Fetal Megakaryocytes Research Articles

Effect of Thrombopoietin Receptor Agonist on Pregnant Mice.

Thrombopoietin receptor agonists (TPO-RAs) are an effective treatment for refractory immune thrombocytopenia (ITP). However, the use of TPO-RAs is limited for ITP in pregnant women due to concerns about fetal toxicity. In this study, we examined the effects of romiplostim, one of the TPO-RAs, on pregnant mice. The mice were injected subcutaneously with romiplostim (1, 5, 10, 30, and 100 μg/kg) on gestational days (GD) 1, 8, and 15. We evaluated maternal and fetal platelet and megakaryocyte counts (MK), fetal weight at birth, placental morphology, and miscarriage rates. Romiplostim increased platelet and MK counts in pregnant mice at all doses and in fetuses at doses above 10 µg/kg. Fetal weight at birth was slightly reduced at a dose of 100 μg/kg, but there were no significant differences in placental weight, spiral artery wall thickness, placental growth factor signal changes, or the rate of resorption at that dosage. The dose of romiplostim used clinically for ITP patients (1–10 μg/kg) did not show any adverse effects on pregnant mice. Although the results of the present study are encouraging, until there are more conclusive data, the use of romiplostim should be evaluated individually in severe, life-threatening cases, and all relevant ethical aspects should be considered.

Megakaryopoiesis and Platelet-Innate Immune Cell Interactions Are Developmentally Regulated

Megakaryocyte endoreplication, maturation and platelet production have all been reported to be affected by developmental stage (Elagib, Exp Hematol 2018). Additionally, we have recently found that P-selectin expression and function are altered in fetal and neonatal murine platelets (Stolla, Blood Adv 2019), suggesting that both megakaryopoiesis and platelet function are developmentally regulated. The developmental regulation of megakaryopoiesis can have important implications regarding platelet production in premature and full-term neonates, platelet regeneration following cord blood transplantation, and the in vitro generation of platelets from iPS cells (Elagib, Exp Hematol 2018). To further explore differences between fetal and adult megakaryocytes, we analyzed primary murine megakaryocytes in the fetal liver and adult bone marrow, and found that fetal megakaryocytes have lower average ploidy as well as increased cell size and initial accumulation of surface GP1bbeta expression at 2N and 4N stages. These latter findings indicate that cytoplasmic maturation begins at lower ploidy stages in fetal versus adult murine megakaryocytes. Consistent with findings in cultured human cord blood versus adult blood-derived progenitors (Liu, Blood 2015), we found lower ploidy and earlier cytoplasmic maturation in cultures of fetal versus adult megakaryocytes. These findings suggest the existence of a cell intrinsic developmental program of megakaryocyte maturation. Global gene expression studies of cultured megakaryocytes derived from fetal liver versus adult bone marrow progenitors revealed differential expression of Gata1, Cyclin D1, Cyclin D2, and Igf2bp3, supporting their proposed roles in fetal versus adult megakaryopoiesis (Klusmann, Genes Dev 2010; Liu, Blood 2011; Elagib, J Clin Invest 2017). Igf1R transcripts were increased in fetal megakaryocytes consistent with their sensitivity to IGF signaling (Klusmann, Genes Dev 2010). In addition, we found increased expression of Lin28b, a negative regulator of Let7 miRNAs, as well as multiple downstream targets of Let7 miRNAs in fetal liver-derived megakaryocytes. Induction of Lin28b expression in adult mice (Zhu, Cell 2011) led to increased expression of Gata1, Cyclin D1, Igf2bp3 and IGF1R in bone marrow-derived megakaryocytes. Interestingly, adult megakaryocyte average ploidy was decreased and the sensitivity to IGF1R inhibition was increased by Lin28b induction, suggesting that the Lin28b axis regulates key aspects of fetal versus adult megakaryopoiesis. The largest category of upregulated genes in adult versus fetal megakaryocyte transcriptomes was immune-related genes, including PPBP, a neutrophil chemotactic cytokine, and beta2M, which we have recently shown to mediate platelet-monocyte interactions (Hilt, JCI Insight 2019). Consistent with the transcriptomic data, neonatal platelets express less MHCI on their surface (an indirect indication of less platelet beta2M protein expression) and release less PPBP post-activation when compared to adult platelets. Activated neonatal platelets induce less neutrophil migration and activation when compared to activated adult platelets. In addition, the releasates from activated neonatal versus adult platelets differentially altered the balance of proinflammatory versus proreparative signaling in monocytes. Finally, the induction of Lin28b expression in adult mice resulted in platelets that express less MHCI, release less PPBP, and induce less neutrophil chemotaxis. Taken together, our studies support the hypothesis that megakaryocyte maturation and resulting platelet-immune cell interactions are developmentally regulated, in part, through a Lin28b-mediated program. Disclosures Palis: Rubius Therapeutics: Consultancy.

GATA-2 Reinforces Megakaryocyte Development in the Absence of GATA-1

GATA-2 is an essential transcription factor that regulates multiple aspects of hematopoiesis. Dysregulation of GATA-2 is a hallmark of acute megakaryoblastic leukemia in children with Down syndrome, a malignancy that is defined by the combination of trisomy 21 and a GATA1 mutation. Here, we show that GATA-2 is required for normal megakaryocyte development as well as aberrant megakaryopoiesis in Gata1 mutant cells. Furthermore, we demonstrate that GATA-2 indirectly controls cell cycle progression in GATA-1-deficient megakaryocytes. Genome-wide microarray analysis and chromatin immunoprecipitation studies revealed that GATA-2 regulates a wide set of genes, including cell cycle regulators and megakaryocyte-specific genes. Surprisingly, GATA-2 also negatively regulates the expression of crucial myeloid transcription factors, such as Sfpi1 and Cebpa. In the absence of GATA-1, GATA-2 prevents induction of a latent myeloid gene expression program. Thus, GATA-2 contributes to cell cycle progression and the maintenance of megakaryocyte identity of GATA-1-deficient cells, including GATA-1s-expressing fetal megakaryocyte progenitors. Moreover, our data reveal that overexpression of GATA-2 facilitates aberrant megakaryopoiesis.

Negative Paracrine Effect of Platelet Factor 4 on Megakaryopoiesis Occurs through Lipoprotein Related Protein Receptor-1 on Megakaryocytes.

Platelet Factor 4 (PF4) is a paracrine inhibitor of megakaryopoiesis in vivo, with platelet counts inversely related to platelet PF4 levels in mice. Duration of thrombocytopenia after chemotherapy-induced thrombocytopenia (CIT) was directly related to platelet PF4 levels. Defining the molecular basis by which PF4 has this effect may have clinical implications in patients with CIT. Based on a recent observation that PF4 could activate endothelial cells through the Lipoprotein Related Protein Receptor-1 (LRP-1), we investigated whether a similar receptor might be involved in the negative paracrine effect of PF4 on megakaryopoiesis. We found that the addition of receptor-associated protein (RAP), a general inhibitor of members of the LRP family of receptors by binding to the alpha subunit, could block the inhibitory affect on in vitro megakaryocyte (MEG) colony number per plated marrow cells in a specific and dose-dependent fashion. Thus, at 0.2 μM RAP could prevent ∼50% of the inhibitory affect of the addition of 20 μg/mL of recombinant PF4 and at 0.4 μM RAP, the inhibitory effect of the added PF4 was completely abolished (p = 0.01 of no RAP vs. 0.4 μM RAP). Since a number of LRP receptors are inhibited by RAP, we repeated these studies using a specific anti-LRP-1 monoclonal antibody that blocks LRP-1 activation by binding to its alpha subunit (American Diagnostic). We have previously shown that transgenic mice overexpressing human PF4 by 6-fold have a 50% decrease in MEG-containing colonies in the above colony assay relative to WT littermates due to increase release of PF4 from developing megakaryocytes. We now found that the number of colonies derived from these high PF4 marrows increased to that seen in WT littermates when 25 μg/mL of the anti-LRP-1 antibody was included (p = 0.01). Since there are no prior studies showing that LRP-1 is expressed on human and murine bone marrow and murine fetal liver-derived MEGs, we performed RT-PCR on the appropriate RNAs as well as flow cytometry for the presence of LRP-1. RT-PCR of MEG RNA shows that LRP-1 message is present in MEGS. By flow cytometry, LRP-1 is clearly present on both human and murine MEGs. These studies support that PF4 inhibits megakaryopoiesis through LRP-1 on developing megakaryocytes. Studies were also done on the megakaryocyte-erythroid progenitor cell line G1ME. These cells do not express LRP-1, but when stimulated to differentiate, LRP-1 is detectable by flow cytometry on maturing megakaryocytes, suggesting that PF4 exerts its negative paracrine effect on developing MEGS and not on earlier progenitor cells. Consistent with this finding, we observed that in mice with different levels of platelet PF4, MEG content in the marrow is inversely related to platelet PF4 levels, but the size and ploidy are not correlated to PF4 content. Thus, we believe that we have defined the receptor system involved as well as the target cell of the observed inhibitory effect of PF4 on megakaryopoiesis. Such studies on the mechanism by which PF4 exerts this negative paracrine effect should lead to a more complete understanding of the regulation of megakaryopoiesis and may offer novel strategies to intervene in clinical thrombocytopenia, especially CIT.

GATA1-Mediated Megakaryocyte Differentiation and Growth Control Can Be Uncoupled and Mapped to Different Domains in GATA1.

The zinc finger transcription factor GATA1 plays a critical non-redundant role in megakaryopoiesis in mice and human patients with both germline and acquired GATA1 mutations. Germline deletion of the mouse regulatory element, HSI (ΔneoΔHS mice), specifically reduces megakaryocyte GATA1 expression to 5% of wild-type levels. ΔneoΔHS mice are viable but have defective megakaryocyte maturation and growth control. Mutant megakaryocytes fail to shed proplatelets in vitro and the platelet count is ~5 fold lower in mutant mice. Moreover, mutant megakaryocyte precursors hyperproliferate in vivo and in vitro.Here, we show there is a specific ~10-fold increase in abnormal CD41+ FcγRlowc-kit+CD9+ megakaryocyte-erythroid progenitor cells in fetal liver. These progenitors form abnormally large colonies in vitro and are unable to form proplatelets. In contrast, earlier myeloid progenitors (the common myeloid progenitor CMP) were present at expected frequencies and differentiated to all non-megakaryocyte myeloid lineages normally. Consistent with this GATA1 expression was selectively reduced in the abnormal progenitors and immature megakaryocyte precursors.We then used these GATA1-deficient primary megakaryocyte progenitors as a cellular substrate to define the amino acid domains within GATA1 required to facilitate proplatelet release and retard immature megakaryocyte growth. Previous data suggested that the bi-functional GATA1 zinc fingers could bind both DNA and interact with protein partners. The C-terminal zinc finger is essential for binding to all GATA sites whereas the N-terminal finger helps binding to a subset of GATA sites and mediates interaction with the critical GATA1 cofactor, FOG-1. Finally, the N-terminal 84 amino acids of GATA1 are important for megakaryopoiesis. Neonates and children with Down Syndrome (DS) can develop megakaryocyte leukaemia acquire mutations in GATA1 exon 2 in fetal life that result in the production of a short form of GATA1 protein (GATA1s) lacking the N-terminal 84 amino acids. In this study, we have systematically dissected GATA1 domains required for platelet release and control of megakaryocyte growth by using retroviral-mediated gene transfer to ectopically express modified GATA1 molecules in primary GATA1-deficient fetal megakaryocyte CD41+FcγRlowc-kit+CD9+progenitors. In addition to the GATA1-FOG-1 interaction and C-terminal zinc finger, two domains within the first N-terminal 110 amino acids are required for platelet release. To restrict megakaryocyte growth, two domains are required; the C-terminal zinc finger and residues in the N-terminus between amino acids 54–110. Importantly, interaction with FOG-l is not required to restrain megakaryocyte growth. Furthermore, we showed that chicken GATA1 was able to rescue both, platelet release and growth control. As only a few residues are conserved in the N-terminus between mouse and chicken GATA1, this potentially pinpoints critical functional residues in the N-terminus. In summary, distinct GATA1 domains regulate terminal megakaryocyte gene expression leading to platelet release and restrain megakaryocyte growth. Analysis of distinct mutations in the N-terminus and a modified version of GATA1 that is crippled in its interaction with FOG-1 shows platelet production and megakaryocyte growth can be uncoupled. Finally, these results have implications for how GATA1s may function in megakaryocytic leukemia in DS.

GATA1-mediated megakaryocyte differentiation and growth control can be uncoupled and mapped to different domains in GATA1.

The DNA-binding hemopoietic zinc finger transcription factor GATA1 promotes terminal megakaryocyte differentiation and restrains abnormal immature megakaryocyte expansion. How GATA1 coordinates these fundamental processes is unclear. Previous studies of synthetic and naturally occurring mutant GATA1 molecules demonstrate that DNA-binding and interaction with the essential GATA1 cofactor FOG-1 (via the N-terminal finger) are required for gene expression in terminally differentiating megakaryocytes and for platelet production. Moreover, acquired mutations deleting the N-terminal 84 amino acids are specifically detected in megakaryocytic leukemia in human Down syndrome patients. In this study, we have systematically dissected GATA1 domains required for platelet release and control of megakaryocyte growth by ectopically expressing modified GATA1 molecules in primary GATA1-deficient fetal megakaryocyte progenitors. In addition to DNA binding, distinct N-terminal regions, including residues in the first 84 amino acids, promote platelet release and restrict megakaryocyte growth. In contrast, abrogation of GATA1-FOG-1 interaction leads to loss of differentiation, but growth of blocked immature megakaryocytes is controlled. Thus, distinct GATA1 domains regulate terminal megakaryocyte gene expression leading to platelet release and restrain megakaryocyte growth, and these processes can be uncoupled.

CD226 is expressed on the megakaryocytic lineage from hematopoietic stem cells/progenitor cells and involved in its polyploidization

In this study, we examined the expression of CD226 on megakaryocytic, granulocytic and erythroid lineage from hematopoietic stem cells/progenitor cells in adult and fetus and its potential role in megakaryocytic maturation. CD34(+) cells from adult and fetus were induced to differentiate toward the megakaryocytic lineage by thrombopoietin (TPO) and the granulocytic lineage by granulocyte colony-stimulating factor (G-CSF), respectively. Mononuclear cells from fetal liver and CD34(+) cells from adult were induced to differentiate toward erythroid-lineage by erythropoiesis (EPO). We investigated the expression of CD226 and lymphocyte function associated antigen-1 (LFA-1) (CD11a) during hemopoiesis. We also studied the effect of CD226 monoclonal antibody (MoAb) and LFA-1 MoAb on megakaryocyte with antibody cross-liking technique. CD34(+) cells from adult and fetus and TPO-induced CD41(+) cells all expressed CD226 molecule. CD226 was not expressed on erythroid progenitor cells and erythroblasts and most cells of granulocytic lineage although G-CSF induced a significant increase of the expression of CD226 on CD34(+) cells in early period of time. CD226 MoAb acts on megakaryocytes by inducing intracellular calcium mobilization. The expression of LFA-1 decreased significantly at late stage of differentiation and maturation of fetal megakaryocytes whereas the expression of LFA-1 on adult megakaryocytes retained at a high level. CD226 MoAb in combination with LFA-1 MoAb shifted the ploidy of generated megakaryocytes from adult-derived CD34(+) cells to higher classes significantly although CD226 and LFA-1 MoAb slightly increased the ploidy of the generated megakaryocytes individually. CD226 MoAb or LFA-1 MoAb or CD226 MoAb plus LFA-1 MoAbs did not increase the ploidy of the generated megakaryocytes from fetus-derived CD34(+) cells. CD226 molecules play an important role in maturation of the megakaryocytes in combination with LFA-1.

Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin-induced differentiation.

Cord blood (CB) has successfully been used as a stem cell source for haemopoietic reconstitution. However, a significant delay in platelet engraftment is consistently found in CB versus adult peripheral blood (PB) or bone marrow transplants. We sought to determine whether or not CB megakaryocytes have reached terminal maturation and, hence, full thrombopoietic potential. A comparative analysis of megakaryocytes cultured from either CB or PB progenitors in the presence of thrombopoietin (TPO) showed a similar differentiation response, although proliferation was 2.4 times higher in CB than in PB cells. Importantly, the TPO-induced ploidy level was notably different: whereas 82.7% of CB megakaryocytes remained diploid (2N) at the end of the culture, more than 50% of PB megakaryocytes had reached a DNA content equal to or higher than 4N. Western blot and flow cytometry analyses revealed that only polyploid PB megakaryocytes expressed cyclins E, A and B, whereas cyclin D3 was detected in both fetal and adult megakaryocytic nuclei. These data suggest that establishment of endomitotic cycles is impaired in CB megakaryocytes, associated with a differential regulation of G1/S cell cycle factors. We believe that the relative immaturity of fetal megakaryocytes could be a contributing factor to the delayed platelet engraftment in cord blood transplantation.

Analysis of Megakaryocyte Ploidy in Fetal Bone Marrow Biopsies Using a New Adaptation of the Feulgen Technique to Measure DNA Content and Estimate Megakaryocyte Ploidy from Biopsy Specimens

Platelet counts in newborns are similar to those of adults and children. However, newborn infants admitted to intensive care nurseries have a high prevalence of thrombocytopenia. The mechanisms responsible for the increased susceptibility to thrombocytopenia are not known. In addition, some studies have documented functional abnormalities in newborn platelets. In an effort to understand differences between platelets in newborns and in adults, we examined megakaryocyte ploidy in bone marrow from fetuses and compared it with bone marrow from adults, using a modified Feulgen stain to measure DNA of individual megakaryocytes. Faced with small fixed tissue samples, we developed a technique for use on bone marrow biopsies to estimate megakaryocyte ploidy and compared the results obtained with this method to those obtained from bone marrow aspirates. This study demonstrated that the overall mean ploidy of fetal megakaryocytes is decreased compared with adults. Additionally, fetal megakaryocyte ploidy increases as megakaryocyte maturation increases, but not to the same extent that adult megakaryocyte ploidy increases with megakaryocyte maturation. Over the gestational period studied, there was no relationship between gestational age and mean ploidy. The small size, shift to a less mature population, and decreased ploidy of fetal megakaryocytes indicate that there are differences in the post mitotic phase of megakaryocyte development in the fetus. Such differences may be related to quantitative and qualitative platelet abnormalities in the newborn. Understanding the physiology and regulation of megakaryocytopoiesis in the fetus and newborn will be valuable in determining the pathophysiologic basis of platelet dysfunction in the newborn.

In vitro growth of human fetal CD34+ cells in the presence of various combinations of recombinant cytokines under serum-free culture conditions.

CD34+ cells were purified from midtrimester human fetal blood and adult bone marrow samples and seeded in serum-free fibrin-clot cultures in order to evaluate the number and the responsiveness to recombinant cytokines of pluripotent (CFU-GEMM), erythroid (BFU-E), megakaryocyte (BFU-meg and CFU-meg) and granulocyte/macrophage (CFU-GM) haemopoietic progenitor cells. The number of the different haemopoietic progenitors/1 x 10(3) CD34+ cells, except CFU-meg, was significantly higher in fetal blood than in adult bone marrow in cultures stimulated by any combination of cytokines including interleukin-3 (IL-3), granulocyte/macrophage colony stimulating factor (GM-CSF) or stem cell factor (SCF) plus erythropoietin (Epo). Nevertheless, whereas adult BFU-E showed a maximal growth in the presence of Epo plus IL-3 or Epo plus SCF, fetal BFU-E showed an optimal growth in the presence of Epo alone, the sensitivity of fetal BFU-E to suboptimal concentrations of Epo being approximately 10-15-fold higher than that of adult BFU-E. Addition of optimal concentrations of IL-3, GM-CSF or SCF, alone or in various combinations, to Epo-containing cultures induced a significant increase in both the number and size of fetal CFU-GEMM, and CFU-GM, and a parallel decrease of fetal BFU-E. Finally, SCF potently synergized with IL-3 in increasing the growth of both classes of fetal megakaryocyte progenitors, BFU-meg and CFU-meg.

Presence and characteristics of circulating megakaryocyte progenitor cells in human fetal blood

The in vitro growth of early (burst-forming unit-megakaryocyte [BFU- meg]) and late (colony-forming unit-megakaryocyte [CFU-meg]) megakaryocyte progenitors was investigated in midtrimester human fetal blood and compared with adult bone marrow. Most of the experiments were performed in a serum-free fibrin-clot assay, using purified hematopoietic progenitor (CD34+) cells. High BFU-meg and CFU-meg levels were found in human fetal blood, with a clear prevalence of BFU-meg (BFU-meg:CFU-meg ratio, 2.5:1), at variance with adult bone marrow, in which mature CFU-meg predominate (BFU-meg:CFU-meg ratio, 0.6:1). Fetal and adult megakaryocyte progenitors had a similar phenotypic profile for the expression of CD34, HLA-DR, and glycoprotein-complex IIB-IIIA. However, fetal BFU-meg were larger in size (number of megakaryocytic elements per colony) than adult BFU-meg, but were usually composed by only one or two foci of development. On the other hand, fetal and adult CFU-meg were similar in both morphology and size. Fetal megakaryocyte progenitors appeared earlier in culture and had an increased proliferative activity as demonstrated by the higher number of megakaryocyte progenitors in S phase with respect to adult CFU-meg and BFU-meg. Finally, fetal megakaryocyte progenitors displayed a higher sensitivity to stimulatory cytokines, in particular recombinant interleukin-3, than adult megakaryocyte progenitors, whereas they were inhibited by purified transforming growth factor-beta 1 in a similar fashion to adult megakaryocyte progenitors.

Presence and Characteristics of Circulating Megakaryocyte Progenitor Cells in Human Fetal Blood

AbstractThe in vitro growth of early (burst-forming unit-megakaryocyte [BFU-meg]) and late (colony-forming unit-megakaryocyte [CFU-meg]) megakaryocyte progenitors was investigated in midtrimester human fetal blood and compared with adult bone marrow. Most of the experiments were performed in a serum-free fibrinclot assay, using purified hematopoietic progenitor (CD34+) cells. High BFU-meg and CFU-meg levels were found in human fetal blood, with a clear prevalence of BFU-meg (BFU-meg:CFU-meg ratio, 2.5:1), at variance with adult bone marrow, in which mature CFU-meg predominate (BFU-meg:CFU-meg ratio, 0.6:1). Fetal and adult megakaryocyte progenitors had a similar phenotypic profile for the expression of CD34, HLA-DR, and glycoprotein-complex IIB-IIIA. However, fetal BFU-meg were larger in size (number of megakaryocytic elements per colony) than adult BFU-meg, but were usually composed by only one or two foci of development. On the other hand, fetal and adult CFU-meg were similar in both morphology and size. Fetal megakaryocyte progenitors appeared earlier in culture and had an increased proliferative activity as demonstrated by the higher number of megakaryocyte progenitors in S phase with respect to adult CFU-meg and BFU-meg. Finally, fetal megakaryocyte progenitors displayed a higher sensitivity to stimulatory cytokines, in particular recombinant interleukin-3, than adult megakaryocyte progenitors, whereas they were inhibited by purified transforming growth factor-β1 in a similar fashion to adult megakaryocyte progenitors.