Bone is comprised of separate inner endosteal and outer periosteal compartments, each with distinct contributions to bone physiology and each maintaining separate pools of cells due to physical separation by the bone cortex. While the skeletal stem cell giving rise to endosteal osteoblasts has been extensively studied, the identification of a periosteal stem cell has been elusive 1 – 5 . Here, we identify a periosteal stem cell (PSC) present in the long bones and calvarium of mice that displays clonal multipotency, self-renewal and sits at the apex of a differentiation hierarchy. Single cell and bulk transcriptional profiling show that PSCs display distinct transcriptional signatures in comparison with both other skeletal stem cells and mature mesenchymal cells. While other skeletal stem cells form bone via an initial cartilage template using the endochondral pathway 4 , 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 PSCs is present in the human periosteum, raising the possibility that PSCs are attractive targets for drug and cellular therapy for skeletal disorders. Moreover, the identification of PSCs provides evidence that bone contains multiple pools of stem cells, each with distinct physiologic functions.

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Upon activation in vitro, only a fraction of the bulk human T helper cell cultures secret the hallmark Th1/2 cytokines (IFN‐γ for Th1 and IL‐4 for Th2, respectively). It is uncertain whether these IFN‐γ–/IL‐4– cells are differentiated Th1 or Th2 cells. Here, we have characterized live IFN‐γ+, IL‐4+ and IFN‐γ–/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 (IFNγ, IL4, and IL5). However, they were comparable with the IFN‐γ+ or IL‐4+ cells in the expression levels of other Th1/Th2 marker genes (GATA3, Tbet, and IL12Rβ2). 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‐γ or IL‐4 producers and the non‐producers, further underscoring the similarity between these two groups.

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.