Christian A. Di Buduo

Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies

We present a programmable bioengineered 3-dimensional silk-based bone marrow niche tissue system that successfully mimics the physiology of human bone marrow environment allowing us to manufacture functional human platelets ex vivo. Using stem/progenitor cells, megakaryocyte function and platelet generation were recorded in response to variations in extracellular matrix components, surface topography, stiffness, coculture with endothelial cells, and shear forces. Millions of human platelets were produced and showed to be functional based on multiple activation tests. Using adult hematopoietic progenitor cells our system demonstrated the ability to reproduce key steps of thrombopoiesis, including alterations observed in diseased states. A critical feature of the system is the use of natural silk protein biomaterial allowing us to leverage its biocompatibility, nonthrombogenic features, programmable mechanical properties, and surface binding of cytokines, extracellular matrix components, and endothelial-derived proteins. This in turn offers new opportunities for the study of blood component production ex vivo and provides a superior tissue system for the study of pathologic mechanisms of human platelet production.

The importance of calcium in the regulation of megakaryocyte function

Platelet release by megakaryocytes is regulated by a concert of environmental and autocrine factors. We previously showed that constitutively released adenosine diphosphate by human megakaryocytes leads to platelet production. Here we show that adenosine diphosphate elicits, in human megakaryocytes, an increase in cytosolic calcium concentration, followed by a plateau, which is lowered in the absence of extracellular calcium, suggesting the involvement of Store-Operated Calcium Entry. Indeed, we demonstrate that megakaryocytes express the major candidates to mediate Store-Operated Calcium Entry, stromal interaction molecule 1, Orai1 and canonical transient receptor potential 1, which are activated upon either pharmacological or physiological depletion of the intracellular calcium pool. This mechanism is inhibited by phospholipase C or inositol-3-phosphate receptor inhibitors and by a specific calcium entry blocker. Studies on megakaryocyte behavior, on extracellular matrix proteins that support proplatelet extension, show that calcium mobilization from intracellular stores activates signaling cascades that trigger megakaryocyte adhesion and proplatelet formation, and promotes extracellular calcium entry which is primarily involved in the regulation of the contractile force responsible for megakaryocyte motility. These findings provide the first evidence that both calcium mobilization from intracellular stores and extracellular calcium entry specifically regulate human megakaryocyte functions.

The secret life of a megakaryocyte: emerging roles in bone marrow homeostasis control

Megakaryocytes are rare cells found in the bone marrow, responsible for the everyday production and release of millions of platelets into the bloodstream. Since the discovery and cloning, in 1994, of their principal humoral factor, thrombopoietin, and its receptor c-Mpl, many efforts have been directed to define the mechanisms underlying an efficient platelet production. However, more recently different studies have pointed out new roles for megakaryocytes as regulators of bone marrow homeostasis and physiology. In this review we discuss the interaction and the reciprocal regulation of megakaryocytes with the different cellular and extracellular components of the bone marrow environment. Finally, we provide evidence that these processes may concur to the reconstitution of the bone marrow environment after injury and their deregulation may lead to the development of a series of inherited or acquired pathologies.

Constitutively released adenosine diphosphate regulates proplatelet formation by human megakaryocytes

Background The interaction of adenosine diphosphate with its P2Y1 and P2Y12 receptors on platelets is important for platelet function. However, nothing is known about adenosine diphosphate and its function in human megakaryocytes. Design and Methods We studied the role of adenosine diphosphate and P2Y receptors on proplatelet formation by human megakaryocytes in culture. Results Megakaryocytes expressed all the known eight subtypes of P2Y receptors, and constitutively released adenosine diphosphate. Proplatelet formation was inhibited by the adenosine diphosphate scavengers apyrase and CP/CPK by 60-70% and by the P2Y12 inhibitors cangrelor and 2-MeSAMP by 50-60%, but was not inhibited by the P2Y1 inhibitor MRS 2179. However, the active metabolites of the anti-P2Y12 drugs, clopidogrel and prasugrel, did not inhibit proplatelet formation. Since cangrelor and 2-MeSAMP also interact with P2Y13, we hypothesized that P2Y13, rather than P2Y12 is involved in adenosine diphosphate-regulated proplatelet formation. The specific P2Y13 inhibitor MRS 2211 inhibited proplatelet formation in a concentration-dependent manner. Megakaryocytes from a patient with severe congenital P2Y12 deficiency showed normal proplatelet formation, which was inhibited by apyrase, cangrelor or MRS 2211 by 50-60%. The platelet count of patients with congenital delta-storage pool deficiency, who lack secretable adenosine diphosphate, was significantly lower than that of patients with other platelet function disorders, confirming the important role of secretable adenosine diphosphate in platelet formation. Conclusions This is the first demonstration that adenosine diphosphate released by megakaryocytes regulates their function by interacting with P2Y13. The clinical relevance of this not previously described physiological role of adenosine diphosphate and P2Y13 requires further exploration.

Involvement of TGFβ1 in autocrine regulation of proplatelet formation in healthy subjects and patients with primary myelofibrosis

Megakaryocytes release platelets into the bloodstream by elongating proplatelets. In this study, we showed that human megakaryocytes constitutively release Transforming Growth Factor β1 and express its receptors. Importantly, Transforming Growth Factor β1 downstream signaling, through SMAD2/3 phosphorylation, was shown to be active in megakaryocytes extending proplatelets, indicating a type of autocrine stimulation on megakaryocyte development. Furthermore, inactivation of Transforming Growth Factor β1 signaling, by the receptor inhibitors SB431542 and Stemolecule ALK5 inhibitor, determined a significant decrease in proplatelet formation. Recent studies indicated a crucial role of Transforming Growth Factor β1 in the pathogenesis of primary myelofibrosis. We demonstrated that primary myelofibrosis-derived megakaryocytes expressed increased levels of bioactive Transforming Growth Factor β1; however, higher levels of released Transforming Growth Factor β1 did not lead to enhanced activation of downstream pathways. Overall, these data propose Transforming Growth Factor β1 as a new element in the autocrine regulation of proplatelet formation in vitro. Despite the increase in Transforming Growth Factor β1 this mechanism seems to be preserved in primary myelofibrosis.

Clinical and pathogenic features of ETV6-related thrombocytopenia with predisposition to acute lymphoblastic leukemia

ETV6-related thrombocytopenia is an autosomal dominant thrombocytopenia that has been recently identified in a few families and has been suspected to predispose to hematologic malignancies. To gain further information on this disorder, we searched for ETV6 mutations in the 130 families with inherited thrombocytopenia of unknown origin from our cohort of 274 consecutive pedigrees with familial thrombocytopenia. We identified 20 patients with ETV6-related thrombocytopenia from seven pedigrees. They have five different ETV6 variants, including three novel mutations affecting the highly conserved E26 transformation-specific domain. The relative frequency of ETV6-related thrombocytopenia was 2.6% in the whole case series and 4.6% among the families with known forms of inherited thrombocytopenia. The degree of thrombocytopenia and bleeding tendency of the patients with ETV6-related thrombocytopenia were mild, but four subjects developed B-cell acute lymphoblastic leukemia during childhood, resulting in a significantly higher incidence of this condition compared to that in the general population. Clinical and laboratory findings did not identify any particular defects that could lead to the suspicion of this disorder from the routine diagnostic workup. However, at variance with most inherited thrombocytopenias, platelets were not enlarged. In vitro studies revealed that the maturation of the patients’ megakaryocytes was defective and that the patients have impaired proplatelet formation. Moreover, platelets from patients with ETV6-related thrombocytopenia have reduced ability to spread on fibrinogen. Since the dominant thrombocytopenias due to mutations in RUNX1 and ANKRD26 are also characterized by normal platelet size and predispose to hematologic malignancies, we suggest that screening for ETV6, RUNX1 and ANKRD26 mutations should be performed in all subjects with autosomal dominant thrombocytopenia and normal platelet size.

Thrombopoietin/TGF-β1 Loop Regulates Megakaryocyte Extracellular Matrix Component Synthesis

Extracellular matrix (ECM) components initiate crucial biochemical and biomechanical cues that are required for bone marrow homeostasis. In our research, we prove that a peri‐cellular matrix composed primarily of type III and type IV collagens, and fibronectin surrounds human megakaryocytes in the bone marrow. The data we collected support the hypothesis that bone marrow megakaryocytes possess a complete mechanism to synthesize the ECM components, and that thrombopoietin is a pivotal regulator of this new function inducing transforming growth factor‐β1 (TGF‐β1) release and consequent activation of the downstream pathways, both in vitro and in vivo. This activation results in a dose dependent increase of ECM component synthesis by megakaryocytes, which is reverted upon incubation with JAK and TGF‐β1 receptor specific inhibitors. These data are pivotal for understanding the central role of megakaryocytes in creating their own regulatory niche within the bone marrow environment. Stem Cells 2016;34:1123–1133

Revealing eltrombopag’s promotion of human megakaryopoiesis through AKT/ERK-dependent pathway activation

Eltrombopag is a small, non-peptide thrombopoietin mimetic that has been approved for increasing platelet count not only in immune thrombocytopenia and Hepatitis C virus-related thrombocytopenia, but also in aplastic anemia. Moreover, this drug is under investigation for increasing platelet counts in myelodysplastic syndromes. Despite current clinical practice, the mechanisms governing eltrombopag’s impact on human hematopoiesis are largely unknown, in part due to the impossibility of using traditional in vivo models. To investigate eltrombopag’s impact on megakaryocyte functions, we employed our established in vitro model for studying hematopoietic stem cell differentiation combined with our latest 3-dimensional silk-based bone marrow tissue model. Results demonstrated that eltrombopag favors human megakaryocyte differentiation and platelet production in a dose-dependent manner. These effects are accompanied by increased phosphorylation of AKT and ERK1/2 signaling molecules, which have been proven to be crucial in regulating physiologic thrombopoiesis. These data further clarify the different mechanisms of action of eltrombopag when compared to romiplostim, which, as we have shown, induces the proliferation of immature megakaryocytes rather than platelet production, due to the unbalanced activation of AKT and ERK1/2 signaling molecules. In conclusion, our research clarifies the underlying mechanisms that govern the action of eltrombopag on megakaryocyte functions and its relevance in clinical practice.

Hyaluronan based hydrogels provide an improved model to study megakaryocyte–matrix interactions

Hyaluronan (HA) is a glycosamminoglican involved in cell biology as well as a relevant polymer for tissue engineering and regenerative medicine. Megakaryocytes (Mks) are immersed in a mesh of extracellular matrix (ECM) components that regulate their maturation in the bone marrow (BM) and the release of platelets into the bloodstream. While fibrous ECMs such as collagens and fibronectin have been demonstrated to differently regulate Mk function and platelet release, the role of HA, that fills the majority of the BM extracellular interstitial space, has not been investigated so far. Here we demonstrated that, although human Mks express HA receptors, they are not affected by HA in terms of in vitro differentiation, maturation and platelet formation. Importantly, chemical properties of HA were exploited to generate hydrogels with entrapped ECMs that represent a useful model to more closely mimic the tridimensional characteristics of the BM environment for studying Mk function. In conclusion, in this work we demonstrated that HA is an ideal candidate for a 3D ex vivo model of human BM ECM component environment.

Abnormal proplatelet formation and emperipolesis in cultured human megakaryocytes from gray platelet syndrome patients

The Gray Platelet Syndrome (GPS) is a rare inherited bleeding disorder characterized by deficiency of platelet α-granules, macrothrombocytopenia and marrow fibrosis. The autosomal recessive form of GPS is linked to loss of function mutations in NBEAL2, which is predicted to regulate granule trafficking in megakaryocytes, the platelet progenitors. We report the first analysis of cultured megakaryocytes from GPS patients with NBEAL2 mutations. Megakaryocytes cultured from peripheral blood or bone marrow hematopoietic progenitor cells from four patients were used to investigate megakaryopoiesis, megakaryocyte morphology and platelet formation. In vitro differentiation of megakaryocytes was normal, whereas we observed deficiency of megakaryocyte α-granule proteins and emperipolesis. Importantly, we first demonstrated that platelet formation by GPS megakaryocytes was severely affected, a defect which might be the major cause of thrombocytopenia in patients. These results demonstrate that cultured megakaryocytes from GPS patients provide a valuable model to understand the pathogenesis of GPS in humans.