Department of Histology and Embryology, Faculty of Medicine MU pvanhara@med.muni.cz Tissue concept and classification Petr Vaňhara, PhD FOUNDING FATHERS OF HISTOLOGY − DISCOVERY OF CELLS 1665 Robert Hooke Anthony van Leeuwenhoek ESSENTIAL TECHNOLOGY • nálevníci • orální bakterie (Selenomonády) • spermatozoa • krvinky • svalová vlákna • histologická barvení 1674-1683 „I see different structures in hunan body. I do not need a microscope to distinguish 21 types! I will call them tissues. In a diseased body the tissues have altered, abnormal structure! FOUNDING FATHERS OF HISTOLOGY − TISSUES So different tissues! Xavier Bichat, 1799 Theodor Schwann Cells are the basic units of any organism ▪ New cells origin only from other cells ▪ Cells exchange energy (open thermodynamic system) ▪ Genetic information is inherited in new generations ▪ Chemical and structural composition of cells is generally identical FOUNDING FATHERS OF HISTOLOGY − MODERN CELL TEHORY Matthias Jacob Schleiden J.E.P. Omnis cellula e cellula! Rudolf Wirchow Robert Remak Cells are the basic units of any organism CELL AND TISSUE VARIABILITY IN A MULTICELLULAR BODY - 6  1013 CELLS of 200 different types - cells form functional, three-dimensional, organized aggregations of morphologically similar cells and their products and derivatives - TISSUES - tissues constitute ORGANS and organ systems Myocardium TISSUES AND ORGANS Parenchyma: functional component of a tissue (liver, lung, pancreatic, kidney parenchyma) Stroma: surrounding, essential supportive tissue Parenchyma Stroma Example: LIVER Parenchyma: - Hepatocytes - Sinusoids and adjacent structures Stroma: - Connective tissue and adjacent structures - Vessels - Nerves - Bile ducts TISSUES AND ORGANS Epithelium Muscle Nerve Connective Based on morphology and function: Myofibrils → contraction Mesoderm – skeletal muscle, myocard, mesenchyme – smooth muscles Rarely ectoderm (eg. m. sphincter a m. dilatator pupillae) Neurons and neuroglia Reception and transmission of electric signals Ectoderm, rarely mesoderm (microglia) Dominant extracellular matrix Connective tissue, cartilage, bone… Mesenchyme Continual, avascular layers of cells with different function, oriented to open space, with specific junctions and minimum of ECM and intercellular space. Derivates of all three germ layers CONTEMPORARY TISSUE CLASSIFICATION ▪ classical histological definition is based on microscopic visualization Functional, three-dimensional, organized aggregation of morphologically similar cells, their products and derivatives TISSUE DEFINITION What is a tissue? Proliferation Differentation Migration Apoptosis Tissue patterns BASIC PRINCIPLES OF HISTOGENESIS How to build a tissue? ? Knoblich JA. Asymmetric cell division during animal development. 2001. Nat Rev Mol Cell Biol Differentiation Self-renewal Stem cells are capable of differentiation and self-renewal Asymmetric division Proliferation Formation of functional types FUNCTIONAL CELL TYPES DIFFERENTIATE FROM STEM CELLS Stem cells are essential STEM CELLS DIFFERENTIATION Totipotent - Constitute all cells of the body incl. extraembryonic tissues - Zygote and early stages Pluripotent - All cells in the body except for trophoblast - Blastocyst – Inner cell mass - ICM (embryoblast) - Embryonic stem cells Multipotent - Give rise to various cell types of a particular tissue - Mesenchymal SC, hematopoietic SC http://www.embryology.ch/anglais/evorimplantation/furchung01.html Oligo- a unipotent - One or several cell types – hematopoietic, tissue precursors for renewal of intestinal epithelia, etc. STEM CELLS Tissue (adult) stem cells - regeneration and renewal of tissue - GIT, CNS, mesenchymal tissues - regenerative medicine, cancer biology Embryonic stem cells (ESCs) - derived from embryoblast (ICM) of preimplantation blastocyst - pluripotent - model of early embryogenesis and histogenesis, regenerative medicine STEM CELLS IN ORGANISM H. Clevers J. Thompson Induced pluripotent stem cells (iPSc) - adult differentiated cell (fibroblast) is reprogrammed into pluripotent state - differentiation into desired cell type - regenerative medicine, cell and gene therapy Nobel prize 2012 Disease modelling Drug testing Tissue replacement … STEM CELLS AS RESEARCH TOOLS hESCs hiPSCs iPSCs SHARE FUNDAMENTAL PROPERTIES WITH hESCs Age-related macular degeneration neovascularisation Clinical trial hiPSCs Retinal pigment epithelium STEM CELLS AS THERAPY Cancer stem cells - solid tumor is always heterogeneous - small population of cells with stem cell character can repopulate tumor tissue after cytotoxic therapy Tissue stem cells Renewal Low frequency (<1%) Quiescence Multipotency Long life Resistence Tumorigenicity Proliferation capacity Cancer stem cells STEM CELLS AS FOES • Induction of differentiation • Terminal differentiation • Determination and commitment -blast -cyte eg. myeloblast e.g. granulocyte CELL DIFFERENTIATION Essential terminology doi:10.1038/nrg3209 DIFFERENTIATION IS DRIVEN BY GENE TRANSCRIPTION Essential mechanisms 1 doi:10.1038/nature10523 TISSUE DIFFER IN THEIR GENETIC AND EPIGENETIC PROFILES Adhesion molecules Growth factors ECM components Cell interactions Metabolites Immunity Inflammation CELLS CAN CREATE UNIQUE MICROENVIRONMENT Essential mechanisms 2 • Embryonic development • Intercellular interaction • Space organization (dimensionality) • Gradient of morphogenes • Epigenetic profile • Gene expression dynamics • Partial pressure of gases • ECM composition • Mechanical stimulation • Perfusion and interstitial flows • Local immunity response • Metabolites Huge number of biological and physically-chemical parameters MICROENVIRONMENT REGULATES TISSUE FUNCTION STEM CELL NICHE HEMATOPOIETIC NICHE Apoptóza Regenerace Senescence Patologická změna MICROENVIRONMENT IS NECESSARY FOR TISSUE HOMEOSTASIS Apoptosis Regeneration Senescence Transformation MICROENVIRONMENT IS NECESSARY FOR TISSUE HOMEOSTASIS MICROENVIRONMENT MIGHT BE CLINICALLY IMPORTANT MICROENVIRONMENT IS IMPORTANT FOR PATHOGENESIS https://www.pathologyoutlines.com/topic/livercirrhosis.html Fibrotic Infections Metabolic disorders Autoimmune disorders Cholestasis Alcohol Genetics Abnormal tissue microenvironemnt Healthy Normal tissue microenvironment Example: liver Stroma Parenchyma Stroma Parenchyma Loss of functional parenchyma Deposition of ECM Activation of (myo)fibroblasts) Dysplasia Inflammation GENERAL TISSUE COMPOSITION ECM Signaling moleculesCells+ + Tissue = MOLECULAR PRINCIPLES OF HISTOGENESIS Essential mechanisms 3 LEWIS WOLPERT’S FRENCH FLAG MODEL Expression patterns of gap and pairrule genes in Drosophila embryos. DOI: 10.1007/s10577-006-1068-z Eric Francis Wieschaus is an American evolutionary developmental biologist and 1995 Nobel Prize-winner. Three short lectures on embryonic patterning DROSOPHILA Eric Wieschaus WHY DO TIGERS HAVE STRIPES? Reaction–diffusion system TISSUE PATTERNS ARE DRIVEN BY GRADIENTS OF MORPHOGENES Belousov-Zabotinsky doi:10.1038/sj.hdy.6800872 Hox genes Highly conserved family of transcription regulators that determine body polarity, orientation and axis Tissue differentiation along anterioposterior axis Human (39 genes) Cluster Chromosome # Hox genes HoxA 7 11 HoxB 17 10 HoxC 12 9 HoxD 2 9 HOX COMPLEX TEMPORO-SPATIAL EXPRESSSION OF MORPHOGENES DRIVES FINAL LOCALIZATION, ORIENTATION AND MORPHOLOGY OF TISSUES AND ORGANS MANIPULATING AER ALTERS INSTRUCTIONS FOR LIMB DEVELOPMENT MORPHOGENES FROM AER AND ZPA DEFINES LIMB FORMATION http://courses.biology.utah.edu/bastiani/3230/DB%20Lecture/Lectures/b14Limb.html HOX PATTERN DRIVES TRANSCRIPTIONAL RESPONSE HOX Vascularisation Fgf Shh … Proliferation Thalidomid STORY OF THALIDOMID Thalidomid embryopathy • phocomelia • amelia • anophtalmia/microphtalmia • abnormal kidneys, heart, GIT, genitalia Frances Oldham Kelsey, FDA USA Ectoderm MesodermEndoderm Trilaminar germ disc (3rd week) HISTOGENESIS AND ORGANOGENESIS ▪ Connective tissue of head ▪ Cranium, dentin ▪ Skeletal muscle of trunk and limbs except cranium ▪ Dermis of skin ▪ Muscles of head ▪ Urogenital system + ducts, glands and gonads ▪ Visceral muscle and connective tissue ▪ Serous membranes of pleura, peritoneum and pericardium ▪ Blood cells, leukocytes ▪ Cardiovascular and lymphatic system ▪ Spleen ▪ Adrenal cortex ▪ GIT epithelium except oral cavity and part of anal canal ▪ Extramural glands of GIT ▪ Epithelium of bladder ▪ Epithelium of respiratory system ▪ Thyroid gland, parathyroid glands, thymus ▪ Tonsils ▪ Epithelium of cavum tympani and Eustachian tube ▪ Epidermis, hair nails, cutaneous and mammary glands ▪ Corneal epithelium and lens of eye ▪ Enamel of teeth ▪ Internal ear ▪ Anterior pituitary gland ▪ Epithelium of oral cavity and part of anal canal ▪ Neural tube and derivatives - CNS - Retina - Posterior pituitary gland - Pineal body ▪ Neural crest and derivatives: - Cranial and sensory ganglia and nerves - Schwann cells - adrenal medulla - Enteroendocrinne cells - Melanocytes - Head mesenchyme and connective tissue - Odontoblasts SurfaceectodermNeuroectoderm headParaxialIntermediateLateral EndodermEctoderm Mesoderm EMBRYONIC DEVELOPMENT CONNECTIVE TISSUE Not just a tissue glue… Mechanical and biological properties → surrounds other tissues, allows compartmentalization, provides support, defines physicochemical environment, brings immunological support, provides storage of energy, ... CONNECTIVE TISSUE Extracellular matrix • Fibrous component ‒ collagen fibers (prototypically col. I, II) ‒ reticular ‒ elastic • Amorphous component (amorphous ground substance) Complex matrix consisting of ‒ glycosaminoglycans ‒ glycoproteins ‒ proteoglycans Specific composition dependens on a tissue type (connective  ligament  cartilage  bone) Cells • Connective tissue – permanent and transient cell populations (e.g. fibroblasts/myofibroblasts, immune cells, adipocytes, adult stem cells) • Cartilage – chondroblasts/chondrocytes • Bone – osteoblasts/osteocytes/osteoclasts GENERAL COMPOSITION OF CONNECTIVE TISSUE CLASSSIFICATION OF CONNECTIVE TISSUE Collagen Structure Function and distribution Loose collagen CT Abundant ground substance, few collagen fibers with random arrangement Microvascularisation Innervation Irregular dense collagen CT Few ground substance, few cells, many collagen fibers, random arrangement Mechanically resistant organ capsules Regular dense collagen CT Tightly arranged collagen fibers with fibroblasts intercalated between them Part of musculoskeletal system. Tendons, ligaments Embryonic Mesenchyme Undifferentiated cells uniformly dispersed in ground substance, few collagen fibers Undifferentiated progenitors Wharton’s jelly Viscous amorphous matrix with collagen fibers. ECM-producing stromal cells with MSC properties. Matrix of umbilical cord Special Reticular CT Network of collagen III fibers and reticular cells Support of hematopoietic and lymphatic cells Elastic Rich in elastic fibers Lig. flava, lig. vocale. Lung interstitium, flexible support to elastic arteries and aorta Adipose Adipocytes Energy storage (white fat), heat production (brown fat) Cartilage Chondroblasts, chondrocytes Mechanical support Bone Osteoblasts, ostecoytes, osteoclasts Mechanical support, calcium and phospate metabolism Blood See lecture on blood & hematopoiesis this semester - Fibroblasts/fibrocytes/myofibroblasts - Heparinocytes - Macrophages of CT = histiocytes - Plasma cells - Lymphocytes - Adipocytes - Adult stem cells Extracelular matrix - Fibrous - Amorphous ground substance Cells • Permanent • Migratory - CT Macrophages = histiocytes - Plasma cells - Lymphocytes, granulocytes - Heparinocytes - … GENERAL COMPOSITION OF CONNECTIVE TISSUE PROPER + • Mesenchymal stem cells differentiate to many cells of CT GENERAL COMPOSITION OF CONNECTIVE TISSUE PROPER • Mesenchymal stem cells are important for tissue engineering GENERAL COMPOSITION OF CONNECTIVE TISSUE PROPER FIBROBLAST https://www.sciencephoto.com/media/1232046/view/fibroblast-tem ECM ECM of connective tissue is produced by fibroblasts (chondrocytes, osteoblasts). However, specific ECM can be produced by any cell of our body (eg. epithelial and muscle cells producing basal lamina). ECM composition determines tissue properties Collagen fibers - family of fibrous proteins encoded by >35 genes (2013) - polymer – subunit = tropocollagen; triple helix - different structural and mechanical properties (strength, elasticity, pliability…) - most abundant protein in human body ( 30% dry weight) EXTRACELLULAR MATRIX – FIBROUS COMPONENT • Polyribosomes bind to RER and synthetise peptide chains 1 a 2 (~250 AA, 28kDa) • In RER peptide chains are modified (hydroxylation of proline and lysine – co-factor vitamin C) Chains assemble into triple helix - procollagen • In GA, procollagen is further modified and secreted from cells COLLAGEN COLLAGEN further study: https://www.ncbi.nlm.nih.gov/books/NBK507709/ Procollagen is then modified to tropocollagen (by procollagenpeptidase) Tropocollagen is organized to higher fibrillar structures in ECM (fibrils, fibers) Individual collagen molecules are connected (lysyloxidases) COLLAGEN Type Localization Structure Main function I Bone, tendons, meniscus, dentin, dermis, capsules of organs, loose CT 90% of type I Fibrils (75nm) – fibers (1-20m) Resilience in pull II Hyaline and elastic cartilage Fibrils (20nm) Resilience in pressure III Skin, veins, smooth muscles, uterus, liver, spleen, kidney, lung Like I, high content of proteoglycans and glycoprotiens, reticular network Shape formation IV Basal lamina of epithelium and endtohelium, basal membranes No fibrils or fibers Mechanical support V Lamina of muscle cells and adipocytes, fetal membranes Like IV VI Interstitial tissue, chondrocytes – adhesion Connecting dermis and epidermis VII Basal membrane of epithelium VIII Some endothelia (Cornea) IX, X Growth plate, hypertrophic and mineralized cartilage Growth of bones, mineralization COLLAGEN AZAN HES HE COLLAGEN IN LIGHT MICROSCOPE Julian Voss-Andreae "Unraveling Collagen" 2005 Orange Memorial Park Sculpture Garden, City of South San Francisco, CA COLLAGEN IN ART • less abundant than collagen • polymer – tropoelastin • minimal tensile resistance, loss of elasticity if overstretched • reduction of hysteresis = allow return back to original state after mechanic change ELASTIC FIBERS ELASTIC FIBERS Elastic fibers Elastin Collagen ELASTIC FIBERS Elastic fibers • Similarly to collagen, elastin precursors are secreted and polymerize • Deposition of elastin aggregate along fibers of protein fibrillin • Amount of fibrillin (nonelastic) and elastin (elastic) determines elasticity of CT Fibrillin Elastin Elastin • collagen 3D meshwork • bone marrow, spleen, lymphatic nodules • microenvironment for e.g. hematopoietic stem cells and progenitors RETICULAR FIBERS RETICULAR CONNECTIVE TISSUE Amorphous extracellular matrix Colorless, transparent, homogenous substance consisting of glycosaminglycans, proteoglycans and structural glycoproteins EXTRACELLULAR MATRIX – GROUND SUBSTANCE linear polysaccharides composed of two disaccharide subunits – uronic acid and hexosamine glucosamin or galactosamin glucuronic or iduronic acid polysaccharides rich in hexosamines = acid mukopolysaccharides GLYCOSAMINOGLYCANS • lineární polysacharidy tvořené disacharidovými podjednotkami - kyselinou uronovou a hexosaminem GYLCOSAMINOGLYCANS https://doi.org/10.3390/cancers15010266 Glycosaminoglycan Localization Hyaluronic acid Umbilical cord, synovial fluid, fluid of corpus vitreum, cartilage Chondroitinsulphate Cartilage, bone, cornea, skin, notochord, aorta Dermatansulphate Skin, ligaments, adventitia of aorta Heparansulphate Aorta, lungs, liver, basal membranes Keratansulphate Iris, cartilage, nucleus pulposus, anulus fibrosus They bind to protein structures (except for hyaluronic acid) GLYCOSAMINOGLYCANS — protein + dominant linear saccharide component — proteoglycan aggregates — water-binding, volume dependent of hydratation — aggrecan (cartilage) — syndecan — fibroglycan PROTEOGLYCANS • dominant protein + branched saccharide component • interaction between cells and ECM ― fibronectin – connects collagen fibers and glykosaminoglycans, cell adhesion and migration ― laminin – basal lamina – epithelial integrity ― chondronectin – cartilage – adhesion of chondrocytes to collagen (J. Nutr. 136:2123-2126, 2006) STRUCTURAL GLYCOPROTEINS Glycoproteins vs. proteoglycans STRUCTURAL GLYCOPROTEINS COMPOSITION OF ECM ECM – SUMMARY - Embryonic mesenchyme and Wharton’s jelly of umbilical cord - Areolar (loose collagen, interstitial) CT - Dense collagen regular/irregular CT - Elastic CT - Reticular CT HISTOLOGICAL CLASSIFICATION OF CT PROPER Embryonic mesenchyme Wharton’s jelly LOOSE COLLAGEN CT • Permanent fibroblasts, macrophages (histiocytes), occasionally adipocytes • Other transient cell types (leukocytes) • Collagen and elastic fibers • Amorphous ground substance is dominant • Most abundant type of CT • Rich vascularization and innervation • Walls of hollow organs, interstitium, mucosal and submucosal CT http://medsci.indiana.edu/a215/virtualscope/docs/chap2_1.htm DENSE COLLAGEN CT • Adipocytes, fibroblasts, reticular, collagen and elastic fibers, capillaries • White and brown adipose tissue ADIPOSE TISSUE • adipocytes are actively formed until 2nd year of life • no innervations, but rich vascularisation • adipocytes with only one lipid droplet • leptin (adipokinins) WHITE ADIPOSE TISSUE • fetus and children up to 1st year of life • fast source of energy • typical localization – between shoulder blades, axilla, mediastinum, around kidneys, pancreas, small intestine • small cells with numerous fat droplets BROWN ADIPOSE TISSUE • Mesenchyme = loose tissue between germ layers • Complex network of star- or spindle-shaped cells • Jelly-like amorphous ground substance http://www.mun.ca/biology/desmid/brian/BIOL3530/DB_Ch02/DBNModel.html DAY 12 of embryonic development EMBRYONIC ORIGIN OF CONNECTIVE TISSUE Connective BoneCartilage Mesenchyme DERIVATIVES OF MESENCHYME DERIVATIVES OF MESENCHYME MESENCHYMAL STEM CELLS DOI: 10.3389/fsurg.2015.00001 APPLICATIONS OF MESENCHYMAL STEM CELLS https://doi.org/10.3389/fbioe.2020.00043 FURTHER STUDY http://www.histology.med.muni.cz Thank you for attention pvanhara@med.muni.cz