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Tendon injuries cause prolonged disability and never recover completely. Current mechanistic understanding of tendon regeneration is limited. Here, we use single-cell transcriptomics to identify a tubulin polymerization-promoting protein family member 3-expressing ( Tppp3 + ) cell population as potential tendon stem cells. Through inducible lineage tracing, we demonstrate that these cells can generate new tenocytes and self-renew upon injury. A fraction of Tppp3 + cells expresses platelet-derived growth factor receptor alpha ( Pdfgra ). Ectopic platelet-derived growth factor-AA (PDGF-AA) protein induces new tenocyte production while inactivation of Pdgfra in Tppp3 + cells blocks tendon regeneration. These results support Tppp3 + Pdgfra + cells as tendon stem cells. Unexpectedly, Tppp3 − Pdgfra + fibro-adipogenic progenitors coexist in the tendon stem cell niche and give rise to fibrotic cells, revealing a clandestine origin of fibrotic scars in healing tendons. Our results explain why fibrosis occurs in injured tendons and present clinical challenges to enhance tendon regeneration without a concurrent increase in fibrosis by PDGF application. Using single-cell transcriptomics and in vivo injury models, Harvey et al. identify a Tppp3 + Pdgfra + stem cell population in the tendon sheath and demonstrate the role of PDGFRα signalling in regeneration and fibrosis.

Oligodendrocytes have been considered as a functionally homogeneous population in the central nervous system (CNS). We performed single-cell RNA sequencing on 5072 cells of the oligodendrocyte lineage from 10 regions of the mouse juvenile and adult CNS. Thirteen distinct populations were identified, 12 of which represent a continuum from Pdgfra⁺ oligodendrocyte precursor cells (OPCs) to distinct mature oligodendrocytes. Initial stages of differentiation were similar across the juvenile CNS, whereas subsets of mature oligodendrocytes were enriched in specific regions in the adult brain. Newly formed oligodendrocytes were detected in the adult CNS and were responsive to complex motor learning. A second Pdgfra⁺ population, distinct from OPCs, was found along vessels. Our study reveals the dynamics of oligodendrocyte differentiation and maturation, uncoupling them at a transcriptional level and highlighting oligodendrocyte heterogeneity in the CNS.

Avapritinib (AYVAKIT™) is a potent and selective tyrosine kinase inhibitor of platelet-derived growth factor receptor alpha (PDGFRA) and KIT activation loop mutants. It is being developed by Blueprint Medicines for the treatment of gastrointestinal stromal tumours (GIST), solid tumours and systemic mastocytosis. Avapritinib is approved in the USA for PDGFRA exon 18 (including D842V) mutant GIST and is undergoing regulatory assessment in the USA as a 4th-line treatment for GIST. Avapritinib is also undergoing regulatory assessment in the EU for PDGFRA D842V mutant GIST. This article summarizes the milestones in the development of avapritinib leading to this first approval for the treatment of adults with unresectable or metastatic GIST harbouring a PDGFRA exon 18 mutation, including PDGFRA D842V mutations. Clinical development of avapritinib is also underway for the treatment of systemic mastocytosis and late-stage solid tumours in several countries.

Kidney fibrosis is the hallmark of chronic kidney disease progression; however, at present no antifibrotic therapies exist 1 – 3 . The origin, functional heterogeneity and regulation of scar-forming cells that occur during human kidney fibrosis remain poorly understood 1 , 2 , 4 . Here, using single-cell RNA sequencing, we profiled the transcriptomes of cells from the proximal and non-proximal tubules of healthy and fibrotic human kidneys to map the entire human kidney. This analysis enabled us to map all matrix-producing cells at high resolution, and to identify distinct subpopulations of pericytes and fibroblasts as the main cellular sources of scar-forming myofibroblasts during human kidney fibrosis. We used genetic fate-tracing, time-course single-cell RNA sequencing and ATAC–seq (assay for transposase-accessible chromatin using sequencing) experiments in mice, and spatial transcriptomics in human kidney fibrosis, to shed light on the cellular origins and differentiation of human kidney myofibroblasts and their precursors at high resolution. Finally, we used this strategy to detect potential therapeutic targets, and identified NKD2 as a myofibroblast-specific target in human kidney fibrosis. A range of techniques are used to investigate the molecular landscape of chronic kidney disease, and the results suggest that distinct populations of pericytes and fibroblasts are the main source of myofibroblasts in kidney fibrosis.

Diffuse midline gliomas with histone H3 lysine27-to-methionine mutations (H3K27M-glioma) are an aggressive type of childhood cancer with few options for treatment. Filbin et al. used a single-cell sequencing approach to study the oncogenic programs, genetics, and cellular hierarchies of H3K27M-glioma. Tumors were mainly composed of cells resembling oligodendrocyte precursor cells, whereas differentiated malignant cells were a smaller fraction. In comparison with other gliomas, these cancers had distinct oncogenic programs and stem cell–like profiles that contributed to their stable tumor-propagating potential. The analysis also identified a lineage-specific marker that may be useful in developing therapies. Science , this issue p. 331 Single-cell analyses of H3K27M glioma defines a putative developmental hierarchy that differs from other gliomas. Gliomas with histone H3 lysine27-to-methionine mutations (H3K27M-glioma) arise primarily in the midline of the central nervous system of young children, suggesting a cooperation between genetics and cellular context in tumorigenesis. Although the genetics of H3K27M-glioma are well characterized, their cellular architecture remains uncharted. We performed single-cell RNA sequencing in 3321 cells from six primary H3K27M-glioma and matched models. We found that H3K27M-glioma primarily contain cells that resemble oligodendrocyte precursor cells (OPC-like), whereas more differentiated malignant cells are a minority. OPC-like cells exhibit greater proliferation and tumor-propagating potential than their more differentiated counterparts and are at least in part sustained by PDGFRA signaling. Our study characterizes oncogenic and developmental programs in H3K27M-glioma at single-cell resolution and across genetic subclones, suggesting potential therapeutic targets in this disease.

The platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases consists of two receptors, PDGFRα and PDGFRβ, that homodimerize and heterodimerize upon ligand binding. Here, we tested the hypothesis that differential internalization and trafficking dynamics of the various PDGFR dimers underlie differences in downstream intracellular signaling and cellular behavior. Using a bimolecular fluorescence complementation approach, we demonstrated that PDGFRα/β heterodimers are rapidly internalized into early endosomes. We showed that PDGFRα/β heterodimer activation does not induce downstream phosphorylation of ERK1/2 and significantly inhibits cell proliferation. Further, we identified MYO1D as a protein that preferentially binds PDGFRα/β heterodimers and demonstrated that knockdown of MYO1D leads to retention of PDGFRα/β heterodimers at the plasma membrane, increased phosphorylation of ERK1/2 and increased cell proliferation. Collectively, our findings impart valuable insight into the molecular mechanisms by which specificity is introduced downstream of PDGFR activation to differentially propagate signaling and generate distinct cellular responses. Here they show that heterodimerization of the receptor tyrosine kinases PDGFRa and PDGFRb negatively affects downstream ERK1/2 signaling and cellular proliferation, due in part to rapid internalization mediated by binding to the unconventional myosin protein MYO1D.

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a non enzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44+, CD55+, CD73+, CD105+, CD140a+, CD140b+, and CD201+ cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

Ischemic stroke is a major cause of disability in adults. Early treatment with thrombolytics and/or thrombectomy can significantly improve outcomes; however, following these acute interventions, treatment is limited to rehabilitation therapies. Thus, identification of therapeutic strategies that can help restore brain function in the post-acute phase remains a major challenge. Here we report that genetic or pharmacologic inhibition of the PDGF-CC/PDGFRα pathway, which has previously been implicated in stroke pathology, significantly reduced myofibroblast expansion in the border of the fibrotic scar and improved outcome in a sensory-motor integration test after experimental ischemic stroke. This was supported by gene expression analyses of cerebrovascular fragments showing upregulation of profibrotic/proinflammatory genes, including genes of the TGF pathway, after ischemic stroke or intracerebroventricular injection of active PDGF-CC. Further, longitudinal intravital 2-photon imaging revealed that inhibition of PDGFRα dampened the biphasic pattern of stroke-induced vascular leakage and enhanced vascular perfusion in the ischemic lesion. Importantly, we found PDGFRα inhibition to be effective in enhancing functional recovery when initiated 24 hours after ischemic stroke. Our data implicate the PDGF-CC/PDGFRα pathway as a crucial mediator modulating post-stroke pathology and suggest a post-acute treatment opportunity for patients with ischemic stroke targeting myofibroblast expansion to foster long-term CNS repair.

In vertebrates, the sclerotome is a transient embryonic structure that gives rise to various tissue support cells, including fibroblasts. However, how fibroblast precursors are guided to diverse tissues remain poorly understood. Using zebrafish, our lab has previously shown that sclerotome-derived cells undergo extensive migration to generate distinct fibroblast subtypes, including tenocytes along the myotendinous junction and fin mesenchymal cells in the fin fold. The pan-fibroblast gene platelet-derived growth factor receptor a ( pdgfra ), which has been implicated in cell migration across various contexts, is specifically expressed in the sclerotome and its descendants. Loss of functional Pdgfra in a pdgfra gene-trap mutant results in severe defects in the migration of sclerotome-derived cells, leading to a dose-dependent loss of tenocytes and fin mesenchymal cells. By combining live imaging and mosaic labeling with a membrane-bound dominant-negative tool, we demonstrate that Pdgfra acts cell-autonomously to regulate the migration of sclerotome-derived cells. In the absence of ligand pdgfab , which is expressed in the medial somite, sclerotome-derived cells fail to migrate medially, resulting in a loss of tenocytes, although they can migrate normally toward the fin fold and generate fin mesenchymal cells. Strikingly, localized expression of Pdgfab in pdgfab mutants can direct the migration of sclerotome-derived cells to both normal and ectopic locations, suggesting a chemoattractive role for the Pdgfab ligand. Together, our results demonstrate that Pdgfab/Pdgfra-mediated chemoattraction guides the migration of sclerotome-derived fibroblast precursors to specific locations, where they diversify into distinct fibroblast subtypes.

Bone formation in mammals requires continuous production of osteoblasts throughout life. A common molecular marker for all osteogenic mesenchymal progenitors has not been identified. Here, by lineage-tracing experiments in fetal or postnatal mice, we discover that Gli1 + cells progressively produce osteoblasts in all skeletal sites. Most notably, in postnatal growing mice, the Gli1 + cells residing immediately beneath the growth plate, termed here “metaphyseal mesenchymal progenitors” (MMPs), are essential for cancellous bone formation. Besides osteoblasts, MMPs also give rise to bone marrow adipocytes and stromal cells in vivo. RNA-seq reveals that MMPs express a number of marker genes previously assigned to mesenchymal stem/progenitor cells, including CD146/Mcam, CD44, CD106/Vcam1, Pdgfra, and Lepr. Genetic disruption of Hh signaling impairs proliferation and osteoblast differentiation of MMPs. Removal of β-catenin causes MMPs to favor adipogenesis, resulting in osteopenia coupled with increased marrow adiposity. Finally, postnatal Gli1 + cells contribute to both chondrocytes and osteoblasts during bone fracture healing. Thus Gli1 marks mesenchymal progenitors responsible for both normal bone formation and fracture repair. Skeletal progenitors in postnatal mice are highly heterogeneous. Using lineage tracing and RNA-seq the authors show that Gli1 + cells give rise to all osteoblasts in mice, including those required for healing of bone fractures.