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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.

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 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.

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).

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the US. Unfortunately, recent clinical trials using immunotherapy targeting the highly immunosuppressive tumor microenvironment have showed disappointing results, as there is no method to predict which patients will respond to therapy. More recently, the development of single cell technology has allowed for in-depth profiling at the cellular level using small amounts of tissue, raising the potential to develop precision medicine tools at time of endoscopic fine needle biopsy. Results: We performed single-cell RNA sequencing (scRNAseq) on endoscopic fine needle biopsies from 10 PDAC tumors at time of diagnostic endoscopic biopsy or 6 surgically resected tissues. We also sequenced 3 adjacent or normal pancreas tissues. We captured 8,521 cells from 3 surgical normal adjacent samples and 46,244 cells from PDAC tumors. Mapping of putative interactions between ligands and receptors demonstrated upregulation of key signaling pathways, including Hedgehog, NOTCH, and chemokine signaling within myeloid, epithelial, T, and NK cells. Differential expression analysis in cytotoxic CD8 T cells of PDA patients revealed increased expression of genes involved in T cell activation (GZMB, GZMA), exhaustion (GZMK, EOMES) as well as immune checkpoint pathway upregulation when compared to cytotoxic T cells in adjacent normal pancreatic tissue. Among the most significantly increased genes in CD8 T cells of PDAC tumor was the immune checkpoint TIGIT. Upon further analysis of the CD8 T cells, we found TIGIT was almost exclusively expressed in exhausted CD8 T cells, while other checkpoints such as PD-1 and LAG3 were equally distributed across effector and exhausted T cell populations. Interestingly, we were able to capture patient-specific heterogeneity of gene expression in T cells, suggesting the possibility of individualized T cell gene signatures present in PDAC tumors. We used mass cytometry and immunostaining to validate our transcript-based findings. Conclusion: Overall, we have successfully performed robust in-depth profiling using single-cell sequencing of PDAC tumors from fine needle biopsies. TIGIT, but not other immune checkpoints, correlates with T cells exhaustion in tumors, revealing an important biological function of this relatively understudied checkpoint. Analysis of our results identified patient-specific heterogeneity of key signaling pathways in different cell compartments of PDAC tumors that have to potential to be leveraged for precision medicine. Citation Format: Nina Steele, Eileen Carpenter, Samantha Kemp, Veerin Sirihorachai, Stephanie The, Lawrence Delrosario, Jenny Lazarus, El-ad Amir, Valerie Gunchick, Carlos Espinoza, Samantha Bell, Lindsey Harris, Valerie Irizarry-Negron, Dan Paglia, Justin Macchia, Fatima Lima, Angel Ka Yan Chu, Heather Schofield, Erik Jan Wamsteker, Richard Kwon, Allison Schulman, Anoop Prabhu, Ryan law, Arjun Sondhi, Katelyn Donahue, Hari Nathan, Clifford Cho, Michelle Anderson, Vaibhav Sahai, Costas Lyssiotis, Benjamin Allen, Arvind Rao, Weiping Zou, Filip Bednar, Timothy Frankel, Marina Pasca di Magliano. Multimodal mapping of the immune landscape in human pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3442.

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