No abstract

There is a growing consensus that clinical practice in the areas of bone marrow (BM) transplantation and gene therapy will rely on the ex vivo expansion of hematopoietic cells. We report here on the development of continuously perfused culture systems (bioreactor systems) that expand human stem and progenitor cells from BM mononuclear cell (MNC) populations obtained without cell enrichment. In three separate experiments, 10 bioreactors were each inoculated with 3 x 10(7) BM MNC from patients undergoing marrow harvest for autologous transplantation. At various times thereafter (between days 6 and 16), duplicate bioreactors were harvested and cells were analyzed. The bioreactors contained three cell populations that were analyzed separately: nonadherent cells; cells that were loosely adherent to the endogenously formed stromal layer; and an adherent cell layer that required trypsinization for removal. Total cell numbers increased continuously to give an overall 10-fold (range, 8- to 11-fold) expansion by day 14. The adherent stromal layer significantly expanded to more than 2 x 10(7) cells, but remained less than 6% of the total cell population. Colony-forming unit-granulocyte-macrophage (CFU-GM) numbers expanded 21-fold (range, 12- to 34-fold) by day 14 and, because this expansion was greater than that for total cells, CFU-GM were enriched by as much as fourfold by day 14. Burst-forming unit-erythroid (BFU-E) numbers peaked earlier than did CFU-GM numbers, with a 12-fold (range, 6- to 18-fold) expansion obtained on day 8. In contrast to CFU- GM, which were predominantly nonadherent, BFU-E were more evenly distributed between the three cell populations. Stem cell activity was measured by the long-term culture-initiating cell (LTC-IC) limiting dilution assay. The number of LTC-IC per reactor consistently increased with time in all cultures, resulting in a 7.5-fold (range, 3.4- to 9.8- fold) expansion. In summary, more than 3 billion cells, containing 12 million CFU-GM, were reproducibly generated from the equivalent of a 10 to 15 ml BM aspirate. These data indicate that small numbers of BM MNC can be readily expanded ex vivo in continuous perfusion cultures, and that such ex vivo expansion may have direct applications in clinical and experimental BM transplantation.

Hematopoietic cells from the malignant clone in chronic myelogenous leukemia (CML) maintain and expand a proliferative advantage over normal hematopoietic cells within the bone marrow. This advantage is often ameliorated or reversed in vivo by IFNa. Based upon earlier studies suggesting decreased

Bone marrow (BM) stromal fibroblasts produce hematopoietic growth factors (HGFs) in response to inflammatory mediators such as tumor necrosis factor-alpha or interleukin-1 alpha (IL-1 alpha). In the absence of such inflammatory stimuli, production of HGFs by BM stromal cells has been problematic and controversial. In vivo, however, basal hematopoiesis maintains blood counts within a normal homeostatic range even in the absence of inflammation, and HGFs are required for progenitor cell differentiation in vitro. To better ascertain the contribution of BM stromal fibroblasts to basal hematopoiesis, we therefore studied HGF production in quiescent BM stromal fibroblasts by three sensitive assays: serum-free bioassay, enzyme-linked immunosorbent assay, and reverse transcriptase polymerase chain reaction. Stromal fibroblasts were cultured in the presence or absence of normal human serum to determine if serum factor(s) present in the noninflammatory (basal) state induce secretion of HGFs. Human serum was found to induce or enhance transcription and secretion of granulocyte- macrophage colony-stimulating factor (GM-CSF) and enhance secretion of constitutively expressed IL-6. In contrast, no secretion of either granulocyte-CSF (G-CSF) or IL-3 was found. These data indicate that factors in normal human serum are active in enhancing GM-CSF and IL-6 production by stromal fibroblasts and suggest that these growth factors contribute to the maintainance of normal, basal hematopoiesis in vivo.

The hematopoietic stimulatory activities of human recombinant IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF) were directly compared using highly enriched human bone marrow progenitor target cells. IL-3 supported a larger number of erythroid and megakaryocytic progenitor cells than did GM-CSF, while GM-CSF supported more myeloid progenitors. IL-3 directly stimulated the division and migration of primitive erythroid burst forming units, while GM-CSF merely sustained their net survival in culture without promoting division and expansion. IL-3 promoted the formation of larger numbers of multipotential granulocyte-erythroid-macrophage-megakaryocyte colony forming unit-derived colonies than did GM-CSF. These data indicate that human IL-3 and GM-CSF have overlapping but distinct hematopoietic activities, and suggest a potential role for the clinical application of combined IL-3/GM-CSF therapy.