Evaluation of human hemopoietic stem cell assays for transplantation and gene therapy

Hemopoietic stem cell transplantation has become an important treatment modality for various hematological and non-hematological diseases, e.g. leukemia, lymphoma, congenital immunodeficiencies, autoimmune disease as well as solid tumors. In addition, the use of hemopoietic stem cell transplantation to induce donor-specific tolerance for organ transplantation is explored. Due to increased use of graft manipulation prior to transplantation, including tumor cell purging, stem cell expansion or gene therapy, there is a strong need for in vitro assays able to assess the number and the quality of human in vivo repopulating stem cells. The stromasupported cobblestone area fOrming cell (CAFC) assay allows for determination of the frequency of progenitor cell subsets in various hemopoietic materials. Additionally, the stromasupported flask long-term culture colony-forming cell (LTC-CFC) assay provides means to assess the quality of the graft by determining the ability of the progenitor cells in the graft to produce progeny. Both assays have been extensively studied in the murine model and good correlations have been established with in vivo assays for transient and permanent engrafting stem cell subsets (1-5). The work described in this thesis aims to clarify whether the CAFC and LTC-CFC assays provide a reliable measure for the human in vivo repopulating stem cells as well. In order to accomplish this, we have determined CAFC and LTC-CFC subsets in human mobilized peripheral blood (MPB) and bone marrow (BM) grafts, and correlated them with clinical parameters reflecting reconstitution of transplanted patients. In addition, data from in vitro assays were compared with those generated in a humanized immunodeficient mouse model.