Cord Blood-derived Megakaryocytes Research Articles

CDK8/19 inhibition triggers a switch from mitosis to endomitosis in cord blood megakaryocytes.

Neonatal and adult megakaryocytes differ in proliferative capacity and ploidy levels, and neonatal and adult platelets differ in function, gene expression, and protein content. The mechanisms underlying these differences are incompletely understood. CDK8 and CDK19 are transcriptional kinases part of the CDK-mediator complex, which regulates gene transcription in a cell-specific manner. We discovered that cortistatin A, a potent highly selective inhibitor of CDK8/CDK19, significantly reduced cell expansion and increased ploidy in cord blood-derived megakaryocytes. These phenotypic changes were associated with gene expression changes that partially overlapped developmentally regulated genes. These findings might have relevance for the management of developmental megakaryocyte disorders.

Sequence-specific 2'-O-methoxyethyl antisense oligonucleotides activate human platelets through glycoprotein VI, triggering formation of platelet-leukocyte aggregates.

Antisense oligonucleotides (ASO) are DNA-based, disease-modifying drugs. Clinical trials with 2'-O-methoxyethyl (2’MOE) ASO have shown dose- and sequence-specific lowering of platelet counts according to two phenotypes. Phenotype 1 is a moderate (but not clinically severe) drop in platelet count. Phenotype 2 is rare, severe thrombocytopenia. This article focuses on the underlying cause of the more common phenotype 1, investigating the effects of ASO on platelet production and platelet function. Five phosphorothioate ASO were studied: three 2’MOE sequences; 487660 (no effects on platelet count), 104838 (associated with phenotype 1), and 501861 (effects unknown) and two CpG sequences; 120704 and ODN 2395 (known to activate platelets). Human cord bloodderived megakaryocytes were treated with these ASO to study their effects on proplatelet production. Platelet activation (determined by surface P-selectin) and platelet-leukocyte aggregates were analyzed in ASO-treated blood from healthy human volunteers. None of the ASO inhibited proplatelet production by human megakaryocytes. All the ASO were shown to bind to the platelet receptor glycoprotein VI (KD ~0.2-1.5 μM). CpG ASO had the highest affinity to glycoprotein VI, the most potent platelet-activating effects and led to the greatest formation of platelet-leukocyte aggregates. 2’MOE ASO 487660 had no detectable platelet effects, while 2’MOE ASOs 104838 and 501861 triggered moderate platelet activation and SYKdependent formation of platelet-leukocyte aggregates. Donors with higher platelet glycoprotein VI levels had greater ASO-induced platelet activation. Sequence-dependent ASO-induced platelet activation and platelet-leukocyte aggregates may explain phenotype 1 (moderate drops in platelet count). Platelet glycoprotein VI levels could be useful as a screening tool to identify patients at higher risk of ASO-induced platelet side effects.

KIFC1 Mediates Centrosome Clustering Prior to Proplatelet Formation from Megakaryocytes

Platelets are specialized anucleate cells that circulate in the blood and serve to prevent bleeding and minimize blood vessel injury. In addition to their hemostatic functions, platelets participate in wound healing, angiogenesis, inflammation, and immunity, and are therefore central players in both maintaining normal physiology and in disease pathogenesis. Platelets are derived from their precursor cells, megakaryocytes (MKs), that reside principally in the bone marrow. During maturation, MKs undergo an altered cell cycle called endomitosis in which they replicate their DNA but avoid cell division, resulting in polyploid MKs with amplified microtubule (MT)-organizing centers called centrosomes. Subsequently, MT-dependent forces are responsible for extending long cytoplasmic protrusions called proplatelets into sinusoidal blood vessels, eventually giving rise to circulating platelets. Despite progress in elucidating key steps of platelet production, there is a conspicuous lack of understanding of what triggers mature, polyploid MKs to undergo the MT rearrangements required for proplatelet production. Using live cell imaging of mouse fetal liver-derived MKs expressing fluorescent b1-tubulin, we have identified a novel MT-based structure in MKs termed a monospindle. Our data suggest that monospindles result from polyploid MKs clustering multiple centrosomes into a centralized MT-organizing center during mitosis, leading to an enlarged array of MTs oriented towards the cell cortex. These structures were also apparent in mouse bone marrow- and human cord blood-derived MKs, suggesting that monospindle formation is a general phenomenon in MKs. Interestingly, a higher percentage of MKs contained monospindles at a timepoint directly prior to proplatelet production (50%) compared to when proplatelets were actively being produced (22%), indicating a possible role in initiating proplatelet formation. Centrosome clustering in cancer cells is mediated by the MT-based mitotic motor protein, KIFC1. Consistently, we found that small molecule inhibition of KIFC1 decreased the percentage of MKs containing monospindles (55% ctrl vs. 6% KIFC1 inhibitor). Strikingly, KIFC1 inhibitor treatment also drastically reduced the percentage of MKs producing proplatelets (peak proplatelet formation: 40% ctrl vs. 5% KIFC1 inhibitor), suggesting that KIFC1-mediated centrosome clustering into monospindles is important for proplatelet production. To test how KIFC1 contributes to these phenotypes, we assessed its expression at different timepoints by Western blot and detected increased KIFC1 levels in more mature MKs preceding proplatelet formation. Cell sorting of MKs into distinct ploidy populations followed by Western blot showed that KIFC1 expression increased with higher ploidy. Thus, our results lead us to suggest a working model in which elevated KIFC1 levels in mature MKs drive monospindle formation to trigger proplatelet formation. Investigating the role of KIFC1-mediated monospindle formation for initiating proplatelet formation could yield a coherent, molecular understanding of how mature, polyploid MKs reorganize MTs for proplatelet production. In addition, these data will help inform basic cell biology, as there are important parallels between centrosome clustering in MKs and cancer cells. Finally, our findings could yield novel therapeutic strategies for treating patients with thrombocytopenia (low platelet counts) by directly stimulating platelet production from mature MKs residing in the bone marrow. Disclosures Italiano: Platelet Biogenesis: Employment, Equity Ownership; Ionis Research Funding: Research Funding.

Comparative Transcriptome Analyses of Laser-Captured Human Primary Megakaryocytes with Platelets from the Same Healthy Donors and from Cord Blood Derived Megakaryocytes

Anucleated platelets have a rich repertoire of RNAs, many of which have provided insights into megakaryocyte-platelet biology and have been proposed as disease biomarkers. Although it is generally believed that the platelet transcriptome is a reflection of the megakaryocyte (MK) RNA content and is transported into platelets during pro-platelet production, this is a poorly studied concept. Healthy human bone marrow (BM) is difficult to access and because MKs represent <0.05% of nucleated cells in BM, making it technically challenging to obtain a pure population. Cultured MKs from CD34-positive umbilical vein cord blood have advantages of being human and initially primary cells, but it is unknown how well the transcriptomes of differentiating MKs in culture correlate with primary BM MKs. Microdissection of BM aspirates by laser capture scanning microscopy can overcome purity limitations by isolating virtually 100% MKs. The goals of this study were to assess the transcriptome correlations between laser-captured primary human MKs (LC-MKs) and both (1) platelets from the same individuals who donated the BM and (2) cord blood derived MKs (CB-MKs).BM aspirates and peripheral blood were collected from three healthy donors. Mature BM MKs were identified by a Board certified Hematologist using standard morphologic criteria, and approximately 200 MKs were microdissected by laser capture scanning microscopy from each aspirate. Peripheral blood platelets were isolated from the same donors followed by leukocyte and erythrocyte depletion. Separately, CD34-positive cells were isolated from umbilical vein cord blood, cultured under standard MK-producing conditions with thrombopoietin and stem cell factor, and CD61-positive CB-MKs were isolated at day 6, 9 and 13 in three independent experiments. Total RNA was extracted from LC-MKs, platelets and CB-MKs and subjected to next-generation RNA-sequencing (total of 15 samples: 5 cell types from 3 independent experiments). The Table summarizes the RNA-seq read numbers generated and the number of protein-coding genes determined for each sample. Notably, the LC-MK RNA yielded fewer reads because the input (estimated in the low picogram range) was less than the other samples. Several known platelet transcripts, including PPBP, PF4, B2M, TMSB4X and OST4 were abundant in our LC-MKs RNA-sequencing analysis. All samples exhibited high expression (1,721-74,923 FPKM) of mitochondrial transcripts.To assess RNA correlations across cell types, we only considered transcripts expressed in both comparison groups. mRNAs from LC-MKs were statistically significantly correlated with mRNAs from both platelets and day 13 CD41a-CD42a-positive CB-MKs. These correlations were highest for the most abundantly expressed (FPKM>50) transcripts (Spearman rank correlation Rho = 0.71 for LC-MKs vs. platelets, and Rho = 0.67 for LC-MKs vs. day 13 CB-MKs). However, even the lower expressed transcripts (using the standard threshold of FPKM>0.3) showed moderate correlation (Rho = 0.37-0.42, p value <E-13). Regardless of transcript abundance, the platelet transcriptome displayed higher correlations with LC-MKs than did CB-MKs. Of the 400 mRNAs common to all LC-MKs samples, 23 were not identified in any of the platelet samples and 9 were not identified in any of the d13 CB-MKs. A secondary goal was to compare transcriptome profiles of CB-MKs during differentiation. Not surprisingly, mRNAs from cultured CB-MKs showed higher correlation when their time in culture was shorter than longer (Rho = 0.87 for d13 vs. d9; 0.81 for d9 vs. d6; and 0.64 for d13 vs. d6). During CB-MK differentiation, 825 transcripts were up-regulated whereas 2725 transcripts were down-regulated.In summary, we provide the first report of transcript profiling from normal, primary human MKs. Peripheral blood platelet RNA transcript levels were representative of MK transcripts from the same subjects, especially if abundantly expressed; cultured MK mRNAs correlated strongly with LC-MKs, while platelet RNAs from the same donor showed an even stronger correlation. mRNA expression differences between platelets and BM MKs could provide insights into thrombopoiesis and may be due to (1) regulated transcript partitioning from MK to platelets, (2) differential RNA endocytosis or exocytosis or (3) RNA degradation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Functionally Distinct Cell Populations Characterize Cord Blood Derived Megakaryocytes

A fundamental understanding of platelet biology requires functional assessment of megakaryocyte-platelet expressed genes and transcripts. The need for gene functional studies has recently increased with unbiased genome wide associations with platelet phenotypes. Animal models with an over-expressed or knocked-out gene have been particularly valuable systems, although species differences may preclude optimal evaluation. Assessing protein function in human platelets can be accomplished with pharmacologic inhibitors, although inhibitor availability and specificity can plague this approach. Studies with primary human megakaryocytes (MKs) are greatly challenged by inaccessibility, low yields and contamination with other cells. Cultured human megakaryocyte-like cell lines (e.g., Meg-01, HEL and others) have proven valuable, but these cells poorly recapitulate platelet physiology. Increasingly, investigators are using cultured human MKs to study gene function. CD34+ hematopoietic stem cells from human cord blood are routinely used to generate megakaryocytes in culture. This system has advantages of being human, relatively easy to obtain, cultured from primary cells and able to be genetically manipulated. Although cord blood-derived megakaryocytes (CBDMs) have proven useful for studying aspects of megakaryocyte/platelet biology, there is little information about the heterogeneous cell populations observed in CBDMs. In the course of our studies with CBDMs we observed multiple distinct cell populations by flow cytometry. Similar populations were also observed in the megakaryocytic Meg-01 cell line. The largest cells (called P1) were the most abundant (consisting of nearly 100% of cells) at day 3 in culture. P2 cells, which are smaller and more granular than P1, appeared at day 6 and by day 13 were ~50% of the total. P3 appeared at day 6 and are the smallest, with size and granularity roughly similar to platelets; by day 13 these were ~30% of the total. P1 but not P2 nor P3 became CD61/CD41/CD42 positive and CD34 negative over 13 days in culture. Ninety-seven percent and 93% of P2 and P3 cells, respectively, were phosphatidyl serine (PS) positive, whereas 93% of P1 cells were PS negative. Electron microscopy revealed many typical features of bone marrow MKs in the PS negative (P1) cells, including large size, polyploid nucleus, mitochondria and immature granules. However, the demarcation membrane system was poorly developed. Virtually all of the PS positive (P2) cells were apoptotic, lacked granules and had no discernable nuclei. Purification of P1 and P2 populations followed by re-culturing revealed that P1 gives rise to both the P2 and P3 populations, whereas P2 gave rise to no other population. CD34+ cells cultured with the pan-caspase inhibitor Z-VAD-FMK developed a much smaller proportion of P2 cells with a corresponding increase in P1 cells. Stimulation of these 3 populations with collagen related peptide, thrombin, protease activated receptor 1-activating peptide (PAR1-AP) and PAR4-AP showed strong integrin activation in P1 cells, but not in P2 nor P3 cells. To assess which population was susceptible to genetic manipulation, day 3 cultures were infected with a lentiviral construct containing a shRNA to talin. Talin knockdown prevented agonist-induced integrin activation in P1 cells and as expected, had no effect on the P2 or P3 population. In summary, these data indicate that only a portion of CBDMs are functional and only the P1 population should be used to assess MK gene function. Over time, the majority of CBDMs become apoptotic and the smaller P3, platelet-like particles have minimal response to agonist compared to peripheral blood platelets. These results may partially explain the difficulties in generating functional platelets in vitro and are consistent with the need to restrain apoptosis for successful megakaryocytopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Actin inhibition increases megakaryocyte proplatelet formation through an apoptosis-dependent mechanism.

BackgroundMegakaryocytes assemble and release platelets through the extension of proplatelet processes, which are cytoplasmic extensions that extrude from the megakaryocyte and form platelets at their tips. Proplatelet formation and platelet release are complex processes that require a combination of structural rearrangements. While the signals that trigger the initiation of proplatelet formation process are not completely understood, it has been shown that inhibition of cytoskeletal signaling in mature megakaryocytes induces proplatelet formation. Megakaryocyte apoptosis may also be involved in initiation of proplatelet extension, although this is controversial. This study inquires whether the proplatelet production induced by cytoskeletal signaling inhibition is dependent on activation of apoptosis.MethodsMegakaryocytes derived from human umbilical cord blood CD34+ cells were treated with the actin polymerization inhibitor latrunculin and their ploidy and proplatelet formation were quantitated. Apoptosis activation was analyzed by flow cytometry and luminescence assays. Caspase activity was inhibited by two compounds, ZVAD and QVD. Expression levels of pro-survival and pro-apoptosis genes were measured by quantitative RT-PCR. Protein levels of Bcl-XL, Bax and Bak were measured by western blot. Cell ultrastructure was analyzed by electron microscopy.ResultsActin inhibition resulted in increased ploidy and increased proplatelet formation in cultured umbilical cord blood-derived megakaryocytes. Actin inhibition activated apoptosis in the cultured cells. The effects of actin inhibition on proplatelet formation were blocked by caspase inhibition. Increased expression of both pro-apoptotic and pro-survival genes was observed. Pro-survival protein (Bcl-xL) levels were increased compared to levels of pro-apoptotic proteins Bak and Bax. Despite apoptosis being activated, the megakaryocytes underwent minimal ultrastructural changes during actin inhibition.ConclusionsWe report a correlation between increased proplatelet formation and activation of apoptosis, and that the increase in proplatelet formation in response to actin inhibition is caspase dependent. These findings support a role for apoptosis in proplatelet formation in this model.

Anagrelide platelet‐lowering effect is due to inhibition of both megakaryocyte maturation and proplatelet formation: insight into potential mechanisms

Anagrelide represents a treatment option for essential thrombocythemia patients. It lowers platelet counts through inhibition of megakaryocyte maturation and polyploidization, although the basis for this effect remains unclear. Based on its rapid onset of action, we assessed whether, besides blocking megakaryopoiesis, anagrelide represses proplatelet formation (PPF) and aimed to clarify the underlying mechanisms. Exposure of cord blood-derived megakaryocytes to anagrelide during late stages of culture led to a dose- and time-dependent inhibition of PPF and reduced proplatelet complexity, which were independent of the anagrelide-induced effect on megakaryocyte maturation. Whereas anagrelide was shown to phosphorylate cAMP-substrate VASP, two pharmacologic inhibitors of the cAMP pathway were completely unable to revert anagrelide-induced repression in megakaryopoiesis and PPF, suggesting these effects are unrelated to its ability to inhibit phosphodiesterase (PDE) 3. The reduction in thrombopoiesis was not the result of down-regulation of transcription factors which coordinate PPF, while the myosin pathway was identified as a candidate target, as anagrelide was shown to phosphorylate the myosin light chain and the PPF phenotype was partially rescued after inhibition of myosin activity with blebbistatin. The platelet-lowering effect of anagrelide results from impaired megakaryocyte maturation and reduced PPF, both of which are deregulated in essential thrombocythemia. These effects seem unrelated to PDE3 inhibition, which is responsible for anagrelide's cardiovascular side-effects and antiplatelet activity. Further work in this field may lead to the potential development of drugs to treat thrombocytosis in myeloproliferative disorders with an improved pharmacologic profile.

Rho kinase inhibition drives megakaryocyte polyploidization and proplatelet formation through MYC and NFE2 downregulation

The processes of megakaryocyte polyploidization and demarcation membrane system (DMS) formation are crucial for platelet production, but the mechanisms controlling these processes are not fully determined. Inhibition of Rho kinase (ROCK) signalling leads to increased polyploidization in umbilical cord blood-derived megakaryocytes. To extend these findings we determined the effect of ROCK inhibition on development of the DMS and on proplatelet formation. The underlying mechanisms were explored by analysing the effect of ROCK inhibition on the expression of MYC and NFE2, which encode two transcription factors critical for megakaryocyte development. ROCK inhibition promoted DMS formation, and increased proplatelet formation and platelet release. Rho kinase inhibition also downregulated MYC and NFE2 expression in mature megakaryocytes, and this down-regulation correlated with increased proplatelet formation. Our findings suggest a model whereby ROCK inhibition drives polyploidization, DMS growth and proplatelet formation late in megakaryocyte maturation through downregulation of MYC and NFE2 expression.

In vitro Biological Characteristics of Human Cord Blood-derived Megakaryocytes

Introduction: Umbilical cord blood (CB) has been used as an alternative source for haematopoietic stem cell transplantation (HSCT) in recent years. However, delayed platelet recovery is frequently associated with CB HSCT. Megakaryocytes (Mk) are the specialised precursors of platelets and they are among the rarest haemopoietic cell types. Despite the rapid expansion of our knowledge of megakaryopoiesis in recent years, many questions, such as the molecular regulatory mechanisms in Mk differentiation and maturation, platelet formation and release, remain unanswered in CB-derived megakaryopoiesis. Variations can be seen from the literature by individual investigators using different approaches for Mk-specific differentiation and maturation induction. The development of in vitro culture methods to obtain sufficient numbers of Mks from readily available haematopoietic stem cells is of value for both basic research and clinical applications. Materials and Methods: The CD34+ cells from cord blood samples were cultured in serum-free medium with haematopoietic growth factors (GFs), such as IL-3, stem cell factor (SCF), and thrombopoietin (Tpo). The differentiation of Mk was monitored using Mk- and platelet-specific monoclonal antibodies and flow cytometric analysis. The morphology of the cultured cells was studied by both light and electronic microscopy (LM and EM). The involvement of the human Notch gene family members was studied by real time-polymerase chain reaction (RT-PCR). Maturation of the cultured Mks was studied using flow cytometric analysis for both platelet-specific surface markers and enodomitosis. Platelet activation was assessed in the cytoplasmic fragments harvested from the cultures. Results: Specific Mk differentiation of &gt;70% resulted from a 2-step culture approach using IL-3, SCF and Tpo for 7 days followed by Tpo only for another 14 days. RT-PCR showed high-level expression of both Notch-1 and its ligand, Jagged-1, in the cultured Mks. Limited levels of polyploidy (&gt;4N, endomitosis, EnM) were observed in the cultured Mks. The results also showed that the cytoplasmic fragments from the cultures responded to platelet activation reagents, including ADP and collagen, marked by upregulation of platelet-specific activation markers, such as CD62P (P-selectin) and PAC-1 (gpαIIbβ3). Conclusion: The methods used in this study are specific for differentiation of Mk from CB CD34+ cell, which can partially mature and produce functional platelets in vitro. This approach for human Mk differentiation could be further optimised and may be adapted on larger scales for clinical purposes.