unit” refers to an output means that visually or digitally conveys the diagnostic results or disease prediction results derived from the judgment unit to the user.
  • the detection unit may be comprised of a display, monitor, printer, data transmission module, or other such component, and may enable visualization of the results or linkage to an electronic medical record system.
  • a composition for diagnosing a bone disease comprising an agent for measuring the number of any one or more cells selected from the group consisting of fibrochondrocytes (FC), chondrogenic lineage cells (CLC), uncommitted stromal cells (USC), and adipogenic lineage cells (ALC).
  • FC fibrochondrocytes
  • CLC chondrogenic lineage cells
  • USC uncommitted stromal cells
  • ALC adipogenic lineage cells
  • the term “formulation for measuring cell count” refers to a composition comprising an antibody, a staining reagent, a fluorescent probe, a marker protein, or a combination thereof, which is used to detect and quantify a specific cell subtype (e.g., fibrochondrocytes, chondrocytes, adipocytes, etc.).
  • the formulation may be used in conjunction with a flow cytometer, immunofluorescence staining, immunohistochemistry, fluorescence microscopy, or other equipment or techniques capable of measuring cell count.
  • the agent for measuring comprises an agent for measuring the expression level of any one or more proteins selected from the group consisting of SFRP4, SOX9, FOXO1, CTHRC1, FZD1, NXN, PRICKLE1, WNT5A, BICC1, IGFBP6, VGLL4, RSPO3, FZD8 and SFRP1, or a gene encoding the same, and the agent for measuring further comprises an agent for measuring the expression level of any one or more proteins selected from the group consisting of PPP2R3A, DKK3, GREM1, FERMT1, GRB10, APCDD1L, ALPK2, BMP2, TPBG, LZTS2 and FZD6, or a gene encoding the same, and the diagnostic composition is provided, wherein the measurement of the number of cells is measured from a biological tissue of a target individual.
  • the biological tissue may be used for experiments such as histological analysis (H&E staining, alizarin red staining, etc.), image analysis (microCT), and transcriptome analysis (RNA sequencing) after undergoing a preprocessing process such as fixation, decalcification, and freezing.
  • H&E staining alizarin red staining, etc.
  • microCT image analysis
  • RNA sequencing transcriptome analysis
  • the bone disease is osteoporosis, osteopenia, osteomalacia, rickets, Paget's disease of bone, osteonecrosis, osteonecrosis of the femoral head (ONFH), Legg-Calve-Perthes disease, Kienbock's disease, Preiser's disease, Scheuermann's disease, osteomyelitis, tuberculous osteomyelitis, Pott's disease, osteosarcoma, chondrosarcoma, Ewing's sarcoma, metastatic bone cancer.
  • the present invention provides a diagnostic composition for at least one of osteoarthritis, osteodysplasia, osteomyelitis, osteopenia, osteomyelitis, osteoarthritis, osteomyelitis metastasis, osteophyte formation, osteopenia, osteopenia, osteomyelitis, osteopenia, osteopenia, osteopenia, osteopenia metastasis ...
  • the diagnostic composition further comprises a means for detecting the ratio of fibrochondrocytes (FC) and chondrogenic lineage cells (CLC) to uncommitted stromal cells (USC).
  • FC fibrochondrocytes
  • CLC chondrogenic lineage cells
  • USC uncommitted stromal cells
  • FCs fibrochondrocytes
  • CLCs chondrogenic lineage cells
  • USCs uncommitted stromal cells
  • the means may include, for example, antibodies, fluorescently labeled probes, primers, or probes that bind to marker genes or surface markers specifically expressed in each cell subtype, and may be used in conjunction with a flow cytometer, an immunofluorescence microscope, or a qPCR device. More specifically, the means may include biomolecules or analysis devices that can quantify the number of each cell population by targeting fibrochondrocyte-specific markers, chondrogenic lineage cell-specific markers, and undifferentiated stromal cell-specific markers.
  • a diagnostic composition wherein the agent capable of measuring the expression level comprises a primer or probe that specifically binds to at least one gene selected from the group consisting of SFRP4, SOX9, FOXO1, CTHRC1, FZD1, NXN, PRICKLE1, WNT5A, BICC1, IGFBP6, VGLL4, RSPO3, FZD8 and SFRP1, and the agent capable of measuring the expression level is an antibody, peptide, aptamer or compound that specifically binds to at least one protein selected from the group consisting of SFRP4, SOX9, FOXO1, CTHRC1, FZD1, NXN, PRICKLE1, WNT5A, BICC1, IGFBP6, VGLL4, RSPO3, FZD8 and SFRP1.
  • primer or probe that specifically binds to one or more genes refers to an oligonucleotide that can complementarily bind to a nucleic acid sequence of a specific gene (e.g., SFRP4, FOXO1, RSPO3, etc.) and selectively detect the expression level or presence of the gene.
  • the primer or probe can be used, for example, in reverse transcription polymerase chain reaction (reverse transcription PCR), real-time quantitative PCR (qPCR), digital PCR (dPCR), RNA in situ hybridization, or microarray analysis.
  • reverse transcription PCR reverse transcription polymerase chain reaction
  • qPCR real-time quantitative PCR
  • dPCR digital PCR
  • RNA in situ hybridization or microarray analysis.
  • Such primers or probes can target one or more of exons, introns, 5' or 3' untranslated regions (UTRs) of the gene, and are designed to minimize non-specific binding through specific binding.
  • LNA Locked nucleic acids
  • LNA nucleosides contain common nucleic acid bases of DNA and RNA and can form base pairs according to the Watson-Crick base pairing rule. However, due to the molecule's "locking" caused by the methylene bridge, LNA does not form an ideal shape in Watson-Crick binding.
  • LNA When LNA is included in a DNA or RNA oligonucleotide, LNA can pair more quickly with a complementary nucleotide chain, thereby increasing the stability of the double helix.
  • an antibody, peptide, aptamer or compound that specifically binds to one or more proteins refers to a biorecognition molecule that can detect the presence of a specific protein (e.g., SFRP4, FOXO1, RSPO3, etc.) or affect its biological function by selectively binding to an epitope or functional structure of the protein.
  • the biorecognition molecule may include a monoclonal antibody, a polyclonal antibody, a humanized antibody, an antibody fragment (Fab, scFv, etc.), a synthetic or derived peptide, a nucleotide-based aptamer, or a small molecule compound.
  • the biorecognition molecule may be used for various analysis and diagnostic purposes such as ELISA, immunohistochemistry (IHC), flow cytometry, Western blot, biosensor or targeted therapeutic agent development, and may be designed or selected to have high specificity and binding affinity for the protein.
  • aptamer refers to an oligonucleotide or peptide molecule, and the general content of aptamers is described in detail in the literature [Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine”. J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. 95(24): 142727(1998)].
  • a "primer” refers to a fragment that recognizes a target gene sequence, and includes a pair of forward and reverse primers, but is preferably a pair of primers that provide analysis results with specificity and sensitivity. High specificity can be achieved when the nucleic acid sequence of the primer is a sequence that does not match a non-target sequence present in the sample, thereby amplifying only the target gene sequence containing the complementary primer binding site and not causing non-specific amplification.
  • probe means a substance that can specifically bind to a target substance to be detected in a sample, and means a substance that can specifically confirm the presence of the target substance in the sample through said binding.
  • the type of probe is not limited to a substance commonly used in the art, but is preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA, and most preferably PNA.
  • the probe includes a biomaterial derived from or similar to a living organism or manufactured in vitro, and may be, for example, an enzyme, a protein, an antibody, a microorganism, an animal or plant cell and organ, a nerve cell, DNA, and RNA.
  • DNA includes cDNA, genomic DNA, and oligonucleotides
  • RNA includes genomic RNA, mRNA, and oligonucleotides
  • proteins may include antibodies, antigens, enzymes, peptides, etc.
  • a kit for diagnosing a bone disease comprising a bone disease composition, which comprises an agent for measuring the number of at least one cell selected from the group consisting of a fibrochondrocyte (FC), a chondrogenic lineage cell (CLC), an uncommitted stromal cell (USC), and an adipogenic lineage cell (ALC).
  • FC fibrochondrocyte
  • CLC chondrogenic lineage cell
  • USC uncommitted stromal cell
  • AAC adipogenic lineage cell
  • a bone disease diagnosis device comprising: a measuring unit for measuring the number of at least one cell selected from the group consisting of fibrochondrocytes (FC), chondrogenic lineage cells (CLC), uncommitted stromal cells (USC), and adipogenic lineage cells (ALC) in a biological sample isolated from a target individual; and a detection unit for detecting the ratio of fibrochondrocytes (FC) and chondrogenic lineage cells (CLC) to uncommitted stromal cells (USC) based on the number of cells measured by the measuring unit; a judgment unit for determining that the target individual is likely to have a bone disease or is corresponding to a bone disease when the ratio increases compared to a control group; and an output unit for outputting the result determined by the judgment unit.
  • FC fibrochondrocytes
  • CLC chondrogenic lineage cells
  • USC uncommitted stromal cells
  • AAC adipogenic lineage cells
  • a device for diagnosing bone diseases which is at least one of osteodystrophy, osteodystrophy (OD), osteodystrophy (OD), osteopenia, osteomyelitis, osteoarthritis, osteomyelitis, osteopenia, osteomyelitis metastasis, osteophyte formation, osteopenia, osteopenia, osteopenia, osteopenia, osteopenia, osteopenia metastasis ...
  • a composition for predicting the stage of bone disease comprising an agent for measuring the number of uncommitted stromal cells (USC) or adipogenic lineage cells (ALC).
  • USC uncommitted stromal cells
  • ALC adipogenic lineage cells
  • the composition for predicting the stage of a bone disease comprises an agent for measuring the expression level of any one or more proteins selected from the group consisting of RSPO3, ANKRD6, PRICKLE2, FZD5, GPC3, FZD8, FZD4, SFRP1, and MAGI2, or a gene encoding the same
  • the composition for predicting the stage of a bone disease further comprises an agent for measuring the expression level of any one or more proteins selected from the group consisting of DAAM2, FZD3, FRZB, and PRICKLE1, or a gene encoding the same, and the measurement of the number of cells is measured from a biological tissue of a target individual, and the composition for predicting the stage of a bone disease is provided.
  • the composition for predicting the stage of a bone disease is provided, wherein the stage is one of 1, 2, 3A, 3B and 4 according to the ARCO (Association Research Circulation Osseous) classification criteria, and the diagnostic composition further comprises a means for detecting the ratio of adipogenic lineage cells (ALC) to uncommitted stromal cells (USC).
  • ALC adipogenic lineage cells
  • USC uncommitted stromal cells
  • ADCs adipogenic lineage cells
  • USCs uncommitted stromal cells
  • the means may include, for example, antibodies, fluorescently labeled probes, primers, or probes that bind to marker genes or surface markers specifically expressed in each cell subtype, and may be used in conjunction with a flow cytometer, an immunofluorescence microscope, or a qPCR device. More specifically, the means may include biomolecules or analytical devices that can quantify the number of each cell population by targeting markers specific to adipogenic lineage cells and undifferentiated stromal cells.
  • a composition for predicting the stage of a bone disease comprising a primer or probe that specifically binds to at least one gene selected from the group consisting of RSPO3, ANKRD6, PRICKLE2, FZD5, GPC3, FZD8, FZD4, SFRP1, and MAGI2, and the agent capable of measuring the expression level is an antibody, peptide, aptamer, or compound that specifically binds to at least one protein selected from the group consisting of RSPO3, ANKRD6, PRICKLE2, FZD5, GPC3, FZD8, FZD4, SFRP1, and MAGI2.
  • kits for predicting the stage of a bone disease which comprises a composition for predicting the stage of a bone disease, the composition comprising a preparation for measuring the number of uncommitted stromal cells (USC) and adipogenic lineage cells (ALC).
  • USC uncommitted stromal cells
  • ALC adipogenic lineage cells
  • the kit provides a kit for predicting bone disease stage, which is an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.
  • a kit for predicting bone disease stage which is an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.
  • a method for providing information necessary for predicting the stage of a bone disease comprising the step of measuring the number of uncommitted stromal cells (USC) and adipogenic lineage cells (ALC).
  • USC uncommitted stromal cells
  • ALC adipogenic lineage cells
  • the method for providing the information further comprises a step of measuring the expression level of any one or more proteins selected from the group consisting of RSPO3, ANKRD6, PRICKLE2, FZD5, GPC3, FZD8, FZD4, SFRP1, and MAGI2, or a gene encoding the same; and a method for providing information necessary for predicting the stage of a bone disease is provided, wherein the method for providing the information further comprises a step of measuring the expression level of any one or more proteins selected from the group consisting of DAAM2, FZD3, FRZB, and PRICKLE1, or a gene encoding the same; and the method for providing the information further comprises a step of detecting the ratio of adipogenic lineage cells (ALC) to uncommitted stromal cells (USC); And if the ratio increases compared to the control group, a step of determining that the target individual is likely to have or corresponds to a high-stage bone disease; A method for providing information necessary for predicting the stage of a bone
  • a diagnostic device for predicting the stage of a bone disease comprising: a measuring unit for measuring the number of at least one cell selected from the group consisting of uncommitted stromal cells (USC) and adipogenic lineage cells (ALC) in a biological sample isolated from a target individual; and a detection unit for detecting the ratio of adipogenic lineage cells (ALC) to uncommitted stromal cells (USC) based on the number of cells measured by the measuring unit; a judgment unit for determining that the target individual is likely to have or corresponds to a high-stage bone disease when the ratio increases compared to a control group; and an output unit for outputting the result determined by the judgment unit.
  • a measuring unit for measuring the number of at least one cell selected from the group consisting of uncommitted stromal cells (USC) and adipogenic lineage cells (ALC) in a biological sample isolated from a target individual
  • ALC adipogenic lineage cells
  • USC uncommitted stromal cells
  • a diagnostic device for predicting the stage of a bone disease comprising: a measuring unit for measuring the expression level of at least one protein selected from the group consisting of RSPO3, ANKRD6, PRICKLE2, FZD5, GPC3, FZD8, FZD4, SFRP1, and MAGI2, or a gene encoding the same, in a biological sample isolated from a target individual; and a judgment unit for determining that the target individual is likely to have or corresponds to a high-stage bone disease when the expression level of the protein or the gene encoding the same measured by the measuring unit increases compared to a control group; and an output unit for outputting the result determined by the judgment unit; wherein the high-stage bone disease corresponds to stages 3B and 4 according to the ARCO classification criteria.
  • the bone disease is osteoporosis, osteopenia, osteomalacia, rickets, Paget's disease of bone, osteonecrosis, osteonecrosis of the femoral head (ONFH), Legg-Calve-Perthes disease, Kienbock's disease, Preiser's disease, Scheuermann's disease, osteomyelitis, tuberculous osteomyelitis, Pott's disease, osteosarcoma, chondrosarcoma, Ewing's sarcoma, metastatic bone cancer.
  • a diagnostic device for predicting the stage of bone disease which is at least one of osteoarthritis, osteodysplasia, osteomyelitis, osteopenia, osteomyelitis metastasis, osteophyte formation imperfecta, osteopetrosis, hypophosphatasia, fibrous dysplasia, osteoarthritis, and diffuse idiopathic skeletal hyperostosis (DISH), is provided.
  • the present invention provides a new cellular and molecular mechanism of disease progression by elucidating spatially compartmentalized stromal cell subtypes and the WNT signaling network between them within osteonecrosis of the femoral head (ONFH) lesions.
  • the present invention enables disease diagnosis, stage prediction, and therapeutic target discovery based on the upstream regulatory role of ALC and WNT signature.
  • the present invention proposes fate reprogramming beyond the existing structure-centered diagnosis and treatment approach, and suggests the possibility of precision diagnosis or intervention strategies based on this.
  • Figure 1 illustrates that ONFH lesions are divided into necrotic and borderline areas, with distinct imaging and histological characteristics.
  • Figure 2 illustrates transcriptome differences between control, necrotic, and borderline samples using bulk RNA-seq analysis.
  • PCA Principal component analysis
  • FIG. 1 A Venn diagram showing DEGs in necrotic vs. control, borderline vs. control, and necrotic vs. borderline comparisons.
  • GSEA analysis between necrotic vs. control revealed that epithelial-mesenchymal transition (EMT) was the most significantly enriched pathway among the Hallmark pathways.
  • GSEA analysis between borderline vs. necrotic samples revealed adipogenesis as the top pathway.
  • (f-g) Enrichment score plots showing the contribution of core genes to the EMT and adipogenesis pathways, respectively.
  • Figure 3 confirms the increase in FCs and CLCs in ONFH using single-cell RNA-seq analysis.
  • FCs fibrochondrocytes
  • ALCs adipose cells
  • USCs undifferentiated stromal cells
  • OLCs osteocytes
  • CLCs chondrocytes
  • FIG. 4 shows that WNT signaling was identified as a key pathway involved in ONFH pathogenesis in single-cell and bulk RNA-seq integrated analyses.
  • GOBP biological process
  • Figure 5 shows that ALC-derived signals act on different stromal cell subtypes and are associated with ONFH stage progression.
  • CellChat analysis shows that ALC has the highest sender-to-receiver ratio, suggesting that it acts as a key upstream signaler.
  • FC WNT signature score average z-score
  • Figure 6 is a representative H&E staining image used to define the cell density grade of tissue sections.
  • Figure 7 shows single-cell transcriptome analysis demonstrating stromal lineage-specific gene programs and expression differences in ONFH.
  • Figure 8 illustrates that ALC acts as an upstream regulator of WNT-related stromal remodeling in ONFH.
  • Figure 9 compares the expression levels of specific genes (RSPO3, PRICKLE1, FZD8, SFRP1) belonging to the ALCs-Wnt gene family between the control group and the ONFH patient group, showing that the expression of these genes significantly increases in the ONFH patient group compared to the control group, and in particular, RSPO3 and FZD8 show a tendency to gradually increase according to the stage of the disease.
  • specific genes RSPO3, PRICKLE1, FZD8, SFRP1
  • Figure 10 To assess regional shifts in stromal subtype balance, normalized cell counts (i.e., cell counts divided by the number of USCs in the same sample) were used to calculate cell type ratios per sample. Two ratios, (FC + CLC)/USC and ALC/USC, were evaluated, and the Wilcoxon rank-sum test was used to compare ratios between ONFH and ARCO stage 3A and between ONFH and ARCO stage 4 groups.
  • HOA hip osteoarthritis
  • FNF femoral neck fracture
  • Microcomputed tomography (microCT), hematoxylin-eosin (H&E), and alizarin red staining were performed on femoral head specimens from patients with osteonecrosis of the femoral head (ONFH), osteoarthritis of the hip (HOA), and femoral neck fracture (FNF) and cadaveric controls.
  • MicroCT analysis was used to evaluate the architecture of the subchondral bone tissue and identify focal calcifications. After fixation and decalcification, tissue sections were stained with H&E to evaluate tissue architecture and stromal remodeling, and alizarin red staining to identify calcium deposition. Stromal cellularity was semiquantitatively assessed according to histological criteria on H&E-stained sections.
  • Cellular density was scored as follows: A score of 0 indicates an acellular or nearly acellular area, a score of 1 indicates a low cell density with sparsely scattered stromal cells, a score of 2 indicates an intermediate cell density with increased but non-aggregated stromal cells, and a score of 3 indicates a high cell density with densely packed stromal cells. These criteria were used to quantify and compare stromal cell density between necrotic areas, marginal regions, and control areas (see Figure 6).
  • Regions of interest were divided into necrotic, borderline, and structurally preserved regions based on gross morphology and microCT findings, and these were used as indicators for subsequent transcriptomic analyses.
  • RNA samples were collected from the necrotic and border areas of ONFH femoral head specimens, and control samples were obtained from the femoral heads and femoral necks of HOA or FNF patients.
  • Total RNA was extracted using TRIzol reagent (Invitrogen, San Diego, CA, USA), and transcriptome profiling was performed by bulk RNA sequencing (QuantSeq 3' mRNA-Seq service, Ebiogen, Seoul, Korea). RNA quality assessment was performed using a TapeStation4000 system (Agilent, Santa Clara, CA, USA).
  • cDNA libraries were constructed using oligo-dT primer sets with Illumina-compatible sequences tethered to the 5' end (QuantSeq 3' mRNA-Seq Library Prep Kit FWD, Lexogen, Vienna, Austria), and reverse transcription was performed. High-throughput sequencing was performed using the Illumina NextSeq 550 platform (San Diego, CA, USA) in a single-end read mode. The resulting raw reads were trimmed with BBDuk and aligned to the human reference genome (hg19) using Bowtie2. Read counts were generated using Bedtools, and after normalization, differentially expressed genes (DEGs) were derived using EdgeR.
  • DEGs differentially expressed genes
  • PCA Principal component analysis
  • GO gene ontology enrichment analysis
  • RNA-sequencing data (Figshare, https:/doi.org/10.6084/m9.figshare.19243722) obtained from alcohol-induced ONFH patients were downloaded and processed using the Seurat package for integrated analysis. Data were normalized using SCTransform, and dataset integration was performed using Harmony to correct for batch effects between samples. Dimensionality reduction was performed first using principal component analysis (PCA), followed by Uniform Manifold Approximation and Projection (UMAP) for visualization. Cell clustering was performed using the FindNeighbors and FindClusters functions in Seurat.
  • PCA principal component analysis
  • UMAP Uniform Manifold Approximation and Projection
  • FCs fibrochondrocytes
  • ALCs adipogenic lineage cells
  • CLCs chondrogenic lineage cells
  • OLCs osteogenic lineage cells
  • USCs uncommitted stromal cells
  • FCs fibrochondrocytes
  • MicroCT image analysis showed that calcium deposition increased in the necrotic areas of the femoral head compared to the cadaveric control group, particularly in the non-trabecular area.
  • the altered calcium deposition was observed as radiolucent foci surrounded by sclerotic rims (Fig. 1a).
  • Histological analysis using hematoxylin-eosin (H&E) staining showed that cellularity was highest in the necrotic areas, intermediate in the border areas, and low in the normal samples (Fig. 1b).
  • Quantitative analysis confirmed that the difference in cellularity between each area was statistically significant (Fig. 1c).
  • Alizarin Red staining revealed dense, amorphous calcium aggregates within necrotic and border areas, which were interpreted as representing ectopic or non-trabecular mineralization rather than organized bone matrix (Fig. 1d). Notably, fibrochondrocyte-like cells were abundant within these calcified areas.
  • Transcriptome analysis revealed EMT in necrotic areas and adipogenic differentiation in border areas.
  • PCA principal component analysis
  • Fig. 2a Hierarchical clustering analysis visualized as a heatmap also highlighted the presence of distinct gene expression signatures at each site (Fig. 2b).
  • DEGs differentially expressed genes
  • Fig. 2c Venn diagram analysis of differentially expressed genes (DEGs) revealed common or site-specific molecular signatures between necrotic vs. control and border vs. necrotic regions (Fig. 2c).
  • GSEA Gene set enrichment analysis revealed that epithelial-mesenchymal transition (EMT) was significantly enriched in necrotic regions compared to controls (Fig.
  • EMT-related genes were functionally classified into two modules: one was a mesenchymal program that generates matrix (e.g., COL1A1, PLOD1), and the other was a program associated with remodeling and fibrotic activation (e.g., FN1, MMP14, VEGFC) (Fig. 2h).
  • matrix e.g., COL1A1, PLOD1
  • remodeling and fibrotic activation e.g., FN1, MMP14, VEGFC
  • adipogenesis-related genes expressed in the border region were functionally classified into three modules: adipogenic (e.g., ADIPOQ, PPARG), inflammatory (e.g., IL6, CXCL2), and metabolic stress-related (e.g., HSPA5, DDIT3), each of which showed different expression patterns in each region (Fig. 2i).
  • adipogenic e.g., ADIPOQ, PPARG
  • inflammatory e.g., IL6, CXCL2
  • metabolic stress-related e.g., HSPA5, DDIT3
  • FCs fibrochondrocytes
  • CLCs chondrocyte-like cells
  • mesenchymal stromal cells mesenchymal stromal cells, endothelial cells, lymphoid cells, and myeloid cells (Fig. 7a). The identity of each lineage was confirmed based on the expression of representative marker genes: mesenchymal stromal cells were defined by COL1A1 and ACTA2, endothelial cells by PECAM1, lymphoid cells by CD3D, and myeloid cells by LYZ (Fig. 7b).
  • FCs fibrochondrocytes
  • ALCs adipogenic lineage cells
  • CLCs chondrogenic lineage cells
  • OLCs osteogenic lineage cells
  • USCs uncommitted stromal cells
  • Each cluster represents a unique marker gene signature, which is presented as a heatmap (Fig. 3b).
  • Comparison of ONFH and control samples revealed that the proportions of FC and CLC were significantly increased in ONFH, as evidenced by the ratio distribution plot (Fig. 3c) and the chi-square test for relative abundance (Fig. 3d).
  • chondrogenic differentiation-related marker genes such as COL9A1, AMTN, and CNMD, were upregulated in ONFH-derived cells, suggesting that fibrochondral differentiation was promoted under pathological conditions (Fig. 7d).
  • Integrated single-cell and bulk RNA sequencing analyses identified spatially discrete Wnt pathway involvement patterns in ONFH.
  • RNA-sequencing single-cell RNA-sequencing (scRNA-seq)-based marker genes as the input gene set and expression datasets derived from bulk RNA-sequencing (DEG rankings, e.g., necrotic vs. control, borderline vs. necrotic).
  • DEG rankings e.g., necrotic vs. control, borderline vs. necrotic.
  • Each marker gene set served as a cell-type-specific signature, allowing for deconvolution of transcriptional programs observed in necrotic or borderline regions at the cell-type level.
  • necrotic and control areas revealed a significant enrichment of marker genes for FCs and CLCs, suggesting a predominance of fibrocartilage and chondrogenic differentiation transcriptional programs in necrotic lesions (Fig. 4a).
  • a heatmap of core genes enriched in FCs also revealed the same trend, with these genes being highly expressed in necrotic areas, moderately expressed in border areas, and lowly expressed in controls (Fig. 4b).
  • Wnt signature scores were calculated for each stromal cell cluster using Seurat's AddModuleScore function. FC-specific Wnt gene-based scores were highest in FCs, and ALC-specific Wnt gene-based scores were highest in ALCs, confirming that each Wnt program is selectively activated within its respective cell type (Fig. 4i).
  • ALC-derived WNT signaling is associated with stromal cell differentiation and ONFH progression.
  • FC and ALC Wnt signature scores were calculated and analyzed.
  • GSEA Gene set enrichment analysis
  • FCs fibrochondrocytes
  • ALCs adipose-like cells
  • Wnt signature score was calculated, summarizing the overall expression levels of subtype-specific Wnt-related genes within each sample.
  • Raw RNA-seq count data were normalized using the log2 counts-per-million (logCPM) method implemented in the edgeR package. This normalization compensates for differences in library sizes between samples by adjusting the number of sequencing reads, enabling meaningful comparisons of gene expression.
  • the log transformation expressed the relative abundance of each gene on a log2 scale, reducing the influence of extreme values in the variance.
  • a z-score transformation was applied to each gene's logCPM value using R's scale function. The z-score represents how many standard deviations a gene's expression in a given sample deviates from the mean expression across all samples. This transformation ensures that all genes contribute equally to the composite score, regardless of their absolute expression levels.
  • the z-score logCPM values of the subtype-specific Wnt pathway genes defined in Figures 4d and 4h were averaged to derive a Wnt signature score for each sample.
  • This composite score reflects the coordinated activity of the subtype-specific Wnt signaling pathway and can be used for cross-sample comparisons and correlation analyses with disease severity.
  • FC Wnt score calculated based on the FC-specific Wnt-related genes defined in Fig. 4d, significantly distinguished ONFH samples from controls but did not correlate with disease severity (ARCO stage) (Figs. 5d-e).
  • ALC Wnt score based on the ALC-specific genes defined in Fig. 4h not only distinguished ONFH from controls but also showed a positive correlation with ARCO stage (Figs. 5f-g).
  • FC-related genes such as SFRP4, SOX9, FOXO1, and CTHRC1
  • ALC-related genes such as RSPO3, ANKRD6, PRICKLE2, and FZD5
  • This analytical strategy also known as metagene or signature score analysis, is widely used in transcriptome studies to summarize differential expression patterns following clustering or enrichment analysis. This not only allows for robust quantification of signaling pathway activity and standardized comparisons between samples, but can also be easily applied to other signaling pathways and cellular contexts.
  • ALC-derived Wnt signaling contributes to disease progression by regulating stromal cell differentiation and shaping spatial signaling dynamics within the femoral head.

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Abstract

La présente invention concerne l'élucidation de changements de distribution spatiale parmi des sous-types de cellules stromales associées à la progression pathologique de la nécrose aseptique de la tête fémorale (ONFH) et un mécanisme de régulation de voies de signalisation WNT, et le diagnostic et la prédiction de stade de maladies sur la base de ceux-ci. En particulier, il a été découvert que l'ALC agit en tant que régulateur en amont de la différenciation stromale, et il est suggéré que des scores de signature WNT à base de FC et d'ALC peuvent être utilisés en tant qu'indicateurs pour le diagnostic ou la prédiction de stade de maladies, ce qui fournit une nouvelle stratégie pour le diagnostic précis de l'ONFH et l'intervention d'un traitement à base de cellules stromales.
PCT/KR2025/007902 2024-06-11 2025-06-10 Composition pour diagnostiquer la nécrose aseptique de la tête fémorale ou prédire le stade de nécrose aseptique de la tête fémorale Pending WO2025258968A1 (fr)

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KR20230029691A (ko) * 2020-05-29 2023-03-03 더 락커펠러 유니버시티 류마티스 관절염 플레어의 마커 및 세포 선행물질
KR102622158B1 (ko) * 2020-08-05 2024-01-08 가톨릭대학교 산학협력단 골관절염 진단용 조성물 및 골관절염 진단을 위한 정보 제공방법
WO2024114336A1 (fr) * 2022-11-30 2024-06-06 北赛泓升(北京)生物科技有限公司 Procédé de préparation de microtissu cartilagineux dérivé de la différenciation de cellules souches pluripotentes humaines et son utilisation

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KR101725025B1 (ko) 2016-11-25 2017-04-10 서울대학교산학협력단 무형성골질환 진단용 miRNA 마커

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JP5548972B2 (ja) * 2007-02-09 2014-07-16 国立大学法人大阪大学 退行性疾患の予防用又は治療用の薬剤のスクリーニング方法
JP2019136042A (ja) * 2013-12-02 2019-08-22 国立大学法人京都大学 Ｆｇｆｒ３病の予防および治療剤ならびにそのスクリーニング方法
KR101822731B1 (ko) * 2016-02-17 2018-02-02 충북대학교 산학협력단 파골세포 분화를 촉진하는 ｍｍｐ－９의 용도
KR20230029691A (ko) * 2020-05-29 2023-03-03 더 락커펠러 유니버시티 류마티스 관절염 플레어의 마커 및 세포 선행물질
KR102622158B1 (ko) * 2020-08-05 2024-01-08 가톨릭대학교 산학협력단 골관절염 진단용 조성물 및 골관절염 진단을 위한 정보 제공방법
WO2024114336A1 (fr) * 2022-11-30 2024-06-06 北赛泓升(北京)生物科技有限公司 Procédé de préparation de microtissu cartilagineux dérivé de la différenciation de cellules souches pluripotentes humaines et son utilisation

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