ISOLATION OF MURINE FETAL HEMATOPOIETIC PROGENITOR CELLS AND SELECTIVE FRACTIONATION OF VARIOUS ERYTHROID PRECURSORS

Abstract

Hemopoietic progenitor cells (colony- and cluster-forming cells in semisolid agar) were purified from light density CBA murine fetal liver cells using fluorescein-conjugated pokeweed mitogen (PWM) and a rhodamine-conjugated antineutrophil serum sandwich (.alpha.N) and 3-parameter fluorescence-activated cell sorting. All clonable progenitor cells were highly enriched (36- to 50-fold) in PWM-positive (> channel 15), .alpha.N-negative (< channel 30) fractions with relatively high intensity (> channel 100) low angle light scatter. No separation was achieved between different types of progenitor cells (granulocyte-macrophage and erythroid colony-forming cells). The enriched fraction was a pure population of large, basophilic, undifferentiated blast cells, and in agar cultures stimulated with colony-stimulating factors up to 90% of the enriched cells were hemopoietic progenitor cells capable of varying levels of clonal proliferation. Further fractionation based on increasing fluorescence with PWM separated into discrete populations, nonproliferative morphologically recognizable erythroid cells, late erythroid progenitor cells (day 2 CFU-E) and cells forming pure or mixed erythroid burst colonies. In addition, the majority of pluripotential hemopoietic stem cells (CFU-S) were clearly separated from progenitor cells forming colonies in vitro. The present techniques provide suitable numbers of enriched progenitor cells for a variety of biologic and biochemical studies.