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Lecture 4

Lecture 4 - September 24 - LIFESCI 2A03

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McMaster University
Margaret Fahnestock

LECTURE 4 LIFESCI 2A03 Stem Cells September 24, 2013  First identification of stem cells: 1963 o Canadians Jim Till and Ernest McCulloch prove the existence of stem cells (Toronto)  Paper: Cytological Demonstration of the Clonal Nature of Spleen Colonies Derived from Transplanted Mouse Marrow Cells  Derived cells from bone marrow and identified some characteristics of these cells  Cells had potential to become many types of different cells and undergo mitosis to produce more of themselves (renew self to create another stem cell) and then other cell can differentiate o Siminovitch, L., E.A. McCulloch, A.J. Till  Paper: Journal of Cellular and Comparative Physiology  Demonstrates that these progenitor cells were capable of self-renewal  Stem Cells – distinguished from other cell types by two important characteristics 1. They are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of dormancy 2. Under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions  Classes of Stem Cells o Embryonic stem cells (pluripotent) o Adult (or somatic) stem cells (multipotent or unipotent)  Upon fertilization, the resulting zygote is totipotent o Totipotent – the ability to create all cells in the body including extra-embryonic cell types; more potential than pluripotent (pluripotent does not include extra-embryonic cell types) o Will go on to create every cell in the body in addition to the placenta and extra-embryonic tissues  Blastocyst – hollow ball of cells containing the trophoblast cells that will form the placenta and the inner cell mass (ICM) that will develop into the embryo o At genetic level – every cell has the same DNA; can differ in expression o Twins occur when at stage 2, the embryo split apart o Early Blastocyst – cells have become restricted; totipotent cell from fertilization has broken into 2 different types  Extra-embryonic tissue (blue)  Inner Cell Mass; only go on to form embryo; pluripotent (pink)  Each cell has potential to become an entire organism o Figure: 5 Day old, pre-implantation embryo  Inner Cell Mass o Blastocyst – 5 days post-fertilization  Outer layer of blastocyst will become placenta and supporting tissues  Inner Cell Mass will become embryo and thus can form all cells and tissues of body o Forms of ICM: Ectoderm, Endoderm, Mesoderm  Blastocyst becomes gastrula  Division causes ICM to become a flattened disk which can be categorized into 3 types  Ectoderm – external layer; go on to form skin of epidermis, neuron of brain, and pigment cells  limited to specialized cell types (multipotent)  Mesoderm – middle layer; go on to form cardiac muscle, skeletal muscle cells, tubule cell of the kidney, RBCs and smooth muscle in the gut  Endoderm – internal layer; go on to form pancreatic cell, thyroid cell and lung cell (alveolar cell)  Some germ cells become sperm and egg cells – totipotent because cells that can form entire organisms are eventually made (however, they are haploid) 1 LECTURE 4 LIFESCI 2A03  Pluripotent cells develop into all the three germ layers  Embryonic Stem Cells (ESC) o Undifferentiated cells of the inner cell mass o Capable of dividing without differentiating for a prolonged period of time (when in culture) o Under certain conditions are capable of developing into every cell type from the three germ layers o Pluripotent 2 LECTURE 4 LIFESCI 2A03 o First identification of Mouse ESC – pluripotent cells derived from mouse embryos  Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells; Martin, G, 1981  Establishment in culture of pluripotent cells from mouse embryos o Embryonic stem cells can be cultured in vitro  Thomason, J.A et al. 1998. Embryonic stem cell lines derived from human blastocysts  Requirements – culture medium and a feeder layer of mouse skin cells  Compare cells derived directly from inner cell mass with cells cultured in petri dish  looks the same  Allowed for 8 generations – took cells out of culture  looks the same as inner cell mass  Small spherical cell, little cytoplasm compared to size of nucleus, no membrane structure, no set of proteins that change structure of cell  Figure: A + C = Cell from ICM; B + D = Cultures ESC o Creating ESC Lines  Rather than generating these de nova, embryonic stem cell lines are maintained through sub culturing – the passage of cells to fresh plates where they can continue to divide  Do not use new cells; use descendants from original experiments  The ICM cells are derived form embryos fertilized in vitro in the course of reproductive therapies/infertility treatments  Rid of trophoblast – only culture inner cell mass; grow in culture dish  Mitotic division; transfer to new culture dish  Dormancy, induce cell division, induce differentiation o Cultured ESC  test whether they are pluripotent (since ESC are pluripotent)  Criteria that characterize ESC as pluripotent 1. Long term culture with cell division (immortal cell lines) o Immortal referring to lineage, not individual cell; cells can divide, descendants can divide and descendants can divide indefinitely 2. Markers of undifferentiated cells a. Gene expression  Express same proteins and genes as embryonic stem cells b. Cell surface proteins 3. Testing for pluripotency a. Inducing differentiation in cell culture  Potential t become any cell type (ectoderm, endoderm, mesoderm); test in culture and in living organism (results in teratoma) b. Teratoma formation – mass of non-cancerous cells that divide and differentiation  Teratoma formation in mice o Teratoma – tumors of multiple lineages containing tissue derived from the three germ layers endoderm, mesoderm and ectoderm o Not fully functional system, but part of it – integrates into the systems, not functional o Significance – assay for pluripotency; used to see whether cell in question is actually pluripotent 3 LECTURE 4 LIFESCI 2A03 o Figure A) Gut B) Neural epithelium C) Bone D) Cartilage E) Striated muscle F) Capillary of kidney o Different from carcinomas – uncontrolled until cause damage; metastatic; escape and harm other tissues; definitely detrimental, may develop inside a tissue o Teratoma – benign, not metastatic, reach maximum size, generally produced not in a tissue  Stem cells are the only cells in the body that are dividing; differ
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