Date of Award

1980

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pathology and Laboratory Medicine

College

College of Graduate Studies

Abstract

The erythropoietin (Ep) dose response of erythroid colony-forming units (CFU-e) and erythroid burst-forming units (BFU-e) from rabbit bone were similar to murine erythroid precursor Ep requirements. Bone marrow and peripheral blood contained a continuum of erythroid precursors at differing stages of maturation. The earliest BFU-e were assayed on day 10. CFU-e were observed in bone marrow, but not in peripheral blood. The most mature erythroid precursors observed in peripheral blood were intermediate BFU-e, assayed on day 5. A linear relationship exists between the number of nucleated bone marrow cells plated and the number of 3 day colonies and 10 day bursts observed. The 10 day bursts were composed of erythroid cells at all stages of maturation and these cells contained normal adult hemoglobins. The methylcellulose assay was then used to characterize the hemopoietic tissue in ossicles induced by demineralized allogeneic bone matrix (DBM) and to investigate the mechanisms of action of burst-promoting activity (BPA). DBM, implanted in muscle, induces the formation of an ossicle within which hemopoietic tissue develops. Analyses of ossicle marrow in vitro demonstrated the presence of committed hemopoietic precursors; colony-forming units in culture (CFU-c), CFU-e and BFU-e by 6 weeks postimplantation. The time courses of colony and burst formation by erythroid precursors in ossicle and femoral marrow were similar. Induction of hemolytic anemia by phenylhydrazine hydrochloride at six weeks post DBM implantation showed that the ossicle marrow was responsive to systemic erythropoietic stimuli The DBM implant is a unique model for studying the development of hemopoietic microenvironments within bone. Rabbit bone marrow conditioned media (BMCM) was found to contain potent erythroid BPA. In order to further characterize the mechanisms of action of BPA and to improve the quantitation or BPA, we studied the effects of BMCM on the number of bursts, cells per burst and 59Fe incorporation into heme. Examination of erythroid precursors at different maturational stages revealed that the sensitivity of erythroid precursors to BPA decreases with maturity. Delayed addition of BMCM to cultures demonstrated a requirement for BPA during the early stages of burst formation. BMCM did not enhance granulocyte/macrophage colony formation. The enhancement of heme synthesis by BMCM was routinely much greater (range, 7- to 109-fold) than the increase in burst number (range, 1- to 2-fold). The latter observation suggested that BPA might increase the size of bursts in addition to augmenting burst number. Simultaneous measurement of cell number and 59Fe incorporation in individual bursts showed a strong correlation between these parameters. In this experiment, the total enhancement of cell number and 59Fe incorporation was 6.6- and 8.0-fold, respectively. These results suggest that a major effect of BMCM BPA is to promote cell division during the early phase of burst formation.

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