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Engineering human bone marrow stromal cells

Weber, Matthew Charles
http://rave.ohiolink.edu/etdc/view?acc_num=case1055867071

Abstract Details

1991, Doctor of Philosophy, Case Western Reserve University, Pathology.
Bone marrow stromal cells regulate normal and leukemic hematopoietic cells through both soluble and cell surface-associated molecules. To molecularly probe the complexities of stromal cell hemoregulation, stable gene transfer capabilities were developed for a model human bone marrow stromal cell line, KM-102, using Epstein-Barr virus (EBV) and BK virus (BKV) episomal expression vectors. Antisense expression technology was applied to distinguish the function of individual soluble factors among the variety of hematopoietic cytokines produced by KM-102 stromal cells. Granulocyte/macrophage colony stimulating factor (GM-CSF) served as a model soluble cytokine. Supernatants from KM-102 cells stably transfected with an antisense GM-CSF episomal expression vector no longer possessed detectable levels of GM-CSF or colony stimulating activity (CSA). Significantly, GM-CSF inhibition unmasked a hematopoietic progenitor cell inhibitory activity. These data suggest that GM-CSF functions in a dominant mode as the major CSA produced by KM-102 and point to a hierarchical relationship among stimulatory and inhibitory hematopoietic factors produced by stromal cells. KM-102 stromal cell hemoregulatory effects mediated through cell contact-dependent interactions were studied in coculture experiments with human my eloid leukemia cell lines. In response to the chemical inducer, 1α, 25-dihydroxyvitamin D3, HL-60 and PLB-985 leukemic cells tightly bound to KM-102 cells were inhibited from differentiating, while their loosely adherent and nonadherent counterparts differentiated normally. These data suggest that in clinical settings, stromal cells may render leukemic cells refractory to differentiation induction therapy. Intercellular adhesion molecule-1 (ICAM-1) served as a model cell surface molecule in our KM-102 stromal cell transfection analyses. This surface molecule is known to play a dominant role in certain intercellular adhesion phenomena involving hematopoietic cells. Antisense and sense ICAM-1 expression experiments did not support a dominant role for ICAM-1 in mediating KM-102 stromal cell:hematopoietic cell interactions. Lastly, the stromal cell surface was engineered using a chimeric gene expression strategy to artificially direct hematopoietic cellular interactions. A chimeric variant of the colony stimulating factor, M-CSF, incorporating a glycoinositolphospholipid (GPL) for membrane anchorage, was expressed on the surface of KM-102 cells. This cytokine, when tethered to the cell surface, functions as a potent cellular adhesin, permitting a several-fold enhancement in cell binding to M-CSF receptor expressing cellular targets. This gene transfer strategy, and derivative protein transfer strategies, can now be used for elucidating the mechanisms of stromal cell hemoregulatory phenomena mediated through cell-cell contact, as well as for engineering the cell surface phenotypes of stromal cells to be used in cell-based therapies.
Mark Tykocinski (Advisor)
251 p.
stromal cells; KM-102; CSF; bone marrow

Recommended Citations

Citations

  • Weber, M. C. (1991). Engineering human bone marrow stromal cells [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1055867071

    APA Style (7th edition)

  • Weber, Matthew. Engineering human bone marrow stromal cells. 1991. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1055867071.

    MLA Style (8th edition)

  • Weber, Matthew. "Engineering human bone marrow stromal cells." Doctoral dissertation, Case Western Reserve University, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=case1055867071

    Chicago Manual of Style (17th edition)

case1055867071
610
© 1991, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.