Human megakaryocytic progenitors (CFU‐M) assayed in methylcellulose: Physical characteristics and requirements for growth

Abstract

The basic culture requirements and several physical characteristics were defined for megakaryocytic colony-forming cells (CFU-M) from normal human marrow growing in methylcellulose. Ficoll-hypaque separated mononuclear cells from human marrow gave rise to megakaryocytic colonies in the presence of normal human plasma and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). Their identity as megakaryocytic colonies was confirmed by immunofluorescence staining with a monoclonal antibody to human factor VIII antigen and by electron microscopy of individually harvested colonies. Demonstration of the single-cell origin of the colonies was provided by analysis of the glucose-6-phosphate dehydrogenase (G-6-PD) enzyme type of individually harvested colonies grown from a G-6-PD heterozygote. The colonies grew best in heparinized or citrated plasma as opposed to serum. Detailed studies suggested that platelet-release products were responsible for this difference. Tritiated thymidine suicide studies showed that the percentage of CFU-M in DNA synthesis was 23 +/- 8% (n = 10). The modal velocity sedimentation rate of CFU-M was 4.9 +/- 0.6 mm/hr (n = 4) while that of concurrently studied granulocyte/macrophage colony-forming cells (CFU-GM) was 5.7 +/- 0.5 mm/hr. Examination of the PHA-LCM dose-response characteristics suggested the presence in the conditioned medium of an inhibitor to megakaryocyte colony growth which was partially removed by chromatography of the medium on Sephadex G-100. The resulting conditioned medium increased the cloning efficiency for CFU-M compared with that with crude PHA-LCM (15.3 +/- 7.0 and 8.2 +/- 5.3/10(5) marrow cells, respectively).

Single cell origin of multilineage colonies in culture. Evidence that differentiation of multipotent progenitors and restriction of proliferative potential of monopotent progenitors are stochastic processes.

“…A representative time sequence of colony formation from a single cell to a multilineage colony and the companion May-Grunwald-Giemsa preparation are depicted in Fig. 1 A-D ( 17) suggested that the use of unfrozen human plasma and platelet-poor plasma supports megakaryocyte colony formation better than serum.…”

Enhancement of megakaryocytopoiesis by Campath-1G-treated natural killer cells

“…In the maturation. [14][15][16][17][18][19][20][21][22][23][24][25][26]39,43 Campath-1G is a T lymphocytespecific moAb that is routinely employed to reduce GVHD absence of PICS-J negligible numbers of MK colonies were observed. None of the CM significantly stimulated MK in allogeneic BMT.…”

“…Our ation. [19][20][21][22]25,26 We have previously demonstrated that Campath-1G-treated T lymphocytes or purified CD4 + T cells data demonstrate that resting and Campath-1G-treated NK may be involved in the immunomodulation of significantly enhance the number of early burst-forming units (BFU-MK) and late colony-forming units (CFU-MK) megakaryocytopoiesis. Keywords: natural killer cells; Campath-1G; megakaryoderived from BM-non adherent mononuclear cells.…”

Tumor necrosis factor type alpha stimulates human endothelial cells to produce granulocyte/macrophage colony-stimulating factor.

“…The nonadherent low-density marrow mononuclear cells were T-lymphocyte depleted by the E-rosette technique using neuraminidase-treated sheep erythrocytes. The nonadherent T-cell-depleted marrow cells were cultured at a concentration of 5 x 104 cells per ml of a medium (Flow Laboratories) containing 1% methylcellulose, 20% (vol/vol) fetal calf serum [or 25% (vol/vol) human plasma for megakaryocyte colony assays], 1% deionized bovine serum albumin (not used in megakaryocyte colony assay) (Reheis, Phoenix, AZ), i0-4 M 2-mercaptoethanol, and penicillin/streptomycin (14,15). Human urinary erythropoietin, partially purified in our laboratory to a specific activity of 200 units/mg, was added at a concentration of 1 unit/ml on day 4 of culture (16).…”

“…Phytohemagglutinin-stimulated lymphocyte conditioned medium (PHA-LCM) (15) served as a positive control. Colonies were counted on day 13 (14,15 Trypsin Digestion of the Growth Factor. One milliliter of ECM or ECMa was digested with 50 units of agarose-bound trypsin (Sigma) for 20 min at 30°C.…”