Role of NG2 expressing cells in murine terminal phalanx regeneration
Research using the adult mammalian model shows that regeneration in the limb is limited to the distal most portion of the terminal phalanx. Recent studies suggest that the cellular contributions made to the regenerating system are lineage restricted and that the niche bone marrow hematopoietic stem cell population’s contributions are minimal. These studies however, do not address other residing populations within the bone marrow, specifically the mesenchymal and endothelial stem cell populations. One of the residing populations, the reputed pericyte or perivascular cells, possesses the ability to differentiate into multiple other cell types. To assess the potential contribution of perivascular cells to the regeneration competency of the terminal phalanges, we began by identifying perivascular cells within the terminal phalanx by using two accepted pericyte markers: nerve-glial antigen 2 (NG2) and endosialin (TEM1). Using NG2 and TEM1 in conjunction with vascular marker Tie2 in the Tie2-EGFP murine reporter line, we confirm a large number of perivascular cells in the bone marrow’s unusually well-developed and organized vasculature and a lower density within the connective tissue microvasculature; implicating a great potential contribution from the bone marrow. Post-amputation, we observe a large population of NG2+ and TEM1+ cells within the regenerating blastema region. Co-immunohistochemical studies reveal the blastema have cells that co-express osteogenic and pericyte markers; strongly suggestive of a transdifferentiation event. We attempt to confirm our hypotheses made in our initial assessment by utilizing two independent cell tracing studies: a DiI labeling of the bone marrow of the terminal phalanx to identify a marrow derived cellular contribution to the regenerate and a genetic fate tracing study using transgenic NG2CreERTamR26REYFP mice to confirm a transdifferentiation event. Using a novel in vivo method , we DiI-label the bone marrow content before amputation and trace DiI labeled bone marrow derived cellular contributions to the regenerate. DiI labeled cells were observed within the blastema expressing either endothelial, perivascular, or osteogenic markers, confirming the bone marrow contributes multiple cell types during the regeneration process. Using a similar experimental design, we genetically label the terminal phalanx NG2 expressing cells using systemic tamoxifen induction of NG2CreERTamR26REYFP mice. We fate trace the initially labeled population during blastema formation and re-differentiation and observed transdifferentiation events of the perivascular cells into two distinctive lineages, endothelial and osteoprogenitor cells. Establishing a direct correlation between peri-vasculature and re-differentiation, we address NG2/perivascular necessity with a series of temporal loss of function studies using a blocking antibody (iNG2). We implant iNG2 soaked microcarrier beads into various regions of the terminal phalanx and during different stages of the regeneration process. The experiments confirm the necessity of NG2 expression for distal bone elongation, as well as ascertain the temporal nature of the NG2 expression in different microenvironments. These results establish the importance of NG2+ cells in the bone marrow during early stages of regeneration, with early iNG2 bone marrow implantation resulting in a complete failure of the regeneration process. In an attempt to rescue this iNG2 failed regeneration we employ an established position-specific fibroblast cell line that displays a surprising plasticity as a cell-based therapeutic. Through a series of RNAi lentiviral transfection of inhibitors of the TGFβ-BMP pathway we induce osteogenic plasticity in the line. These results reveal regeneration competency associated with the mammalian terminal phalanx is in part due to the ability to recruit local perivascular multipotent populations, which has great translational relevancy.