Morphological and ultrastructural study of megakaryocytopoiesis in liquid cultures of hamster spleen

Choudhury, Chandra; Katsnelson, Irena; Arnold, Eugene A.

doi:10.1182/blood.v65.5.1282.1282

Blood

1985

DOI: 10.1182/blood.v65.5.1282.1282

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Morphological and ultrastructural study of megakaryocytopoiesis in liquid cultures of hamster spleen

Chandra Choudhury

Irena Katsnelson

Eugene A. Arnold

Abstract: A long-term liquid culture system of hemopoietic tissue derived from adult hamster spleen has been described. These primary liquid cultures can maintain stem cell proliferation and differentiation for more than three months without secondary repopulation. A characteristic of the liquid cultures is the formation of clusters of hemopoietic cells around adherent stromal cells. Some islands were composed exclusively of megakaryocytes and adherent cells. Isolation of these clusters of differentiating megakaryocytes… Show more

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1998

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Cited by 3 publications

References 19 publications

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Hemopoiesis in long-term stroma-dependent cultures from lymphoid tissue: Production of cells with myeloid/dendritic characteristics

O’Neill

1998

In Vitro Cell.Dev.Biol.-Animal

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A long-term stroma-dependent culture system (LTC) has been developed which continuously produces hemopoietic cells providing an in vitro system for the study of cell differentiation. These nonadherent cell populations contain a large subpopulation of dendritic cells (DC). LTC producing DC were easily generated from spleen, but could also be established from bone marrow (BM) and lymph node with less success. It was difficult to establish DC-producing LTC from thymus. The properties of splenic and thymic stroma have been compared. Spleen stroma developed more complicated networks of fibroblasts, endothelial cells, macrophages, and DC. Thymic stromal monolayers were dominated by epithelial cells and fibroblasts, with a lower proportion of macrophages and endothelial cells. They had a relatively sparse structure of cell networks compared with spleen stroma. Cells with dendritiform morphology first appeared in cultures by 2-3 wk. The majority of cells produced were large cells which expressed DC-specific cell surface markers, major histocompatibility complex (MHC) Class II molecules, and the CD80/CD86(B7) costimulator. A high proportion of cells also expressed myeloid cell markers. No T or B lymphoid cells or granulocytes were present in the cultures. LTC continued to produce nonadherent cells resembling myeloid/DC for long periods, even after passage of stromal cells and stem cells at about 3-4 mo. after culture establishment. The LTC system offers potential to study the in vitro differentiation of myeloid/DC.

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Hemopoiesis in long-term stroma-dependent cultures from lymphoid tissue: Production of cells with myeloid/dendritic characteristics

O’Neill

1998

In Vitro Cell.Dev.Biol.-Animal

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Cell cycle status of stromal cells in long‐term haematopoietic cultures

Choudhury

Sparks

1991

Cell Proliferation

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This study was performed to further define the mechanism by which the stromal micro-environment regulates haematopoiesis. In long-term marrow cultures the interactions between stromal cells and haematopoietic cells can be investigated at the cellular level. Long-term marrow cultures from hamsters do not require repopulation or addition of hydrocortisone and are suitable for investigation of cell kinetics. The cellular kinetics of haematopoietic and stromal cells, as studied by tritiated thymidine ([3H]dT) incorporation, revealed that DNA synthesis occurred in both the non-adherent and the adherent cells. In established cultures the adherent stromal cells were predominantly in a quiescent non-cycling state: less than 2% adherent cells incorporated [3H]dT within 5 h. Removal of the supernatant cells did not affect the labelling index of adherent cells, since the labelling indices at the 50-75 h time point were 14.3% and 12.5% in the presence and absence of supernatant cells respectively. An apparent stimulus for stromal cells to incorporate [3H]dT was attachment or adhesion. Following replating of supernatant cells of long-term marrow cultures, 23.3% of the reformed adherent layer cells were labelled compared with 12-14% in cultures with previously formed unmobilized adherent cells (P less than 0.01). The data indicate that adherent cells are not required to synthesize DNA for maintenance of haematopoiesis in established long-term marrow cultures, and that recruitment into the cell cycle has an independent mechanism that is not influenced by feed-back from the supernatant cells.

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Immunohistochemical localization of membrane and alpha-granule proteins in plastic-embedded mouse bone marrow megakaryocytes and murine megakaryocyte colonies

Stenberg

Beckstead

McEver

et al. 1986

Blood

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Using an immunoperoxidase technique that permits optimal antigen localization at the light microscope level, we have detected two platelet alpha-granule constituents and three platelet membrane glycoproteins in mouse bone marrow megakaryocytes and in murine megakaryocyte colonies grown in soft agar culture for three to seven days. Using polyclonal antibodies prepared against human platelet proteins, we have demonstrated labeling for von Willebrand factor, fibrinogen, and the membrane glycoproteins IIIa and GMP-140 in both bone marrow megakaryocytes and megakaryocyte colonies after seven days of culture. Using monoclonal antibodies to membrane glycoproteins IIb and GMP-140, we have demonstrated label in mouse bone marrow megakaryocytes. Granulocyte and macrophage colonies were negative for each of these markers. Murine bone marrow megakaryocytes and megakaryocyte colonies demonstrated a similar enzyme histochemical pattern: weakly positive for alpha-naphthyl acetate esterase and negative for chloroacetate esterase. These data indicate that megakaryocytes grown in soft agar culture express many of the same glycoproteins as bone marrow megakaryocytes. Furthermore, the ability of antibodies directed against human platelet membrane glycoproteins to identify murine megakaryocyte glycoproteins indicates that these constituents have been highly conserved during evolution.

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Morphological and ultrastructural study of megakaryocytopoiesis in liquid cultures of hamster spleen

Cited by 3 publications

References 19 publications

Hemopoiesis in long-term stroma-dependent cultures from lymphoid tissue: Production of cells with myeloid/dendritic characteristics

Hemopoiesis in long-term stroma-dependent cultures from lymphoid tissue: Production of cells with myeloid/dendritic characteristics

Cell cycle status of stromal cells in long‐term haematopoietic cultures

Immunohistochemical localization of membrane and alpha-granule proteins in plastic-embedded mouse bone marrow megakaryocytes and murine megakaryocyte colonies

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