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Nature Neuroscience Aug 2021The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and...
The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and emotional skills into adulthood. Although mature cerebellar neuroanatomy is well studied, understanding of its developmental origins is limited. In this study, we systematically mapped the molecular, cellular and spatial composition of human fetal cerebellum by combining laser capture microscopy and SPLiT-seq single-nucleus transcriptomics. We profiled functionally distinct regions and gene expression dynamics within cell types and across development. The resulting cell atlas demonstrates that the molecular organization of the cerebellar anlage recapitulates cytoarchitecturally distinct regions and developmentally transient cell types that are distinct from the mouse cerebellum. By mapping genes dominant for pediatric and adult neurological disorders onto our dataset, we identify relevant cell types underlying disease mechanisms. These data provide a resource for probing the cellular basis of human cerebellar development and disease.
Topics: Cerebellum; Fetus; Humans; Laser Capture Microdissection; Neurogenesis; Single-Cell Analysis; Transcriptome
PubMed: 34140698
DOI: 10.1038/s41593-021-00872-y -
Nature Methods Oct 2023Single-cell proteomics by mass spectrometry is emerging as a powerful and unbiased method for the characterization of biological heterogeneity. So far, it has been...
Single-cell proteomics by mass spectrometry is emerging as a powerful and unbiased method for the characterization of biological heterogeneity. So far, it has been limited to cultured cells, whereas an expansion of the method to complex tissues would greatly enhance biological insights. Here we describe single-cell Deep Visual Proteomics (scDVP), a technology that integrates high-content imaging, laser microdissection and multiplexed mass spectrometry. scDVP resolves the context-dependent, spatial proteome of murine hepatocytes at a current depth of 1,700 proteins from a cell slice. Half of the proteome was differentially regulated in a spatial manner, with protein levels changing dramatically in proximity to the central vein. We applied machine learning to proteome classes and images, which subsequently inferred the spatial proteome from imaging data alone. scDVP is applicable to healthy and diseased tissues and complements other spatial proteomics and spatial omics technologies.
Topics: Animals; Mice; Proteome; Mass Spectrometry; Proteomics; Laser Capture Microdissection
PubMed: 37783884
DOI: 10.1038/s41592-023-02007-6 -
Journal of Visualized Experiments : JoVE Feb 2021The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an...
The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps. It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection. Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.
Topics: Animals; Brain; Brain Mapping; Male; Mice, Inbred C57BL; Microdissection; Mice
PubMed: 33645582
DOI: 10.3791/61941 -
Methods in Molecular Biology (Clifton,... 2016The new opportunities of modern assays of molecular biology can only be exploited fully if the results can be accurately correlated to the tissue phenotype under...
The new opportunities of modern assays of molecular biology can only be exploited fully if the results can be accurately correlated to the tissue phenotype under investigation. This is a general problem of non-in situ techniques, whereas results from in situ techniques are often difficult to quantify. The use of bulk tissue, which is not precisely characterized in terms of histology, has long been the basis for molecular analysis. It has, however, become apparent, that this simple approach is not sufficient for a detailed analysis of molecular alterations, which might be restricted to a specific tissue phenotype (e.g., tumor or normal tissue, stromal or epithelial cells). Microdissection is a method to provide minute amounts of histologically characterized tissues for molecular analysis with non-in situ techniques and has become an indispensable research tool. If tissue diversity is moderate and negligible, manual microdissection can be an easy and cost-efficient method of choice. In contrast, the advantage of laser microdissection is a very exact selection down to the level of a single cell, but often with a considerable time exposure to get enough material for the following analyses. The latter issue and the method of tissue preparation needed for laser microdissection are the main problems to solve if RNA, highly sensitive to degradation, shall be analyzed. This chapter focuses on optimized procedures for manual microdissection and laser microdissection to analyze RNA of malignant and nonmalignant prostate tissue.
Topics: Benzoxazines; Coloring Agents; Equipment Design; Gene Expression Regulation, Neoplastic; Humans; Laser Capture Microdissection; Male; Prostate; Prostatic Neoplasms; RNA; RNA Stability; Staining and Labeling
PubMed: 26667453
DOI: 10.1007/978-1-4939-3204-7_2 -
Proteomics May 2021Multiple applications of proteomics in life and health science, pathology and pharmacology, require handling size-limited cell and tissue samples. During proteomic... (Review)
Review
Multiple applications of proteomics in life and health science, pathology and pharmacology, require handling size-limited cell and tissue samples. During proteomic sample preparation, analyte loss in these samples arises when standard procedures are used. Thus, specific considerations have to be taken into account for processing, that are summarised under the term microproteomics (μPs). Microproteomic workflows include: sampling (e.g., flow cytometry, laser capture microdissection), sample preparation (possible disruption of cells or tissue pieces via lysis, protein extraction, digestion in bottom-up approaches, and sample clean-up) and analysis (chromatographic or electrophoretic separation, mass spectrometric measurements and statistical/bioinformatic evaluation). All these steps must be optimised to reach wide protein dynamic ranges and high numbers of identifications. Under optimal conditions, sampling is adapted to the studied sample types and nature, sample preparation isolates and enriches the whole protein content, clean-up removes salts and other interferences such as detergents or chaotropes, and analysis identifies as many analytes as the instrumental throughput and sensitivity allow. In the suggested review, we present and discuss the current state in μP applications for processing of small number of cells (cell μPs) and microscopic tissue regions (tissue μPs).
Topics: Laser Capture Microdissection; Mass Spectrometry; Proteins; Proteomics; Specimen Handling
PubMed: 33547857
DOI: 10.1002/pmic.202000318 -
Current Protocols in Molecular Biology Oct 2015Laser microdissection (LM) offers a relatively rapid and precise method of isolating and removing specified cells from complex tissues for subsequent analysis of their...
Laser microdissection (LM) offers a relatively rapid and precise method of isolating and removing specified cells from complex tissues for subsequent analysis of their RNA, DNA, protein or metabolite content, thereby allowing assessment of the role of different cell types in the normal physiological or disease processes being studied. In this unit, protocols for the preparation of mammalian frozen tissues, fixed tissues, and cytologic specimens for LM, including tissue freezing, tissue processing and paraffin embedding, histologic sectioning, cell processing, hematoxylin and eosin staining, immunohistochemistry, and image-guided cell targeting are presented. Also provided are recipes for generating lysis buffers for the recovery of nucleic acids and proteins. The Commentary section addresses the types of specimens that can be utilized for LM and approaches to staining of specimens for cell visualization. Emphasis is placed on the preparation of tissue or cytologic specimens as this is critical to effective LM.
Topics: Animals; Humans; Laser Capture Microdissection; Mammals; Single-Cell Analysis; Specimen Handling
PubMed: 26423586
DOI: 10.1002/0471142727.mb25a01s112 -
Kidney International Apr 2019Bulk-tissue RNA-Seq is increasingly being used in the study of physiological and pathophysiological processes in the kidney; however, the presence of multiple cell types...
Bulk-tissue RNA-Seq is increasingly being used in the study of physiological and pathophysiological processes in the kidney; however, the presence of multiple cell types in kidney tissue complicates data interpretation. We addressed the question of which cell types are represented in whole-kidney RNA-Seq data in order to identify circumstances in which bulk-kidney RNA-Seq can be successfully interpreted. We carried out RNA-Seq in mouse whole kidneys and in microdissected renal tubule segments. To aid in the interpretation of the data, we compiled a database of cell-type selective protein markers for 43 cell types believed to be present in kidney tissue. The whole-kidney RNA-Seq analysis identified transcripts corresponding to 17,742 genes, distributed over 5 orders of magnitude of expression level. Markers for all 43 curated cell types were detectable. Analysis of the cellular makeup of mouse and rat kidney, calculated from published literature, suggests that proximal tubule cells account for more than half of the mRNA in a kidney. Comparison of RNA-Seq data from microdissected proximal tubules with data from whole kidney supports this view. RNA-Seq data for cell-type selective markers in bulk-kidney samples provide a valid means to identify changes in minority-cell abundances in kidney tissue. Because proximal tubules make up a substantial fraction of whole-kidney samples, changes in proximal tubule gene expression can be assessed presumptively by bulk-kidney RNA-Seq, although results could potentially be complicated by the presence of mRNA from other cell types.
Topics: Animals; Biomarkers; Gene Expression Profiling; Kidney; Mice; Microdissection; RNA, Messenger; RNA-Seq; Rats; Transcriptome
PubMed: 30826016
DOI: 10.1016/j.kint.2018.11.028 -
Methods in Cell Biology 2019Metanephric organ culture, or ex vivo embryonic kidney culture, was developed in the mid-twentieth century as a means to understand the development of the mammalian...
Metanephric organ culture, or ex vivo embryonic kidney culture, was developed in the mid-twentieth century as a means to understand the development of the mammalian kidney and was used in early studies of polycystic kidney disease to explore mechanisms of renal cyst initiation by non-genetic factors. Following the identification of cystogenic genes, a resurgence of the use of metanephric organ culture occurred and has yielded insight into basic mechanisms of cystic dilation; facilitated identification of pathogenic pathways and potential therapeutic targets; and provided a system for evaluating therapeutic agents. This chapter provides detailed, step-by-step protocols with rationale and tips for the establishment, maintenance and treatment of metanephric organ cultures, and for performance of the most commonly employed secondary analyses of these cultures.
Topics: Animals; Culture Media; Cyclic AMP; Disease Models, Animal; Embryo, Mammalian; Female; Humans; Intravital Microscopy; Kidney; Mice; Microdissection; Microscopy, Fluorescence; Organ Culture Techniques; Polycystic Kidney Diseases
PubMed: 31395378
DOI: 10.1016/bs.mcb.2019.04.018 -
Proteomics Sep 2020The problem with cancer tissue is that its intratumoral heterogeneity and its complexity is extremely high as cells possess, depending on their location and function,... (Review)
Review
The problem with cancer tissue is that its intratumoral heterogeneity and its complexity is extremely high as cells possess, depending on their location and function, different mutations, different mRNA expression and the highest intricacy in the protein pattern. Prior to genomic and proteomic analyses, it is therefore indispensable to identify the exact part of the tissue or even the exact cell. Laser-based microdissection is a tried and tested technique able to produce pure and well-defined cell material for further analysis with proteomic and genomic techniques. It sheds light on the heterogeneity of cancer or other complex diseases and enables the identification of biomarkers. This review aims to raise awareness for the reconsideration of laser-based microdissection and seeks to present current state-of-the-art combinations with omic techniques.
Topics: Genome; Genomics; Humans; Laser Capture Microdissection; Neoplasms; Proteomics
PubMed: 32578340
DOI: 10.1002/pmic.202000077 -
Current Opinion in Urology Jan 2023To review noteworthy research from the last 2 years on surgical management of azoospermia. (Review)
Review
PURPOSE OF REVIEW
To review noteworthy research from the last 2 years on surgical management of azoospermia.
RECENT FINDINGS
The recommended treatments for nonobstructive and obstructive azoospermia have not appreciably changed. However, recent level-1 evidence has reinforced superiority of micro-dissection testicular sperm extraction over sperm aspiration in men with nonobstructive azoospermia, and several studies have identified genetic and other clinical factors that may aid in selecting candidates for testicular sperm extraction. Machine learning technology has shown promise as a decision support system for patient selection prior to sperm retrieval as well a tool to aid in sperm identification from testis tissue.
SUMMARY
Most men with obstructive azoospermia who desire fertility can be offered either surgical reconstruction or sperm retrieval. For men with nonobstructive azoospermia, sperm retrieval with microdissection testicular sperm extraction remains the gold standard treatment. Uncovering more genetic causes of nonobstructive azoospermia may aid in properly counseling and selecting patients for microdissection testicular sperm extraction. Neural networks and deep learning may have a future role in patient selection for surgical sperm retrieval and postprocedural sperm identification.
Topics: Humans; Male; Azoospermia; Semen; Sperm Retrieval; Microdissection; Testis; Retrospective Studies
PubMed: 36301052
DOI: 10.1097/MOU.0000000000001055