Synovium and infrapatellar fat pad share common mesenchymal progenitors and undergo coordinated changes in osteoarthritis.

Osteoarthritis (OA) affects multiple tissues in the knee joint, including the synovium and intra-articular adipose tissue (IAAT) that are attached to each other. However, whether these two tissues share the same progenitor cells and hence function as a single unit in joint homeostasis and diseases is largely unknown. Single-cell transcriptomic profiling of synovium and infrapatellar fat pad (IFP), the largest IAAT, from control and OA mice revealed five mesenchymal clusters and predicted mesenchymal progenitor cells (MPCs) as the common progenitors for other cells: synovial lining fibroblasts (SLFs), myofibroblasts (MFs), and preadipocytes 1 and 2. Histologic examination of joints in reporter mice having Dpp4-CreER and Prg4-CreER that label MPCs and SLFs, respectively, demonstrated that Dpp4+ MPCs reside in the synovial sublining layer and give rise to Prg4+ SLFs and Perilipin+ adipocytes during growth and OA progression. After OA injury, both MPCs and SLFs gave rise to MFs, which remained in the thickened synovium at later stages of OA. In culture, Dpp4+ MPCs possessed mesenchymal progenitor properties, such as proliferation and multilineage differentiation. In contrast, Prg4+ SLFs did not contribute to adipocytes in IFP and Prg4+ cells barely grew in vitro. Taken together, we demonstrate that the synovium and joint fat pad are one integrated functional tissue sharing common mesenchymal progenitors and undergoing coordinated changes during OA progression.

Both synovium and intra-articular adipose tissue (IAAT) in knee joint play a critical role in joint health and osteoarthritis (OA) progression. Recent single-cell RNA-sequencing studies have been performed on the mouse and human synovium. However, IAATs residing in close proximity to the synovium have not been studied yet. Our study reveals mesenchymal cell heterogeneity of synovium/infrapatellar fat pad (Syn/IFP) tissue and their OA responses. We identify Dpp4+ multipotent progenitors as a source that give rise to Prg4+ lining layer fibroblasts in the synovium, adipocytes in the IFP, and myofibroblasts in the OA Syn/IFP tissue. Our work demonstrates that Syn/IFP is a functionally connected tissue that shares common mesenchymal progenitors and undergoes coordinated OA changes. This novel insight advances our knowledge of previously understudied joint tissues and provides new directions for drug discovery to treat joint disorders.

Induced regeneration of articular cartilage - identification of a dormant regeneration program for a non-regenerative tissue.

A mouse organoid culture model was developed to regenerate articular cartilage by sequential treatment with BMP2 and BMP9 (or GDF2) that parallels induced joint regeneration at digit amputation wounds in vivo. BMP9-induced chondrogenesis was used to identify clonal cell lines for articular chondrocyte and hypertrophic chondrocyte progenitor cells from digit fibroblasts. A protocol that includes cell aggregation enhanced by BMP2 followed by BMP9-induced chondrogenesis resulted in the differentiation of organized layers of articular chondrocytes, similar to the organization of middle and deep zones of articular cartilage in situ, and retained a differentiated phenotype following transplantation. In addition, the differentiation of a non-chondrogenic connective tissue layer containing articular chondrocyte progenitor cells demonstrated that progenitor cell sequestration is coupled with articular cartilage differentiation at a clonal level. The studies identify a dormant endogenous regenerative program for a non-regenerative tissue in which fibroblast-derived progenitor cells can be induced to initiate morphogenetic and differentiative programs that include progenitor cell sequestration. The identification of dormant regenerative programs in non-regenerative tissues such as articular cartilage represents a novel strategy that integrates regeneration biology with regenerative medicine.

Microcomputed tomography staging of bone histolysis in the regenerating mouse digit.

Humans and mice have the ability to regenerate the distal digit tip, the terminal phalanx (P3) in response to amputation. What distinguishes P3 regeneration from regenerative failure is formation of the blastema, a proliferative structure that undergoes morphogenesis to regenerate the amputated tissues. P3 regeneration is characterised by the phases of inflammation, tissue histolysis and expansive bone degradation with simultaneous blastema formation, wound closure and finally blastemal differentiation to restore the amputated structures. While each regenerating digit faithfully progresses through all phases of regeneration, phase progression has traditionally been delineated by time, that is, days postamputation (DPA), yet there is widespread variability in the timing of the individual phases. To diminish variability between digits during tissue histolysis and blastema formation, we have established an in-vivo method using microcomputed tomography (micro CT) scanning to identify five distinct stages of the early regeneration response based on anatomical changes of the digit stump. We report that categorising the initial phases of digit regeneration by stage rather than time greatly diminishes the variability between digits with respect to changes in bone volume and length. Also, stages correlate with the levels of cell proliferation, osteoclast recruitment and osteoprogenitor cell recruitment. Importantly, micro CT staging provides a means to estimate open versus closed digit wounds. We demonstrate two spatially distinct and stage specific bone repair/regeneration responses that occur during P3 regeneration. Collectively, these studies showcase the utility of micro CT imaging to infer the composition of radiolucent soft tissues during P3 blastema formation. Specifically, the staging system identifies the onset of cell proliferation, osteoclastogenesis, osteoprogenitor recruitment, the spatial initiation of de novo bone formation and epidermal closure.

Hyaline cartilage differentiation of fibroblasts in regeneration and regenerative medicine.

Amputation injuries in mammals are typically non-regenerative; however, joint regeneration is stimulated by BMP9 treatment, indicating the presence of latent articular chondrocyte progenitor cells. BMP9 induces a battery of chondrogenic genes in vivo, and a similar response is observed in cultures of amputation wound cells. Extended cultures of BMP9-treated cells results in differentiation of hyaline cartilage, and single cell RNAseq analysis identified wound fibroblasts as BMP9 responsive. This culture model was used to identify a BMP9-responsive adult fibroblast cell line and a culture strategy was developed to engineer hyaline cartilage for engraftment into an acutely damaged joint. Transplanted hyaline cartilage survived engraftment and maintained a hyaline cartilage phenotype, but did not form mature articular cartilage. In addition, individual hypertrophic chondrocytes were identified in some samples, indicating that the acute joint injury site can promote osteogenic progression of engrafted hyaline cartilage. The findings identify fibroblasts as a cell source for engineering articular cartilage and establish a novel experimental strategy that bridges the gap between regeneration biology and regenerative medicine.

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification.

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (µCT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo µCT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

BMP9 stimulates joint regeneration at digit amputation wounds in mice.

A major goal of regenerative medicine is to stimulate tissue regeneration after traumatic injury. We previously discovered that treating digit amputation wounds with BMP2 in neonatal mice stimulates endochondral ossification to regenerate the stump bone. Here we show that treating the amputation wound with BMP9 stimulates regeneration of a synovial joint that forms an articulation with the stump bone. Regenerated structures include a skeletal element lined with articular cartilage and a synovial cavity, and we demonstrate that this response requires the Prg4 gene. Combining BMP2 and BMP9 treatments in sequence stimulates the regeneration of bone and joint. These studies provide evidence that treatment of growth factors can be used to engineer a regeneration response from a non-regenerating amputation wound.

miR-150 regulates obesity-associated insulin resistance by controlling B cell functions.

Adipose tissue resident B cells account for more than 20% of stromal cells within visceral adipose tissues; however, their functions in the adipose tissue niche are poorly elucidated. Here we report that miR-150 modulates adipose tissue function by controlling activation of B cells and their interactions with other immune cells. miR-150KO mice displayed exacerbated obesity-associated tissue inflammation and systemic insulin resistance, which is recapitulated by adoptive transfer of B cells, but not purified immunoglobulin, into obese B(null) mice. Using purified cell populations, we found that enhanced proinflammatory activation of adipose tissue T cells and macrophages was due to miR-150KO B cells action but not cell-autologous mechanisms. miR-150KO B cells displayed significantly enhanced antigen presentation upon stimulation, ultimately leading to elevated inflammation and insulin resistance, compared to wild type B cells. Knockdown of identified miR-150 target genes, Elk1, Etf1 or Myb attenuated B cell action by altering B cell receptor pathways and MHCII cell surface presentation. Our results demonstrate a critical role for miR-150 in regulating B cell functions in adipose tissue which ultimately regulate both metabolic and immunologic homeostasis in the adipose tissue niche.