Project description:The development of alternatives for autologous bone grafts is a major focus of bone tissue engineering. To produce living implants, the use of skeletal stem and progenitor cells (SSPCs) are envisioned as key ingredients. SSPCs can be obtained from bone marrow, adipogenic tissue, dental pulp and periosteum. Human periosteum-derived cells (hPDCs) exhibit a number of progenitor characteristics and have well-documented in vivo bone formation capacity. Here, we have characterized and compared hPDCs derived from tibia with new sources of hPDCs, i.e. from maxilla and mandible (craniofacial hPDCs, as a potential source for tissue engineered implants for craniofacial applications.
Project description:Bone regeneration is a highly efficient process allowing scarless healing after injury. The periosteum, the outer layer of bones, is a critical source of skeletal stem/progenitor cells (SSPCs), as well as immune, endothelial and neural cells during bone repair. In our study, we generated a single-nuclei atlas of the murine periosteal response to bone fracture. We generated single nuclei datasets from uninjured periosteum and from injured periosteum and hematoma/fracture callus at days 5 and 7 post-injury from wild-type mice.
Project description:Periosteum is a major source of skeletal stem/progenitor cells (SSPCs) during bone repair. However, the cellular composition of the periosteum is poorly characterized. Here, we provide single-cell RNAseq data of periosteal cells isolated by explant culture at steady state and at day 3 post-tibial fracture. We found that periosteal cell populations are heterogeneous containing several sub-populations of SSPCs. Upon fracture, SSPCs are activated by leaving their mesenchymal state, and engaging into fibrogenesis prior to chondrogenesis.
Project description:Mesenchymal stromal cells (hMSCs) are advancing into the clinic but the therapeutic efficacy of hMSCs faces the problem of donor variability. In bone tissue engineering, no reliable markers have been identified which are able to predict the bone-forming capacity of hMSCs prior to implantation. To this end, we isolated hMSCs from 62 donors and characterized systematically their in vitro lineage differentiation capacity, gene expression signature and in vivo capacity for ectopic bone formation. Our data confirms the large variability of in vitro differentiation capacity which did not correlate with in vivo ectopic bone formation. Using DNA microarray analysis of early passage hMSCs we identified a diagnostic bone-forming classifier. In fact, a single gene, CADM1, strongly correlated with the bone-forming capacity of hMSCs and could be used as a reliable in vitro diagnostic marker. Furthermore, data mining of genes expressed correlating with in vivo bone formation represented involvement in neurogenic processes and Wnt signaling. We will apply our data set to predict therapeutic efficacy of hMSCs and to gain novel insight in the process of bone regeneration. Our bio-informatics driven approach may be used in other fields of cell therapy to establish diagnostic markers for clinical efficacy.
Project description:Bone regeneration relies on the activation of skeletal stem cells (SSCs) that still remain poorly characterized. Here, we show that periosteum contains SSCs with high bone regenerative potential compared to bone marrow stromal cells/skeletal stem cells (BMSCs) in mice. Although periosteal cells (PCs) and BMSCs are derived from a common embryonic mesenchymal lineage, post-natally PCs exhibit greater clonogenicity, growth and differentiation capacity than BMSCs. During bone repair, PCs can efficiently contribute to cartilage and bone, and integrate long-term after transplantation. Molecular profiling uncovers genes encoding Periostin and other extracellular matrix molecules associated with the enhanced response to injury of PCs. Periostin gene deletion impairs PC functions and fracture consolidation. Periostin-deficient periosteum cannot reconstitute a pool of PCs after injury demonstrating the presence of SSCs within periosteum and the requirement of Periostin in maintaining this pool. Overall our results highlight the importance of analyzing periosteum and PCs to understand bone phenotypes.
Project description:The shape of the human face is largely genetically determined, but the genetic drivers of craniofacial morphology remain poorly understood. In particular, little is known about the contributions of gene regulatory sequences active in the developing face to craniofacial morphology. Here we used a combination of epigenomic profiling, in vivo characterization of more than 200 craniofacial candidate enhancer sequences in transgenic mice, and targeted deletion experiments to examine the role of distant-acting enhancers in craniofacial development. We identified complex regulatory landscapes with thousands of enhancers genome-wide that drive a remarkable spatial complexity of in vivo expression patterns. The ChIP-seq experiments in this entry was the basis for the genome-wide analysis of craniofacial enhancers and served as the source for substantialin vivo characterization via transgenic reporter mice and for enhancer knockout experiments. p300 ChIP-seq experiment on mouse embryonic tissue (e11.5)
Project description:Endochondral ossification (EO) is the natural route for the regeneration of large and mechanically challenged bone defects. Regeneration occurs via a fibrocartilagenous phase which turns into bone upon vascularization and the formation of a transient collagen type X extra cellular matrix. These two critical initiator of EO are mediated by Hedgehog proteins. We investigated a tissue engineering approach using Sonic Hedgehog (Shh) as a pleiotropic factor regulating the in vitro formation of a vascularized bone tissue precursor for in vivo endochondral bone formation. The tissue engineered graft was formed using human mesenchymal stem cells and prevascularized using human umbilical vein endothelial cells. We show that Shh induced, in vitro, the maturation of the engineered vascular network along with the expression of collagen type X which resulted, in vivo, in an improved vascularization and the rapid formation of large amounts of osteoids through EO. Osteoids further matured into, currently unmatched, clinically relevant amount of lamellar bone including osteoclasts, bone lining cells and bone marrow-like cavities. This result suggests that Hh is a master regulator of EO allowing for the formation of complex tissues with considerable therapeutic potential for bone regeneration. The effect of Cyclopamine on expression of Hedgehog, angiogenesis and axon guidance marker genes was analyzed by seeding a coculture of 92% hMSCs and 8% huvEC supplemented or not in cyclopamine, for 12 days
Project description:The shape of the human face is largely genetically determined, but the genetic drivers of craniofacial morphology remain poorly understood. In particular, little is known about the contributions of gene regulatory sequences active in the developing face to craniofacial morphology. Here we used a combination of epigenomic profiling, in vivo characterization of more than 200 craniofacial candidate enhancer sequences in transgenic mice, and targeted deletion experiments to examine the role of distant-acting enhancers in craniofacial development. We identified complex regulatory landscapes with thousands of enhancers genome-wide that drive a remarkable spatial complexity of in vivo expression patterns. The ChIP-seq experiments in this entry was the basis for the genome-wide analysis of craniofacial enhancers and served as the source for substantialin vivo characterization via transgenic reporter mice and for enhancer knockout experiments.
Project description:Endochondral ossification (EO) is the natural route for the regeneration of large and mechanically challenged bone defects. Regeneration occurs via a fibrocartilagenous phase which turns into bone upon vascularization and the formation of a transient collagen type X extra cellular matrix. These two critical initiator of EO are mediated by Hedgehog proteins. We investigated a tissue engineering approach using Sonic Hedgehog (Shh) as a pleiotropic factor regulating the in vitro formation of a vascularized bone tissue precursor for in vivo endochondral bone formation. The tissue engineered graft was formed using human mesenchymal stem cells and prevascularized using human umbilical vein endothelial cells. We show that Shh induced, in vitro, the maturation of the engineered vascular network along with the expression of collagen type X which resulted, in vivo, in an improved vascularization and the rapid formation of large amounts of osteoids through EO. Osteoids further matured into, currently unmatched, clinically relevant amount of lamellar bone including osteoclasts, bone lining cells and bone marrow-like cavities. This result suggests that Hh is a master regulator of EO allowing for the formation of complex tissues with considerable therapeutic potential for bone regeneration.
Project description:Craniofacial development involves regulation of a compendium of transcription factors, signaling molecules and epigenetic regulators. Histone deacetylases (HDACs) are involved in the regulation of cell proliferation, differentiation and homeostasis across a wide range of tissues, such as brain, cardiovascular system, muscular system, and skeletal system. However, functional role of Hdac4 during craniofacial development is still unclear. In this study, we investigated the effects of Hdac4 knockout in craniofacial skeletal development by conditionally disrupting the Hdac4 gene in cranial neural crest cells (CNCCs) using Cre-mediated recombination. Mice deficient in Hdac4 in CNCCs-derived osteoblasts demonstrated a dramatic decrease in bone formation in frontal bone. In vitro pre-osteoblasts (MC3T3-E1 cells) lacking Hdac4 exhibited reduced proliferation activity in association with dysregulation of cell cycle-related genes. These findings suggest that Hdac4 acts partially as a regulator of craniofacial skeletal development by positively regulating proliferation of CNCCs-derived osteoblasts.