Development of Murine Anterior Interbody and Posterolateral Spinal Fusion Techniques.

BACKGROUND: Multiple animal models have previously been utilized to investigate anterior fusion techniques, but a mouse model has yet to be developed. The purpose of this study was to develop murine anterior interbody and posterolateral fusion techniques. METHODS: Mice underwent either anterior interbody or posterolateral spinal fusion. A protocol was developed for both procedures, including a description of the relevant anatomy. Samples were subjected to micro-computed tomography to assess fusion success and underwent biomechanical testing with use of 4-point bending. Lastly, samples were fixed and embedded for histologic evaluation. RESULTS: Surgical techniques for anterior interbody and posterolateral fusion were developed. The fusion rate was 83.3% in the anterior interbody model and 100% in the posterolateral model. Compared with a control, the posterolateral model exhibited a greater elastic modulus. Histologic analysis demonstrated endochondral ossification between bridging segments, further confirming the fusion efficacy in both models. CONCLUSIONS: The murine anterior interbody and posterolateral fusion models are efficacious and provide an ideal platform for studying the molecular and cellular mechanisms mediating spinal fusion. CLINICAL RELEVANCE: Given the extensive genetic tools available in murine disease models, use of fusion models such as ours can enable determination of the underlying genetic pathways involved in spinal fusion.

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.

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.

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.

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.

SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts.

Hedgehog signaling is essential for bone formation, including functioning as a means for the growth plate to drive skeletal mineralization. However, the mechanisms regulating hedgehog signaling specifically in bone-forming osteoblasts are largely unknown. Here, we identified SLIT and NTRK-like protein-5(Slitrk5), a transmembrane protein with few identified functions, as a negative regulator of hedgehog signaling in osteoblasts. Slitrk5 is selectively expressed in osteoblasts and loss of Slitrk5 enhanced osteoblast differentiation in vitro and in vivo. Loss of SLITRK5 in vitro leads to increased hedgehog signaling and overexpression of SLITRK5 in osteoblasts inhibits the induction of targets downstream of hedgehog signaling. Mechanistically, SLITRK5 binds to hedgehog ligands via its extracellular domain and interacts with PTCH1 via its intracellular domain. SLITRK5 is present in the primary cilium, and loss of SLITRK5 enhances SMO ciliary enrichment upon SHH stimulation. Thus, SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts that may be attractive as a therapeutic target to enhance bone formation.

Specimen Preparation for Single-Cell Sequencing Analysis of Skeletal Cells.

Recent work emphasizes that bone comprises numerous mesenchymal cell types each with different biologic functions, and deconvoluting the functions of these cells requires technical approaches with single-cell resolution, such as single-cell RNA sequencing (scRNA-seq). A critical step in conducting a successful single-cell sequencing study of bone is generation of a single-cell suspension of skeletal cells while preserving cell viability. Here we describe a method to prepare single-cell suspensions from skeletal tissue in preparation for single-cell sequencing studies. Also included are optional steps for fluorescence-activated cell sorting (FACS) of skeletal cells, which allows subsequent scRNA-seq studies to be focused on specific populations of interest.

Adult stem cells and regenerative medicine-a symposium report.

Adult stem cells are rare, undifferentiated cells found in all tissues of the body. Although normally kept in a quiescent, nondividing state, these cells can proliferate and differentiate to replace naturally dying cells within their tissue and to repair its wounds in response to injury. Due to their proliferative nature and ability to regenerate tissue, adult stem cells have the potential to treat a variety of degenerative diseases as well as aging. In addition, since stem cells are often thought to be the source of malignant tumors, understanding the mechanisms that keep their proliferative abilities in check can pave the way for new cancer therapies. While adult stem cells have had limited practical and clinical applications to date, several clinical trials of stem cell-based therapies are underway. This report details recent research presented at the New York Academy of Sciences on March 14, 2019 on understanding the factors that regulate stem cell activity and differentiation, with the hope of translating these findings into the clinic.

The Unmixing Problem: A Guide to Applying Single-Cell RNA Sequencing to Bone.

Bone is composed of a complex mixture of many dynamic cell types. Flow cytometry and in vivo lineage tracing have offered early progress toward deconvoluting this heterogeneous mixture of cells into functionally well-defined populations suitable for further studies. Single-cell sequencing is poised as a key complementary technique to better understand the cellular basis of bone metabolism and development. However, single-cell sequencing approaches still have important limitations, including transcriptional effects of cell isolation and sparse sampling of the transcriptome, that must be considered during experimental design and analysis to harness the power of this approach. Accounting for these limitations requires a deep knowledge of the tissue under study. Therefore, with the emergence of accessible tools for conducting and analyzing single-cell RNA sequencing (scRNA-seq) experiments, bone biologists will be ideal leaders in the application of scRNA-seq to the skeleton. Here we provide an overview of the steps involved with a single-cell sequencing analysis of bone, focusing on practical considerations needed for a successful study. © 2019 American Society for Bone and Mineral Research.

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.

Targeting skeletal endothelium to ameliorate bone loss.

Recent studies have identified a specialized subset of CD31hiendomucinhi (CD31hiEMCNhi) vascular endothelium that positively regulates bone formation. However, it remains unclear how CD31hiEMCNhi endothelium levels are coupled to anabolic bone formation. Mice with an osteoblast-specific deletion of Shn3, which have markedly elevated bone formation, demonstrated an increase in CD31hiEMCNhi endothelium. Transcriptomic analysis identified SLIT3 as an osteoblast-derived, SHN3-regulated proangiogenic factor. Genetic deletion of Slit3 reduced skeletal CD31hiEMCNhi endothelium, resulted in low bone mass because of impaired bone formation and partially reversed the high bone mass phenotype of Shn3-/- mice. This coupling between osteoblasts and CD31hiEMCNhi endothelium is essential for bone healing, as shown by defective fracture repair in SLIT3-mutant mice and enhanced fracture repair in SHN3-mutant mice. Finally, administration of recombinant SLIT3 both enhanced bone fracture healing and counteracted bone loss in a mouse model of postmenopausal osteoporosis. Thus, drugs that target the SLIT3 pathway may represent a new approach for vascular-targeted osteoanabolic therapy to treat bone loss.

Na(+) /H(+) exchanger 1 (NHE1) function is necessary for maintaining mammary tissue architecture.

BACKGROUND: The mammary gland is an ideal model to study the link between form and function in normal tissue. Perhaps as interesting as the cues necessary to generate this structure are the signals required to maintain its branched architecture over the lifetime of the organism, since likely these pathways are de-regulated in malignancies. Previously, we have shown that the Na(+) /H(+) exchanger 1 (NHE1), a critical regulator of intracellular pH, was necessary for mammary branching morphogenesis. Here we provide strong evidence that NHE1 function is also necessary for maintaining mammary branched architecture. RESULTS: Inhibition of NHE1 with 5-N-Methy-N-isobutyl amiloride (MIA) on branched structures resulted in a rapid (within 24 hr) and reversible loss of branched architecture that was not accompanied by any overt changes in cell proliferation or cell death. NHE1 inhibition led to a significant acidification of intracellular pH in the branched end buds that preceded a number of events, including altered tissue polarity of myoepithelial cells, loss of NHE1 basal polarity, F-actin rearrangements, and decreased E-cadherin expression. CONCLUSIONS: Our results implicate NHE1 function and intracellular pH homeostasis as key factors that maintain mammary tissue architecture, thus, indirectly allowing for mammary function as a milk-providing (form) and -producing (function) gland.

First ayurvedic approach towards green drugs: anti cervical cancer-cell properties of Clerodendrum viscosum root extract.

The concept of Ayurvedic expert guided drug discovery and development is defined and put to test systematically for the first time in literature. Western Science has explored only ~5% of the approximately 25,000 species of higher plants for drug leads. The ancient medical science of Ayurveda has however employed a much larger spectrum of plants for clinical treatment. Clerodendrum viscosum (CV), a commonly growing weed in the Indian subcontinent has been employed by S. Nirmalananda (Ayurvedic expert) for the treatment of cervical cancer. Here we isolate and characterize a water extract fraction (Cv-AP) from the root of CV and evaluate its anticervical cancer cell bioactivity. Our results indicate that Cv-AP possesses pro-apoptotic, anti-proliferative, and anti-migratory activity in a dose-dependent fashion against cervical cancer cell lines. In contrast, primary fibroblasts (control healthy cells), when exposed to similar concentrations of this extract, fail to undergo apoptosis and remain relatively unaffected. These findings suggest that Clerodendrum viscosum (CV) is a readily available source of components with potent anti-cancer activity and selective bioactivity against cervical cancer cells. The major component in CV-AP was identified as a glycoprotein via SDS Page and Concanavalin-A binding studies. This study serves to illustrate that systematic collaboration with Ayurveda is a practical and powerful strategy in drug discovery and development.

Dendrimer-curcumin conjugate: a water soluble and effective cytotoxic agent against breast cancer cell lines.

Curcumin, which is derived from the plant Curcuma longa, has received considerable attention as a possible anti-cancer agent. In cell culture, curcumin is capable of inducing apoptosis in cancer cells at concentrations that do not affect normal cells. One draw-back holding curcumin back from being an effective anti-cancer agent in humans is that it is almost completely insoluble in water and therefore has poor absorption and subsequently poor bioavailability. Here we have generated a number of curcumin derivatives (tetrahydro-curcumin, curcumin mono-carboxylic acid, curcumin mono-galactose, curcumin mono-alkyne and dendrimer-curcumin conjugate) to test whether any of them display both cytotoxicity and water solubility. Of those tested only dendrimer-curcumin conjugate exhibited both water solubility and cytotoxicity against SKBr3 and BT549 breast cancer cells. When compared to curcumin dissolved in DMSO, dendrimer-curcumin conjugate dissolved in water was significantly more effective in inducing cytotoxicity, as measured by the MTT assay and effectively induced cellular apoptosis measured by caspase-3 activation. Since dendrimer-curcumin conjugate is water soluble and capable of inducing potent cytotoxic effects on breast cancer cell lines, it may prove to be an effective anti-cancer therapy to be used in humans.

Nongenomic Mechanisms of PTEN Regulation.

A large amount of data supports the view that PTEN is a bona fide tumor suppressor gene. However, recent evidence suggests that derailment of cellular localization and expression levels of functional nonmutated PTEN is a determining force in inducing abnormal cellular and tissue outcomes. As the cellular mechanisms that regulate normal PTEN enzymatic activity resolve, it is evident that deregulation of these mechanisms can alter cellular processes and tissue architecture and ultimately lead to oncogenic transformation. Here we discuss PTEN ubiquitination, PTEN complex formation with components of the adherens junction, PTEN nuclear localization, and microRNA regulation of PTEN as essential regulatory mechanisms that determine PTEN function independent of gene mutations and epigenetic events.

Intracellular pH regulation by Na⁺/H⁺ exchanger-1 (NHE1) is required for growth factor-induced mammary branching morphogenesis.

Regulation of intracellular pH (pHi) and protection against cytosolic acidification is primarily a function of the ubiquitous plasma membrane Na+/H+exchanger-1 (NHE1), which uses a highly conserved process to transfer cytosolic hydrogen ions (H+) across plasma membranes in exchange for extracellular sodium ions (Na+). Growth factors, which are essential regulators of morphogenesis, have also been found to be key activators of NHE1 exchanger activity; however, the crosstalk between both has not been fully evaluated during organ development. Here we report that mammary branching morphogenesis induced by transforming growth factor-alpha (TGFα) requires PI3K-dependent NHE1-activation and subsequent pHi alkalization. Inhibiting NHE1 activity after TGFα stimulation with 10 µM of the NHE1-specific inhibitor N-Methyl-N-isobutyl Amiloride (MIA) dramatically disrupted branching morphogenesis, induced extensive proliferation, ectopic expression of the epithelial hyper-proliferative marker Keratin-6 and sustained activation of MAPK. Together these findings indicate a novel developmental signaling cascade involving TGFα>PI3K>NHE1>pHi alkalization, which leads to a permissible environment for MAPK negative feedback inhibition and thus regulated mammary branching morphogenesis.