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.     

Maturation and regulation of megakaryocytopoiesis.

The in vitro cloning technique for detecting megakaryocyte precursor cells was employed to compare stimuli known to influence megakaryocytopoiesis. Preparations of thrombopoietic stimulating factor (TSF) did not directly stimulate the growth of megakaryocyte colonies (CFU-m) but increased the frequency of CFU-m when TSF was added to the cultures with a constant amount of megakaryocyte colony stimulating factor. Platelets or platelet homogenates did not influence the frequency of CFU-m or the size of individual colonies. Analysis of cell surface properties of megakaryocytes obtained either by isolation from bone marrow or from in vitro colonies revealed species differences. The possibility that megakaryocytopoiesis and platelet release are regulated both within the marrow as well as by humoral factors is discussed.     

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.     

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.     

巨核细胞生成调控相关细胞因子及其作用研究进展

造血干细胞分化生成巨核细胞是一个十分复杂的过程,包括造血干细胞动员及其向巨核系祖细胞分化,巨核系祖细胞增殖、分化生成未成熟巨核细胞,巨核细胞的成熟和血小板释放等过程。研究发现,造血干细胞动员及其向各系细胞分化的大部分过程都在一种称为"龛"的结构中进行,多种龛内信号分子参与了造血干细胞的动员和分化调控。该文对造血干细胞龛内参与造血干细胞动员和分化生成巨核细胞的几种重要细胞因子及其调控作用进行综述。     

Human megakaryocytopoiesis--normal and abnormal.

Regulation of megakaryocyte and platelet production remains poorly understood. In culture system two separate activities are needed for maximum production of megakaryocyte progenitors: promotor of clonal expansion and promoter of maturation, other growth factors and cells also contribute to regulation of megakaryocytopoiesis. Increased proliferation of megakaryocytes is observed in myeloproliferative disorders and idiopathic thrombocytopenic purpura, and decreased proliferation is found in aplastic anaemia and hypomegakaryocytic thrombocytopenia. Dysmegakaryocytopoiesis is present in myelodysplastic syndromes and acute leukaemia, and a proliferation of immature megakaryocytes in acute megakaryoblastic leukaemia. Increased understanding of human megakaryocytopoiesis is beginning to help in rational clinical management.     

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.     

In vivo inhibition of megakaryocyte and platelet production by platelet factor 4 in mice.

The in vivo effect of human platelet factor 4 (PF4) on murine megakaryocytopoiesis and thrombopoiesis was studied. Administration of PF4 induced a dose-dependent decrease in the numbers of megakaryocytes and their progenitor cells (CFU-MK), continuing for 1 week after the injection. However, the size of megakaryocytes and their colonies was not changed following PF4 injection. Platelet levels were significantly decreased at days 3-4. The number of CFU-GM was decreased at days 1-2. White blood cells and hemoglobin were unaffected by PF4. These data indicate that PF4 inhibits megakaryocyte and platelet production in vivo by acting on the early stage of megakaryocyte development.     

A biological and computational model of megakaryocyte development as a stochastic branching process

The purpose of this paper is to describe a model of megakaryocytopoiesis as a branching process with stochastic processes regulating critical control points of differentiation along the stem cell megakaryocyte platelet axis. Progress of cells through these critical control points are regulated by transitional probabilities, which in turn are regulated by influences such as growth factors. The critical control points include transition of resting megakaryocytic stem cells (CFU-meg) into proliferating stem cells, the cessation of cytokinesis, and the cessation of DNA synthesis. A computerized computational method has been developed for directly fitting the stochastic branching model to colony growth data. The computational model has allowed transitional probabilities to be derived from colony size data. The model provides a unifying explanation for much of the heterogeneity of stages of maturation within populations of megakaryocytes and is fully compatible with historical data supporting the stochastic nature of hematopoietic stem cell regulation and with modern molecular concepts about control of the cell cycle.     

The relationship between megakaryocyte ploidy and platelet volume

The origins and biologic significance of platelet heterogeneity in general, and platelet volume heterogeneity in particular, have been controversial scientific issues during the past decade. Although it has generally been held that specific megakaryocyte properties, especially ploidy level, are important determinants of platelet volume, the precise relationship between megakaryocyte properties and platelet properties is not well defined. The physiologic processes that specifically determine the relationship between megakaryocyte ploidy and platelet volume are unclear, and understanding of these processes has been further complicated due to the multiplicity of experimental and clinical models used to study the problem. Although it is generally true that increases in megakaryocyte ploidy are associated with increases in megakaryocyte volume, it is not well established that platelet volume is also increased during normal or abnormal thrombopoiesis as a direct result of a change in the ploidy level. Reexamination of earlier studies and some recent investigations suggest that changes in platelet volume and megakaryocyte ploidy are in fact dissociated in response to experimental thrombocytopenia. Critical review of the literature concerning the relationship between megakaryocyte ploidy and platelet volume reveals a limited number of conclusions that are well substantiated and emphasizes the relative lack of understanding about the events governing the complex process of platelet production and platelet heterogeneity.     

Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells

Megakaryocytopoiesis and thrombocytopoiesis result from the interactions between hematopoietic progenitor cells, humoral factors, and marrow stromal cells derived from mesenchymal stem cells (MSCs) or MSCs directly. MSCs are self-renewing marrow cells that provide progenitors for osteoblasts, adipocytes, chondrocytes, myocytes, and marrow stromal cells. MSCs are isolated from bone marrow aspirates and are expanded in adherent cell culture using an optimized media preparation. Culture-expanded human MSCs (hMSCs) express a variety of hematopoietic cytokines and growth factors and maintain long-term culture-initiating cells in long-term marrow culture with CD34(+) hematopoietic progenitor cells. Two lines of evidence suggest that hMSCs function in megakaryocyte development. First, hMSCs express messenger RNA for thrombopoietin, a primary regulator for megakaryocytopoiesis and thrombocytopoiesis. Second, adherent hMSC colonies in primary culture are often associated with hematopoietic cell clusters containing CD41(+) megakaryocytes. The physical association between hMSCs and megakaryocytes in marrow was confirmed by experiments in which hMSCs were copurified by immunoselection using an anti-CD41 antibody. To determine whether hMSCs can support megakaryocyte and platelet formation in vitro, we established a coculture system of hMSCs and CD34(+) cells in serum-free media without exogenous cytokines. These cocultures produced clusters of hematopoietic cells atop adherent MSCs. After 7 days, CD41(+) megakaryocyte clusters and pro-platelet networks were observed with pro-platelets increasing in the next 2 weeks. CD41(+) platelets were found in culture medium and expressed CD62P after thrombin treatment. These results suggest that MSCs residing within the megakaryocytic microenvironment in bone marrow provide key signals to stimulate megakaryocyte and platelet production from CD34(+) hematopoietic cells.     

Characterization of human megakaryocytic colony formation in human plasma

We have analysed the contribution to megakaryocyte colony formation in methylcellulose made by human plasma, serum, media conditioned by phytohemagglutinin (PHA) stimulated leukocytes (PHA-LCM), erythropoietin (EPO) preparations, and platelets. The culture system was used as a bioassay for megakaryocyte colony stimulating activity (Meg-CSA) in plasma samples of patients with perturbed megakaryocytopoiesis. Preparations of heparinized platelet-poor plasma yielded the most consistent results. Platelet-poor plasma of normal subjects will at best facilitate the occasional growth of small megakaryocyte colonies. Colony frequency and size are reproducibly enhanced in the presence of PHA-LCM as a source of exogenous Meg-CSA. Commercially available EPO preparations may vary in their content of activities that influence megakaryocyte colony formation. Addition of these preparations to cultures that contain plasma and PHA-LCM usually does not enhance colony formation. In contrast to platelet-poor plasma, platelet rich plasma and serum are less supportive of megakaryocyte colony growth. It is suggested that this loss of activity may be related to the release of inhibitors by activated platelets or alternatively caused by absorption of activities by platelets. Plasma samples from patients with megakaryocytopoietic dysfunction may contain components that promote colony formation without addition of PHA-LCM or EPO. This phenomenon is consistently observed for patients with severe aplastic anemia and bone marrow transplant recipients after completion of their ablative preparative regimen.     

Role of induced genetic instability in the mutagenic effects of chemicals and radiation

Recent studies have demonstrated that cells exposed to ionizing radiation or alkylating agents can develop prolonged genetic instability. Induced genetic instability is manisested in multiple ways, including delayed reproductive death, an increased rate of point mutations, and an increased rate of chromosome rearrangements. In many respects these changes are similar to the genetic instability associated with cancer and some human genetic diseases. Therefore, as with cancer cells, multiple mechanisms may be involved, some occuring in the early stages and some in the later stages. The high percentage of cells that develop induced genetic instability after exposure to stress, and the prolonged period over which the instability occurs, indicates that the instability is not in response to residual damage in the DNA or mutations in specific genes. Instead, changes affecting most of the exposed cells, such as epigenetic alterations in gene expression or chain reactions of chromosome rearrangements, are a more likely explanation. Learning more about the mechanisms involved in this process is essential for understanding the consequences of exposure of cells to ionizing radiation or alkylating agents.     

Role of natural killer cells, in comparison with T lymphocytes and monocytes, in the regulation of normal human megakaryocytopoiesis in vitro

Natural killer (NK) cells are thought to play an important role in host defense against virus-infected and neoplastic cells. Recent reports suggest that these cells may also influence developmental events in the course of normal erythropoiesis and granulopoiesis. The role of NK cells in the regulation of normal human megakaryocytopoiesis has not been reported, but clinical observations suggest that NK cell effects on megakaryocyte progenitors might differ from those of other cell lineages. We therefore carried out in vitro studies designed to assess the influence of NK cells on the growth of autologous megakaryocyte colony-forming units (CFU-Meg). To provide a frame of reference for these experiments, the effect of T lymphocytes, and monocyte-macrophages (M luminal diameter) on autologous CFU-Meg cloning efficiency was also studied. Purified immune effector cells were co-cultured in plasma clots with both unseparated, and progenitor cell-enriched marrow mononuclear cells (MNC) at target to effector cell ratios varying from 100:1 to 1:1. Resulting megakaryocyte colonies were enumerated by indirect fluorescence microscopy by using a rabbit anti-human platelet glycoprotein antiserum as probe for cells of the megakaryocyte lineage. The addition of NK cells to both unseparated (n = 12), and progenitor-enriched (n = 3) MNC at target to effector cell ratios of 2:1 and 1:1 resulted in a significant (p less than 0.05) augmentation in CFU-Meg-derived colony formation. Augmentation of colony formation was blocked by incubating the NK cells in Leu-11b monoclonal antibody. Stimulation appeared to be carried out by the production of a soluble growth factor which was detectable in NK cell-conditioned medium. Exposure of NK cells for 18 hr to highly purified or recombinant gamma-interferon (500 U/10(6) cells), a putative NK cell stimulator, neither increased nor abrogated the stimulatory effect. The latter could be accomplished, however, by centrifuging (200 X G for 5 min), and then preincubating the target and effector cells together for 3 hr before plating. At no time was significant inhibition of CFU-Meg demonstrated. In contrast to these results, when tested at the same ratios, and under the same conditions, no consistent effect on CFU-Meg cloning efficiency could be demonstrated by the addition of whole T cells, T cell subsets, or M luminal diameter. These results suggest that NK cells could play a role in the basal regulation of megakaryocytopoiesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional characterization of hNUDC as a novel accumulator that specifically acts on in vitro megakaryocytopoiesis and in vivo platelet production

Human NUDC (hNUDC) has been previously described as a human homolog of a fungal nuclear migration protein. It is a multifunctional interactive protein that forms an association with the microtubule motor complex in a variety of cells. Our recent studies demonstrated that hNUDC could bind specifically to the thrombopoietin receptor (Mpl) and suggest a potential role for hNUDC in megakaryocytopoiesis and thrombopoiesis. The present study is designed to define its biological activity. We demonstrate that the recombinant hNUDC significantly increases megakaryocyte maturation in serum-free liquid-cultured human CD34(+) cells and stimulates colony formation in serum-free semi-solid cultures. Flow cytometry analyses also confirm the stimulatory effect of hNUDC on megakaryocyte polyploidization and in vitro platelet production. In vivo experiments further demonstrate that the administration of hNUDC substantially enhance the number of circulating platelets in normal mice.     

Mouse mutants and phenotypes: accessing information for the study of mammalian gene function

Recent advances in high-throughput gene targeting and conditional mutagenesis are creating new and powerful resources to study the in vivo function of mammalian genes using the mouse as an experimental model. Mutant ES cells and mice are being generated at a rapid rate to study the molecular and phenotypic consequences of genetic mutations, and to correlate these study results with human disease conditions. Likewise, classical genetics approaches to identify mutations in the mouse genome that cause specific phenotypes have become more effective. Here, we describe methods to quickly obtain information on what mutant ES cells and mice are available, including recombinase driver lines for the generation of conditional mutants. Further, we describe means to access genetic and phenotypic data that identify mouse models for specific human diseases.     

Ability of plasma from patients with immune thrombocytopenic purpura (ITP) to promote the growth of megakaryocyte progenitors from normal bone marrow.

The ability of plasma from ITP patients (before and after splenectomy) to support the growth of megakaryocyte progenitors was compared with that from healthy subjects. Plasma Factor Index-Megakaryocyte PFI-Mk (ITP) which expressed resultant colony growth was significantly lower before splenectomy, but it normalized after splenectomy. (PFI-Mk) (ITP) did not relate neither to megakaryocyte nor to platelet counts. A positive correlation has been observed between megakaryocyte and platelet numbers in healthy subjects and in ITP patients after splenectomy, but not before splenectomy. The proportion of immature megakaryocytes was markedly higher in ITP marrow before splenectomy. This study indicates, that in ITP apart from antibodies directed to platelets and megakarocytes a low plasma stimulatory activity affected megakaryocytopoiesis.     

Regulation of murine megakaryocyte size and ploidy by non-platelet-dependent mechanisms in radiation-induced megakaryocytopenia

Megakaryocytic macrocytosis was evaluated in mice after irradiation with 6.5 Gy 60Co gamma rays. During the second and third months after sublethal irradiation, one or more of the following abnormalities of thrombocytopoiesis was present: thrombocytopenia, megakaryocytopenia, macromegakaryocytosis, a shift to higher ploidies, and enlargement of cells within ploidy groups. After transfusion-induced thrombocytosis, reductions in megakaryocyte size were delayed or absent relative to non-irradiated mice, and there was more of a tendency to shift to lower values for megakaryocyte ploidy. Mice with radiation-induced megakaryocytopenia failed to show rebound thrombocytosis during recovery from immunothrombocytopenia, in spite of further increases in megakaryocyte size and ploidy. The findings support the hypotheses that numbers of megakaryocytes may influence the regulation of megakaryocytopoiesis even when there is an excess of platelets and that ploidy distribution is not the sole determinant of the average size of a population of megakaryocytes. After irradiation, persistent megakaryocytopenia may not severely affect platelet production under steady-state conditions, but the ability of the marrow to respond to homeostatic regulation is compromised.     

Human umbilical cord blood-derived stromal cells: Multifaceted regulators of megakaryocytopoiesis

It has been demonstrated that stromal cell precursors exist in human umbilical cord blood. After being cultured in vitro, these cells are called human umbilical cord blood-derived stromal cells (hUCBDSCs). However, the role of hUCBDSCs in hematopoiesis is still unclear. We have previously shown that hUCBDSCs are superior to human bone marrow stromal cells (hBMSCs) at enhancing the expansion of megakaryocyte colony forming units (CFU-Meg). Based on this observation, we postulated that hUCBDSCs might promote megakaryocytopoiesis. To test this hypothesis, we developed a megakaryocyte/hUCBDSC co-culture model and a hematopoietic microenvironment injury model in nude mice. We explored the ability and mechanisms by which hUCBDSCs promoted the proliferation of megakaryocytes in vitro, and we also explored their capacity to restore the hematopoietic microenvironment in vivo. As expected, hUCBDSCs were more effective than hBMSCs at enhancing the proliferation of megakaryocyte lines from HEL cells and restoring megakaryocytopoiesis in a hematopoietic microenvironment injury model in nude mice. Thrombopoietin (TPO) and stromal cell derived factor-1 (SDF-1) are two of the key factors underlying this capacity. We also found that gap junction intercellular communication (GJIC) between HEL cells and hUCBDSCs might be partially absent. Our data provide the first evidence that hUCBDSCs play a regulatory role during megakaryocytopoiesis, which might be important for designing treatments for patients with megakaryocytic injury.     

Bardet–Biedl Syndrome ciliopathy is linked to altered hematopoiesis and dysregulated self‐tolerance

Bardet–Biedl Syndrome (BBS) is a pleiotropic genetic disease caused by the dysfunction of primary cilia. The immune system of patients with ciliopathies has not been investigated. However, there are multiple indications that the impairment of the processes typically associated with cilia may have influence on the hematopoietic compartment and immunity. In this study, we analyze clinical data of BBS patients and corresponding mouse models carrying mutations in Bbs4 or Bbs18. We find that BBS patients have a higher prevalence of certain autoimmune diseases. Both BBS patients and animal models have altered red blood cell and platelet compartments, as well as elevated white blood cell levels. Some of the hematopoietic system alterations are associated with BBS‐induced obesity. Moreover, we observe that the development and homeostasis of B cells in mice is regulated by the transport complex BBSome, whose dysfunction is a common cause of BBS. The BBSome limits canonical WNT signaling and increases CXCL12 levels in bone marrow stromal cells. Taken together, our study reveals a connection between a ciliopathy and dysregulated immune and hematopoietic systems.