The Expression of Cell Cycle Cyclins in a Human Megakaryoblast Cell Line Exposed to Simulated Microgravity.

The study of the physiological and pathophysiological processes under extreme conditions facilitates a better understanding of the state of a healthy organism and can also shed light on the pathogenesis of diseases. In recent years, it has become evident that gravitational stress affects both the whole organism and individual cells. We have previously demonstrated that simulated microgravity inhibits proliferation, induces apoptosis, changes morphology, and alters the surface marker expression of megakaryoblast cell line MEG-01. In the present work, we investigate the expression of cell cycle cyclins in MEG-01 cells. We performed several experiments for 24 h, 72 h, 96 h and 168 h. Flow cytometry and Western blot analysis demonstrated that the main change in the levels of cyclins expression occurs under conditions of simulated microgravity after 96 h. Thus, the level of cyclin A expression showed an increase in the RPM group during the first 4 days, followed by a decrease, which, together with the peak of cyclin D, may indicate inhibition of the cell cycle in the G2 phase, before mitosis. In addition, based on the data obtained by PCR analysis, we were also able to see that both cyclin A and cyclin B expression showed a peak at 72 h, followed by a gradual decrease at 96 h. STED microscopy data also confirmed that the main change in cyclin expression of MEG-01 cells occurs at 96 h, under simulated microgravity conditions, compared to static control. These results suggested that the cell cycle disruption induced by RPM-simulated microgravity in MEG-01 cells may be associated with the altered expression of the main regulators of the cell cycle. Thus, these data implicate the development of cellular stress in MEG-01 cells, which may be important for proliferating human cells exposed to microgravity in real space.

An age-progressive platelet differentiation path from hematopoietic stem cells causes exacerbated thrombosis.

Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis.

Polyamine release and vesicular polyamine transporter expression in megakaryoblastic cells and platelets.

Polyamines not only play essential roles in cell growth and function of living organisms but are also released into the extracellular space and function as regulators of chemical transduction, although the cells from which they are released and their mode of release are not well understood. The vesicular polyamine transporter (VPAT), encoded by the SLC18B1 is responsible for the vesicular storage of spermine and spermidine, followed by their vesicular release from secretory cells. Focusing on VPAT will help identify polyamine-secreting cells and new polyamine functions. In this study, we investigated the possible involvement of VPAT in vesicular release of polyamines in MEG-01 clonal megakaryoblastic cells and platelets. RT-PCR, western blotting, and immunohistochemistry revealed VPAT expression in MEG-01 cells. MEG-01 cells secreted polyamines upon A23187 stimulation in the presence of Ca2+, which is temperature-dependent and sensitive to bafilomycin A1. A23187-induced polyamine secretion from MEG-01 cells was reduced by treatment with reserpine, VPAT inhibitors, or VPAT RNA interference. Platelets also expressed VPAT, displaying a punctate distribution, and released spermidine upon A23187 and thrombin stimulation. These findings have demonstrated VPAT-mediated vesicular polyamine release from MEG-01 cells, suggesting the presence of similar vesicular polyamine release mechanisms in platelets.

Efficient megakaryopoiesis and platelet production require phospholipid remodeling and PUFA uptake through CD36.

Lipids contribute to hematopoiesis and membrane properties and dynamics; however, little is known about the role of lipids in megakaryopoiesis. Here we show that megakaryocyte progenitors, megakaryocytes and platelets present a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake as well as de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in low platelets. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production and that changes in dietary fatty acids may be a viable target to modulate platelet counts.

Silencing of p53 and CDKN1A establishes sustainable immortalized megakaryocyte progenitor cells from human iPSCs.

Platelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology.

The in vitro effects of hepatoblastoma cells on the growth and differentiation of blasts in transient abnormal myelopoiesis associated with Down syndrome.

Transient abnormal myelopoiesis (TAM) in neonates with Down syndrome, which spontaneously resolves within several weeks or months after birth, may represent a special form of leukemia developing in the fetal liver (FL). To explore the role of hepatoblasts, one of the major constituents of the FL hematopoietic microenvironment, in the pathogenesis of TAM, we investigated the influence of a human hepatoblastoma cell line, HUH-6, on the in vitro growth and differentiation of TAM blasts. In a coculture system with membrane filters, which hinders cell-to-cell contact between TAM blasts and HUH-6 cells, the growth and megakaryocytic differentiation of TAM blast progenitors were increased in the presence of HUH-6 cells. The culture supernatant of HUH-6 cells contained hematopoietic growth factors, including stem cell factor (SCF) and thrombopoietin (TPO). The neutralizing antibody against SCF abrogated the growth-stimulating activity of the culture supernatant of HUH-6 cells, demonstrating that, among the growth factors produced by HUH-6 cells, SCF may be the major growth stimulator and that TPO may be involved in megakaryocytic differentiation, rather than growth, of TAM blasts. This suggests that hepatoblasts function in the regulation of the growth and differentiation of TAM blasts in the FL through the production of hematopoietic growth factors, including SCF and TPO, and are involved in the leukemogenesis of TAM.

Yolk sac, but not hematopoietic stem cell-derived progenitors, sustain erythropoiesis throughout murine embryonic life.

In the embryo, the first hematopoietic cells derive from the yolk sac and are thought to be rapidly replaced by the progeny of hematopoietic stem cells. We used three lineage-tracing mouse models to show that, contrary to what was previously assumed, hematopoietic stem cells do not contribute significantly to erythrocyte production up until birth. Lineage tracing of yolk sac erythromyeloid progenitors, which generate tissue resident macrophages, identified highly proliferative erythroid progenitors that rapidly differentiate after intra-embryonic injection, persisting as the major contributors to the embryonic erythroid compartment. We show that erythrocyte progenitors of yolk sac origin require 10-fold lower concentrations of erythropoietin than their hematopoietic stem cell-derived counterparts for efficient erythrocyte production. We propose that, in a low erythropoietin environment in the fetal liver, yolk sac-derived erythrocyte progenitors efficiently outcompete hematopoietic stem cell progeny, which fails to generate megakaryocyte and erythrocyte progenitors.

Engineered PD-L1-Expressing Platelets Reverse New-Onset Type 1 Diabetes.

The pathogenesis of Type 1 diabetes (T1D) arises from the destruction of insulin-producing ß-cells by islet-specific autoreactive T cells. Inhibition of islet-specific autoreactive T cells to rescue ß-cells is a promising approach to treat new-onset T1D. The immune checkpoint signal axis programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) can effectively regulate the activity of T cells and prevent autoimmune attack. Here, megakaryocyte progenitor cells are genetically engineered to overexpress PD-L1 to produce immunosuppressive platelets. The PD-L1-overexpressing platelets (designated PD-L1 platelets) accumulate in the inflamed pancreas and may suppress the activity of pancreas autoreactive T cells in newly hyperglycemic non-obese diabetic (NOD) mice, protecting the insulin-producing ß-cells from destruction. Moreover, PD-L1 platelet treatment also increases the percentage of the regulatory T cells (Tregs) and maintains immune tolerance in the pancreas. It is demonstrated that the rescue of ß-cells by PD-L1 platelets can effectively maintain normoglycemia and reverse diabetes in newly hyperglycemic NOD mice.

Establishment of a megakaryoblastic cell line for conventional assessment of platelet calcium signaling.

Platelet function tests utilizing agonists or patient serum are generally performed to assess platelet activation ex vivo. However, inter-individual differences in platelet reactivity and donor requirements make it difficult to standardize these tests. Here, we established a megakaryoblastic cell line for the conventional assessment of platelet activation. We first compared intracellular signaling pathways using CD32 crosslinking in several megakaryoblastic cell lines, including CMK, UT-7/TPO, and MEG-01 cells. We confirmed that CD32 was abundantly expressed on the cell surface, and that intracellular calcium mobilization and tyrosine phosphorylation occurred after CD32 crosslinking. We next employed GCaMP6s, a highly sensitive calcium indicator, to facilitate the detection of calcium mobilization by transducing CMK and MEG-01 cells with a plasmid harboring GCaMP6s under the control of the human elongation factor-1α promoter. Cells that stably expressed GCaMP6s emitted enhanced green fluorescent protein fluorescence in response to intracellular calcium mobilization following agonist stimulation in the absence of pretreatment. In summary, we have established megakaryoblastic cell lines that mimic platelets by mobilizing intracellular calcium in response to several agonists. These cell lines can potentially be utilized in high-throughput screening assays for the discovery of new antiplatelet drugs or diagnosis of disorders caused by platelet-activating substances.

Regulation of bone metabolism by megakaryocytes in a paracrine manner.

Megakaryocytes (MKs) play key roles in regulating bone metabolism. To test the roles of MK-secreted factors, we investigated whether MK and promegakaryocyte (pro-MK) conditioned media (CM) may affect bone formation and resorption. K562 cell lines were differentiated into mature MKs. Mouse bone marrow macrophages were differentiated into mature osteoclasts, and MC3T3-E1 cells were used for osteoblastic experiments. Bone formation was determined by a calvaria bone formation assay in vivo. Micro-CT analyses were performed in the femurs of ovariectomized female C57B/L6 and Balb/c nude mice after intravenous injections of MK or pro-MK CM. MK CM significantly reduced in vitro bone resorption, largely due to suppressed osteoclastic resorption activity. Compared with pro-MK CM, MK CM suppressed osteoblastic differentiation, but stimulated its proliferation, resulting in stimulation of calvaria bone formation. In ovariectomized mice, treatment with MK CM for 4 weeks significantly increased trabecular bone mass parameters, such as bone volume fraction and trabecular thickness, in nude mice, but not in C57B/L6 mice. In conclusion, MKs may secrete anti-resorptive and anabolic factors that affect bone tissue, providing a novel insight linking MKs and bone cells in a paracrine manner. New therapeutic agents against metabolic bone diseases may be developed from MK-secreted factors.

Prolonged inhibition and incomplete recovery of mitochondrial function in oxazolidinone-treated megakaryoblastic cell lines.

Thrombocytopenia is commonly seen in patients receiving linezolid for >14 days. Linezolid is a reversible inhibitor of mitochondrial function in various cell types. This study investigated the inhibitory effects of linezolid and tedizolid, and their potential recovery on (i) CYTox I expression (subunit I of cytochrome c-oxidase; encoded by the mitochondrial genome), (ii) cytochrome c-oxidase activity and (iii) mitochondrial respiration (Seahorse bioanalysis) in two megakaryocytic cell lines [UT-7 WT (human acute megakaryoblastic leukaemia cells) and UT-7 MPL (transduced to express the thrombopoietin receptor)]. Cells were exposed to linezolid (0.5-25 mg/L) or tedizolid (0.1-5 mg/L) for up to 5 days and recovery followed after drug removal. Both oxazolidinones caused concentration- and time-dependent inhibition of CYTox I expression, cytochrome c-oxidase activity and mitochondrial spare capacity. On electron microscopy, mitochondria appeared dilated with a loss of cristae. Globally, tedizolid exerted stronger effects than linezolid. While CYTox I expression recovered completely after 6 days of drug washout, only partial (linezolid) or no (tedizolid) recovery of cytochrome c-oxidase activity, and no rescue of mitochondrial spare capacity (after 3 days) was observed. Thus, and in contrast to previous studies using a variety of cell lines unrelated to megakaryocytic lineages, the inhibitory effects exerted by oxazolidinones on the mitochondrial function of megakaryoblastic cells appear to be particularly protracted. Given the dynamics of platelet production and destruction, these results may explain why oxazolidinone-induced thrombocytopenia is one of the most common side effects in patients exposed to these antibiotics.

Low iron promotes megakaryocytic commitment of megakaryocytic-erythroid progenitors in humans and mice.

The mechanisms underlying thrombocytosis in patients with iron deficiency anemia remain unknown. Here, we present findings that support the hypothesis that low iron biases the commitment of megakaryocytic (Mk)-erythroid progenitors (MEPs) toward the Mk lineage in both human and mouse. In MEPs of transmembrane serine protease 6 knockout (Tmprss6-/-) mice, which exhibit iron deficiency anemia and thrombocytosis, we observed a Mk bias, decreased labile iron, and decreased proliferation relative to wild-type (WT) MEPs. Bone marrow transplantation assays suggest that systemic iron deficiency, rather than a local role for Tmprss6-/- in hematopoietic cells, contributes to the MEP lineage commitment bias observed in Tmprss6-/- mice. Nontransgenic mice with acquired iron deficiency anemia also show thrombocytosis and Mk-biased MEPs. Gene expression analysis reveals that messenger RNAs encoding genes involved in metabolic, vascular endothelial growth factor, and extracellular signal-regulated kinase (ERK) pathways are enriched in Tmprss6-/- vs WT MEPs. Corroborating our findings from the murine models of iron deficiency anemia, primary human MEPs exhibit decreased proliferation and Mk-biased commitment after knockdown of transferrin receptor 2, a putative iron sensor. Signal transduction analyses reveal that both human and murine MEP have lower levels of phospho-ERK1/2 in iron-deficient conditions compared with controls. These data are consistent with a model in which low iron in the marrow environment affects MEP metabolism, attenuates ERK signaling, slows proliferation, and biases MEPs toward Mk lineage commitment.

The GABA receptor GABRR1 is expressed on and functional in hematopoietic stem cells and megakaryocyte progenitors.

GABRR1 is a rho subunit receptor of GABA, the major inhibitory neurotransmitter in the mammalian brain. While most investigations of its function focused on the nervous system, its regulatory role in hematopoiesis has not been reported. In this study, we found GABRR1 is mainly expressed on subsets of human and mouse hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MkPs). GABRR1-negative (GR-) HSCs led to higher donor-derived hematopoietic chimerism than GABRR1-positive (GR+) HSCs. GR+ but not GR- HSCs and MkPs respond to GABA in patch clamp studies. Inhibition of GABRR1 via genetic knockout or antagonists inhibited MkP differentiation and reduced platelet numbers in blood. Overexpression of GABRR1 or treatment with agonists significantly promoted MkP generation and megakaryocyte colonies. Thus, this study identifies a link between the neural and hematopoietic systems and opens up the possibility of manipulating GABA signaling for platelet-required clinical applications.

Enabling Large-Scale Ex Vivo Production of Megakaryocytes from CD34+ Cells Using Gas-Permeable Surfaces.

Patients suffering from acute or sustained thrombocytopenia require platelet transfusions, which are entirely donor-based and limited by challenges related to storage and fluctuating supply. Developing cell-culture technologies will enable ex vivo and donor-independent platelet production. However, critical advancements are needed to improve scalability and increase megakaryocyte (Mk) culture productivity. To address these needs, we evaluated Mk production from mobilized peripheral blood CD34+ cells cultured on a commercially available gas-permeable silicone rubber membrane, which provides efficient gas exchange, and investigated the use of fed-batch media dilution schemes. Starting with a cell-surface density of 40 × 103 CD34+ cells per cm2 (G40D), culturing cells on the membrane for the first 5 days and employing media dilutions yielded 39 ± 19 CD41+ CD42b+ Mks per input CD34+ cell by day 11-a 2.2-fold increase compared with using standard culture surfaces and full media exchanges. By day 7, G40D conditions generated 1.5-fold more CD34+ cells and nearly doubled the numbers of Mk progenitors. The increased number of Mk progenitors coupled with media dilutions, potentially due to the retention of interleukin (IL)-3, increased Mk production in G40D. Compared with controls, G40D had higher viability, yielded threefold more Mks per milliliter of media used and exhibited lower mean ploidy, but had higher numbers of high-ploidy Mks. Finally, G40D-Mks produced proplatelets and platelet-like-particles that activate and aggregate upon stimulation. These results highlight distinct improvements in Mk cell-culture and demonstrate how new technologies and techniques are needed to enable clinically relevant production of Mks for platelet generation and cell-based therapies.

CD45 expression discriminates waves of embryonic megakaryocytes in the mouse.

Embryonic megakaryopoiesis starts in the yolk sac on gestational day 7.5 as part of the primitive wave of hematopoiesis, and it continues in the fetal liver when this organ is colonized by hematopoietic progenitors between day 9.5 and 10.5, as the definitive hematopoiesis wave. We characterized the precise phenotype of embryo megakaryocytes in the liver at gestational day 11.5, identifying them as CD41++CD45-CD9++CD61+MPL+CD42c+ tetraploid cells that express megakaryocyte-specific transcripts and display differential traits when compared to those present in the yolk sac at the same age. In contrast to megakaryocytes from adult bone marrow, embryo megakaryocytes are CD45- until day 13.5 of gestation, as are both the megakaryocyte progenitors and megakaryocyte/erythroid-committed progenitors. At gestational day 11.5, liver and yolk sac also contain CD41+CD45+ and CD41+CD45- cells. These populations, and that of CD41++CD45-CD42c+ cells, isolated from liver, differentiate in culture into CD41++CD45-CD42c+ proplatelet-bearing megakaryocytes. Also present at this time are CD41-CD45++CD11b+ cells, which produce low numbers of CD41++CD45-CD42c+ megakaryocytes in vitro, as do fetal liver cells expressing the macrophage-specific Csf receptor-1 (Csf1r/CD115) from MaFIA transgenic mice, which give rise poorly to CD41++CD45-CD42c+ embryo megakaryocytes both in vivo and in vitro In contrast, around 30% of adult megakaryocytes (CD41++CD45++CD9++CD42c+) from C57BL/6 and MaFIA mice express CD115. We propose that differential pathways operating in the mouse embryo liver at gestational day 11.5 beget CD41++CD45-CD42c+ embryo megakaryocytes that can be produced from CD41+CD45- or from CD41+CD45+ cells, at difference from those from bone marrow.

Stem cell factor and NSC87877 combine to enhance c-Kit mediated proliferation of human megakaryoblastic cells.

Enhancement of hematopoietic stem cells (HSCs) proliferation is a central aim in bone marrow transplantation (BMT). A stem cell factor (SCF) and c-Kit mediated extracellular signaling trigger proliferation of HSCs. This signaling is negatively regulated by protein tyrosine phosphatases (PTPs), SHP-1 and SHP-2. Although NSC87877 (N) is known to inhibit SHP-1/SHP-2, c-Kit-mediated HSCs proliferation by inhibiting SHP-1/SHP-2 has not been reported. This study investigated the combined effect of SCF (S) and N in c-Kit mediated proliferation and underlying mechanisms. The growth of human megakaryoblastic cell line, MO7e and HSCs, upon treatment with S and N alone, and in combination was assessed by PrestoBlue staining. The expression of c-Kit, phosphorylated c-Kit, SHP-1/SHP-2 and HePTP inhibition using S and N treatment were evaluated in the MO7e cells. Megakaryoblast cell proliferation was determined by quantification of Ki-67+, S-phase, BrdU+ and CFDA-SE+ cells using flow cytometry. The combination of S and N leads to enhanced cell growth compared with either S or N alone. Collectively, the results reveal a novel mechanism by which S in combination with N significantly enhances proliferation of human megakaryoblast cells. The pretreatment of N before S enhances proliferation of cells than S alone. This promising combination would likely play an essential role in enhancing the proliferation of cells.

Cytokine control of megakaryopoiesis.

Platelets are anuclear blood cells required for haemostasis and are implicated in other processes including inflammation and metastasis. Platelets are produced by megakaryocytes, specialized cells that are themselves generated by a process of controlled differentiation and maturation of bone-marrow stem and progenitor cells. This process of megakaryopoiesis involves the coordinated interplay of transcription factor-controlled cellular programming with extra-cellular cues produced locally in supporting niches or as circulating factors. This review focuses on these external cues, the cytokines and chemokines, that drive production of megakaryocytes and support the terminal process of platelet release. Emphasis is given to thrombopoietin (Tpo), the major cytokine regulator of steady-state megakaryopoiesis, and its specific cell surface receptor, the Mpl protein, including normal and pathological roles as well as clinical application. The potential for alternative or supplementary regulatory mechanisms for platelet production, particularly in times of acute need, or in states of infection or inflammation are also discussed.