section #8: an introduction to stem cells ward g. walkup iv

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  • Section #8: An Introduction to Stem CellsWard G. Walkup IV

  • Overview of Section #8 SlidesHi All, below is an overview of my section slidesSlide #4: what a stem cell isSlides #5-6: adult vs. embryonic stem cellsSlides #7-13: toti, multi and pluripotency of stem cellsSlides #14-15: induced pluripotent stem cellsSlides #16-30: different types of cloning#17-20: molecular cloning#21-26: reproductive cloning#27-30: therapeutic cloningSlides #31-37: Muotri PaperSlides #38 on: extra unused stuff on neural differentiation

  • Todays section material will present an overview of stem cells, covering the following topics:What a stem cell isAdult versus embryonic stem cellsMulti, pluri, totipotency and induced pluripotentcy of stem cellsDifferent types of cloning (Molecular, Reproductive & Therapeutic)Generation of mice with chimeric brainsOverview of Section Materials

  • What is a Stem Cell?http://www.patentbaristas.com/wp/wp-content/uploads/2007/04/stem-cells.jpg & Biology 1, 2008 LectureCells that:Have the capacity for self-renewal and ability to divide for indefinite periods in cultureCan give rise to two or more specialized cells (Unrestricted potential for differentiation)

  • Embryonic stem cellsPrimitive (undifferentiated) cells derived from a 5-day preimplantation embryo that have the potential to become a wide variety of specialized cell types

    Adult (Somatic) stem cellsAn undifferentiated cell found in a differentiated tissue that can:Renew itselfDifferentiate to give rise to all the specialized cell types of tissue from which it originatedScientists do not agree about whether or not adult stem cells may give rise to cell types other than those of the tissue from which they originateEmbryonic Stem Cells

  • Types of Stem Cells (Adult & Embryonic)ADD SLOW SLIDEhttp://www.nature.com/ncb/journal/v3/n9/fig_tab/ncb0901-e205_F1.htmlEmbryonic stem cell has unrestricted potential for differentiationAdult stem cells are lineage restricted

  • Toti, Pluri and Multipotency of Stem CellsPluripotent A stem cell able to give rise to all cell types that make up the body, but not extra embryonic tissues such as amnion, chorion and other placental componentsAn embryonic stem cell is an example of a pluripotent cellPluripotency can be demonstrated by:Ability of the progeny from a single cell to form derivatives of all three embryonic germ layers (Endo-, Ecto- and Mesoderm)Ability to generate a teratoma after injection into an immunosuppressed mouse

  • Toti, Pluri and Multipotency of Stem CellsPluripotent A stem cell able to give rise to all cell types that make up the body, but not extra embryonic tissues such as amnion, chorion and other placental componentsAn embryonic stem cell is an example of a pluripotent cellPluripotency can be demonstrated by:Ability of the progeny from a single cell to form derivatives of all three embryonic germ layers (Endo-, Ecto- and Mesoderm)Ability to generate a teratoma after injection into an immunosuppressed mouse

  • Toti, Pluri and Multipotency of Stem CellsPluripotent A stem cell able to give rise to all cell types that make up the body, but not extra embryonic tissues such as amnion, chorion and other placental componentsAn embryonic stem cell is an example of a pluripotent cellPluripotency can be demonstrated by:Ability of the progeny from a single cell to form derivatives of all three embryonic germ layers (Endo-, Ecto- and Mesoderm)Ability to generate a teratoma after injection into an immunosuppressed mousehttp://www.biology-online.org/articles/embryonic_stem_cells_prospects/figures.html

  • Toti, Pluri and Multipotency of Stem CellsPluripotent A stem cell able to give rise to all cell types that make up the body, but not extra embryonic tissues such as amnion, chorion and other placental componentsAn embryonic stem cell is an example of a pluripotent cellPluripotency can be demonstrated by:Ability of the progeny from a single cell to form derivatives of all three embryonic germ layers (Endo-, Ecto- and Mesoderm)Ability to generate a teratoma after injection into an immunosuppressed mousehttp://erl.pathology.iupui.edu/C604/GENE826.HTM

  • TotipotentA stem cell able to give rise to all the cell types that make up the body, plus all of the cell types that make up extra embryonic tissues, such as the placentaA zygote is an example of a totipotent cellMultipotentA stem cell able to develop into more than one type of cell in the bodyAn adult (Somatic) stem cell can be an example of a multipotent cell(Multipotent)Biology 150Development Lecture Slides, Fall 2006CaltechToti, Pluri and Multipotency of Stem Cells

  • (Multipotent)Biology 150, Development Lecture Slides, Fall 2006, CaltechToti, Pluri and Multipotency of Stem Cells

  • (Multipotent)Biology 150, Development Lecture Slides, Fall 2006, CaltechToti, Pluri and Multipotency of Stem Cells

  • Adult cells (e.g. Fibroblasts) reprogrammed to an embryonic stem cell-like state by forced expression of specific genesExpression of the transcription factors Oct4, Sox2, c-Myc & Klf4, under lentiviral and retroviral promoters causes reprogrammingMouse and human iPSCs have been generatediPSCs have been shown to have important characteristics of pluripotent stem cells:Expression of stem cell markersAbility to form tumors containing cells from all 3 germ layersAbility to contribute to many different tissues when injected into mouse embryos at an early stage in dvelopmentinduced Pluripotent Stem Cells (iPSCs)

  • Generating iPSCsTransfectLentiviral & Retroviral Vectors Containing:

  • Cloning is an umbrella term traditionally used by scientists to describe different processes for duplicating biological materialA basic understanding of the different types of cloning is paramount for taking an informed stance on current public policy issuesThree types of cloning will be discussed in the upcoming slides:Recombinant DNA Technology or DNA/Molecular CloningReproductive CloningTherapeutic CloningMolecular, Reproductive and Therapeutic Cloning

  • Refers to the transfer of a DNA fragment of interest from one organism to a self-replicating genetic elementThe most common self-replicating genetic element is extra-chromosomal DNA known as a plasmidTypically a five part process:A DNA fragment containing the gene of interest is isolated from chromosomal DNA using restriction enzymesThe gene of interest is united with a plasmid that has been cut using the same restriction enzymesThe gene of interest and plasmid are covalently linked using DNA ligase, to yield a recombinant DNA moleculeThe recombinant DNA is moved into a host cell, to allow for propagation using the host cells enzymatic machineryHost cells containing the recombinant DNA are selected for and identifiedMolecular Cloning

  • Molecular Cloning1.2. & 3.Adapted From: Alberts et al. Molecular Biology of the Cell, 2002, Figure 8-30, p 501

  • Molecular Cloning4.5.Adapted From: Alberts et al. Molecular Biology of the Cell, 2002, Figure 8-31, p 501

  • Uses of Molecular Cloning: Deconvolution of complex biological and biochemical processes by studying components in isolationExamples: Metabolic pathways, Translation, TranscriptionGenetic engineering of organismsExamples: Bioremediation, crop engineering, heterologous production of pharmaceuticalsIdentification of evolutionary relationshipsExamples: DNA sequencing of genomes and individual genesMolecular Cloning

  • Refers to technology used to generate an animal that has the same nuclear DNA (Somatic cell nucleus) as another currently, or previously existing animalCan use a technique known as Somatic Cell Nuclear Transfer (SCNT) to perform reproductive cloning*Genetic material from the nucleus of an adult egg is transfered to an egg whose nucleus, and thus its genetic material, has been removed (enucleated egg)The reconstructed egg containing DNA from the donor cell is treated with chemicals or electric current to stimulate cell divisionOnce the embryo reaches a suitable stage, it is transferred to the uterus of a female host, where it continues to grow until birthReproductive Cloning

  • Stuff

  • Animals created using nuclear transfer are not truly identical cones of the donor animal!Only the clones chromosomal DNA is the same as the donorSome of the clones genetic material comes from mitochondria located in the cytoplasm of the enucleated eggInterestingly, acquired mutations in mitochondri