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Growth properties and genetic manipulation of murine hemopoietic stem cells

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Title: Growth properties and genetic manipulation of murine hemopoietic stem cells
Author: Pawliuk, Robert James
Degree Doctor of Philosophy - PhD
Program Medical Genetics
Copyright Date: 1996
Abstract: The development of recombinant retroviral vectors able to transfer exogenous genetic material into hemopoietic target cells has played a pivotal role in our current understanding of hemopoiesis and has played a pioneering role in the field of gene therapy. However, with the efficiency of gene transfer to murine stem cells only 15% the power of recombinant retroviral gene transfer is currently severely compromised by the efficiency of retroviral infection. To optimize the utility of recombinant retroviruses, the human CD24 cell surface antigen was developed as a dominant selectable marker in a retroviral vector to enable the identification and selection of retrovirally transduced murine bone marrow cells, including those with long term in vivo repopulating ability. Following infection of day 45-FU treated murine bone marrow cells and selection of retrovirally transduced cells using an anti-CD24 antibody and Fluorescence Activated Cell Sorting (FACS), functional analysis of selected CD24+ cells demonstrated the presence of hemopoietic cells at various stages of development, including in vitro clonogenic progenitors, day 12 CFU-S, and cells with totipotent long-term repopulating potential. Further experiments demonstrated the ability to regenerate the hemopoietic systems of myeloablated recipient mice with cells derived exclusively from provirally marked stem cells and that the transferred CD24 gene was expressed in various phenotypically defined populations of cells in vivo including marrow stem cell candidates defined by the Sca+Lin" cell surface phenotype. Thus, CD24 can be utilized not only as a selectable marker but also as a means to track and phenotype transduced cells and their progeny in vitro and in vivo. To provide information on the recovery of hemopoietic stem cells following bone marrow transplant, irradiated recipient mice were injected with various numbers of day 14.5 fetal liver or day 45-FU adult bone marrow estimated to contain 10, 100 or 1000 Competitive Repopulating Units (CRU). Analysis of the femoral marrow of primary recipients showed complete recovery of bone marrow cellularity and clonogenic progenitor content and a near full recovery of day 12 CFU-S numbers irrespective of the number or origin of the cells initially transplanted. While the recovery of donor-cell-derived CRU was incomplete in all cases, fetal liver was markedly superior to those from adult bone marrow. Moreover, proviral integration analysis of mice receiving retrovirally transduced CD24+ selected bone marrow cells provided evidence for a >300-fold clonal amplification of a single transduced stem cell. These studies have provided procedures for the selection, tracking and phenotyping of murine bone marrow cells, including those with competitive long term lympho-myeloid repopulating ability. The availability of such procedures should increase the power of retroviral marking studies, and be advantageous in studies aimed at the genetic manipulation of hemopoietic stem cells and their progeny, as well as in the development of vectors able to optimize the expression of transferred genes in specific target cells of interest for use in human gene therapy trials. Moreover, these findings set the stage for attempts to enhance hemopoietic stem cell regeneration post-transplant by the administration of exogenous agents or the expression of intracellular factors that may enhance the regenerative potential of stem cells.
URI: http://hdl.handle.net/2429/6134
Series/Report no. UBC Retrospective Theses Digitization Project [http://www.library.ubc.ca/archives/retro_theses/]

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