Dissecting The Origins Of Human Embryonic Skeletal Stem And Progenitor Cells

Skeletal stem cells(SSCs)are a group of tissue specific stem cells that continuously generate chondrocyte,osteoblast and stromal cell types,and are capable of self-renewing.As a key event of developmental skeletogenesis,increasing studies revealed that skeletal stem cells played a key function in skeletal development and injury repair.Its emergence,phenotypic enrichment and biological functions have received wide attention in the recent years.Through prospective isolation and genetic lineage tracing studies,multi-potent and self-renewal skeletal stem cells were identified in growth plate,resting zone and periosteum of long bone in mice.In addition,a latest research indicated that skeletal stem cells existed in the growth plate of human17-week-old fetal long bone.However,the origins,molecular characteristics and regulation mechanisms of skeletal stem cells remained to be elucidated.Firstly,we selected and analyzed human limb buds at 5 weeks post conception(5WPC),as well as human limb long bones at 8 weeks post conception(8 WPC)before and after the occurence of the primary ossification center.Integrated analysis of the limb bud and long bone samples revealed 16 subsets.Among them,mesenchyme and epithelium subsets were enriched in limb buds,and we mapped these subsets along proximal-distal and anterior-posterior axis via known characteristic marker genes.The results showed that long bones consisted of chondrocyte and osteo-progenitors.In addition,the results of unsupervised hierarchical clustering analysis,principal component analysis and pseudotime analysis by RNA velocity showed that osteo-chondrogenic progenitor(OCP)that highly expressed TWIST2 and PDGFRA in the limb buds and long bones,indicating the possibility of the earliest skeletal stem and progenitor cells.Secondly,the data of further hierarchical clustering analysis of embryonic long bone osteo-chondrogenic progenitor subsets demonstrated that 3 populations of stem and progenitor cells,including TWIST2⁺limb bud-derived mesenchymal cell(LBDMC),bone marrow stromal cell(BMSC)that highly expressed CXCL12 and PDGFRA,and a group of embryonic skeletal stem and progenitor cells(e SSPC)that were capable of differentiating into chondrocytes and osteo-progenitors.By transcriptional regulation network analysis,we deciphered the embryonic skeletal stem and progenitor cells subset enriched transcription factor FOXP1/2.The results of immunofluorescence staining further showed that most of FOXP1/2 positive cells were distributed in outer layer of perichondrium and inner part of the renascent primary ossification center(POC).It was in concert with the murine data that chondrocyte progenitors were identified in the outer layer of perichondrium.Afterwards,by virtue of perorming differential gene screening,we defined a set of surface markers to enrich embryonic skeletal stem and progenitor cells:PDGFRA^low/-PDPN⁺CADM1⁺.Phenotypic embryonic skeletal stem and progenitor cells were capable of strong colony-forming unit-fibrobalst(CFU-F)ability in vitro.Serial colony-forming unit-fibrobalst and differentiation experiments in vitro and in vivo showed that e SSPCs were capable of self-renewing and generating osteo-chondrogenic lineage cells,while no hematopoiesis supporting ability.Moreover,we analyzed calvaria bones of 8 WPC human embryos,and found a group of neural crest derived cells(NCDCs)with similar phenotype of PDGFRA^low/-PDPN⁺CADM1⁺.Neural crest derived cells were also highly expressing FOXP1/2 transcriptional network and mostly garthered around the sagittal suture and perichondrium of the calvaria bones,indicating of its key role in calvaria development and membranous ossification.Taken together,this study revealed the single cell atlas of mapping early human embryonic skeletogeneis.To the best of our knowledge,it was the first investigation focused on the origin and heterogeneity of human skeletal stem cell,identifying a group of e SSPCs from the level of transcriptomes and biological functions.The study unraveled the cellular heterogeneity and lineage hierarchy during human embryonic skeletogenesis,which was of great significance to deepen the understanding of human skeletogenesis and injury repair mechanism,and might help develop novel cellular therapies to promote bone and cartilage regeneration.