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:NextGen Consortium: Functional Genomics of Platelet Aggregation Using iPS and Derived Megakaryocytes
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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:Signaling by thrombopoietin (TPO) in complex with its receptor, c-MPL, is critical for hematopoietic stem cell (HSC) homeostasis and platelet generation. Here we show that TA-316, a novel chemically synthesized c-MPL agonist (CMA), is useful for ex vivo platelet generation from human-induced pluripotent stem (iPS) cell-derived immortalized megakaryocyte progenitor cell lines (imMKCLs). Moreover, the generation is clinically applicable, because self-renewal expansion and platelet release is tightly controllable. TA-316 but not eltrombopag, another CMA, promoted both the self-renewal and maturation of imMKCLs, leading to more than a twofold higher platelet production than that achieved with recombinant human TPO (rhTPO). Interestingly, TA-316 seemed to favor MK-biased differentiation from bone marrow CD34+ HSC/progenitors and imMKCLs through the upregulation of vascular endothelial growth factor A and fibroblast growth factor 2. This result suggests TA-316 could facilitate the development of an efficient and useful system to expand platelets from imMKCLs.
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Project description:MicroRNAs (miRNAs) regulate cell physiology by altering protein expression, but the biology of platelet miRNAs is largely unexplored. We tested whether platelet miRNA levels were associated with platelet reactivity by genome-wide profiling using platelet RNA from 19 healthy subjects. We found that human platelets express 284 miRNAs. Unsupervised hierarchical clustering of miRNA profiles resulted in 2 groups of subjects that appeared to cluster by platelet aggregation phenotypes. Seventy-four miRNAs were differentially expressed (DE) between subjects grouped according to platelet aggregation to epinephrine, a subset of which predicted the platelet reactivity response. Using whole genome mRNA expression data on these same subjects, we computationally generated a high-priority list of miRNA-mRNA pairs in which the DE platelet miRNAs had binding sites in 3'UTRs of DE mRNAs, and the levels were negatively correlated. Three miRNA-mRNA pairs (miR-200b:PRKAR2B, miR-495:KLHL5 and miR-107:CLOCK) were selected from this list and all 3 miRNAs knocked down protein expression from the target mRNA. Reduced activation from platelets lacking PRKAR2B supported these findings. In summary, (1) platelet miRNAs are able to repress expression of platelet proteins, (2) miRNA profiles are associated with and may predict platelet reactivity, and (3) bioinformatic approaches can successfully identify functional miRNAs in platelets. Total RNA from the platelets of 19 donors was harvested and labeled with Hy3. Reference RNA (a pool of all samples) was labeled with Hy5. This submission represents the miRNA expression component of the study.
2011-09-26 | E-GEOD-27917 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) regulate cell physiology by altering protein expression, but the biology of platelet miRNAs is largely unexplored. We tested whether platelet miRNA levels were associated with platelet reactivity by genome-wide profiling using platelet RNA from 19 healthy subjects. We found that human platelets express 284 miRNAs. Unsupervised hierarchical clustering of miRNA profiles resulted in 2 groups of subjects that appeared to cluster by platelet aggregation phenotypes. Seventy-four miRNAs were differentially expressed (DE) between subjects grouped according to platelet aggregation to epinephrine, a subset of which predicted the platelet reactivity response. Using whole genome mRNA expression data on these same subjects, we computationally generated a high-priority list of miRNA-mRNA pairs in which the DE platelet miRNAs had binding sites in 3'UTRs of DE mRNAs, and the levels were negatively correlated. Three miRNA-mRNA pairs (miR-200b:PRKAR2B, miR-495:KLHL5 and miR-107:CLOCK) were selected from this list and all 3 miRNAs knocked down protein expression from the target mRNA. Reduced activation from platelets lacking PRKAR2B supported these findings. In summary, (1) platelet miRNAs are able to repress expression of platelet proteins, (2) miRNA profiles are associated with and may predict platelet reactivity, and (3) bioinformatic approaches can successfully identify functional miRNAs in platelets.
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Project description:Tumor-educated platelets (TEPs) are a potential method of liquid biopsy for the diagnosis and monitoring of cancer. However, the mechanism underlying tumor education of platelets is not known, and transcripts associated with TEPs are often not tumor-associated transcripts. We demonstrate that direct tumor transfer of transcripts to circulating platelets is an unlikely source of the TEP signal. We use CDSeq, a latent Dirichlet allocation algorithm, to deconvolute the TEP signal in blood samples from patients with glioblastoma. We demonstrate that a significant proportion of transcripts in the platelet transcriptome are derived from non-platelet cells, and the use of this algorithm allows the removal of contaminant transcripts. Furthermore, we used the results of this algorithm to demonstrate that TEPs represent a subset of more activated platelets, which also contain transcripts normally associated with non-platelet inflammatory cells, suggesting that these inflammatory cells, possibly in the tumor microenvironment, transfer transcripts to platelets that are then found in circulation. Our analysis suggests a useful and efficient method of processing TEP transcriptomic data to enable the isolation of a unique TEP signal associated with specific tumors.
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