Endosteal stem cells at the bone-blood interface: A double-edged sword for rapid bone formation: Bone marrow endosteal stem cells provide a robust source of bone-making osteoblasts both in normal and abnormal bone formation.

Endosteal stem cells are a subclass of bone marrow skeletal stem cell populations that are particularly important for rapid bone formation occurring in growth and regeneration. These stem cells are strategically located near the bone surface in a specialized microenvironment of the endosteal niche. These stem cells are abundant in young stages but eventually depleted and replaced by other stem cell types residing in a non-endosteal perisinusoidal niche. Single-cell molecular profiling and in vivo cell lineage analyses play key roles in discovering endosteal stem cells. Importantly, endosteal stem cells can transform into bone tumor-making cells when deleterious mutations occur in tumor suppressor genes. The emerging hypothesis is that osteoblast-chondrocyte transitional identities confer a special subset of endosteal stromal cells with stem cell-like properties, which may make them susceptible for tumorigenic transformation. Endosteal stem cells are likely to represent an important therapeutic target of bone diseases caused by aberrant bone formation.

Bone marrow endosteal stem cells dictate active osteogenesis and aggressive tumorigenesis.

The bone marrow contains various populations of skeletal stem cells (SSCs) in the stromal compartment, which are important regulators of bone formation. It is well-described that leptin receptor (LepR)+ perivascular stromal cells provide a major source of bone-forming osteoblasts in adult and aged bone marrow. However, the identity of SSCs in young bone marrow and how they coordinate active bone formation remains unclear. Here we show that bone marrow endosteal SSCs are defined by fibroblast growth factor receptor 3 (Fgfr3) and osteoblast-chondrocyte transitional (OCT) identities with some characteristics of bone osteoblasts and chondrocytes. These Fgfr3-creER-marked endosteal stromal cells contribute to a stem cell fraction in young stages, which is later replaced by Lepr-cre-marked stromal cells in adult stages. Further, Fgfr3+ endosteal stromal cells give rise to aggressive osteosarcoma-like lesions upon loss of p53 tumor suppressor through unregulated self-renewal and aberrant osteogenic fates. Therefore, Fgfr3+ endosteal SSCs are abundant in young bone marrow and provide a robust source of osteoblasts, contributing to both normal and aberrant osteogenesis.

The fate of early perichondrial cells in developing bones.

In endochondral bone development, bone-forming osteoblasts and bone marrow stromal cells have dual origins in the fetal cartilage and its surrounding perichondrium. However, how early perichondrial cells distinctively contribute to developing bones remain unidentified. Here we show using in vivo cell-lineage analyses that Dlx5+ fetal perichondrial cells marked by Dlx5-creER do not generate cartilage but sustainably contribute to cortical bone and marrow stromal compartments in a manner complementary to fetal chondrocyte derivatives under the regulation of Hedgehog signaling. Postnatally, Dlx5+ fetal perichondrial cell derivatives preferentially populate the diaphyseal marrow stroma with a dormant adipocyte-biased state and are refractory to parathyroid hormone-induced bone anabolism. Therefore, early perichondrial cells of the fetal cartilage are destined to become an adipogenic subset of stromal cells in postnatal diaphyseal bone marrow, supporting the theory that the adult bone marrow stromal compartments are developmentally prescribed within the two distinct cells-of-origins of the fetal bone anlage.

Single-Cell Transcriptomic Analysis Reveals Developmental Relationships and Specific Markers of Mouse Periodontium Cellular Subsets.

The periodontium is essential for supporting the functionality of the tooth, composed of diversity of mineralized and non-mineralized tissues such as the cementum, the periodontal ligament (PDL) and the alveolar bone. The periodontium is developmentally derived from the dental follicle (DF), a fibrous tissue surrounding the developing tooth bud. We previously showed through in vivo lineage-tracing experiments that DF contains mesenchymal progenitor cells expressing parathyroid hormone-related protein (PTHrP), which give rise to cells forming the periodontal attachment apparatus in a manner regulated by autocrine signaling through the PTH/PTHrP receptor. However, the developmental relationships between PTHrP+ DF cells and diverse cell populations constituting the periodontium remain undefined. Here, we performed single-cell RNA-sequencing (scRNA-seq) analyses of cells in the periodontium by integrating the two datasets, i.e. PTHrP-mCherry+ DF cells at P6 and 2.3kb Col1a1 promoter-driven GFP+ periodontal cells at P25 that include descendants of PTHrP+ DF cells, cementoblasts, osteoblasts and periodontal ligament cells. This integrative scRNA-seq analysis revealed heterogeneity of cells of the periodontium and their cell type-specific markers, as well as their relationships with DF cells. Most importantly, our analysis identified a cementoblast-specific metagene that discriminate cementoblasts from alveolar bone osteoblasts, including Pthlh (encoding PTHrP) and Tubb3. RNA velocity analysis indicated that cementoblasts were directly derived from PTHrP+ DF cells in the early developmental stage and did not interconvert with other cell types. Further, CellPhoneDB cell-cell communication analysis indicated that PTHrP derived from cementoblasts acts on diversity of cells in the periodontium in an autocrine and paracrine manner. Collectively, our findings provide insights into the lineage hierarchy and intercellular interactions of cells in the periodontium at a single-cell level, aiding to understand cellular and molecular basis of periodontal tissue formation.

Intercellular Interactions of an Adipogenic CXCL12-Expressing Stromal Cell Subset in Murine Bone Marrow.

Bone marrow houses a multifunctional stromal cell population expressing C-X-C motif chemokine ligand 12 (CXCL12), termed CXCL12-abundant reticular (CAR) cells, that regulates osteogenesis and adipogenesis. The quiescent pre-adipocyte-like subset of CXCL12+ stromal cells ("Adipo-CAR" cells) is localized to sinusoidal surfaces and particularly enriched for hematopoiesis-supporting cytokines. However, detailed characteristics of these CXCL12+ pre-adipocyte-like stromal cells and how they contribute to marrow adipogenesis remain largely unknown. Here we highlight CXCL12-dependent physical coupling with hematopoietic cells as a potential mechanism regulating the adipogenic potential of CXCL12+ stromal cells. Single-cell computational analyses of RNA velocity and cell signaling reveal that Adipo-CAR cells exuberantly communicate with hematopoietic cells through CXCL12-CXCR4 ligand-receptor interactions but do not interconvert with Osteo-CAR cells. Consistent with this computational prediction, a substantial fraction of Cxcl12-creER+ pre-adipocyte-like cells intertwines with hematopoietic cells in vivo and in single-cell preparation in a protease-sensitive manner. Deletion of CXCL12 in these cells using Col2a1-cre leads to a reduction of stromal-hematopoietic coupling and extensive marrow adipogenesis in adult bone marrow, which appears to involve direct conversion of CXCL12+ cells to lipid-laden marrow adipocytes without altering mesenchymal progenitor cell fates. Therefore, these findings suggest that CXCL12+ pre-adipocyte-like marrow stromal cells prevent their premature differentiation by maintaining physical coupling with hematopoietic cells in a CXCL12-dependent manner, highlighting a possible cell-non-autonomous mechanism that regulates marrow adipogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Multimodal Mapping of the Tumor and Peripheral Blood Immune Landscape in Human Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by an immune-suppressive tumor microenvironment that renders it largely refractory to immunotherapy. We implemented a multimodal analysis approach to elucidate the immune landscape in PDA. Using a combination of CyTOF, single-cell RNA sequencing, and multiplex immunohistochemistry on patient tumors, matched blood, and non-malignant samples, we uncovered a complex network of immune-suppressive cellular interactions. These experiments revealed heterogeneous expression of immune checkpoint receptors in individual patient's T cells and increased markers of CD8+ T cell dysfunction in advanced disease stage. Tumor-infiltrating CD8+ T cells had an increased proportion of cells expressing an exhausted expression profile that included upregulation of the immune checkpoint TIGIT, a finding that we validated at the protein level. Our findings point to a profound alteration of the immune landscape of tumors, and to patient-specific immune changes that should be taken into account as combination immunotherapy becomes available for pancreatic cancer.