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。     

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.     

Disparate effects of interleukin 11 and thrombopoietin on megakaryocytopoiesis in vitro

The effects of interleukin-11 (IL-11) and thrombopoietin (TPO) on murine megakaryocytopoiesis were studied using a serum-free culture system. Acting alone, both IL-11 and TPO increased the number of acetylcholinesterase (AchE)(+)cells (megakaryocytes), the latter being more potent than the former. TPO, but not IL-11, increased the mean AchE activity per megakaryocyte (AchE activity/megakaryocyte). TPO increased both the number of megakaryocytes with high ploidy, and of those with low ploidy. In contrast, IL-11 increased only the number of megakaryocytes with high ploidy. The effect of TPO on megakaryocyte ploidy was stronger than that of IL-11. Both IL-11 and TPO increased the proportion of large megakaryocytes, but the latter was more potent than the former. While the stimulatory effects of IL-11 and TPO on the number of megakaryocytes were enhanced by IL-3 or stem cell factor (SCF), synergism of IL-11 or TPO with IL-3 or SCF in stimulating AchE activity/megakaryocyte was inconsistent. IL-11 and TPO stimulated the formation of colony-forming units of megakaryocyte in the presence of IL-3, but not alone, with similar maximum colony numbers for both cytokines. Our findings thus demonstrate that IL-11 principally stimulates megakaryocyte maturation rather than the proliferation of megakaryocytes, whereas TPO stimulates both.     

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.     

Induction of megakaryocyte differentiation by a novel pregnancy-specific hormone.

Maturation of megakaryocytes and subsequent platelet release are normally regulated by a network of cytokines, including thrombopoietin and various interleukins. Because abnormal platelet production and activation have been implicated in gestational pathologies, additional pregnancy-specific cytokines may play important roles in the regulation of megakaryocytopoiesis. Consistent with this hypothesis, we have found that the hormone prolactin-like protein E, a placental hormone that we have recently characterized, targets megakaryocytes through a specific cell surface receptor and induces megakaryocyte differentiation through a gp130-dependent signal transduction pathway.     

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.     

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.     

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.     

Wistar Furth Rat Megakaryocytes Lack Dense Compartments and Intercellular Plaques,Membranous Structures Rich in Cytoskeletal Proteins

Wistar Furth (WF) rats have an abnormal thrombopoietic phenotype with morphologically aberrant megakaryocytes, larger than normal mean platelet volume, and platelet alpha-granule protein deficiency. Here, ultrastructural comparisons of WF rat megakaryocytes to those of rats (Wistar) with normal platelet formation during stimulated megakaryocytopoiesis following 5-fluorouracil administration, have revealed a previously unrecognized membrane structure in normal rat megakaryocytes, and two additional abnormalities in WF megakaryocytes. The novel structures were zones of electron density on the cytoplasmic face of apposed plasma membranes of adjoining normal megakaryocytes. These modified focal adhesion-type contacts were distributed at intervals between adjacent megakaryocytes, and were spaced by deposits of extracellular material. These structures also were present between apposed plasma membranes of Wistar rat megakaryocytes in unperturbed marrows, but were absent between megakaryocytes of WF rats. The second WF rat megakaryocyte abnormality is the absence of cytoplasmic dense compartments, another specialized membranous structure that is continuous with the megakaryocyte demarcation membrane system. Both the intercellular plaques and dense compartments of Wistar rat megakaryocytes were found to be rich in cytoskeletal proteins including actin, α-actinin, talin, and vinculin as indicated by ultrastructural immunogold labeling. We hypothesize that an abnormality in cytoskeletal protein function may be responsible for the lack of these structures in the WF rat.     

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.     

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.     

A mathematical model of thrombopoiesis in rats

A mathematical model of thrombopoiesis in rats is presented. This has four compartments; stem cells, megakaryocytes, thrombocytes and thrombopoietin. A high thrombopoietin concentration influences bone marrow proliferation in three ways. Firstly the stem cells are stimulated and a slow increase in megakaryocyte number follows. Secondly there are additional endomitoses in the (early) megakaryocytes resulting in an increase in megakaryocyte volume. Thirdly the megakaryocyte maturation time is shortened. The parameters of the model are determined from experimental values for the normal, maximum and minimum proliferation rates, maturation times and destruction rates. The model is tested by comparing simulated results for acute and chronic thrombocytopenia and thrombocytosis with experimental curves from the literature. The model and data agree within the limits of experimental error. Not all of the thrombopoietic regulatory system is known yet, so some important alternative hypotheses are investigated and compared with the model. Several hypotheses have been excluded in this way.     

A MATHEMATICAL MODEL OF THROMBOPOIESIS IN RATS

A mathematical model of thrombopoiesis in rats is presented. This has four compartments; stem cells, megakaryocytes, thrombocytes and thrombopoietin. A high thrombopoietin concentration influences bone marrow proliferation in three ways. Firstly the stem cells are stimulated and a slow increase in megakaryocyte number follows. Secondly there are additional endomitoses in the (early) megakaryocytes resulting in an increase in megakaryocyte volume. Thirdly the megakaryocyte maturation time is shortened. The parameters of the model are determined from experimental values for the normal, maximum and minimum proliferation rates, maturation times and destruction rates. The model is tested by comparing simulated results for acute and chronic thrombocytopenia and thrombocytosis with experimental curves from the literature. The model and data agree within the limits of experimental error. Not all of the thrombopoietic regulatory system is known yet, so some important alternative hypotheses are investigated and compared with the model. Several hypotheses have been excluded in this way.     

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.     

Human cholinesterase genes localized by hybridization to chromosomes 3 and 16

Summary A cloned human cDNA for cholinesterase (ChE) was used as a probe for in situ hybridization to spread lymphocyte chromosomes to map the structural human CHE genes to distinct chromosomal regions. The recent genetic linkage assignment of the CHE1 locus of the CHE gene to chromosome 3q was confirmed and further refined to 3q21-q26, close to the genes coding for transferrin (TF) and transferrin receptor (TFRC). The CHE1 allele localizes to a 3q region that is commonly mutated and then associated with abnormal megakaryocyte proliferation in acute myelodysplastic anomalies. In view of earlier findings that ChE inhibitors induce megakaryocytopoiesis in culture, this localization may indicate that ChEs are involved in regulating the differentiation of megakaryocytes. A second site for ChEcDNA hybridization was found on chromosome 16q11-q23, demonstrating that the CHE2 locus of the cholinesterase gene, which directs the production of the common C5 variant of serum ChE, also codes for a structural subunit of the enzyme and is localized on the same chromosome with the haptoglobin (HP) gene, both genes being found on the long arm of chromosome 16. The finding of two sites for ChEcDNA hybridization suggests that the two loci coding for human ChEs may include nonidentical sequences responsible for the biochemical differences between ChE variants.     

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.     

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.     

Immature megakaryocytes in the mouse: In vitro relationship to megakaryocyte progenitor cells and mature megakaryocytes

An assay describing conditions for the maturation of single immature megakaryocytes in vitro is reported. Enriched populations of small, relatively immature megakaryocytes have been found to develop into single, mature megakaryocytes by 60 hours in semisolid agar cultures. Continued incubation of these cells did not lead to the formation of colonies within 5–7 days. Maturation was indicated by increasing cell size and cytoplasmic and acetylcholinesterase content. Factors stimulating the development of immature megakaryocytes were found in preparations of human embryonic kidney cell-conditioned media (a source of in vivo Thrombopoietic Stimulatory Factor), peritoneal exudate cell-conditioned medium, lung-conditioned medium, or bone marrow cellular sources of activity (adherent cells or cells that sediment at 5–6 mm hr^-1). Immature megakaryocytes cultured serum free responded to sources of an auxiliary megakaryocyte potentiating activity by developing into single, large megakaryocytes but did not respond to a megakaryocyte colony-stimulating factor devoid of detectable potentiator activity present in WEHl-3-conditioned medium. In contrast, serum-free proliferation of the megakaryocyte progenitor cell required both megakaryocyte colony-stimulating factor and the auxiliary potentiator activity. In the presence of megakaryocyte colony-stimulating factor alone, progenitor cells did not form colonies of easily detectable megakaryocytes. However, groups of cells comprised entirely of small acetylcholinesterase containing immature megakaryocytes were observed, thus establishing that megakaryocyte colony development passes through a stage of immature cells prior to detectable megakaryocyte development and that some acetylcholinesterase-containing cells can undergo cellular division.     

Glycosaminoglycans enhance megakaryocytopoiesis by modifying the activities of hematopoietic growth regulators

We have previously reported that heparin is capable of stimulating in vitro and in vivo megakaryocytopoiesis in mice and has a thrombopoietic effect when given in chronic immune thrombocytopenic purpura and that heparin and several other glycosaminoglycans (GAGs) promote the growth of human megakaryoblastic cell lines in the presence of serum. We show here that GAGs, including heparan sulfate (HS), chondroitin sulfate (CS), dermantan sulfate (DS), and hyaluronic acid (HA), also stimulate in vitro growth of murine megakaryocyte progenitors and augment the diameter of individual megakaryocytes in the presence of serum. However, in a serum-free agar system, the GAGs alone had no effect on megakaryocyte colony formation, suggesting that GAGs cooperate with some serum factor(s) to exert their activity. We also show that heparin significantly potentiates the megakaryocytopoietic activity of C-Mpl ligand and interleukin (IL)-6 but not IL3, GM-CSF, SCF, and Epo. In addition, the GAGs significantly neutralize the inhibitory action of platelet factor 4 (PF4) and transforming growth factor β1 (TGFβ1) on megakaryocyte colony growth. These results demonstrate a stimulating activity of GAGs on megakaryocytopoiesis by modifying the activity of several growth-regulating factors. © 1996 Wiley-Liss, Inc.