The Role Of Cadm1~+ Skeletal Stem Cells In The Progression Of Osteoarthritis

Background and objectiveOsteoarthritis（OA）is a common degenerative joint disorder characterized by articular cartilage degeneration and subchondral bone sclerosis,which severely affects patients’quality of life and cannot be cured at present.Skeletal stem cells（SSC）are tissue-specific stem cells derived from bone and cartilage,capable of self-renewing and differentiating into osteoblasts and chondrocytes,and actively involved in bone development,homeostasis and repair.SSCs are highly heterogeneous cell populations.In recent years,with the development of flow cytometry,single-cell RNA sequencing（sc RNA-seq）and lineage tracing techniques,increasing SSC populations with different localization and function have been identified.It was reported that endogenous SSCs can be activated to promote articular cartilage regeneration,which can be used to repair cartilage damage in osteoarthritis.However,the underlying role of SSCs in the pathogenesis of osteoarthritis has not been investigated so far.Our previous work revealed the origin of human embryonic SSCs via discovering a PDGFRA^low/-PDPN⁺CADM1⁺embryonic skeletal stem/progenitor cell（e SSPC）population,which was mainly localized in the joint and periosteum,with the ability to self-renew and generate osteochondral lineage cells.Cell Adhesion Molecule 1（CADM1）was first reported as a tumor suppressor gene and has recently been identified as a potential marker for bone formation.In the present study,we found that the expression level and distribution of Cadm1 significantly changed in the knees of post-traumatic osteoarthritis（PTOA）mice and patients with OA.In order to investigate whether Cadm1 can be used as an evolutionarily conserved marker for SSCs,and to clarify its role in the progression of OA,the following studies were conducted.Methods1.Time-sequenced sc RNA-seq datasets of mouse limb buds and postnatal long bones were collected from GEO database for further analyses;2.Expression pattern of Cadm1 during mouse long bone development was analyzed at single-cell level;3.Immunohistochemical staining was performed to detect the distribution of Cadm1 in the developing mouse long bone;4.The osteochondrogenic potential of Cadm1⁺SSCs was analyzed by bioinformatics analysis and in vitro assays;5.PTOA mouse model was generated and the mouse tibial plateau was collected for enzymatic digestion,cell sorting and sc RNA-seq.The transcriptional changes of Cadm1⁺SSCs in the progression of OA was analyzed at single-cell level;6.Immunohistochemical staining was used to detect changes in the distribution of Cadm1 during the progression of OA.Results1.Integrated sc RNA-seq analysis of embryonic limb buds and postnatal long bones identified a Cadm1-positive population during mouse long bone development;2.Bioinformatics analysis and in vitro assays showed that the Cadm1-positive population had osteogenic and chondrogenic potential;3.sc RNA-seq analysis showed that the differentiation preference of Cadm1⁺SSCs changed after PTOA,with osteoblastic potential elevated and chondrogenic potential decreased;4.Immunohistochemical staining showed that the expression and distribution of Cadm1 changed with the progression of OA.ConclusionCadm1 marks both human and mouse SSC populations,and is involved in the pathological progression of osteoarthritis through its altered differentiation preference.