Date of Award

1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Makio Ogawa

Second Advisor

Perry V. Halushka

Third Advisor

Eric Lacy

Fourth Advisor

Pamela Pharr

Fifth Advisor

Greg Warr

Abstract

Studies of B-lymphopoiesis have long been hindered by the lack of assays capable of supporting the differentiation and identification of lymphohemopoietic and early committed B-lymphoid progenitors. The clonal identification of cell-cycle dormant lymphohemopoietic progenitors from mice treated with 5 flurouracil (5-FU) became possible with the development of the two-step assay for lymphohemopoietic progenitors. Studies presented in this dissertation are centered around elucidation of the processes of differentiation of lymphohemopoietic progenitors in an attempt to determine the sequence of events occurring in divergence of the B-lymphoid and myeloid lineages. To address this question a single-step assay capable of supporting the differentiation of proliferating lymphohemopoietic and early committed B-lymphoid progenitors was developed. This assay makes use of semisolid methylcellulose culture, in situ microscopic examination and flow cytometric analysis to identify progenitors with lymphohemopoietic and B-lymphoid potential. Using this assay the lymphohemopoietic progenitors present in normal mice were characterized, and of these, approximately 75% were found to be sensitive to the cytotoxic effects of 5-FU. The proliferation and commitment of primitive lymphohemopoietic progenitors was then examined, and found to proceed through 3 stages a lymphohemopoietic proliferative stage, a commitment stage and an early committed B­ lymphoid proliferative stage. Finally using a modified version of the two-step assay, the lineage potential of individual lineage negative Ly 6A/E (Sca-1) positive progenitors from normal mice were examined. Assuming the culture system is equally permissive for the differentiation of all myeloid lineages, the results support a model of random restriction of lineage potential with a late divergence of the B-lymphoid and the myeloid lineages, possibly resulting in the formation of a bipotential B-lymphoid/macrophage progenitor.

Rights

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