Requirement of Pdgfrα+ cells for calvarial bone repair.

Platelet-derived growth factor receptor α (PDGFRα) is often considered as a general marker of mesenchymal cells and fibroblasts, but also shows expression in a portion of osteoprogenitor cells. Within the skeleton, Pdgfrα+ mesenchymal cells have been identified in bone marrow and periosteum of long bones, where they play a crucial role in participating in fracture repair. A similar examination of Pdgfrα+ cells in calvarial bone healing has not been examined. Here, we utilize Pdgfrα-CreERTM;mT/mG reporter animals to examine the contribution of Pdgfrα+ mesenchymal cells to calvarial bone repair through histology and single-cell RNA sequencing (scRNA-Seq). Results showed that Pdgfrα+ mesenchymal cells are present in several cell clusters by scRNA-Seq, and by histology a dramatic increase in Pdgfrα+ cells populated the defect site at early timepoints to give rise to healed bone tissue overtime. Notably, diphtheria toxin-mediated ablation of Pdgfrα reporter+ cells resulted in significantly impaired calvarial bone healing. Our findings suggest that Pdgfrα-expressing cells within the calvarial niche play a critical role in the process of calvarial bone repair.

Integrated transcriptomics of human blood vessels defines a spatially controlled niche for early mesenchymal progenitor cells.

Human blood vessel walls show concentric layers, with the outermost tunica adventitia harboring mesenchymal progenitor cells. These progenitor cells maintain vessel homeostasis and provide a robust cell source for cell-based therapies. However, human adventitial stem cell niche has not been studied in detail. Here, using spatial and single-cell transcriptomics, we characterized the phenotype, potential, and microanatomic distribution of human perivascular progenitors. Initially, spatial transcriptomics identified heterogeneity between perivascular layers of arteries and veins and delineated the tunica adventitia into inner and outer layers. From this spatial atlas, we inferred a hierarchy of mesenchymal progenitors dictated by a more primitive cell with a high surface expression of CD201 (PROCR). When isolated from humans and mice, CD201Low expression typified a mesodermal committed subset with higher osteogenesis and less proliferation than CD201High cells, with a downstream effect on canonical Wnt signaling through DACT2. CD201Low cells also displayed high translational potential for bone tissue generation.

TrkA + sensory neurons regulate osteosarcoma proliferation and vascularization to promote disease progression.

Bone pain is a presenting feature of bone cancers such as osteosarcoma (OS), relayed by skeletal-innervating peripheral afferent neurons. Potential functions of tumor-associated sensory neurons in bone cancers beyond pain sensation are unknown. To uncover neural regulatory functions, a chemical-genetic approach in mice with a knock-in allele for TrkA was used to functionally perturb sensory nerve innervation during OS growth and disease progression. TrkA inhibition in transgenic mice led to significant reductions in sarcoma-associated sensory innervation and vascularization, tumor growth and metastasis, and prolonged overall survival. Single-cell transcriptomics revealed that sarcoma denervation was associated with phenotypic alterations in both OS tumor cells and cells within the tumor microenvironment, and with reduced calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF) signaling. Multimodal and multi-omics analyses of human OS bone samples and human dorsal root ganglia neurons further implicated peripheral innervation and neurotrophin signaling in OS tumor biology. In order to curb tumor-associated axonal ingrowth, we next leveraged FDA-approved bupivacaine liposomes leading to significant reductions in sarcoma growth, vascularity, as well as alleviation of pain. In sum, TrkA-expressing peripheral neurons positively regulate key aspects of OS progression and sensory neural inhibition appears to disrupt calcitonin receptor signaling (CALCR) and VEGF signaling within the sarcoma microenvironment leading to significantly reduced tumor growth and improved survival. These data suggest that interventions to prevent pathological innervation of osteosarcoma represent a novel adjunctive therapy to improve clinical outcomes and survival.

The Mohawk homeobox gene represents a marker and osteo-inhibitory factor in calvarial suture osteoprogenitor cells.

The regeneration of the mammalian skeleton's craniofacial bones necessitates the action of intrinsic and extrinsic inductive factors from multiple cell types, which function hierarchically and temporally to control the differentiation of osteogenic progenitors. Single-cell transcriptomics of developing mouse calvarial suture recently identified a suture mesenchymal progenitor population with previously unappreciated tendon- or ligament-associated gene expression profile. Here, we developed a Mohawk homeobox (MkxCG; R26RtdT) reporter mouse and demonstrated that this reporter identifies an adult calvarial suture resident cell population that gives rise to calvarial osteoblasts and osteocytes during homeostatic conditions. Single-cell RNA sequencing (scRNA-Seq) data reveal that Mkx+ suture cells display a progenitor-like phenotype with expression of teno-ligamentous genes. Bone injury with Mkx+ cell ablation showed delayed bone healing. Remarkably, Mkx gene played a critical role as an osteo-inhibitory factor in calvarial suture cells, as knockdown or knockout resulted in increased osteogenic differentiation. Localized deletion of Mkx in vivo also resulted in robustly increased calvarial defect repair. We further showed that mechanical stretch dynamically regulates Mkx expression, in turn regulating calvarial cell osteogenesis. Together, we define Mkx+ cells within the suture mesenchyme as a progenitor population for adult craniofacial bone repair, and Mkx acts as a mechanoresponsive gene to prevent osteogenic differentiation within the stem cell niche.

Mapping Somatosensory Afferent Circuitry to Bone Identifies Neurotrophic Signals Required for Fracture Healing.

The profound pain accompanying bone fracture is mediated by somatosensory neurons, which also appear to be required to initiate bone regeneration following fracture. Surprisingly, the precise neuroanatomical circuitry mediating skeletal nociception and regeneration remains incompletely understood. Here, we characterized somatosensory dorsal root ganglia (DRG) afferent neurons innervating murine long bones before and after experimental long bone fracture in mice. Retrograde labeling of DRG neurons by an adeno-associated virus with peripheral nerve tropism showed AAV-tdT signal. Single cell transcriptomic profiling of 6,648 DRG neurons showed highest labeling across CGRP+ neuron clusters (6.9-17.2%) belonging to unmyelinated C fibers, thinly myelinated Aδ fibers and Aß-Field LTMR (9.2%). Gene expression profiles of retrograde labeled DRG neurons over multiple timepoints following experimental stress fracture revealed dynamic changes in gene expression corresponding to the acute inflammatory ( S100a8 , S100a9 ) and mechanical force ( Piezo2 ). Reparative phase after fracture included morphogens such as Tgfb1, Fgf9 and Fgf18 . Two methods to surgically or genetically denervate fractured bones were used in combination with scRNA-seq to implicate defective mesenchymal cell proliferation and osteodifferentiation as underlying the poor bone repair capacity in the presence of attenuated innervation. Finally, multi-tissue scRNA-seq and interactome analyses implicated neuron-derived FGF9 as a potent regulator of fracture repair, a finding compatible with in vitro assessments of neuron-to-skeletal mesenchyme interactions.

Isolation of Perivascular Mesenchymal Progenitor Cells from Human Adipose Tissue by Flow Cytometry.

Perivascular cells represent an in vivo counterpart of mesenchymal stromal/stem cells that populate the outer layer of blood vessels. Pericytes in capillaries and microvessels and adventitial cells of large arteries and veins give rise to stem/progenitor cells when isolated and cultured in vitro. These cells have been considered candidate cell types for cell therapy. Adipose tissue, being highly vascularized, dispensable, and easily accessed, is a viable option to obtain perivascular cells for use in research and in clinical trials. Here, we describe our established protocol to extract perivascular cells from human fat through fluorescence-activated cell sorting, which allows for the isolation of defined populations of progenitor cells with high reproducibility.

Tppp3+ synovial/tendon sheath progenitor cells contribute to heterotopic bone after trauma.

Heterotopic ossification (HO) is a pathological process resulting in aberrant bone formation and often involves synovial lined tissues. During this process, mesenchymal progenitor cells undergo endochondral ossification. Nonetheless, the specific cell phenotypes and mechanisms driving this process are not well understood, in part due to the high degree of heterogeneity of the progenitor cells involved. Here, using a combination of lineage tracing and single-cell RNA sequencing (scRNA-seq), we investigated the extent to which synovial/tendon sheath progenitor cells contribute to heterotopic bone formation. For this purpose, Tppp3 (tubulin polymerization-promoting protein family member 3)-inducible reporter mice were used in combination with either Scx (Scleraxis) or Pdgfra (platelet derived growth factor receptor alpha) reporter mice. Both tendon injury- and arthroplasty-induced mouse experimental HO models were utilized. ScRNA-seq of tendon-associated traumatic HO suggested that Tppp3 is an early progenitor cell marker for either tendon or osteochondral cells. Upon HO induction, Tppp3 reporter+ cells expanded in number and partially contributed to cartilage and bone formation in either tendon- or joint-associated HO. In double reporter animals, both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells gave rise to HO-associated cartilage. Finally, analysis of human samples showed a substantial population of TPPP3-expressing cells overlapping with osteogenic markers in areas of heterotopic bone. Overall, these data demonstrate that synovial/tendon sheath progenitor cells undergo aberrant osteochondral differentiation and contribute to HO after trauma.

ZIC1 Dictates Osteogenesis Versus Adipogenesis in Human Mesenchymal Progenitor Cells Via a Hedgehog Dependent Mechanism.

Numerous intrinsic factors regulate mesenchymal progenitor commitment to a specific cell fate, such as osteogenic or adipogenic lineages. Identification and modulation of novel intrinsic regulatory factors represent an opportunity to harness the regenerative potential of mesenchymal progenitors. In the present study, the transcription factor (TF) ZIC1 was identified to be differentially expressed among adipose compared with skeletal-derived mesenchymal progenitor cells. We observed that ZIC1 overexpression in human mesenchymal progenitors promotes osteogenesis and prevents adipogenesis. ZIC1 knockdown demonstrated the converse effects on cell differentiation. ZIC1 misexpression was associated with altered Hedgehog signaling, and the Hedgehog antagonist cyclopamine reversed the osteo/adipogenic differentiation alterations associated with ZIC1 overexpression. Finally, human mesenchymal progenitor cells with or without ZIC1 overexpression were implanted in an ossicle assay in NOD-SCID gamma mice. ZIC1 overexpression led to significantly increased ossicle formation in comparison to the control, as assessed by radiographic and histologic measures. Together, these data suggest that ZIC1 represents a TF at the center of osteo/adipogenic cell fate determinations-findings that have relevance in the fields of stem cell biology and therapeutic regenerative medicine.

Aldehyde Dehydrogenase, a Marker of Normal and Malignant Stem Cells, Typifies Mesenchymal Progenitors in Perivascular Niches.

Innate mesenchymal stem cells exhibiting multilineage differentiation and tissue (re)generative-or pathogenic-properties reside in perivascular niches. Subsets of these progenitors are committed to either osteo-, adipo-, or fibrogenesis, suggesting the existence of a developmental organization in blood vessel walls. We evaluated herein the activity of aldehyde dehydrogenase, a family of enzymes catalyzing the oxidation of aldehydes into carboxylic acids and a reported biomarker of normal and malignant stem cells, within human adipose tissue perivascular areas. A progression of ALDHLow to ALDHHigh CD34+ cells was identified in the tunica adventitia. Mesenchymal stem cell potential was confined to ALDHHigh cells, as assessed by proliferation and multilineage differentiation in vitro of cells sorted by flow cytometry with a fluorescent ALDH substrate. RNA sequencing confirmed and validated that ALDHHigh cells have a progenitor cell phenotype and provided evidence that the main isoform in this fraction is ALDH1A1, which was confirmed by immunohistochemistry. This demonstrates that ALDH activity, which marks hematopoietic progenitors and stem cells in diverse malignant tumors, also typifies native, blood vessel resident mesenchymal stem cells.

Radical polymerization inside living cells.

Polymerization reactions conducted inside cells must be compatible with the complex intracellular environment, which contains numerous molecules and functional groups that could potentially prevent or quench polymerization reactions. Here we report a strategy for directly synthesizing unnatural polymers in cells through free radical photopolymerization using a number of biocompatible acrylic and methacrylic monomers. This offers a platform to manipulate, track and control cellular behaviour by the in cellulo generation of macromolecules that have the ability to alter cellular motility, label cells by the generation of fluorescent polymers for long-term tracking studies, as well as generate a variety of nanostructures within cells. It is remarkable that free radical polymerization chemistry can take place within such complex cellular environments. This demonstration opens up a multitude of new possibilities for how chemists can modulate cellular function and behaviour and for understanding cellular behaviour in response to the generation of free radicals.

Scaffolds in vascular regeneration: current status.

An ideal vascular substitute, especially in <6 mm diameter applications, is a major clinical essentiality in blood vessel replacement surgery. Blood vessels are structurally complex and functionally dynamic tissue, with minimal regeneration potential. These have composite extracellular matrix (ECM) and arrangement. The interplay between ECM components and tissue specific cells gives blood vessels their specialized functional attributes. The core of vascular tissue engineering and regeneration relies on the challenges in creating vascular conduits that match native vessels and adequately regenerate in vivo. Out of numerous vascular regeneration concerns, the relevance of ECM emphasizes much attention toward appropriate choice of scaffold material and further scaffold development strategies. The review is intended to be focused on the various approaches of scaffold materials currently in use in vascular regeneration and current state of the art. Scaffold of choice in vascular tissue engineering ranges from natural to synthetic, decellularized, and even scaffold free approach. The applicability of tubular scaffold for in vivo vascular regeneration is under active investigation. A patent conduit with an ample endothelial luminal layer that can regenerate in vivo remains an unanswered query in the field of small diameter vascular tissue engineering. Besides, scaffolds developed for vascular regeneration, should aim at providing functional substitutes for use in a regenerative approach from the laboratory bench to patient bedside.