Junko Suda

Multilineage expression of haemopoietic precursors with an abnormal clone in idiopathic myelofibrosis.

Summary. To clarify the lineage involvement of haemopoietic progenitor cells in idiopathic myelofibrosis (MF), simultaneous analysis of morphology and chromosomes were performed on single colonies from a 70‐year‐old Japanese woman with typical presentation of MF. The cytogenetic analysis of unstimu‐lated peripheral blood cells revealed three cell lines 47.XX, −6, +9, +der(6)t (1;6)(lqter→1q21::6p21→6qter), 46,XX, −6, +der(6) and 46.XX. Of 91 metaphases examined, the numbers of each cell line were 43, 41 and 7, respectively. Blood mononuclear cells were plated at 2 × 103 or 1 × 104/ml in methylcellulose medium containing phytohaemagglutinin‐stimulated leucocyte conditioned medium and erythropoietin. On days 10,12 and 14 of culture, 47 individual colonies were lifted and served for morphological and cytogenetic examination. Morphological examination revealed that the colonies contained neutrophils (n), macrophages (m), basophils (b), erythrocytes (E) and megakaryocytes (M). Twenty‐two of the 47 colonies had analysable metaphases, yielding totally 158 metaphases. Twelve colonies contained three or more metaphases. Eight colonies (3bE, IE, 2b, InEM, IbEM) were of the abnormal cell line 46,XX, − 6, + der(6), and four colonies (2E, IbE, ImE) were of the karyotypically normal cell line 46,XX. The other abnormal cell line 47.XX, −6, +9, +der(6) was not detected in any of the metaphases obtained from single colonies. These results provided direct evidence that the pluripotent stem cells were involved in MF.

Establishment of megakaryoblastic cell lines by coinfection of Abelson murine leukemia virus and recombinant SV40-retrovirus.

Murine embryonic cells including yolk sac prepared from 8‐day embryos were co‐infected with Abelson murine leukemia virus (A‐MuLV) and/or a recombinant retrovirus containing large T and small t antigens, and early region of simian virus 40 (M‐SV40). By coinfection with A‐MuLV and M‐SV40, megakaryoblastic cells were obtained in addition to mast cells and fibroblastic cells. However, following infection with A‐MuLV or M‐SV40 alone, no megakaryoblastic cells were detected, although mast cells and/or fibroblastic cells developed. The same results were obtained in several experiments. By single‐cell transfer, 6 acetyl‐cholinesterase (AchE)‐positive clonal cell lines were established. Characteristics of megakaryocytes, such as AchE, glycoproteins lIb and IlIa, and platelet peroxidase were detected in two representative cells (C1 and C8). More significant changes expressing differentiation were observed following treatment with phorbol myristate acetate or pokeweed mitogen‐stimulated murine spleen cell conditioned medium, although release of platelets was not observed. This is the first report showing development of megakaryocytic cells as the result of coinfection with retroviruses.

Myeloid and erythroid lineage expression of haemopoietic progenitors derived from an abnormal clone in erythroleukaemia

Summary. To clarify the lineage involvement of haemopoietic progenitor cells in erythroleukaemia, the morphology and chromosomes of single colonies from a patient with erythroleukaemia were analysed simultaneously. The cytogenetic analysis of bone marrow cells revealed two clones; 44.XY, −7, −12, −17, del (5)(q31),+ Mar and 43,XY, −7, −12, −17, −19,del(5)(q31),+ Mar. Of 40 metaphases examined, there were 34 and six of these clones, respectively. Bone marrow mononuclear cells were plated at 5 × 104/ml in methylcellulose medium containing phytohaemagglutinin‐stimulated leucocyte conditioned medium and erythropoietin. Seventeen colonies, i.e. nine blast cell colonies, four myeloid (Sudan black B‐positive) colonies, and four erythroid (benzidine‐positive) colonies contained analysable metaphases, yielding 102 metaphases in total. Except for chromosome random loss, the karyotype within a colony remained constant. All three types of colonies showed an abnormal clone; 44.XY, − 7, −12, − 17,del(5)(q31),+Mar. From these findings, it is concluded that myeloid and erythroid lineages in erythroleukaemia were derived from the same abnormal clone.

Early B Cell Differentiation from Hematopoietic Stem Cells in the Presence of Stromal Cells and Interleukin-7 (IL-7)

The early stage of the pathway by which lymphocytes differentiate from hematopoietic stem cells was studied at a clonal level, using a stromal cell line (ST-2). IL-3-induced blast colonies, shown to be capable of differentiation into a variety of hematopoietic cells, were used as a source of enriched hematopoietic progenitor cells. These cells were Thy-1+ and B220−. In 7 of 211 wells receiving each blast colony, lymphoid cell and myeloid cell growth was observed on ST-2 layer. The cells were proved to be B lymphocytes by phenotype and immunoglobulin gene rearrangement analysis and by demonstration of surface expression of IgM. The clonal origin of B lymphoid and myeloid lineage cells was confirmed by the generation of both B lymphoid and myeloid cells in the same well following a fluorescence-activate cell sorter (FACS) clone sorting of IL-3-induced blast cells. These results provide evidence that cells of B lymphoid and myeloid lineage can originate from single primitive hematopoieitc stem cells.