A vertebral skeletal stem cell lineage driving metastasis.

Vertebral bone is subject to a distinct set of disease processes from long bones, including a much higher rate of solid tumour metastases1-4. The basis for this distinct biology of vertebral bone has so far remained unknown. Here we identify a vertebral skeletal stem cell (vSSC) that co-expresses ZIC1 and PAX1 together with additional cell surface markers. vSSCs display formal evidence of stemness, including self-renewal, label retention and sitting at the apex of their differentiation hierarchy. vSSCs are physiologic mediators of vertebral bone formation, as genetic blockade of the ability of vSSCs to generate osteoblasts results in defects in the vertebral neural arch and body. Human counterparts of vSSCs can be identified in vertebral endplate specimens and display a conserved differentiation hierarchy and stemness features. Multiple lines of evidence indicate that vSSCs contribute to the high rates of vertebral metastatic tropism observed in breast cancer, owing in part to increased secretion of the novel metastatic trophic factor MFGE8. Together, our results indicate that vSSCs are distinct from other skeletal stem cells and mediate the unique physiology and pathology of vertebrae, including contributing to the high rate of vertebral metastasis.

A multi-stem cell basis for craniosynostosis and calvarial mineralization.

Craniosynostosis is a group of disorders of premature calvarial suture fusion. The identity of the calvarial stem cells (CSCs) that produce fusion-driving osteoblasts in craniosynostosis remains poorly understood. Here we show that both physiologic calvarial mineralization and pathologic calvarial fusion in craniosynostosis reflect the interaction of two separate stem cell lineages; a previously identified cathepsin K (CTSK) lineage CSC1 (CTSK+ CSC) and a separate discoidin domain-containing receptor 2 (DDR2) lineage stem cell (DDR2+ CSC) that we identified in this study. Deletion of Twist1, a gene associated with craniosynostosis in humans2,3, solely in CTSK+ CSCs is sufficient to drive craniosynostosis in mice, but the sites that are destined to fuse exhibit an unexpected depletion of CTSK+ CSCs and a corresponding expansion of DDR2+ CSCs, with DDR2+ CSC expansion being a direct maladaptive response to CTSK+ CSC depletion. DDR2+ CSCs display full stemness features, and our results establish the presence of two distinct stem cell lineages in the sutures, with both populations contributing to physiologic calvarial mineralization. DDR2+ CSCs mediate a distinct form of endochondral ossification without the typical haematopoietic marrow formation. Implantation of DDR2+ CSCs into suture sites is sufficient to induce fusion, and this phenotype was prevented by co-transplantation of CTSK+ CSCs. Finally, the human counterparts of DDR2+ CSCs and CTSK+ CSCs display conserved functional properties in xenograft assays. The interaction between these two stem cell populations provides a new biologic interface for the modulation of calvarial mineralization and suture patency.

Discovery of a periosteal stem cell mediating intramembranous bone formation.

Bone consists of separate inner endosteal and outer periosteal compartments, each with distinct contributions to bone physiology and each maintaining separate pools of cells owing to physical separation by the bone cortex. The skeletal stem cell that gives rise to endosteal osteoblasts has been extensively studied; however, the identity of periosteal stem cells remains unclear1-5. Here we identify a periosteal stem cell (PSC) that is present in the long bones and calvarium of mice, displays clonal multipotency and self-renewal, and sits at the apex of a differentiation hierarchy. Single-cell and bulk transcriptional profiling show that PSCs display transcriptional signatures that are distinct from those of other skeletal stem cells and mature mesenchymal cells. Whereas other skeletal stem cells form bone via an initial cartilage template using the endochondral pathway4, PSCs form bone via a direct intramembranous route, providing a cellular basis for the divergence between intramembranous versus endochondral developmental pathways. However, there is plasticity in this division, as PSCs acquire endochondral bone formation capacity in response to injury. Genetic blockade of the ability of PSCs to give rise to bone-forming osteoblasts results in selective impairments in cortical bone architecture and defects in fracture healing. A cell analogous to mouse PSCs is present in the human periosteum, raising the possibility that PSCs are attractive targets for drug and cellular therapy for skeletal disorders. The identification of PSCs provides evidence that bone contains multiple pools of stem cells, each with distinct physiologic functions.

Identification of a stem cell mediating osteoblast versus adipocyte lineage selection.

Most skeletal fragility disorders are characterized by bone loss with a concurrent gain in marrow adipocytes 1-8. This suggests that a cell that forms adipocytes at the expense of osteoblasts is central to the pathogenesis of skeletal disorders. However, this cellular point of bifurcation between adipocyte and osteoblast differentiation pathways remains unknown. Here, we identify a new cell type defined by co-expression of skeletal stem cell and adipocyte precursor markers, 9-13 (CD24+CD29+ skeletal stem cells (SSCs)), that serves as a key cellular point of bifurcation between the osteoblast and adipocyte differentiation pathways, giving rise to closely related osteoblast and adipocyte lineage-restricted precursors. CD24+CD29+SSCs comprise a small fraction of SSCs, and only this fraction displays full stemness features, including the ability to undergo serial transplantation. In line with serving as the osteoblast/adipocyte bipotent cell, the "bone to fat" tissue remodeling occurring in models of postmenopausal osteoporosis or after high fat diet exposure occur in part by reprogramming these CD24+CD29+SSCs to change their output of lineage-restricted precursors. Lastly, as subcutaneous white adipose tissue displays a similar set of CD24+CD29+ stem cells and related lineage-restricted progenitors, these findings provide a new schema explaining the stem cell basis of bone versus adipose tissue production that unifies multiple mesenchymal tissues.

Discovery of a Vertebral Skeletal Stem Cell Driving Spinal Metastases.

Vertebral bone is subject to a distinct set of disease processes from those of long bones, notably including a much higher rate of solid tumor metastases that cannot be explained by passive blood flow distribution alone. The basis for this distinct biology of vertebral bone has remained elusive. Here we identify a vertebral skeletal stem cell (vSSC), co-expressing the transcription factors ZIC1 and PAX1 together with additional cell surface markers, whose expression profile and function are markedly distinct from those of long bone skeletal stem cells (lbSSCs). vSSCs display formal evidence of stemness, including self-renewal, label retention and sitting at the apex of their differentiation hierarchy. Lineage tracing of vSSCs confirms that they make a persistent contribution to multiple mature cell lineages in the native vertebrae. vSSCs are physiologic mediators of spine mineralization, as genetic blockade of the ability of vSSCs to generate osteoblasts results in defects in the vertebral neural arch and body. Human counterparts of vSSCs can be identified in vertebral endplate specimens and display a conserved differentiation hierarchy and stemness. Multiple lines of evidence indicate that vSSCs contribute to the high rates of vertebral metastatic tropism observed clinically in breast cancer. Specifically, when an organoid system is used to place both vSSCs and lbSSCs in an identical anatomic context, vSSC-lineage cells are more efficient than lbSSC-lineage cells at recruiting metastases, a phenotype that is due in part to increased secretion of the novel metastatic trophic factor MFGE8. Similarly, genetically targeting loss-of-function to the vSSC lineage results in reduced metastasis rates in the native vertebral environment. Taken together, vSSCs are distinct from other skeletal stem cells and mediate the unique physiology and pathology of vertebrae, including contributing to the high rate of metastatic seeding of the vertebrae.

Schistosoma mansoni Infection Is Associated With Increased Monocytes and Fewer Natural Killer T Cells in the Female Genital Tract.

Schistosoma mansoni infection may impair genital mucosal antiviral immunity, but immune cell populations have not been well characterized. We characterized mononuclear cells from cervical brushings of women with and without S mansoni infection. We observed lower frequencies of natural killer T cells and higher frequencies of CD14+ monocytes in infected women.

Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma.

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically comprise less than 1% of the tumor mass. Material derived from bulk tumor contains tumor content at a concentration insufficient for characterization. Therefore, fluorescence activated cell sorting using eight antibodies, as well as side- and forward-scatter, is described here as a method of rapidly separating and concentrating with high purity thousands of HRS cells from the tumor for subsequent study. At the same time, because standard protocols for exome sequencing typically require 100-1,000 ng of input DNA, which is often too high, even with flow sorting, we also provide an optimized, low-input library construction protocol capable of producing high-quality data from as little as 10 ng of input DNA. This combination is capable of producing next-generation libraries suitable for hybridization capture of whole-exome baits or more focused targeted panels, as desired. Exome sequencing of the HRS cells, when compared against healthy intratumor T or B cells, can identify somatic alterations, including mutations, insertions and deletions, and copy number alterations. These findings elucidate the molecular biology of HRS cells and may reveal avenues for targeted drug treatments.

Human T helper (Th) cell lineage commitment is not directly linked to the secretion of IFN-gamma or IL-4: characterization of Th cells isolated by FACS based on IFN-gamma and IL-4 secretion.

Upon activation in vitro, only a fraction of the bulk human T helper cell cultures secret the hallmark Th1/2 cytokines (IFN-gamma for Th1 and IL-4 for Th2, respectively). It is uncertain whether these IFN-gamma-/IL-4- cells are differentiated Th1 or Th2 cells. Here, we have characterized live IFN-gamma+, IL-4+ and IFN-gamma-/IL-4- cells isolated from Th cell cultures treated under Th1 or Th2 polarizing conditions by employing affinity matrix capture technology. RNA samples from the sorted cells were analyzed by real time RT-PCR and microarrays. The double negative cells from either Th1 or Th2 cultures expressed lower levels of Th1/Th2 marker cytokine genes (IFNgamma, IL4, and IL5). However, they were comparable with the IFN-gamma+ or IL-4+ cells in the expression levels of other Th1/Th2 marker genes (GATA3, Tbet, and IL12Rbeta2). Most importantly, these double negative cells were already committed in their Th1/Th2 lineages. Gene expression profiling analysis showed that very few previously identified Th1/Th2 marker genes were differentially expressed between the IFN-gamma or IL-4 producers and the non-producers, further underscoring the similarity between these two groups.