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Using a clonal culture system, we investigated the lymphohematopoietic effects of recombinant interleukin-7 (IL-7) obtained from conditioned media of transfected COS 1 cells. IL-7 alone acted on murine bone marrow cells and supported the formation of B-cell colonies. These colony cells were positive for B220, and some of them were also found to have either IgM or Thy-1. B220+, IgM- cells, but not B220- cells sorted from fresh bone marrow cells were able to form B cell colonies in the presence of IL-7. Thus, IL-7 supported the differentiation of B220+, IgM- cells to B220+, IgM+ cells. B220+, IgM+ cells did not proliferate in the presence of IL-7. IL-7 did not affect the myeloid colony formation supported by IL-3, IL-5, IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), and G-CSF. On the other hand, lymphocyte colony formation was not affected by IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, or G-CSF. Interestingly, IL-1 alpha inhibited IL-7- induced B cell colony formation in a dose-dependent manner, while the same concentration of IL-1 alpha enhanced the myeloid colony formation by IL-3. This reciprocal effect of IL-1 alpha may act on hematopoietic progenitor cells without accessory cells. These data show that IL-7 is a B cell growth factor and that IL-1 alpha may play an important role in differentiation of myeloid and lymphoid lineages.

We studied the effect of purified interleukin-3 (IL-3) and erythropoietin on colony formation by hemopoietic progenitors in serum- free cultures of spleen cells from 5-fluorouracil (5-FU)-treated mice. In the presence of IL-3 alone, most of the multilineage (three or more lineages) colonies did not contain erythroid cells. However, in the presence of IL-3 and erythropoietin, most of the multilineage colonies contained various numbers of erythroid cells. Replating experiments suggest that IL-3 maintains the growth of the progenitor cells, which could differentiate into erythroid cells. Erythropoietin facilitated the terminal differentiation and amplification of erythroid cells, although it did not sustain the growth of multipotential stem cells. Single-cell transfer experiments demonstrate that IL-3 supported the late stages of differentiation of neutrophils, macrophages, eosinophils, and megakaryocytes in the absence of lineage-specific factors. Therefore, IL-3 supports the differentiation of multilineage hemopoietic progenitors, and the terminal differentiation of most hemopoietic lineages, with the exception of the erythroid lineage.

A male neonate with Down's syndrome and congenital myeloproliferative disorder was studied. His blood picture showed the unique coexistence of leukocytosis with matured cells and a large number of blast cells. The in vitro proliferation and differentiation of blast cells into various lineages in the presence of phytohemagglutinin-stimulated leukocyte conditioned medium (PHA-LCM) was examined by using a liquid culture and a methylcellulose culture system. The differentiation of blast cells into myeloid cells was confirmed by specific cytochemical stainings, electron microscopy, and an immunologic study. No specific factors in the plasma of the patient promoted the proliferation or differentiation of blast cells. The cellular composition of colonies grown in methylcellulose culture from single blast cells was studied by a micromanipulation technique. High plating efficiency was observed. Of 136 cultures, 78 showed colony growth. Half of the blast cells were colony-forming cells that could proliferate and differentiate into basophils, neutrophils, eosinophils, macrophages, and erythrocytes in the presence of PHA-LCM. Using the blast cells with a high differentiation capacity to the basophil pathway, we studied the effect of recombinant granulocyte-macrophage colony-stimulating factor (GM- CSF). Recombinant GM-CSF support neutrophils, eosinophils, and macrophages but not typical basophils. These findings of the cell differentiation of blast cells into various kinds of cells in vitro were in agreement with the finding of neutrophilia, eosinophilia, basophilia, and thrombocythemia in this patient.

Blast cell colonies seen in cultures of spleen cells from 5- fluorouracil-treated mice provide a highly enriched population of primitive hemopoietic progenitors. Our recent studies of the differentiation potentials of the paired daughter cells of these progenitors showed different patterns of differentiation in the colonies produced by the separated daughter cells. In this study, we carried out sequential micromanipulation of paired progenitors followed by cytologic examinations of the colonies derived from these progenitors. Of the total 94 evaluable cultures, consisting of three or more colonies, 52 consisted of macrophage colonies and one consisted of megakaryocyte colonies. In the remaining 41 cultures, diverse combinations of colonies revealing heterogeneous compositions of cell lineages were identified. Presumptive genealogic trees of the differentiation of hemopoietic progenitors constructed for the latter group of cultures suggested that monopotent progenitors may be derived from pluripotent progenitors in two ways: (1) directly during one cell division of pluripotent cells or (2) as a result of progressive lineage restriction during successive division of the pluripotent progenitors. The results also suggested that some of the oligopotent progenitors are capable of limited self-renewal.

We present the in vitro differentiation of marrow cells from a patient with Down's syndrome accompanied by megakaryoblastic leukemia into basophils in the presence of phytohemagglutinin-stimulated leukocyte conditioned medium, using a liquid culture and methylcellulose culture system. Identification of basophils was established by metachromatic staining with toluidine blue, transmission electron microscopy, and the presence of histamine. However, these basophils did not release histamine in response to calcium ionophore or chemotactic peptide. Samples from suspension cultures that contained 90% basophils showed chromosomal markers characteristic of leukemic cells (48, XY, +11, +21, t(1;15)) in all examined mitoses. The cellular composition of leukemic colonies grown in methylcellulose culture from single cells was studied using the micromanipulation technique. High plating efficiency and extreme predominance of basophil colonies were observed. In a total 137 cultures, 79 revealed colony growth. Of 59 colonies that were analyzed by cytologic examination, 46 were pure basophil colonies. These basophil colonies showed disperse morphology, similar to that of a normal basophil colony. The clonality of the basophil colonies and skewing of lineage expression were documented from leukemic single-cell cultures. These data showed that leukemic cells have the capacity for differentiation into some lineages that are not expressed in vivo.