Investigation of the Effects of In Vitro Cytokine Exposure on Short and Long Term Reconstituting Haemopoietic Stem and Progenitor Cells in a Murine Model

Holyoake, Tessa Laurie (1996) Investigation of the Effects of In Vitro Cytokine Exposure on Short and Long Term Reconstituting Haemopoietic Stem and Progenitor Cells in a Murine Model. PhD thesis, University of Glasgow.

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There is increasing interest in the possibility of expanding haemopoietic stem and progenitor cells ex vivo. Successful expansion of pluripotent haemopoietic stem cells would extend the use of high dose chemotherapy with autologous rescue to include those patients for whom we are currently unable to harvest sufficient stem cells for transplantation. It is likely that the efficiency of gene transduction would be significantly enhanced under conditions in which stem cells were proliferating. For cancer treatments, using high dose but non-myeloablative chemotherapy, progenitor cell expansion should reduce harvesting requirements and, thereby, tumour cell contamination. Finally, the primary aim of these studies was to expand, ex vivo, those progenitors cells thought to mediate the early phase of engraftment in order to reduce the duration of both neutropenia and thrombocytopenia following bone marrow transplantation (BMT). We have attempted to determine optimum ex vivo culture conditions which allow maximum amplification of murine transient engrafting stem cells. The short term (6 days) incubation of unfractionated bone marrow in liquid culture with Stem Cell Factor (SCF), Interleukin-11 (IL-11), with or without Macrophage Inflammatory Protein-1 alpha (MIP-1alpha) produced a 50 fold amplification of those cells which were shown to rescue lethally irradiated recipients in a BMT model. Following ex vivo expansion, ten to twenty fold fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, bone marrow cells expanded ex vivo resulted in significantly more rapid haemopoietic recovery. Thus, we were able to demonstrate expansion of progenitor cells ex vivo and to assess the engraftment potential of the expanded cells. The results suggested two potential benefits for patients with cancer if similar data could be obtained with human haemopoietic cells. Although we had demonstrated expansion of progenitor cells, it was equally important to assess the effect that ex vivo culture had on those stem cells responsible for long term reconstitution following BMT. In a serial transplantation model, unmanipulated bone marrow was only able to consistently sustain secondary BMT recipients but bone marrow expanded ex vivo sustained quaternary BMT recipients which remained alive and well more than 120 days after BMT. These findings have important implications for transplantation and gene transfer studies since expansion of clonogenic cells accompanied by maintenance of long term reconstituting stem cells will result, not only in improved early engraftment, but also in sustained long-term reconstitution following transplantation and may result in enhanced transduction efficiencies with genes of interest. Following tertiary and quaternary BMT many of the recipients of expanded cells developed B-cell chronic lymphocytic leukaemia (B-CLL). The group transplanted with SCF / IL-11 / MIP-1alpha expanded marrow developed leukaemia earlier and with a greater frequency that those transplanted with SCF / IL-11 expanded cells. The leukaemia was shown to have arisen in host haemopoietic cells rather than in the donor bone marrow which had been expanded ex vivo. The mechanism underlying the development of these leukaemias is not clear and will be the subject of future study within our group.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Ian Pragnell
Keywords: Cellular biology, Immunology
Date of Award: 1996
Depositing User: Enlighten Team
Unique ID: glathesis:1996-76108
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 16:38
Last Modified: 19 Nov 2019 16:38

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