Maintenance of hemopoiesis in long-term bone marrow cultures from S1/S1d and W/Wv mice

Although phenotypically similar, the cellular defects in congenitally anemic mice of genotype W/Wv and S1/S1d are quite different. W/Wv mice have defective hemopoietic stem cells; in contrast, S1/S1d mice have normal stem cells, but their hemopoietic microenvironment cannot support normal differentiation of the stem cells. We also observed defective hemopoiesis as measured by granulocyte-macrophage colony-forming units (GM-CFU) in long-term bone marrow cultures (LTBMC) established with marrow from these mutants, but in contrast to an earlier report we obtained long-term maintenance of hemopoiesis (up to 20 weeks) albeit at a lower level than the control (28% of control for S1/S1d and 23% for W/Wv). These levels probably reflect more accurately the in vivo effects of the mutations than the severe defect reported previously. However, this level of hemopoiesis and the high variability observed in replicate flasks in LTBMC make it difficult to study these mutants in tissue culture.     

Fate of senescent megakaryocytes in the bone marrow

S ummary . Degenerating senescent megakaryocytes have been identified in mouse bone marrow by light and electron microscopy. The ultrastructural changes which occur as degeneration proceeds are characteristic of death by apoptosis, although most cells appear to round up rather than undergo fragmentation. A hitherto unreported finding in degenerating cells was the presence of bundles of approximately 7 nm diameter parallel filaments in nuclei and membrane-bound nuclear fragments. Structurally, they resembled bundles of filaments induced in nuclei with dimethyl sulphoxide and identified as actin. Often a bundle appeared to terminate at the inner membrane. In the cytoplasm the presence of microtubules and centrioles indicates that not all the latter organelles are lost by the megakaryocyte during platelet release. Degenerating senescent megakaryocytes are rare in the marrow of normal mice but increase in frequency during 5-fluorouracil stimulated thrombocytosis. The dying cells are eventually phagocytosed by macrophages, a process that can occur extravascularly, showing that entry of senescent megakaryocytes into the circulation is not necessary for their disposal.     

Low ploidy megakaryocytes in steady-state rat bone marrow

The amount of DNA in individual megakaryocytes (MK) was measured by Feulgen photometry performed on smears of steady-state rat bone marrow. The MK were identified by acetylcholinesterase (ACHE) staining. The method described is particularly suitable in the study of immature MK, hitherto difficult to examine for DNA content. The main MK ploidies were 8N, 16N, and 32N, comprising 33.4% +/- 1.4%, 40.8% +/- 1.4%, and 17.9% +/- 1.3%, respectively, of the total MK numbers. Only 6.7% +/- 1.7% had less than octoploid DNA content. Very few cells were found belonging to the 64N class. Among the immature MK, a relatively large fraction was found between the ploidy levels, indicating a rapid turnover within these ploidy classes. The demonstration of ACHE activity even in MK with a diploid DNA content indicates that the flow from the committed progenitors to differentiated MK occurs at the 2N level.     

Cytokine production by primary bone marrow megakaryocytes

Primary human bone marrow megakaryocytes were studied for their ability to express and release cytokines potentially relevant to their proliferation and/or differentiation. The purity of the bone marrow megakaryocytes was assessed by morphologic and immunocytochemical criteria. Unstimulated marrow megakaryocytes constitutively expressed genes for interleukin-1 beta (IL-1 beta), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha), by the polymerase chain reaction (PCR) and Northern blot analysis. At the protein level, megakaryocytes secreted significant amounts of IL-1 beta (53.6 +/- 3.6 pg/mL), IL-6 (57.6 +/- 15.6 pg/mL), and GM-CSF (24 +/- 4 pg/mL) but not TNF-alpha. Exposure of human marrow megakaryocytes to IL-1 beta increased the levels of IL-6 (87.3 +/- 2.3 pg/mL) detected in the culture supernatants. Transforming growth factor- beta was also able to stimulate IL-6, IL-1 beta, and GM-CSF secretion, but was less potent than stimulation with phorbol-12-myristate-13- acetate (PMA). The secreted cytokines acted additively to maintain and increase the number of colony-forming unit-megakaryocytes colonies (approximately 35%). These studies demonstrate the production of multiple cytokines by isolated human bone marrow megakaryocytes constitutively or stimulated in vitro. The capacity of human megakaryocytes to synthesize several cytokines known to modulate hematopoietic cells supports the concept that there may be an autocrine mechanism operative in the regulation of megakaryocytopoiesis.     

Emperipolesis of marrow cells within megakaryocytes in the bone marrow of sublethally irradiated mice

The incidence of megakaryocytic emperipolesis was studied in the bone marrow of normal and X-irradiated mice. Two groups of mice received total body irradiation with a single dose of 5 Gy and one of the two groups had been treated with a radioprotective drug, ethiofos (WR-2721), before irradiation. Mice from a third group remained unexposed to irradiation and served as controls. The Wright-Giemsa stained bone marrow smears were analyzed every 5 days during a 30-day period, starting 1 day after irradiation. The number of megakaryocytes exhibiting the phenomenon was determined and expressed as an average value for every experimental group. The frequency of megakaryocytic emperipolesis was less than 15% of megakaryocytes from control smears but increased to 34% in mice that had only been irradiated and to 43% when mice were treated with WR-2721 before irradiation. In the last case, i.e., irradiation and treatment with a radioprotective drug, a positive correlation between the macrocytic megakaryocytes and elevated emperipolesis was noted. Under light microscopy, there were no signs of phagocytosis; engulfed cells remained unaltered with their normal structure intact. Granulocytic, erythroid, and lymphoid cells appeared to be the most frequent marrow cells engulfed by mature megakaryocytes. The number of incorporated cells in one megakaryocyte ranged from 1 to 3, though occasionally more than 6 were seen in macrocytic megakaryocytes. Based on our findings and on a review of the associated literature, we believe emperipolesis is an interesting cellular phenomenon related to the fast passage of marrow cells across the marrow-blood barrier, especially through the cytoplasm of megakaryocytes in response to an increased demand for cell delivery. The high demand for cell delivery which occurs after irradiation may cause certain mature bone marrow cells to take a transmegakaryocyte path to enter the circulation of the blood. Irradiation seems to have an immediate effect (observed after 24 h) on emperipolesis, suggesting that a humoral factor is involved in the pathogenesis.     

Long-term human bone marrow cultures

Pronlonged in vitro growth of murine bone marrow has been achieved using a flask culture system. We report the adaption of the technique to the growth of human bone marrow. In this system, CFU-GM and CFU-E are maintained for 4-9 wk. Morphologically recognizable granulopoiesis occurs for 4-6 wk and erythropoiesis for 1.5-2.5 wk. Functional T lymphocytes are maintained for at least 5 wk. The adherent population is composed of macrophages, fibroblast-like cells, and flat pavement- like cells. Fat-containing cells are not prominant. This culture system provides a means to study human hematopoietic cell proliferation and differentiation in vitro, as well as to examine interactions between stromal cells and hematopoietic and lymphoid progenitors.     

Expression of adhesion antigens of human bone marrow megakaryocytes, circulating megakaryocytes and blood platelets.

There is evidence that mature megakaryocytes migrate into sinusoids, enter the blood and fragment in the vascular bed. We wondered whether differences in expression of adhesion antigens could be associated with the egress of megakaryocytes from bone marrow into the peripheral blood or the fragmentation into platelets. Megakaryocytes from human marrow were purified by counterflow centrifugal elutriation followed by a glycoprotein Ib-dependent agglutination procedure. Megakaryocytes from central venous blood and pulmonary arteries were purified by counterflow centrifugal elutriation alone. Adhesion antigens were labelled in an immunohistochemical assay. Both bone marrow megakaryocytes and platelets from healthy volunteers stained > 75% positive for CD36, CD41, CD42, Cdw49b (alpha subunit VLA2), Cdw49e (alpha subunit VLA5), Cdw49f (alpha subunit VLA6) and CD62. Circulating megakaryocytes, although > 75% positive for CD41, had, unlike platelets and bone marrow megakaryocytes, a reduced and remarkable heterogeneous (5-100% positive) labelling with antibodies against Cdw49b, Cdw49e, Cdw49f. These results could be confirmed by comparing the bone marrow megakaryocytes, circulating megakaryocytes and platelets from 7 patients that were recovered and processed at the same time. Morphologically mature, circulating megakaryocytes have, unlike bone marrow megakaryocytes, a heterogeneous expression of adhesion antigens, especially of Cdw49b, Cdw49e, and Cdw49f.     

Cell interactions influencing murine marrow megakaryocytes: nature of the potentiator cell in bone marrow

Auxiliary bone marrow cells are required for optimal murine megakaryocyte colony formation in addition to progenitor cells and a colony stimulating activity (CSA) present in WEHI-3 cell conditioned medium. These auxiliary cells are adherent, with a sedimentation rate of 5.8 mm hr-1 and buoyant density of 1.065-1.078 gcm-3. The activity from bone marrow cells is loss at irradiation doses above 900 rad. Bone marrow cells with these characteristics, and supernatants from lung, bone shafts, and peritoneal exudate cells were all active in enhancing megakaryocyte colony incidences in mouse bone marrow cultures above those stimulated by an obligatory activity in WEHI-3 cell conditioned medium. Certain macrophage cell lines (J774, P388D1) could elaborate the activity. This study confirms that a potentiation activity enhances CSA stimulation of megakaryocyte colony formation. The potentiator is elaborated by bone marrow cells in limiting amounts requiring either high cell concentrations or an exogenous source of the activity for optimal colony growth.     

Differential effects of TGF-beta 1 on lymphohemopoiesis in long-term bone marrow cultures

Latent transforming growth factors beta (TGF-beta) are easily detectable in embryonic and adult hematopoietic tissues and in vitro studies show that they are potent antagonists of lymphopoiesis and myelopoiesis when converted to biologically active form. To learn more about possible roles in hematopoiesis, active TGF-beta 1 was added to cultures prepared to support myeloid cells (Dexter conditions) or B lineage lymphocytes (Whitlock-Witte conditions) and studied in detail. Hematopoiesis was permanently arrested in Dexter cultures treated with 40 pmol/L (1 ng/mL) of active TGF-beta from initiation. In addition, adipogenesis was inhibited in a dose-dependent manner, and adherent layers from treated cultures were defective when recharged with fresh bone marrow cells. Ongoing neutrophil production was terminated in established cultures when addition of the factor was delayed for 8 weeks. In contrast, in experiments with Whitlock-Witte cultures, some of the flasks produced lymphocytes in the continuous presence of TGF- beta 1 (40 pmol/L). Lymphopoiesis was completely arrested by ten-fold higher concentrations, and this was most effective when added at the beginning of culture. Precursors of lymphocytes as well as the microenvironmental elements necessary for supporting their growth survived 2 weeks of cytokine treatment (400 pmol/L) in Dexter cultures. Normal outgrowth of lymphocytes occurred when the cultures were switched to Whitlock-Witte conditions. Surface marker expression on lymphocytes growing in TGF-beta resistant or previously treated cultures was not unusual. These studies demonstrate that TGF-beta is a negative regulator of hematopoiesis in long-term cultures and show that this includes effects on microenvironmental elements. At low concentrations, production of myeloid cells was preferentially affected.     

Comparative analyses of megakaryocytes derived from cord blood and bone marrow

Thrombocytopenia is typically observed in patients undergoing cord blood transplantation. We hypothesized that delayed recovery of the platelet count might be caused by defects in the megakaryocytic differentiation pathway of cord blood progenitors. To test this hypothesis, we compared the features of in vitro megakaryocytopoiesis between cord blood progenitors and those in bone marrow cells after isolation of CD34+ cells as progenitors. The proliferative responses of the progenitors in cord blood are higher than those in bone marrow cells in the presence of interleukin (IL)-3, stem cell factor (SCF) and thrombopoietin (TPO). However, the ability to generate mature megakaryocytes was higher in bone marrow progenitors than in cord blood in the same in vitro culture system, when examined by the expression of CD41, polyploidy and proplatelet formation. Furthermore, an earlier induction of c-mpl protein, a receptor for TPO, was observed in the progenitors from bone marrow than in those from cord blood in the presence of SCF and IL-3. Therefore, the ability to generate mature megakaryocytes in bone marrow progenitors is superior to that in cord blood, and the delayed engraftment of platelets after cord blood transplantation might be attributed to the features of cord blood megakaryocyte progenitors.     

Normal colony stimulating factor (CSF) production by bone marrow stromal cells and abnormal granulopoiesis with decreased CFUc in S1/S1d mice.

The concentration of CFUC and the production of stromal-derived CSF in the femora of S1/S1d mice were determined. There was a lower concentration CFUC and a smaller total number of nucleated cells in the femoral marrow of WCB6. S1/S1d (S1/S1d) mice than in WCB6. +/+/(+/+) mice or in C57B1/6. +/+ (C57Bl) mice. On the other hand stromal-derived CSF production by femora from S1/S1d mice did not differ significantly from that of +/+'s. These observations indicate that the microenvironmental defect of S1/S1d mice results in decreased growth of granulocytic precursors as well as those of erythroid and megakaryocytic cells. This is consistent with the reported decrease in multipotential stem cell proliferation. Stromal cell derived CSF production was normal and could not be implicated in the decreased production of granulocytic precursors.