Session 7 - HSC A Historical Perspective

Session 7 - HSC A Historical Perspective - HSC- a...

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HSC- a historical perspective • Lethal irradiation of civil population in 1945 • Phenotype reproduced in mice • Rescue of phenotype by bone marrow transplantation -spleen colonization is the assay • Transplantation of bone marrow cells suggests there is a cell subtype giving rise to spleen colonies • Developing cell surface antibodies to cells and sort different sets of cells- spleen colonization
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ematopoietic Stem Cells Hematopoietic Stem Cells Session VII /12/07 2/12/07
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Development of HSCs
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Alergic& antiparasite “of bone marrow” clotting
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Sites of hematopoietic activity in e developing embryo Primitive blood starts in YS the developing embryo Blood cells ciculate between the YS and EB Potential of blood cells is restricted/no HSCs found HSCs are in AGM first and in YS second
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The first site of definitive hematopoiesis seems to be the GM but the cellular origin of HSC is unclear although AGM, but the cellular origin of HSC is unclear, although it is agreed that they derive from the mesoderm • Two possible models for the cellular origin of HSC: – 1) HSCs originate in a putative “hemangioblast” in the AGM - common mesodermal precursor of endothelial and blood cells – 2) HSCs originate in already committed endothelial cells, that retain the ability to make HSCs
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Primitive vs definitive hematopoiesis
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Colonization theory of HSCs • AGM is primary site of HSC formation iver thymus spleen and marrow are Liver, thymus, spleen and marrow are thought to be colonies of AGM born HSCs ontroversial) (controversial) • Can you think of research strategies to proach that question systematically? approach that question systematically?
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LCM Microarray to determine eir expression profiles of SC clusters AGM/Fetal their expression profiles of Hematopoietic clusters HSC clusters AGM/Fetal liver/thymus/spleen/BM Find common possibly early HSC genes by overlapping databases with specific lineage marker Determine which genes are expressed Isolate promoter regions of 20 or so genes and make reporter (B-gal) mi at the AGM- should be there first and than at the other sites (BM, etc) Make a Specific Promoter-Cre-ER mouse Induce Cre briefly/transiently in a Rosa 26 mouse at the time when is just in the AGM and track the fate of blue cells
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In Vitro HSCs Assays hort- rm - weeks Short term 3 weeks Solid substrate (methylcellulose/agar/plasma clot) -determine # of colony forming cells CFC (CFU-C) iquid media - ore differentiated cells by flow cytometry (cannot Liquid media score differentiated cells by flow cytometry (cannot determine CFC) Long-term - 5 weeks or more- but there is a lack of mphoid differentiation lymphoid differentiation • LTC-IC (long term colony initiating cell) • On irradiated fibroblast feeders / ability to form multipotent colonies after extended divisions – CAFC - cobblestone area growing cells •O n bone marrow stromal cells- long term colony forming + gy g repopulation of irradiated mice/ but there is no expansion of the stem cell pool in culture
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This note was uploaded on 09/28/2008 for the course BIO 4400 taught by Professor Tumbar,td during the Spring '07 term at Cornell University (Engineering School).

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Session 7 - HSC A Historical Perspective - HSC- a...

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