Two classes of primitive pluripotent hemopoietic progenitor cells: separation by adherence.

Methylcellulose cultures containing mouse marrow cells at low densities and partially purified preparations of erythropoietin and Interleukin-3 were scored after 2 weeks for the presence of macroscopic multilineage colonies (from "primary" CFU-macro GEMM). Whole cultures were then harvested and replanted to assess the number of "secondary" CFU-macro GEMM produced, but not detected, during the primary culture period. In such experiments adherent marrow cells yielded significantly higher numbers of secondary CFU-macro GEMM than did either fresh or nonadherent marrow cells. Removal of macroscopic colonies prior to replating showed that most secondary CFU-macro GEMM were not derived from primary CFU-macro GEMM. In vivo studies also revealed a differential effect of adherence separation on the frequency of day 10 CFU-S, which decreased, by comparison to cells capable of long-term repopulation, which increased. Primary adherent CFU-macro GEMM from 5-fluorouracil (5-FU) treated mice showed an 18-fold higher self-renewal capacity than their counterparts in normal marrow. Nevertheless the majority of secondary CFU-macro GEMM obtained from primary cultures of adherent 5-FU cells were again not derived from primary CFU-macro GEMM. Cells capable of immediately generating large multilineage colonies thus appear to represent an intermediate compartment of pluripotent progenitors whose self-renewal properties, may, however, vary over a considerable range. Our results further suggest that these progenitors are derived ultimately from a more primitive adherent cell whose tendency to begin to divide in vitro is low and whose presence correlates with cells capable of long-term myeloid repopulation in vivo.

Gentamicin reduces the self-renewal capacity of murine pluripotent hemopoietic stem cells.

This study pursues our initial observation of an association between prophylactic treatment of irradiated mice with gentamicin sulfate, and the subsequent low self-renewal capacity exhibited by transplanted marrow CFUS which they host. Either drug treatment of the host before engraftment, or exposure of marrow cells in vitro prior to their inoculation, produced suppressed CFUS self-renewal with respect to paired controls. Characteristics of primary spleen colonies (number, size, histology) were not altered, suggesting a specific deficit in secondary stem cell production or maintenance.

Merocyanine 540 alters the self-renewal capacity of murine pluripotent hemopoietic stem cells.

Merocyanine 540 (MC 540), an impermeant photoreactive dye with a high affinity for the plasma membrane of hemopoietic precursors, was examined for effects on the self-replicative capacity (SR) of CFUS from normal mouse marrow, and on various CFUS subpopulations fractionated by velocity sedimentation. Brief exposure (30 s) of whole marrow to MC 540 resulted in a reduction in overall CFUS self-renewal in primary recipient spleens. Reduced self-renewal was also observed when fractionated CFUS subpopulations were used. Reduced self-renewal was not accompanied by obvious changes in primary spleen colony number or composition. MC 540 may interact with specific superficial membrane sites relevant to the SR process. Upon longer exposure, further MC 540 effects appear restricted to a reactive subpopulation of large (low SR) CFUS. Self-renewal was enhanced in this fractionated pool, without evidence of primary colony reduction, by intermediate staining. This strongly suggests that under certain conditions the dye can alter a fundamental functional property of susceptible stem cells, without their inactivation. Still more prolonged staining apparently leads to the selective elimination of this reactive low SR CFUS subset. A marked reduction in whole marrow-derived spleen colonies was accompanied by an enhanced self-renewal capacity among the survivors. These two MC 540 effects--stem cell modification, and stem cell elimination--may reflect different stages in an ongoing membrane photo-oxidation process.