Distinct molecular profiles of skull bone marrow in health and neurological disorders.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

Spatial proteomics in three-dimensional intact specimens.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.

A guidebook for DISCO tissue clearing.

Histological analysis of biological tissues by mechanical sectioning is significantly time-consuming and error-prone due to loss of important information during sample slicing. In the recent years, the development of tissue clearing methods overcame several of these limitations and allowed exploring intact biological specimens by rendering tissues transparent and subsequently imaging them by laser scanning fluorescence microscopy. In this review, we provide a guide for scientists who would like to perform a clearing protocol from scratch without any prior knowledge, with an emphasis on DISCO clearing protocols, which have been widely used not only due to their robustness, but also owing to their relatively straightforward application. We discuss diverse tissue-clearing options and propose solutions for several possible pitfalls. Moreover, after surveying more than 30 researchers that employ tissue clearing techniques in their laboratories, we compiled the most frequently encountered issues and propose solutions. Overall, this review offers an informative and detailed guide through the growing literature of tissue clearing and can help with finding the easiest way for hands-on implementation.

Human placental trophoblast progenitor cells (hTPCs) promote angiogenesis and neurogenesis after focal cerebral ischemia in rats.

INTRODUCTION: Reduction of blood flow below a threshold value in brain regions locally or globally is called cerebral ischemia and proper treatment requires either the restoration of normal blood flow and/or the administration of neuroprotective therapies. Human trophoblast progenitor cells (hTPCs) give rise to the placenta and are responsible for the invasion and vascular remodeling of the maternal vessels within the uterus. Here, we tested whether hTPCs promoted to differentiate along neural lineages may exhibit therapeutic properties in the setting of cerebral ischemia in vivo. MATERIALS AND METHODS: Cerebral ischemia was generated in rats via middle cerebral artery occlusion and, after 24 h, hTPCs were injected systemically via tail vein. Animals were sacrified at Day 3 or 11. RESULTS: TTC staining indicated that infarct volumes were smaller in hTPC treated animals. Visible myelin recovery was observed in the hTPC injected group with Luxol Fast Blue staining. On Day 11 after hTPC transplantation, DLX5 and VEGF expression, as well as 2 and 10 d after hTPC transplantation, NKX2.2 were significantly increased; while LHX6, Olig1, PDGFRα, VEGFR1 and VEGFR2 showed trends toward improved expression in brain tissue via immunoblot analysis. Neuron-like differentiated cells were positive for both NeuN and Cresyl Violet staining. CONCLUSION: Here, we demonstrate for the first time that hTPCs enhance the expression of angiogenic and neurogenic factors in rat brain after stroke. Transplantation of hTPCs could form the basis of novel therapeutic approaches for the treatment of stroke in the clinical setting.

Contribution of Human Trophoblast Progenitor Cells to Neurogenesis in Rat Focal Cerebral Ischemia Model.

OBJECTIVE: : A decrease in the blood flow below a current level in the brain results in ischemia. Studies demonstrated that human trophoblast progenitor cells (hTPCs) contribute to the treatment of many diseases. Therefore, hTPCs might be a promising source to repair ischemia in cerebral ischemia models. For this purpose, we evaluated the expression of many neurogenesis markers by performing hTPC transplantation after focal cerebral ischemia in rats. METHODS: : hTPCs, isolated from the term placentae, were characterized by immunofluorescent staining and differentiated into neuron-like cells. Differentiation was confirmed with immunostaining of GFAP and NeuN proteins. Cerebral ischemia models were generated in rats via middle cerebral artery occlusion and, after 24 hours, hTPCs were injected via the tail vein. Animals were sacrificed on day 3 or day 11. Immunohistochemical analysis was performed with proteins associated with neurogenesis and neuronal development, such as DLX2, DLX5, LHX6, NGN1, and NGN2, Olig1, Olig2, and PDGFRα. RESULTS: : According to our results, hTPCs may alleviate ischemic damage in the brain and contribute to the neurogenesis after ischemia. CONCLUSIONS: : Based on our findings, this topic should be further investigated as the hTPC-based therapies may be a reliable source that can be used in the treatment of stroke and ischemia.

Whole-mouse clearing and imaging at the cellular level with vDISCO.

Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies.

Human Trophoblast Progenitor Cells Express and Release Angiogenic Factors.

Trophoblast stem cells develop from polar trophectoderm and give rise to the cells that generate the placenta. Trophoblast cells are responsible for the uterine invasion and vascular remodeling during the implantation of the embryo. However this knowledge is not yet to be confirmed for trophoblast progenitor cells (TPCs). In this study, we aimed to demonstrate that human TPCs (hTPCs) express and release angiogenic factors for the first time. TPCs were isolated from the term placenta. Then immunophenotyping was performed by FACS method by analyzing caudal type homeobox 2 (CDX2) and eomesodermin (EOMES). Immunofluorescence staining of CDX2 and EOMES was performed on these cells. Lastly, angiogenesis-related proteins were detected by western blot and ELISA assays. The isolated cells were positive for trophoblast stem cell markers CDX2 and EOMES in 92.5% and 92.7% of cells, respectively showing the characteristics of TPCs. The investigation of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (VEGFR1), and vascular endothelial growth factor receptor 2 (VEGFR2) at protein and mRNA level in comparison with human umbilical vein endothelial cells (HUVECs), revealed that human TPCs (hTPCs) have higher levels of VEGF and VEGFR1 transcripts. Additionally soluble forms of VEGF and VEGFR1 were detected in supernatants of hTPCs. With this information, TPCs seem to be promising for regenerative cell therapies by increasing angiogenesis.