Using a clonal culture system, we investigated the hemopoietic effects of purified recombinant IL-5 obtained from conditioned media of transfected Xenopus oocytes. IL-5 alone acted on untreated bone marrow cells and supported the formation of a small number of colonies, all of which were predominantly eosinophilic. However, it did not support colony formation by spleen cells from 5-FU-treated mice, in which only primitive stem cells had survived, while IL-3 and G-CSF did. Eosinophil-containing colonies were formed from these cells in the presence of IL-5 and G-CSF together. In contrast, G-CSF alone did not support any eosinophil colonies. The eosinophilopoietic effect of IL-5 was dose-dependent, and was neutralized specifically by anti-IL-5 antibody. To exclude the possibility of interactions with accessory cells in the same culture dish, we replated a small number (200 cells/dish) of enriched hemopoietic progenitors, obtained from blast cell colonies, which were formed by cultivation of spleen cells from 5-FU-treated mice in the presence of IL-3 or G-CSF. From these replated blast cells, eosinophil colonies were induced in dishes containing IL-5 but not in those containing G-CSF alone. From these findings, it was concluded that IL-5 did not act on primitive hemopoietic cells, but on blast cells induced by IL-3 or G-CSF. IL-5 specifically facilitated the terminal differentiation and proliferation of eosinophils. In this respect, the role of IL-5 in eosinophilopoiesis seems to be analogous to erythropoietin, which promotes the terminal differentiation and amplification of erythroid cells. Moreover, IL-5 maintained the viability of mature eosinophils obtained from peritoneal exudate cells of the mice infected with parasites, indicating mature functional eosinophils carried IL-5 receptors. The synergistic effects of IL-5 and colony-stimulating factors on the expansion of eosinophils is supposed to contribute to the urgent mobilization of eosinophils at the time of helminthic infections and allergic responses.

We analyzed the differentiation of murine hemopoietic colonies derived from paired progenitors in culture. Single progenitors were isolated by use of a micromanipulation technique from blast cell colonies cultured from the spleens of 5-fluorouracil-treated mice. Eighteen to 24 hr later, the paired progenitors were separated with a micromanipulator and cultured in methylcellulose medium containing erythropoietin and pokeweed-mitogen spleen cell conditioned medium. Six to nine days later, the two colonies derived from the paired progenitors were individually picked and differential counts were performed by using May-Grunwald-Giemsa stain. The abbreviations used here are n, neutrophil; m, macrophage; e, eosinophil; mast, mast cell; M, megakaryocyte; E, erythrocyte. Of a total of 387 pairs that could be evaluated, 68 were pairs of colonies consisting of dissimilar combinations of cell lineages such as m-nmmastEM,Thirty-nine were homologous pairs revealing identical lineage combinations such as nmmastEM, nmmastM, nmmast, mmastEM, nmEM, nine, nmM, mM, and nm lineages. However, in members of some of these pairs, the proportions of the individual cell lineages were significantly different. the remainder were pairs of single lineage colonies. Paired progenitors obtained from the stem cell colonies of normal mice also revealed homologous and nonhomologous expression of the cell lineages. Comparison of lineage expression in colonies derived from single progenitors with the sum of lineages expressed in pairs of colonies derived from single progenitors indicated that the diversity was not due to injury inflicted by micromanipulation. These observations provide experimental data in support of stochastic mechanisms of stem cell differentiation.Several models have been proposed for the mechanisms of differentiation of hemopoietic stem cells. These models include the hemopoietic inductive microenvironment (HIM) model of Trentin (1), the "stem cell competition" model that was advocated most recently by VanZant and Goldwasser (2)

By using a micromanipulator, single cells from blast cell colonies were individually transferred to 35-mm culture dishes for secondary colony formation. When individual colonies appeared to be mature, they were examined for cellular composition by May-Grunwald-Giemsa staining and were replated for determination of unexpressed hemopoietic potentials. We describe here a total of 50 mixed hemopoietic colonies. Seven types of colonies consisting of cells in two different lineages were seeni.e., neutrophil-macrophage, neutrophil-eosinophil, macrophage-eosinophil, macrophage-mast cell, macrophage-megakaryocyte, macrophage-erythrocyte, and erythrocyte-megakaryocyte. Six types of colonies revealed three cell lineages-i.e., neurophmrhage-eosinophilneutroph amast cell, neutrophil-macrophage-erythrocyte, macrophage-mast cell erythrocyte, neutrophi-macrophage-megakaryocyte, and neutrophil-erythrocyte-megakaryocyte lineages. In addition, multilineage colonies expressing terminal differentiation in varying combinations of more than three lineages were present. Replating studies confirmed that the progenitors for many of these colonies are terminally committed to differentiation only in the lineages disclosed by staining. This study, thus, provides a proof for the single-cell origin of mouse hemopoietic colonies expressing various combinations of cell lineages. It also supports the hypothesis that the differentiation of multipotential hemopoietic progenitors is through progressive and stochastic restriction in cell lineages.Recent progress in clonal cell culture techniques has provided methods for growing human and murine multilineage hemopoietic colonies. Several groups of investigators (1-3) have described hemopoietic colonies in culture that reveal terminal differentiation in more than three cell lineages-i.e., granulocyteerythrocyte-macrophage-megakaryocyte colonies. Hemopoietic colonies revealing differentiation in only two or three cell lineages also have been described. Granulocyte-macrophage colonies were the first such colonies to be described (4); subsequently, colonies revealing terminal differentiation in erythrocyte-megakaryocyte lineages (5, 6), granulocyte (neutrophil)-erythrocyte lineages (7), and erythrocyte-eosinophil lineages were described (8). We have described a type of colony containing neutrophils-macrophages-megakaryocytes and lackdng erythroid cells (9). However, evidence for the single-cell origin of the multilineage colonies described in these reports has only been indirect-i.e., application of statistical analysis of cultures of mixtures of male and female cells (7-9).Two attempts have been made to prove the single-cell origin of multilineage hemopoietic colonies by use of single-cell transfer. Johnson and Metcalf (10) described a total of three mixed colonies (a neutrophil-macrophage-blast cell colony, an erythrocyte-neutrophil-blast cell colony, and an erythrocyte-neutrophil-macrophage-blast cell colony) derived from single progenitors obtained from mouse fetal liver. Hara and Noguchi (1...

Several investigators have described hemopoietic colonies expressing multi-lineage differentiation in culture. We recently identified a class of murine hemopoietic progenitors which form blast cell colonies with very high replating efficiencies. In order to clarify further the relationship between progenitors for blast cell colonies and progenitors for the multilineage hemopoietic colonies in culture, we carried out analyses of kinetic and differentiation properties of murine blast cell colonies. Serial observations of the development of blast cell colonies into multilineage (and single lineage) colonies in cultures of spleen cells obtained from 5-fluorouracil (5-FU)-treated mice confirmed the transitional nature of the murine blast cell colonies. The data also suggested that the early pluripotent progenitors are in G0 for variable periods, and that when triggered into cell cycle, they proliferate at relatively constant doubling rates during the early stages of differentiation. The notion that some of the pluripotent progenitors are in G0 was also supported by long-term thymidine suicide studies in which spleen cells were exposed to 3H-thymidine with high specific activity for 5 days in culture, washed, and assayed for surviving progenitors. Comparison of replating abilities of day-7 and day-16 blast cell colonies from normal as well as 5-FU-treated mice indicated that some of the day-7 blast cell colonies are derived from maturer populations of progenitors which are sensitive to 5-FU. In contrast, progenitors for the day-16 blast cell colonies are dormant in cell cycle and were not affected by 5-FU treatment. Previously we reported that progenitors for day-16 blast cell colonies have a significant capacity for self-renewal. These observations suggest the hypothesis that the capability for self-renewal is accompanied by long periods of G0, and that once commitment to differentiation takes place, then active cell division occurs.

We studied the effects of interleukin-3 (IL-3) on colony formation by hemopoietic progenitors in methylcellulose cultures of spleen cells from 5-fluorouracil (FU)-treated mice. Purified IL-3 supported the growth of various types of multilineage colonies including blast cell colonies. The types of colonies were similar to those supported by pokeweed-mitogen spleen cell conditioned medium (PWM-SCM), except that IL-3 supported eosinophil and neutrophil expression better. Delayed addition of IL-3 to cultures 7 days after cell plating decreased the number of colonies to one-half the number in cultures with IL-3 added on day 0. It did not alter the proliferative and differentiation characteristics of late emerging multipotential blast cell colonies. These observations suggest that IL-3 does not trigger hemopoietic progenitors into active cell proliferation but is necessary for their continued proliferation. This permissive role of IL-3 is consistent with a stochastic model of stem cell proliferation which features random entry into cell cycle. IL-3 also supported the growth of multilineage colonies from single cells isolated from blast cell colonies by micromanipulation. This result shows that IL-3 acts directly on multipotential progenitors. Analysis of colonies derived from paired progenitors revealed disparate lineage expression and was in accordance with the stochastic model of stem cell differentiation.