A Study of Hemopoiesis on Sublayer of Peritoneal Cells in the Presence of Hemopoietic Cytokines

We studied the effects of erythropoietin and thrombopoietin on the clonogenic capacity and direction of differentiation of the hemopoietic cells that form colonies on acetate cellulose membrane in the peritoneal cavity of mice. An increased level of erythropoietin in the blood of recipient mice after blood letting led to the appearance of erythroid colonies upon transplantation of syngeneic hemopoietic cells but did not affect the differentiation of transplanted xenogeneic (guinea pig) hemopoietic cells. Erythropoietin transported top the stromal sublayer by a polymeric carrier also induced erythroid differentiation, while thrombopoietin transported in a similar way somewhat enhanced megakaryocytopoiesis.     

A study of hemopoiesis on a sublayer of peritoneal cells in the presence of hemopoietic cytokines

We studied the effects of erythropoietin and thrombopoietin on the clonogenic capacity and direction of differentiation of the hemopoietic cells that form colonies on acetate cellulose membrane in the peritoneal cavity of mice. An increased level of erythropoietin in the blood of recipient mice after blood letting led to the appearance of erythroid colonies upon transplantation of syngeneic hemopoietic cells but did not affect the differentiation of transplanted xenogeneic (guinea pig) hemopoietic cells. Erythropoietin transported top the stromal sublayer by a polymeric carrier also induced erythroid differentiation, while thrombopoietin transported in a similar way somewhat enhanced megakaryocytopoiesis.     

Effect of insulin on murine megakaryocytopoiesis in a liquid culture system

To examine the influence of insulin on megakaryocytopoiesis, we tested its effect on murine bone marrow cultures in a liquid culture system. In the presence of pokeweed mitogen-stimulated spleen cell conditioned medium in culture, insulin markedly enhanced megakaryocyte colony formation and increased the number and size of free megakaryocytes seen after 7 days. Many of the cells in cultures with insulin, however, were classified as immature, since they had a basophilic cytoplasm, a low cytoplasmic/nuclear ratio and low acetylcholinesterase activity. It is suggested that insulin potentiates murine marrow megakaryocytopoiesis in vitro, but that this is not accompanied by differentiation of the cells from the immature to mature state.     

Multiple levels of regulation of megakaryocytopoiesis

A working hypothesis for the regulation of megakaryocytopoiesis is described on the basis of current data. The hypothesis proposes that in vivo megakaryocytes are generated by 1) the expansion of clonable progenitor cells into immature megakaryocytes by locally produced (and regulated) interleukin-3 (IL-3) and 2) the development and maturation of immature megakaryocytes by a dual system; by a lineage specific mechanism involving thrombopoietic stimuli in the steady state and thrombocytopenic conditions, and by a lineage nonspecific mechanism via IL-3 in damaged or reconstituting marrow. The hypothesis predicts that if IL-3 is a significant in vivo regulator of megakaryocyte formation and development, receptor for IL-3 should be present on megakaryocytes and may be vestigially on platelets. Small but significant levels of 125I IL-3 were found to bind to platelets from normal mice. The level of binding on platelets was found to be enhanced sevenfold from mice that had received high levels of irradiation followed by bone marrow transplantation. This contrasted with a twofold increase in the level of binding to platelets from mice made acutely thrombocytopenic with antiplatelet serum. The data suggest that IL-3 may be involved in the in vivo regulation of murine megakaryocytopoiesis and may be a significant factor in rebound thrombopoiesis following bone marrow damage.     

Thrombin-stimulated effects on megakaryocytopoiesis and pulmonary-platelet interactions

The effects of thrombin stimulation on megakaryocytopoiesis and pulmonary-platelet interactions were investigated before and after administration of the compound to 15 mongrel dogs. Each dog served as its own control. Thrombin was given to encourage the traffic of megakaryocytes into the lung and to study the thrombin-stimulated effects on megakaryocytopoiesis in the bone marrow. Our results showed that thrombin increased the numbers of bone marrow cells in general and megakaryocytes (MK) in particular. In addition, the maturation cycle of megakaryocytes was accelerated and the number of MK migrating into the central venous circulation was nearly doubled. Most of the circulating MK ultimately became sequestered in pulmonary capillaries, where platelets were shed into the arterial circulation. We conclude that thrombin has a major stimulatory effect on megakaryocytopoiesis in the bone marrow and that the lung plays an important role as a vascular filter and regulator of circulating platelet count.     

Animal models with inherited hematopoietic abnormalities as tools to study thrombopoiesis

Animals with hereditary abnormalities of hematopoiesis are quite useful in the study of regulatory pathways of megakaryocytopoiesis and platelet formation. Seven such animal models are analyzed here. The Wistar Furth rat has been recently discovered to have reduced platelet number, but large mean platelet volume, and is, therefore, a model of hereditary macrothrombocytopenia. Study of the Wistar Furth rat may help to elucidate the process of platelet formation. Two mouse mutants the S1/S1d and W/Wv, have macrocytic anemia with reduced megakaryocyte number, but normal platelet count. In these mice, the platelet count is maintained by increased platelet production per megakaryocyte. These models demonstrate that factors other than platelet level are monitored in the feedback regulation of megakaryocytopoiesis and platelet production, and further study should lead to a better understanding of the regulation of megakaryocyte size. The Belgrade rat has severe microcytic anemia with decreased megakaryocyte number. Megakaryocyte size is increased, but platelet count is moderately reduced and thus the megakaryocyte-platelet picture resembles that of severe iron deficiency anemia. A more in depth examination of this model should delineate the effects of iron deficiency and hypoxia on megakaryocytopoiesis. The grey collie dog has cyclic hematopoiesis with large asynchronous fluctuations in all blood cell counts at approximately 2-week intervals. Megakaryocytes have not been studied. This model should be a tool to define the relationships between hematopoietic growth factors and differentiation of the various hematopoietic cell lineages. The br/br rabbit has a transient disturbance in fetal megakaryocytopoiesis and brachydactyly due to spontaneous amputation. Further study of this model may provide a better understanding of fetal megakaryocyte development and establish whether an association exists between the abnormal megakaryocytes and the limb amputations. The nude mouse with its severe T-lymphocyte deficiency has been studied to ascertain whether T cells play a regulatory role in normal and acute thrombocytopenia-stimulated megakaryocytopoiesis. The question of whether T cells or their products are responsible for reactive thrombocytosis in chronic inflammation could be examined with this model. These animal mutants have provided and should continue to provide important models for understanding the regulation of megakaryocytopoiesis and platelet production.     

Megakaryocytopoiesis studied in a mathematical model. 4. Regulation of cell flow in a transit population

The role of cells capable of formation of megakaryocytic colonies (CFUm) in regulation of thrombocytopoiesis was studied using a simulation model of megakaryocytopoiesis. The CFUm were shown to be the least differentiated cells involved in regulation of megakaryocytopoiesis in mice upon immune thrombocytopenia and after intravenous injection of hydroxyurea. A correlation was found between the CFUm population productivity and the number of cells in a transit population of the megakaryocytic line which makes it possible to reproduce experimental kinetics in the model. The duration of cell development from CFUm to megakaryocytes is about 3 days.     

Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro.

Megakaryocytopoiesis was selectively inhibited in cultured murine bone marrow cells by a 15-mer oligodeoxynucleotide complementary to the initiator AUG region in butyrylcholinesterase mRNA. Furthermore, conditioned medium from Xenopus oocytes producing recombinant butyrylcholinesterase stimulated megakaryocytopoiesis. These observations implicate butyrylcholinesterase in megakaryocytopoiesis and suggest application of oligodeoxynucleotides for modulating bone marrow development.     

Effects of cyclophosphamide on murine bone marrow and splenic megakaryocyte-CFC,granulocyte-macrophage-CFC,and peripheral blood cell levels

The effect of cyclophosphamide (CY) on megakaryocytopoiesis in mice was examined with assays of megakaryocyte colony-forming cells (Meg-CFC) in bone marrow and spleen and simultaneous determinations of peripheral blood counts, after a single intraperitoneal dose (200 mg/kg) of CY. Significant rebound thrombocytosis (170% of normal) occurred at day 11 after injection with CY, although only modest preceding thrombocytopenia (70% of normal) was observed. After an initial 3–5-day period of suppression, total megakaryocyte colony-forming cells (Meg-CFC) in both bone marrow and spleen of CY-treated mice demonstrated rebound increases at 5 and 7 days, respectively, after administration of the drug. Granulocyte-macrophage colony-forming cells (GM-CFC) exhibited alterations which were similar to those of Meg-CFC, suggesting similar sensitivities of Meg-CFC and GM-CFC to CY. The increase in Meg-CFC in both bone marrow and spleen preceded development of thrombocytosis by 4–6 days. This suggests that increased platelet counts in CY-treated mice are attributable, at least in part, to alterations in feedback mechanisms which control megakaryocytopoiesis, with resultant stimulation of the megakaryocyte progenitor compartment.     

Megakaryocyte colony-stimulating factor (Mk-CSF): its physiologic significance

Megakaryocyte colony-stimulating activity (Mk-CSA) is required for in vitro megakaryocyte colony formation. Its in vivo significance in megakaryocytopoiesis is unknown. We studied 12 patients undergoing bone marrow transplantation (BMT) at our institution. The bone marrow megakaryocyte progenitor cells (CFU-Mk), the serum level of Mk-CSA, and the platelet count on the 28th day after BMT were studied. Patients with elevated Mk-CSA levels had less CFU-Mk in their bone marrow than did patients with a normal or decreased Mk-CSA (p less than 0.01). Animal experiments using murine models have documented that several purified molecules including erythropoietin, multi-CSF and GM-CSF possess Mk-CSA. The in vitro Mk-CSF of WEHI-3-conditioned medium is multi-CSF. The in vivo significance for megakaryocytopoiesis of these factors is not clear. In the human system, Mk-CSA is increased in conditions with decreased bone marrow megakaryocytes. Recombinant human or primate CSFs have in vitro Mk-CSA utilizing both human and murine cells as targets. However, the presence of these activities does not fully explain the Mk-CSA in human serum rich in Mk-CSA. The precise regulation of human blood cell levels and the studies discussed suggest that there is a specific Mk-CSF that responds to in vivo changes in megakaryocyte numbers. Proof of its physiologic role awaits the isolation of a pure factor.     

Regeneration of the caudal and pectoral fins of a bony fish, Gambusia (Haplochilus) schoelleri

The study showed that excision of the caudal fin at basal level was followed by complete regeneration in one and a half months, whereas if it was amputated at mid-fin level, complete regeneration took two months. The findings confirm that the greater the extent of amputation, the faster the rate of regeneration. In the case of the pectoral fin, only part of which was removed, it was found that the fin completely regenerated, with recovery of its original pattern, within two months after amputation.     

New insights into the regulation of human megakaryocytopoiesis

It is apparent that multiple cellular stages and biologic processes can be identified during megakaryocytopoiesis that are potentially subject to control by hematopoietic growth factors and marrow accessory cell populations. Two classes of megakaryocyte progenitor cells, the colony forming unit-megakaryocyte (CFU-MK) and the burst forming unit-megakaryocyte (BFU-MK), have now been detected in normal human bone marrow cells. The BFU-MK by virtue of the greater cellular content of its resultant colonies and the delayed time of appearance of these colonies appears to be a more primitive progenitor cell with a greater proliferative potential than the CFU-MK. A number of hematopoietic growth factors including megakaryocyte colony stimulating factor, (MK-CSF), recombinant erythropoietin (EPO) and granulocyte macrophage colony stimulating factor (GM-CSF) are each capable of increasing cloning efficiency of human megakaryocyte progenitor cells. It is presently unknown whether these factors act directly on the CFU-MK or whether they stimulate marrow accessory cells to elaborate growth factors that influence CFU-MK proliferation. In order to answer this question, the effect of these growth factors on the cloning efficiency of a human megakaryocytic cell line, EST-IU, was examined. Each of these factors was capable of increasing leukemia cell colony formation. One can conclude from these studies that MK-CSF, EPO, and GM-CSF act directly on cells of the megakaryocytic lineage. The physiologic significance of the lineage nonspecific effects of EPO and GM-CSF on megakaryocytopoiesis is yet to be determined. On the basis of these observations, a model of human megakaryocytopoiesis was suggested. Several factors appear able to influence multiple steps in megakaryocytic development, whereas others influence only specific stages or cellular events occurring during megakaryocytopoiesis.     

Co-stimulatory effect of TLR2 and TLR4 stimulation on megakaryocytic development is mediated through PI3K/NF-ĸB and XBP-1 loop

Toll-like receptors (TLRs) are a class of proteins (patterns recognition receptors-PRRs) capable of recognizing molecules frequently found in pathogens (that are so-called pathogen-associated molecular patterns-PAMPs), they play a key role in the initiation of innate immune response by detecting PAMPs. Our findings show that the functional effects of TLRs co-stimulation on megakaryocytopoiesis. A single cell may receive multiple signal inputs and we consider that multiple TLRs are likely triggered during infection by multiple PAMPs that, in turn, might be involved in infection driven megakaryocytopoiesis, and the present study provide the evidence for the megakaryocytic effects of TLRs co-stimulation.     

Cell-to-cell variability in the differentiation program of human megakaryocytes

Differentiation of CD34(+) stem/progenitor cells into megakaryocytes is thought to be a uniform, unidirectional process, in which cells transform step by step from less differentiated precursor stages to more differentiated megakaryocytes. Here we propose the concept and present evidence based on single-cell analysis that differentiation occurs along multiple, partially asynchronous routes. In all CD34(+) cells cultured with thrombopoietin, surface appearance of glycoprotein IIIa (GPIIIa) preceded that of GPIb, indicating that the expression of these glycoproteins occurs in a timely ordered manner. Cellular F-actin content increased in parallel with GPIb expression. Only cells that expressed GPIb were polyploid, pointing to co-regulation of GPIb expression, actin cytoskeleton formation and polyploidization during megakaryocytopoiesis. On the other hand, most progenitor cells responded to thrombin but not to thromboxane A(2) analogue by rises in cytosolic [Ca(2+)](i). The appearance of thromboxane-induced responses during megakaryocytopoiesis was not strictly linked to glycoprotein expression, because cells showed responsiveness either before or after GPIb expression. The same non-strictly sequential pattern was observed for disappearance of the Ca(2+) response by prostacyclin mimetic; in some megakaryocytes it occurred before and in others after GPIb expression. Thus, megakaryocytic differentiation follows along independent routes that are either strictly sequential (GPIIIa and GPIb expression) or proceed at different velocities (Ca(2+) signal regulation).     

Cellular and humoral immunity factors as regulators of regenerative morphogenesis

The published and author's data concerning changes in the immune system during regeneration of various organs are summarized. During the first few hours after partial removal of organs possessing high regeneration capacity, lymphocytes stimulate proliferation of nonlymphoid cells of an organ identical to the operated one; the production of antibodies against thymus-dependent antigen also increases. At the following stages of regeneration, the lymphocytes suppress the cell proliferation and decrease the antigen production. The level of these changes correlates with the level of post operational deficiency of the organ being maximal after total removal of the organ. The functional properties of splenocytes at different stages of regeneration suggest that the high T-helper and T-suppressor activities correlate with stimulation and suppression of non-lymphoid cells proliferation respectively. Culture medium supernatant after cultivation of these lymphocytes also changes the proliferation of hepatocytes. The author considers the impairment of natural immune tolerance caused by deficiency of organ auto-antigens that normally suppress lymphocyte proliferation to be the cause of the changes in lymphocyte activity.     

Mouse models of diseases of megakaryocyte and platelet homeostasis

Platelets are the small anuclear blood cells that are the product of megakaryocytopoiesis, the process of hematopoietic stem cell commitment to megakaryocyte production and the differentiation and maturation of these cells for platelet release. Deregulation or disruption of megakaryocytopoiesis can result in platelet deficiencies, the thrombocytopenias, with attendant risk of hemorrhage or thrombocytosis, a pathological excess of platelet numbers. Mouse models, particularly those engineered to carry genetic alterations modeling mutations associated with human disease, have provided important insights into megakaryocytopoiesis and deregulation of this process in disease. This review focuses on mouse models of diseases of altered megakaryocyte and platelet number, illustrating the profound contribution of these models in validating suspected roles of disease-associated genetic alterations, promoting discovery of new links between genetic mutations and specific diseases, and providing unique tools for better understanding of disease pathophysiology and progression, as well as resources to define drug action or develop new therapeutic strategies.     

Effect of erythroid differentiation factor on megakaryocytic differentiation of L8057, a murine megakaryoblastic leukemia cell line.

To assess the potent effect of erythroid differentiation factor (EDF) on megakaryocytopoiesis, effect of EDF on megakaryocytic differentiation of L8057, a murine megakaryoblastic cell line, was examined. EDF potentiated AchE induction of L8057 in a dose dependent manner. The potency of EDF on megakaryocytic differentiation is comparable to that on erythroid differentiation reported previously. The present results suggest that EDF may play a regulatory role in megakaryocytopoiesis as well as in erythropoiesis.     

The in vivo effects of a culture medium. II. Influence of culture medium administered prior to irradiation on hemopoietic recovery of gamma-irradiated mice.

The culture medium administered to C57Bl/6 mice 18 h and 8 h before a single irradiation (9 Gy) had a radioprotective effect and clearly influenced postirradiation changes in haemopoiesis. Haemopoiesis recovery appeared to be faster in culture medium-pretreated animals than in those irradiated without such pretreatment. By 12-15 days after irradiation, the thymus cortex appeared to be repaired, on day 21 a multiple increase in extramedullary erythropoiesis, myelopoiesis and megakaryocytopoiesis in the red pulp was found and later, on day 28, the lymphopoiesis in the white pulp of spleen was restored. The rate of haemopoiesis proliferation of predominantly myeloid cells which reached a control level on day 28 following irradiation. Consequently, the regenerative processes in blood-forming organs were accompanied by considerable reticulocytosis and complete recovery of neutrophil and platelet counts in the peripheral blood as seen on day 21. Despite a slower rate complete recovery of the total leukocyte count was reached by day 180 after irradiation.     

The global regulatory logic of organ regeneration: circuitry lessons from skin and its appendages

In organ regeneration, the regulatory logic at a systems level remains largely unclear. For example, what defines the quantitative threshold to initiate regeneration, and when does the regeneration process come to an end? What leads to the qualitatively different responses of regeneration, which restore the original structure, or to repair which only heals a wound? Here we discuss three examples in skin regeneration: epidermal recovery after radiation damage, hair follicle fate choice after chemotherapy damage, and wound-induced feather regeneration. We propose that the molecular regulatory circuitry is of paramount significance in organ regeneration. It is conceivable that defects in these controlling pathways may lead to failed regeneration and/or organ renewal, and understanding the underlying logic could help to identify novel therapeutic strategies.     

Altered Expression of Inducible Nitric Oxide Synthase After Sciatic Nerve Injury in Rat

Nitric oxide is known to contribute to neuronal damage as well as to peripheral neuronal regeneration following injury. Sciatic nerve injury is a common and serious complication of intramuscular injections. In order to ascertain the role of inducible nitric oxide synthase (iNOS) in the injured sciatic nerve, we studied the expression of this enzyme by RT-PCR and immunohistochemistry, in a rat model of sciatic nerve injury. In sham-operated control rats iNOS expression was undetectable by immunohistochemistry and its mRNA level was also very low. In contrast, in the experimental group that was subjected to sciatic nerve injury, both mRNA and protein of iNOS were found to be significantly elevated. The protein level of iNOS, as revealed by positive immunostaining, peaked at 7 days post-surgery followed by a decrease. Similarly, the iNOS mRNA levels remained elevated at 1, 3, 7 days but declined to very low level by day 21, after surgery. This study indicates that the increased expression of iNOS after sciatic nerve injury in rats may contribute to nerve regeneration. Thus our results suggest that excessive expression of iNOS after nerve injury is not conducive to nerve regeneration.