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Lecture 040609 Pyle

Lecture 040609 Pyle - MCDB-168 Lecture Embryonic Stem Cells...

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MCDB-168 Lecture: Embryonic Stem Cells Spring 2009 April Pyle, Ph.D. Microbiology, Immunology and Molecular Genetics UCLA Broad Stem Cell Center Main Goals of the Lecture How are human embryonic stem cells(hESCs) and mouse (mESCs) derived? What are the definitions of a pluripotent ESC? ESC importance and biology Know basic concepts about how ESCs can be manipulated to modify gene expression How are mESC different from hESC and how is pluripotency of each assayed? See examples of directed differentiation of ES-cells to specific lineages Reasons for new stem cell derivations and future use in regenerative medicine
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Properties of Pluripotent Stem Cells (Part I) OCT 4 SSEA4 (human) SSEA1 (mouse) Alkaline Phosphatase (AP)
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Markers of Pluripotency are below and are shown with pictures on the previous slide 1. ESCs are positive for OCT4, Alkaline Phosphatase (AP) and mouse- or human- specific SSEA antigens, 2. Can give rise to all cell types in the body, can self renew or give rise to new stem cells every division, 3. Remain karyotypically stable over continuous passage, and 4. Once injected into immunocompromised mice, will form teratomas in which cells from each germ layer can be identified. Properties of Pluripotent Stem Cells (Part II) Types of Pluripotent Stem Cells Donovan and Gearhart Nature 2001 New derivations: Epiblast stem cells Induced Pluripotent Stem Cells
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Embryonal Carcinoma (EC) Cell Research Precedes hESC Research Mature cystic teratoma of the ovary Ovarian yolk sac tumor Testicular teratoma Teratomas (benign) and teratocarcinomas (malignant) most commonly found in gonads composed of differentiated tissues from all 3 germ lineages 1950’s: mouse strain 129 showed incidence of ~1% Tumors arising from a small nest of cells within testes Pluripotent cells: a single cell injected intra- peritoneally can self renew & produce all cell types in teratoma – a stem cell! Rossant lab ppt Derivation of Mouse EC cells clonal cells derived from teratomas could recapitulate the differentiation pattern of the original tumor following injection In culture these clones differentiated into various cell types. Culture of these cells became more predictable with the introduction of mouse embryonic fibroblasts (MEFs) Permitted the subcloning of a pluripotent teratocarcinoma cell lines – could proliferate indefinitely, produce teratomas, form cystic embryoid bodies. Late 1970’s – isolation of human embryonic carcinoma cell lines Retinoic acid treatment neural tissue myocardium chondrocytes endoderm skeletal muscle Rossant lab ppt
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Embryonic stem cells ES cell lines are DERIVED from the inner cell mass, ICM (ES cells are “a culture artifact”, do not exist in the embryo !) Can be stably maintained in culture in a pluripotent state without transformation (compare to tissue specific stem cells, and cell lines from differentiated tissues!) Mouse ES cells but not human can be manipulated in culture and injected to blastocysts to generate genetically
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