Turbulence activates platelet biogenesis to enable clinical scale manufacturing from human iPS cells
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PROVIDER: PRJNA448035 | ENA |
REPOSITORIES: ENA
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Project description:To identify the genes whose expression levels are changed in VerMES and dish culture, we performed global gene expression analysis of immortalized megakaryocyte cell lines delived from hiPSC.
2018-07-12 | GSE112512 | GEO
Project description:Background: While potassium ion (K+) is known to be vital to platelet functions, we have limited knowledge about K+ channels involved in thrombopoiesis from imMKCLs, the iPSC-derived megakaryocyte progenitor cell lines that we have established for ex vivo manufacturing of platelet products for clinical use. Objective: We aimed to elucidate how K+ channels contribute to platelet biogenesis and focused on the role of Ca2+-activated K+ channel KCNN4 (also known as KCa3.1). Methods: Using imMKCLs and human cord blood hematopoietic stem cell-derived megakaryocytes (CB-megakaryocytes), we examined the dynamic changes of intracellular cations during platelet biogenesis. Along with RNA-seq profiling of K+ channels, we investigated the role of KCNN4 by using inhibitors or by direct gene knockdown in proplatelet formation or platelet production. We further examined its relationship with tubulin reorganization, mitochondrial functions, and reactive oxygen species (ROS) levels. Results: Continuous reduction of intracellular K+ levels ([K+]i) was observed during the 6-day of maturing imMKCLs. KCNN4 was expressed at the initiation of platelet generation in megakaryocytes and KCNN4 inhibition resulted in impaired proplatelet formation and reduced platelet productivity in imMKCLs and CB-megakaryocytes, accompanied by decreased [K+]i, diminished mitochondrial membrane potential and an elevated level of ROS. Conclusions: Our findings suggest that the decline of [K+]i via KCNN4 is a key mechanism linking tubulin regulation, ROS, and mitochondrial functions to proplatelet formation and intact thrombopoiesis. This study sheds new light on thrombopoiesis mechanism that contributes to improved ex vivo platelet manufacturing.
2026-04-20 | GSE295199 | GEO
Project description:Thioredoxin-interacting protein (TXNIP) is ubiquitously expressed in blood cells, including hematopoietic stem cells (HSCs), monocytes, and platelets. Here we report a novel finding that TXNIP plays a crucial role in megakaryopoiesis and platelet biogenesis via interacting with GATA1, a transcription factor for megakaryocyte-erythroid differentiation. Txnip-/- mice displayed immature megakaryocytes in the bone marrow with thrombocytopenia, which had gotten worse as the mice aged. Transcriptome analysis revealed that the transcriptional activity of GATA1 was significantly enhanced in the Txnip-/- megakaryocyte precursors (MkPs) than wild type (WT) cells. During megakaryopoiesis in ex vivo, Txnip-/- MkPs remained small in cell size with less mitochondrial mass, and more glycolysis for ATP production, as opposed to the normal megakaryocyte maturation. The effects of TXNIP in megakaryocytes were recapitulated in human cord blood CD34+ HSC-derived differentiation. Taken together, this study demonstrates the importance of spatiotemporal expression of TXNIP in platelet biogenesis. We propose for the first time that TXNIP might play a critical role in determining a lineage between megakaryocytes and erythroid cells from a common megakaryocyte-erythroid progenitor via regulation of transcriptional activity of GATA1.
2022-12-31 | GSE186400 | GEO
Project description:NextGen Consortium: Functional Genomics of Platelet Aggregation Using iPS and Derived Megakaryocytes
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Project description:KCNN4 mediated potassium ion efflux maintains mitochondrial functions leading to platelet biogenesis
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Project description:Expression of thioredoxin-interacting protein (TXNIP) during megakaryopoiesis is indispensable for platelet biogenesis
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Project description:To complement donor-dependent platelet supplies, we previously developed an ex vivo manufacturing system using iPSC-derived expandable megakaryocytes, imMKCLs, and a turbulent flow bioreactor, VerMES, to generate iPSC-derived platelet products (iPSC-PLTs). However, the tank size of VerMES was limited to 10 L (VerMES10). Here we examined the feasibility of scaling up to 50 L (VerMES50) with reciprocal motion by two impellers. Under optimized turbulence parameters corresponding to VerMES10, VerMES50 elicited iPSC-PLTs with intact in vivo hemostatic function but with less production efficiency. This insufficiency was caused by increased defective turbulent flow space. A computer simulation proposed that designing VerMES50 with three impellers or a new bioreactor with a modified rotating impeller and unique structure reduces this space. These findings indicate that large-scale PLT manufacturing from cultured imMKCLs requires optimization of the tank structure in addition to optimal turbulent energy and shear stress.
2024-03-26 | GSE262455 | GEO
Project description:<p>The causal mechanisms of common diseases and their therapies have been only marginally illuminated by genetic variants identified in genome wide association studies (GWAS) utilizing single nucleotide polymorphism (SNPs). Platelet activation pathways reflecting hemostasis and thrombosis are the underlying substrate for many cardiovascular diseases and related acute events. To overcome GWAS limitations, genomic studies are needed that integrate molecular surrogates for platelet-related phenotypes assayed in cell-based models derived from individuals of known genotypes and phenotypes. In our GWAS study of native platelet aggregation phenotypes and aggregation in response to low dose aspirin in 2200 subjects (GeneSTAR, Genetic Study of Aspirin Responsiveness), important genome wide "signals" (p<5x10-8) associated with native platelet aggregation and important "signals" associated with platelet responsiveness to aspirin were identified and replicated. Although we are currently performing functional genomics studies to elucidate our most promising findings in known genes (PEAR1, MET, PIKC3G), most "signals" occurred in intergenic regions or in introns. Mechanistic interpretation is limited by uncertainty as to which gene(s) are up- or down-regulated in the presence of most SNP modifications. In this 3 phase proposal, we will (1) create pluripotent stem cells (iPS) from peripheral blood mononuclear cells, and then differentiate these stem cells into megakaryocytes (2) develop an efficient strategy to produce iPS and megakaryocytes using a novel pooling method, and (3) produce iPS and megakaryocytes from 250 subjects in GeneSTAR (European Americans and African Americans), selected based on specific hypotheses derived from GWAS signals in native and post aspirin platelet function; characterize genetic mRNA transcripts using a comprehensive Affymetrix array; measure protein expression for transcripts of interest using mass spectrometry; examine mRNA and protein expression patterns for each GWAS signal to determine the functional pathway(s) involved in native platelet phenotypes; and examine the functional genomics of variations in responsiveness to aspirin using our prior genotyped and phenotyped population. Precise information about the exact functional processes in megakaryocytes and platelets may lead to innovative and tailored approaches to risk assessment and novel therapeutic targets to prevent first and recurrent cardiovascular and related thrombotic events.</p>
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Project description: Not available
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Project description:The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins
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