Stem Cells and Differentiation PDF

Warning The contents of these slides are the exclusive property of the Instructor and/or granted by third parties (textbooks’ reference for pictures) and are therefore protected by the current regulations governing the Protection of Copyright. All rights are reserved. The reproduction and/or...

Warning The contents of these slides are the exclusive property of the Instructor and/or granted by third parties (textbooks’ reference for pictures) and are therefore protected by the current regulations governing the Protection of Copyright. All rights are reserved. The reproduction and/or diffusion, even partial, by any analogical and/or digital means, without the consent of the rights holder is FORBIDDEN. Any unauthorized use of the above mentioned "Contents" is under the full and exclusive responsibility of the users who will be responsible for it, according to the laws and regulations in force. It is allowed the use of the material for private and study use, however not for profit and without commercial purposes. Stem cells and differentiation What is a stem cell “A stem cell is a cell from the embryo, fetus, or adult that has, under certain conditions, the ability to reproduce itself for long periods or, in the case of adult stem cells, throughout the life of the organism. It also can give rise to specialized cells that make up the tissues and organs of the body.” What is a stem cell In self-renewing adult tissues (high cell turnover rates), cells are continually being born, differentiating, and dying. These tissues contain tissue specific stem cells that contribute to tissue turnover. Stem cells: from the zygote to an adult organism All cells in a multi-cellular organism derive from a single cell, the zygote  What can stem cells do o Build novel tissues and organs (embryo stem cells) o Enable the growth of developing tissues and organs (foetal stem cells) o Support homeostasis and regeneration of adult tissues and organs, enabling their healing (adult stem cells)  Stem cell properties 1. Self-renewal (clonogenity): they can proliferate and renew/replenish their population through extensive cell divisions, sometimes after extended quiescent phases. 2. Differentiation potential: under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells, with specialized functions and morphology.  Stem cell properties Stem cells o Stem cells can divide a virtually unlimited number of times maintaining their undifferentiated status (symmetric Symmetric division cell division) Symmetric division Asymmetric division o Stem cells can give rise to two different daughter cells: one retaining the undifferentiated status, the other one with a committed phenotype that will Committed cell undergo differentiation (asymmetric cell division) Differentia ted cell  Stem cell properties o Stem cells usually do not produce differentiated cells directly, but intermediate cell types (also different) that are usually called progenitors cells o These intermediate cells only go through a limited number of divisions before they differentiate  Stem Cell Classification Stem cells are classified either based on their potency or based on their origin  Stem cell properties The range of potential differentiated fates a stem cell can achieve reduces during development: o Zygote → totipotent o Embryo stem cells → pluripotent o Adult stem cells → multipotent/oligopotent o Somatic cells → unipotent o Terminally differentiated cells → nullipotent, stably morpho functionally specialized phenotype  Totipotent stem cells Early embryo: zygote and its daughter cells up to 3-4 cell divisions (8 cells stage):  Able to divide indefinitely  Differentiate towards any cell lineage to generate all embryo-foetal tissues, including extra embryo membranes  Cells in the morula lose this totipotency  Pluripotent stem cells Embryonic stem cells isolated from the inner cell mass of the blastocyst:  Able to divide and differentiate towards any of the three-germ layer- derived lineages  Cannot give rise to extra embryonal membranes  Multipotent stem cells As the embryo grows these pluripotent cells develop into specialized, multipotent stem cells:  Able to differentiate towards lineages from a single germ layer Hematopoietic stem cells Mesenchymal stromal cells Oligopotent stem cells  Able to differentiate towards a limited number of cell lineages  Unipotent stem cells  Able to divide indefinitely and differentiate towards a single cell lineage  Somatic cell precursors: - Hepatocytes - Satellite cells - Epidermal stem cells  Stem cell niche  Specialized microenvironment surrounding a stem cell population  Contains the appropriate composition and concentration of growth factors, chemical and physical signals, humoral mediators to regulate the balance between proliferation and differentiation properties of stem cells and their progenies  Different niches may have different repertoires of signals, adapted to a specific location and function The promise of regenerative medicine  The ability of adult stem cells to self-renew and produce terminally differentiated cells declines over time, contributing to tissue aging The promise of regenerative medicine  Many tissues are not only self-renewing but also self-repairing to maintain tissue homeostasis  Stem cells offer enormous promises for advancing health and medicine  Used to replace damaged cells and organs or else by supporting the body's intrinsic repair mechanisms  Stem cell sources exploited in applications and clinical trials Mesenchymal stromal cells (MSC) Connective stroma of all connective tissues: ✓ Bone marrow ✓ Cord blood ✓ Adipose tissue ✓ Amniotic fluid ✓ Placenta ✓ Wharton’s jelly ✓ Dental pulp Stem cell sources exploited in applications and clinical trials Induced Pluripotent Stem Cells (iPSC) Induced Pluripotent Stem Cells (iPSC)  The Nobel Prize in Physiology or Medicine 2012 was awarded jointly to Sir John B. Gurdon and Shinya Yamanaka "for the discovery that mature cells can be reprogrammed to become pluripotent"  N publications in PubMed 10000 15000 20000 25000 0 5000 1970 1972 1974 1976 1978 1980 1982 1984 Research time line 1986 1988 1990 1992 1994 1996 1998 2000 Year of Publication 2002 2004 2006 Timeline in stem cell research 2008 2010 2012 2014 2016 2018 Cell isolation and culture  Cell isolation and culture  Removal of cells from a multicellular organism and subsequent growth in a favorable artificial environment  Cell culture represent one of the most valuable technical achievements of the entire study of biology  Cell isolation and culture  The first reported primary cultures were explants of frog neuronal fibers (Harrison et al., 1907) and dissociated sponges (Wilson, 1907) that grew and differentiated over the course of several days.  By the 1950s, the introduction of protease digestion, defined media, and antibiotics made vertebrate cell culture a more widely accessible technique.  Cell isolation and culture ADVANTAGES  The possibility of obtaining large quantities of cells  The possibility of studying numerous and different cellular activities (membrane trafficking, cell movement, cell division, macromolecule synthesis, cell signaling)  The possibility of studying cell differentiation  The possibility of studying the responses to treatment with drugs, hormones, growth factors and other active substances. Cell isolation and culture Tissue Collection Blood Drawing Organ/Tissue Biopsy  Cell isolation and culture Tissue Collection Skin punch biopsy Muscle biopsy Bone marrow biopsy  Cell isolation and culture Cell isolation  Obtaining cell cultures starting from tissue specimens is complex, given that tissues are heterogeneous  Isolation protocols provide for a first phase whose purpose is to separate the different cell types present in the tissue, demolishing the extracellular matrix and the intercellular junctions that keep them together  A first phase of dissociation of the single cells by mechanical techniques (usually proteolytic enzymes) is followed by a separation phase to separate the various cell types present in the cell suspension obtained  Cell isolation and culture Cell isolation Adherent  By exploiting the different abilities of Suspension different cell types to adhere to glass or plastic surfaces  Using selective culture media that promote the growth of only certain cell types  Cell isolation and culture Some definitions…  Normal cells usually divide only a limited number of times before losing their ability to proliferate (senescence) → “finite” cell lines  Some cell lines become immortal (transformation) by acquiring the ability to divide indefinitely → “continuous” cell lines  Cell isolation and culture Some definitions…  Adherent cells grow on the support on which they were seeded (i.e. on the flask wall)  Other cells grow in suspension in the culture medium without adhering to any support  Cell isolation and culture What we need…  Sterile conditions and appropriate equipment  Isolation protocols  Culture protocols  Sterile conditions Cell isolation and culture and appropriate equipment Inverted Microscope Laminar Air Flow Hood CO2 Incubator  Sterile conditions Cell isolation and culture and appropriate equipment Water bath Centrifuge  Cell isolation and culture Cell culture The artificial environment in which the cells are cultured consists of:  Growth medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals)  Growth factors, hormones, and gases (O2, CO2)  Controlled physicochemical conditions (pH, osmotic pressure, temperature)  To prevent contamination of cell cultures by bacteria, low levels of antibiotics are added in the medium  Cell isolation and culture Cell culture  To amplify the cell population, cells can be dissociated and detached from the substrate through enzymatic digestion (trypsin)  Cell isolation and culture Long-term Storage  Cells are suspended with the appropriate cryo- protective agent (e.g., DMSO or glycerol)  Stored at temperatures below -80° up to –130°C until needed  Cell isolation and culture Cell culture  The traditional two-dimensional cultures, in which cells grow on the flat surface of a plate, are being flanked by three-dimensional cultures (3D cultures), in which the cells are grown in a three-dimensional matrix consisting of synthetic and/or natural extracellular material.  Since cells in an organism do not live on flat surfaces, three-dimensional arrays are thought to provide a much more physiological environment for cells in culture.  Furthermore, 3D cultures are also more suitable for studying interactions between cells.  To study the interactions between cells are also extensively used co-culture systems, in which multiple cell types are cultured together. Cell isolation and culture Cell culture Cell Culture 1 Cell Culture 2 Tumor Spheroids  Cell isolation and culture Cell culture  3D Bioprinting is an innovative technique that uses cells and other biocompatible materials as “inks”, also known as bioinks, to print living structures layer-by-layer which mimic the behavior of natural living systems