Ultrastructural analysis of murine megakaryocyte maturation in vitro: comparison of big-cell and heterogeneous megakaryocyte colonies

Two morphologically distinct types of murine megakaryocyte (MK) colonies are present after three to seven days in soft agar culture: (a) "big-cell" colonies composed of ten to 30 large, mature-appearing megakaryocytes and (b) "heterogeneous" colonies consisting of approximately 100 or more cells at various stages of differentiation. Cytochemical and immunocytochemical techniques were used to study MK maturation in colonies as well as normal mouse bone marrow. Acetylcholinesterase (AChE), a specific marker for murine platelets and MK, was found in the perinuclear cisterna, endoplasmic reticulum, and occasionally, Golgi cisternae of MK in three-day big-cell colonies and immature bone marrow MK. MK in seven-day big-cell colonies and mature bone marrow MK showed additional reaction sites in the demarcation membrane system and occasional granules. In seven-day heterogeneous colonies, small cells resembled immature bone marrow MK with respect to AChE localization, whereas large cells corresponded to mature bone marrow MK. With immunogold procedures at the ultrastructural level, polyclonal antibodies against human platelet membrane glycoprotein IIIa and antimouse platelet antiserum labeled bone marrow MK and all MK from colonies grown in soft agar cultures for three to seven days. Granulocytes and macrophages in both bone marrow and soft agar cultures were negative for AChE and these immunocytochemical markers. These data indicate that the pattern of expression of AChE during maturation of MK is similar in vivo and in vitro and demonstrate, when using this marker at the fine-structural level, that a greater range of MK maturational stages is present in heterogeneous colonies than is observed in MK in big-cell colonies. Furthermore, we have confirmed that small cells in heterogeneous colonies are MK and that these colonies are composed solely of MK and their precursors.     

Growth of canine T-lymphocyte colonies in vitro.

Canine lymphocytes from peripheral blood, lymph nodes, thymus and bone marrow were stimulated with phytohemagglutinin-P (PHA) or concanavalin-A (CON-A) to form colonies in methylcellulose. Lymphocytes exposed to mitogens in liquid phase formed clumps the size of colonies. Lymphocyte clumping was eliminated by plating cells directly into methylcellulose, but high concentrations of mitogens (CON-A or PHA is greater than 10 mg/10(6) lymphocytes) were required in order to get subsequent colony formation. Thus, in contrast to published reports, exposure of lymphocytes to mitogen prior to plating was not required for cloning of canine peripheral blood lymphocytes. Colonies from thymus, lymph node, or peripheral blood consisted predominantly of T lymphocytes, whereas cultures from bone marrow also produced colonies with macrophage morphology and surface-adherent colonies with mesenchymal morphology.     

An improved plasma culture system for the production of megakaryocyte colonies in vitro.

A modified culture system has been developed to grow and quantitate megakaryocyte colonies from mouse bone marrow more efficiently than described in other reports. Using this method, it was shown that 30% of CFU-M in normal marrow cells and 70 to 90% of CFU-M in regenerating marrow cells were killed by high specific activity 3H-TdR in vitro. These results indicate that CFU-M are proliferating even in normal adult hemopoietic tissue, but that the proportion of cells that are proliferating is greater in regenerating marrow than in normal intact mice.     

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

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.     

Characteristics of rat megakaryocyte colonies and their progenitors in agar culture

The characteristics of megakaryocyte colonies that develop from megakaryocyte progenitors of rat bone marrow stimulated by rat spleen-conditioned medium (SCM) in agar culture were investigated. Colony frequency was optimal on day 7 and increased relative to both the number of cells plated and the concentration of SCM used. Plating efficiencies averaged 72 +/- 16 megakaryocyte colonies/10(5) cells with 0.1 ml SCM/culture. Colonies were categorized as small cell and big cell. Small-cell colonies had a greater proliferative potential, with a mean of 25 cells/colony. Big-cell colonies averaged 15 cells/colony. The ratio of big-cell to small-cell colonies was 0.69 +/- 0.29. Granulocyte-macrophage colonies, which were also stimulated by SCM, accounted for 70% +/- 15% of the total colonies in the cultures. Cytocidal experiments with tritiated thymidine reduced megakaryocyte colony formation by 45% and granulocyte-macrophage colony formation by 21%. The properties of rat, mouse, and human megakaryocyte progenitors as assayed in vitro are compared.     

Characteristics of murine megakaryocytic colonies in vitro.

Characteristics of murine megakaryocytic colonies and their progenitor cells (CFU-m) were studied in vitro in agar gel. Colony growth required the presence of poke-weed-mitogen-stimulated spleen-conditioned medium. The number of colonies formed was linearly related to both the number of marrow cells plated and the amount of conditioned medium added. In addition, CFU-m were found in both the spleen and peripheral blood. Conditioned medium was also made without plasma, and this resulted in a cloning efficiency greater than that of conditioned medium prepared with plasma. The percentage of CFU-m in DNA synthesis was low (10%), as determined both in vivo and in vitro. Velocity sedimentation revealed that the majority of CFU-m sedimented at 4.3 mm/hr and had a tritiated thymidine (3H-TdR) suicide rate of 1.5 +/- 1.5%. A shoulder on the profile of CFU-m sedimented at approximately 6 mm/hr, with a suicide rate of 79 +/- 2%. Analysis of these data indicated that the majority of CFU-m were not in cycle or were in a long G1 period. The results suggest that CFU-m is a primitive progenitor, possibly closely related to murine splenic colony-forming units (CFU-s), analogous to erythroid bursts and granulocytic colony-forming units.     

Immunofluorescent identification of human megakaryocyte colonies using an antiplatelet glycoprotein antiserum

The development of a satisfactory in vitro assay system for human megakaryocyte colony forming progenitor cells has been delayed by the lack of a suitable marker for cells of human megakaryocyte lineage. For this purpose we raised an antiserum directed against a purified human platelet glycoprotein preparation. In conjunction with indirect immunofluorescent staining of human bone marrow, this antiserum labeled only platelets, megakaryocytes, and an infrequent population of small mononuclear cells. These small mononuclear cells, not otherwise identifiable as members of the megakaryocyte series, constituted 22.9% of the total fluorescein positive nucleated bone marrow cells. This antiserum was also used to label colonies cultured from human peripheral blood mononuclear cells using a modified plasma clot technique. A mean of 123 fluorescein-labeled colonies were cloned per 10(6) mononuclear cells cultured. Granulocyte-macrophage and erythroid burst colonies did not label using this method. No augmentation of colony numbers was found with varying concentrations of erythropoietin, human embryonic kidney cell conditioned media (a source of thrombopoietin), or media conditioned by a human T lymphoblast cell line (a source of both colony stimulating and burst promoting activities). Immunofluorescent labeling for platelet glycoproteins is a convenient phenotypic marker for cells of human megakaryocyte lineage useful in the study of in vitro human megakaryocytopoiesis.     

Characterization of mouse clonogenic megakaryocyte progenitors

Although it has been shown that unfractionated bone marrow, hematopoietic stem cells, common myeloid progenitors, and bipotent megakaryocyteerythrocyte progenitors can give rise to megakaryocyte colonies in culture, monopotent megakaryocyte-committed progenitors (MKP) have never been prospectively isolated from the bone marrow of adult mice. Here, we use a monoclonal antibody to the megakaryocyte-associated surface protein, CD9, to purify MKPs from the c-kit(+)Sca-1(-)IL7Ralpha(-)Thy1.1(-)Lin(-) fraction of adult C57BLKa-Thy1.1 bone marrow. The CD9(+) fraction contained a subset of CD41(+)FcgammaR(lo)CD34(+)CD38(+) cells that represent approximately 0.01% of the total nucleated bone marrow cells. They give rise mainly to colony-forming unit-megakaryocytes and occasionally burst-forming unit-megakaryocytes, with a plating efficiency >60% at the single-cell level. In vivo, MKPs do not have spleen colony-forming activity nor do they contribute to long-term multilineage hematopoiesis; they give rise only to platelets for approximately 3 weeks. Common myeloid progenitors and megakaryocyteerythrocyte progenitors can differentiate into MKPs after 72 h in stromal cultures, indicating that MKPs are downstream of these two progenitors. These isolatable MKPs will be very useful for further studies of megakaryopoiesis as well as the elucidation of their gene expression patterns.     

Growth of macroscopic human megakaryocyte colonies from cord blood in culture with recombinant human thrombopoietin (c-mpl ligand) and the effects of gestational age on frequency of colonies

We investigated the effects of recombinant human thrombopoietin (rhTPO) on the growth of megakaryocytic (MK) colony derived MK progenitors from human cord blood (CB) in vitro and the effects of gestational age on the number of MK colonies. The results demonstrated that rhTPO alone supports the growth of MK colonies and induces not only proliferation but also differentiation of MK progenitors. CB shows a high frequency of MK colonies; most of which are very large and equivalent to high proliferative potential colony-forming unit-megakaryocyte. The colonies could be macroscopically observed as white spots in the culture dish. Preterm neonates showed greater numbers of MK progenitors than term neonates and there was an inverse correlation between gestational age and concentration of MK progenitors of CB. The effects of gestational age was an important factor on the proliferative capacity of MK progenitors and on the response to rhTPO.     

In vitro growth of colonies of mitogen-stimulated mouse T lymphocytes: I. Conditions affecting colony formation; II. Structure of colonies and component cells

Mouse lymph node cells sensitized with PHA or Con A in liquid phase grew into T-cell colonies when seeded in a two-layer soft agar culture system containing the mitogen. The colony cells were of T-cell lineage. This was deduced from their morphology, ultrastructure, positive strain for theta-isoantigen and the fact that no colonies were formed by lymphoid cells from congenitally athymic nude mice. The architecture of the colonies and their component cells was studied by scanning electron microscopy. Clonogenic assay indicated that macrophages are active modulators of T cell proliferation. Colony formation was markedly enhanced by hemolysate and/or amino acid, L-glutamine or L-cystine, added to the culture medium. The largest number of colonies grew when both the liquid and soft agar media were supplemented with hemolysate and one of the amino acids. Under these conditions the minimal seeding level for colony formation could be reduced from 2.0 X 10(5) to 1.6 X 10(4) cells/culture.     

Single platelet and megakaryocyte morpho-dynamics uncovered by multicolor reporter mouse strains in vitro and in vivo

Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte (MK) tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Cre-driven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of MK in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to wild-type controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and MK biology on a single cell level.     

HIV-associated thrombocytopenia: in vitro megakaryocyte colony formation in 10 patients.

In vitro bone-marrow megakaryocyte colony formation was studied in 10 patients with HIV-associated thrombocytopenia to investigate the mechanism of thrombocytopenia. Increased colony formation was observed in 3 patients and decreased growth in 7 patients. No relationship was noted between the growth potential of megakaryocyte progenitors and platelet count, number of CD4+ celts, platelet response to azidothymidine, and platelet count 7 days after culture. In all patients, megakaryocyte morphology was abnormal: blebbing of the membrane and abnormal chromatin with separated lobes of nuclei. Further studies are needed to determine if growth potential of megakaryocyte progenitors is useful in understanding the mechanism of thrombocytopenia in HIV-infected individuals.     

Human megakaryocytes. IV. Growth and characterization of clonable megakaryocyte progenitors in agar

Human megakaryocyte progenitors were cloned in semisolid agar from unfractionated bone marrow cells and recognized by their capability of producing discrete megakaryocyte colonies. Megakaryocyte colonies were identified in situ by immunofluorescence, using antibodies against platelet glycoproteins Ib, IIb, and IIIa, as well as von Willebrand factor (vWf), which are regarded as distinct protein markers for the megakaryocyte-platelet lineage. Megakaryocyte colonies typically contained 20-50 cells arranged in compact configurations, with high nuclear-cytoplasmic ratios, diameters between 10 and 14 micron, and round, oval, or indented nuclei. Colony numbers peaked at days 6 and 7, with a mean of 17.9 megakaryocyte colonies (range, 8-33) per 2 X 10(5) unseparated marrow cells. The in vitro growth characteristics and kinetics of megakaryocytes grown in agar are different from those described for the plasma clot and methylcellulose systems, which suggests selection of distinct progenitor subsets. Consequently, this assay may be a useful complement to other approaches in characterizing the megakaryocyte progenitor population.     

Differential effects of red cells on the formation of normal and neoplastic mouse B lymphocyte colonies in vitro.

Intact sheep red cells potentiated mouse B lymphocyte colony growth in agar but red cell membranes or lysates exhibited no comparable ability. For maximum colony formation red cells had to be present throughout the entire culture period. Red cells added to cultures late in the culture period did not potentiate growth. Intimate contact between colony-forming cells and red cells was not essential for potentiation to occur. Red cell lysates inhibited normal B lymphocyte colony formation in cultures containing intact red cells, but did not inhibit colony formation by cells of the B lymphoid leukemia ABE-8. This differential effect may provide a means of differentiating normal from neoplastic colony-forming B lymphocytes in the mouse. Eight different tumors were also examined. Intact red cells potentiated colony formation by all of them. Lysed red cells did not potentiate the growth of any of the tumor lines. The mastocytoma P815 was the only tumor whose colony formation was inhibited by the addition of intact and lysed red cells.     

Enhanced growth of megakaryocyte colonies in culture in the presence of heparin and fibroblast growth factor

Fibroblast growth factors (FGFs) are a family of mitogenic proteins that promote the division of most mesoderm- and neuroectoderm-derived cells. Interaction with heparin-like molecules appears to be critical for FGFs to exert their activity. We previously reported that FGF-1, FGF-2, and heparin are each capable of stimulating megakaryocytopoiesis in vitro and that heparin has a thrombopoietic effect when administered to patients with chronic immune thrombocytopenic purpura. The present study was designed to determine whether the combination of heparin and FGF results in a synergistic effect on murine megakaryocytopoiesis. In vitro, in both plasma clot and agar serum-free culture systems, FGF-1 and FGF-2 stimulated megakaryocyte colony formation in a dose-dependent fashion. Half-maximal effect of the two FGFs was observed at 1 ng/ml, and their effect was saturating at 20 ng/ml. The addition of heparin to cultures stimulated by FGF-1 or FGF-2 resulted in a 2-fold increase in the number of megakaryocyte colonies compared to the culture containing FGF alone. In the presence of heparin (5 IU/ml), FGF at 1 ng/ml exerted an almost maximal stimulating effect on megakaryocyte colony formation. When murine and human CD34+ cells were used as target cells, a similar interaction between FGF and heparin was observed. The combination of FGF and heparin induced a maximal growth of megakaryocyte colonies from CD34+ cells. These findings demonstrate the synergistic effect of heparin and FGF on megakaryocytopoiesis.     

Endogenous megakaryocyte colonies from peripheral blood in precursor cell cultures of patients with myeloproliferative disorders

Megakaryocyte colony formation, as identified by conventional techniques, was observed in precursor cell cultures from peripheral blood in 8 of 20 consecutive patients with diagnosis of myeloproliferative disease (4/11 patients with polycythemia vera, 3/5 with essential thrombocythemia, 1/2 with primary osteomyelofibrosis and 2 with a myeloproliferative syndrome not further assessable), but not in 50 healthy controls (p less than 0.0001). 7 cultures showed spontaneous erythroid colonies, but were negative for megakaryocyte colonies. Megakaryocyte colony formation was independent of added erythropoietin, plasma or human leukocyte-conditioned medium, but was dependent on the presence of accessory cells. The cells in megakaryocyte colonies had the characteristic morphology of megakaryocytes and stained positively with the IIIa/IIb monoclonal anti-platelet antibody. Thus, megakaryocyte colony formation by precursor cells from peripheral blood in the absence of exogenous stimulating factors seems to be a phenomenon specific for myeloproliferative disease. Differential diagnosis of thrombocythemia may be facilitated by demonstration of endogenous megakaryocyte colony formation, which does not occur in secondary disease.     

Disparate differentiation in mouse hemopoietic colonies derived from paired progenitors.

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, M-nmmastEM, nm-nmmastEM, nmmastM-nmmastEM, M-nmmastM, nmmast-nmmastM, nm-nmmastE, M-nmM, n-nmM, mM-nmM, m-nmmast, nm-nme, me-nm, mM-nm, n-ne, m-mmast, m-mM, M-nm, M-mM, E-nm, m-nm, M-m, etc. Thirty-nine were homologous pairs revealing identical lineage combinations such as nmmastEM, nmmastM, nmmast, mmastEM, nmEM, nme, 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.