http://repub.eur.nl/res/aut/13093/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/23384/ 2001-04-25T00:00:00Z 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. http://repub.eur.nl/res/pub/9191/ 1999-01-01T00:00:00Z Nonobese diabetic/severe combined immune deficiency (NOD/SCID) mouse repopulating cells (SRC) have been proposed to represent a more primitive human stem cell subset than the cobblestone area-forming cell (CAFC) week (wk) 6 or the long-term culture-initiating cell (LTC-IC) wk 5 on the basis of their difference in frequency, phenotype, transfectibility, and multilineage outgrowth potential in immunodeficient recipients. We have assessed the percentage of various progenitor cell populations (colony-forming cell [CFC] and CAFC subsets) contained in unsorted NOD/SCID BM nucleated cells (nc), human umbilical cord blood (UCB) nc, bone marrow (BM) nc, peripheral blood stem cells (PBSC), and CD34(+) selected UCB nc, seeding in the BM and spleen of NOD/SCID mice within 24 hours after transplantation. The seeding efficiency of NOD/SCID BM CAFC wk 5 was median (range) in the spleen 2.9% (0.7% to 4.0%) and in the total BM 8.7% (2.0% to 9.2%). For human unsorted UCB nc, BM nc, PBSC, and CD34(+) UCB cells, the seeding efficiency for CAFC wk 6 in the BM of NOD/SCID mice was 4.4% (3.5% to 6.3%), 0.8% (0.3% to 1.7%), 5.3% (1. 4% to 13.6%), and 4.4% (3.5% to 6.3%), respectively. Using flow cytometry, the percentage CD34(+) UCB cells retrieved from the BM of sublethally or supralethally irradiated NOD/SCID mice was 2.3 (1.4 to 2.8) and 2.5 (1.6 to 2.7), respectively. Because we did not observe any significant differences in the seeding efficiencies of the various stem cell subsets, it may be assumed that the SRC seeding efficiency in NOD/SCID mice is similarly low. Our data indicate that the seeding efficiency of a graft can be of great influence when assessing stem cell frequencies in in vivo repopulation assays.