Dissertation

Self -renewal of human bone marrow -derived hematopoietic stem cells in vitro

Brandt, John E

2000

Overview

The development of protocols for the in vitro expansion of human hematopoietic stem cells (HSC) has been largely based upon optimizing the conditions for proliferation of cells expressing an immunological phenotype associated with native, unmanipulated HSC. The objective of this study was to determine whether the kinetics of functional HSC division in vitro correlated with phenotype. Adult bone marrow-derived CD34+ cells were labeled with carboxyfluorescein diacetate succidimidyl ester, and the kinetics of division of committed and primitive hematopoietic progenitor cells, as well as cells capable of competitive marrow repopulation, were examined for up to five days under conditions demonstrated to produce expanded populations of engraftment-competent HSC. In endothelial cell-based and stromal cell-free cultures, a high proliferative capacity was associated with committed progenitor cells, whereas slower division was characteristic of primitive cobblestone area-forming cells. The ability of marrow CD34 + cells, isolated from the cultures on the basis of prior cell divisions, to competitively repopulate fragments of allogeneic bone implanted into severe combined immunodeficient mice was tested. While committed progenitor cells were capable of one to two divisions within the first thirty hours of expansion culture, cells capable of long-term hematopoietic activity required at least 1.5 days to undergo a single division. The progeny of these cells were characterized by asynchronous subsequent division. By 2.5 days of culture, cells with marrow repopulation activity were capable of one, but not two, divisions. The expression of a CD34+CD38− phenotype, which is associated with native, unmanipulated HSC, did not predict marrow engraftment. Rather, the number of prior divisions was the primary determinant of functional capacity. After five days of culture, marrow repopulation capacity was limited to cells that had undergone four or fewer divisions, despite the continued expression of CD34 by cells that had divided five or more times. The data indicate that engraftment-competent adult marrow HSC are refractory to early division in ex vivo expansion culture, and remain resistant to continued, synchronous division once activated to self-renew. The data further suggest that, in short-term culture, the kinetics of cell cycling may be a more reliable indicator than phenotype of HSC function.

• Publisher: ProQuest Dissertations & Theses
• Subject: Anatomy & physiology / Animals / Cellular biology / Medicine / Physiology / Surgery
• ISBN: 9780493016726, 0493016724