In Vitro Properties of Purified Human Stem Cell Candidates

Abstract

Serum-free cultures supplemented with IL-6, IL-3, steel factor, and erythropoietin support extensive production of erythroid cells from purified bone marrow stem cell candidates which are themselves maintained in number in such cultures. In this study, the mechanism responsible for the observed maintenance of primitive hematopoietic cells in rapidly proliferating suspension cultures was examined. The following models were considered: (1) proliferating cells represent a small minority of cells at start of culture (large quiescent pool), (2) self-renewal of primitive cells (balanced by loss through differentiation and death), and (3) asymmetrical divisions (each division of a primitive cell yielding one equally primitive daughter cell and one less primitive, i.e., committed daughter cell). To discriminate between these various models, the proliferative behavior of purified CD34⁺CD71 cells was studied at a population level using the fluorescent membrane dye PKH26 and at a single cell level by studying cultures of individually sorted CD34⁺CD45RA^loCD71^lo bone marrow stem cell candidates. The results from these experiments indicate that the majority of purified stem cell candidates do not rapidly proliferate in response to the combination of growth factors used and that limited production of CD34⁺ cells compenstates for the loss of such cells in culture. These observations have to be taken into account in the development of clinical useful strategies for the expansion of primitive hematopoietic bone marrow cells ex vivo.