Recent studies to harness the capacity of BMP2 to induce de novo bone formation for tissue and/or limb regeneration resulted in the identification of a novel chondro-osseous progenitor that is primed to engraft into sites of active bone formation, even when delivered systemically. These cells were identified using lineage tracing for glial high affinity glutamate transporter GLAST, which has been shown to be restricted to neural stem cells, astrocytes, and chondrocytes. Transcriptome analysis, using single cell RNAseq, led to the identification of a highly replicating stem cell that appears to undergo an epithelial to mesenchymal transition (EMT) to become a cell that expresses a large number of chondrocyte and osteoblast associated transcripts similar to a recently reported periosteal stem cell. Surprisingly, while the replicating stem cell expressed GLAST as well as neurogranin, another synaptic protein, and β-tubulin 3/TUJ1; whereas mature chondrocytes did not express this protein supporting its neural association. In further support several or all clusters also expressed several Schwann cell markers including N-cadherin, Krox20, and myelin basic protein. Transcriptome analysis also revealed that this cluster is highly expressing H2A.Z and SET methyltransferase with decreasing synthesis in other clusters/cells correlating with pseudotime or cell maturation. The data collectively has led us to hypothesize that Schwann cells upon exposure to BMP2 undergo epigenetic reprogramming and reactivation of primed tissue specific developmental enhancers leading to a neural crest progenitor/stem cell phenotype. Further, these stem/progenitors are then able to undergo re-differentiation into chondrocytes and osteoblasts. To test this hypothesis, we propose to utilize the phospholipid protein 1 (Plp1) promoter driving Cre recombinase and tomato redfloxSTOPflox mouse to be able to initially label Schwann cells and allow for reporter expression in downstream reprogrammed cells. GLAST-TR+ cells obtained 48 hours after delivery of BMP2, will also be included to obtain potential earlier clusters. GLAST-TR+ and/or Plp1-TR+ cells from soft tissues surrounding the region of de novo bone formation 24 hours after delivery of BMP2 and subjected to single cell RNAseq and single cell ATACseq, which will identify changes in chromatin within the cell populations that might reflect reprogramming of the cells to the highly replicative stem cell. The resultant data will be correlated with the scRNAseq data to provide a molecular mechanism for the generation of these cells. From this data one can then start to envision ex vivo reprogramming of cells to form these chondro- osseous progenitors that will home to sites of active bone formation.

The grant proposes to identify potential epigenetic and transcriptional changes in nonmyelinating Schwann cells associated with peripheral nerves that lead to the formation of a novel highly replicating stem cell and chondro-osseous progenitor. Preliminary data suggests delivery of BMP2 may lead to epigenetic reprogramming to form a neural crest stem cell-like state, where they re-differentiate into a chondro-osseous progenitor. We propose to use single cell RNAseq and single cell ATACseq to identify and characterize this mechanism.