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A New Role of NAP1L1 in Megakaryocytes and Human Platelets.

ABSTRACT: Platelets (PLTs) are anucleate and considered incapable of nuclear functions. Contrastingly, nuclear proteins were detected in human PLTs. For most of these proteins, it is unclear if nuclear or alternatively assigned functions are performed, a question we wanted to address for nuclear assembly protein 1like 1 (NAP1L1). Using a wide array of molecular methods, including RNAseq, co-IP, overexpression and functional assays, we explored expression pattern and functionality of NAP1L1 in PLTs, and CD34⁺-derived megakaryocytes (MKs). NAP1L1 is expressed in PLTs and MKs. Co-IP experiments revealed that dihydrolipolylysine-residue acetyltransferase (DLAT encoded protein PDC-E2, ODP2) dynamically interacts with NAP1L1. PDC-E2 is part of the mitochondrial pyruvate-dehydrogenase (PDH) multi-enzyme complex, playing a crucial role in maintaining cellular respiration, and promoting ATP-synthesis via the respiratory chain. Since altered mitochondrial function is a hallmark of infectious syndromes, we analyzed PDH activity in PLTs from septic patients demonstrating increased activity, paralleling NAP1L1 expression levels. MKs PDH activity decreased following an LPS-challenge. Furthermore, overexpression of NAP1L1 significantly altered the ability of MKs to form proplatelet extensions, diminishing thrombopoiesis. These results indicate that NAP1L1 performs in other than nucleosome-assembly functions in PTLs and MKs, binding a key mitochondrial protein as a potential chaperone, and gatekeeper, influencing PDH activity and thrombopoiesis.

SUBMITTER: Freitag M

PROVIDER: S-EPMC9737020 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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A New Role of NAP1L1 in Megakaryocytes and Human Platelets.

Freitag Martin M Schwertz Hansjörg H

International journal of molecular sciences 20221124 23

Platelets (PLTs) are anucleate and considered incapable of nuclear functions. Contrastingly, nuclear proteins were detected in human PLTs. For most of these proteins, it is unclear if nuclear or alternatively assigned functions are performed, a question we wanted to address for nuclear assembly protein 1like 1 (NAP1L1). Using a wide array of molecular methods, including RNAseq, co-IP, overexpression and functional assays, we explored expression pattern and functionality of NAP1L1 in PLTs, and CD ...[more]

PMID: 36499021

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Project description:BackgroundPlatelet transfusion therapy has made a great breakthrough in clinical practice, and the differentiation of human embryonic stem cells (hESCs) to produce functional platelets has become a new potential approach, however, efficient generation of functional platelets still faces great challenges. Here, we presented a novel approach to highly and efficiently generate mature megakaryocytes (MKs) and functional platelets from hESCs.MethodsIn hypoxic conditions, we successfully replicated the maturation process of MKs and platelets in a controlled in vitro environment by introducing an optimal combination of cytokines at various stages of development. This method led to the generation of MKs and platelets derived from hESCs. Subsequently, mature MKs and functional platelets were further comprehensively investigated and characterized using a variety of methodologies, including flow cytometry analysis, RT-qPCR validation, Giemsa-Wright's staining, immunofluorescent staining, RNA transcriptome analysis, and DNA ploidy analysis. Additionally, the in vivo function of platelets was evaluated through the transplantation using thrombocytopenia model mice.ResultsUnder our 3D differentiation conditions with four sequential stages, hESCs could be efficiently induced into mature MKs, with 95% expressing CD41aCD42a or 90% expressing CD41aCD42b, and those MKs exhibited polyploid properties, produced filamentous proplatelet structures and further generated platelets. Furthermore, 95% of platelets showed CD42b+CD62p+ phenotype upon the stimulation with ADP and TRAP-6, while 50% of platelets exhibited the ability to bind PAC-1, indicating that hESC-derived platelets possessed the in vitro functionality. In mice models of thrombocytopenia, hESC-derived platelets effectively restored hemostasis in a manner comparable to human blood-derived platelets. Further investigation on the mechanism of this sequential differentiation revealed that cellular differentiation and molecular interactions during the generation of hESC-derived MKs and platelets recapitulated the developmental trajectory of the megakaryopoiesis and thrombopoiesis.ConclusionsThus, our results demonstrated that we successfully established a highly efficient differentiation of hESCs into mature MKs and functional platelets in vitro. The in vivo functionality of hESC-derived platelets closely resembles that of natural human platelets, thus offering a promising avenue for the development of functional platelets suitable for future clinical applications.

Project description:BackgroundPlatelet-rich plasma (PRP), which is prepared by concentrating platelets in autologous blood, shows efficacy in chronic skin wounds via multiple growth factors. However, it exhibits heterogeneity across patients, leading to unstable therapeutic efficacy. Human induced pluripotent stem cell (iPSC)-derived megakaryocytes and platelets (iMPs) are capable of providing a stable supply, holding promise as materials for novel platelet concentrate-based therapies. In this context, we evaluated the effect of iMPs on wound healing and validated lyophilization for clinical applications.MethodsThe growth factors released by activated iMPs were measured. The effect of the administration of iMPs on human fibroblasts and human umbilical vein endothelial cells (HUVECs) was investigated in vitro. iMPs were applied to dorsal skin defects of diabetic mice to assess the wound closure rate and quantify collagen deposition and angiogenesis. Following the storage of freeze-dried iMPs (FD-iMPs) for three months, the stability of growth factors and their efficacy in animal models were determined.ResultMultiple growth factors that promote wound healing were detected in activated iMPs. iMPs specifically released FGF2 and exhibited a superior enhancement of HUVEC proliferation compared to PRP. Moreover, an RNA-seq analysis revealed that iMPs induce polarization to stalk cells and enhance ANGPTL4 gene expression in HUVECs. Animal studies demonstrated that iMPs promoted wound closure and angiogenesis in chronic wounds caused by diabetes. We also confirmed the long-term stability of growth factors in FD-iMPs and their comparable effects to those of original iMPs in the animal model.ConclusionOur study demonstrates that iMPs promote angiogenesis and wound healing through the activation of vascular endothelial cells. iMPs exhibited more effectiveness than PRP, an effect attributed to the exclusive presence of specific factors including FGF2. Lyophilization enabled the long-term maintenance of the composition of the growth factors and efficacy of the iMPs, therefore contributing to stable supply for clinical application. These findings suggest that iMPs provide a novel treatment for chronic wounds.

Project description:Platelets are essential for hemostatic plug formation and thrombosis. The mechanisms of megakaryocyte (MK) differentiation and subsequent platelet production from stem cells remain only partially understood. The manufacture of megakaryocytes (MKs) and platelets from cell sources including hematopoietic stem cells and pluripotent stem cells have been highlighted for studying the platelet production mechanisms as well as for the development of new strategies for platelet transfusion. The mouse bone marrow stroma cell line OP9 has been widely used as feeder cells for the differentiation of stem cells into MK lineages. OP9 cells are reported to be pre-adipocytes. We previously reported that 3T3-L1 pre-adipocytes differentiated into MKs and platelets. In the present study, we examined whether OP9 cells differentiate into MKs and platelets using MK lineage induction (MKLI) medium previously established to generate MKs and platelets from hematopoietic stem cells, embryonic stem cells, and pre-adipocytes. OP9 cells cultured in MKLI medium had megakaryocytic features, i.e., positivity for surface markers CD41 and CD42b, polyploidy, and distinct morphology. The OP9-derived platelets had functional characteristics, providing the first evidence for the differentiation of OP9 cells into MKs and platelets. We then analyzed gene expressions of critical factors that regulate megakaryopoiesis and thrombopoiesis. The gene expressions of p45NF-E2, FOG, Fli1, GATA2, RUNX1, thrombopoietin, and c-mpl were observed during the MK differentiation. Among the observed transcription factors of MK lineages, p45NF-E2 expression was increased during differentiation. We further studied MK and platelet generation using p45NF-E2-overexpressing OP9 cells. OP9 cells transfected with p45NF-E2 had enhanced production of MKs and platelets. Our findings revealed that OP9 cells differentiated into MKs and platelets in vitro. OP9 cells have critical factors for megakaryopoiesis and thrombopoiesis, which might be involved in a mechanism of this differentiation. p45NF-E2 might also play important roles in the differentiation of OP9 cells into MK lineages cells.

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A New Role of NAP1L1 in Megakaryocytes and Human Platelets.

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A New Role of NAP1L1 in Megakaryocytes and Human Platelets.

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