Differentiating erythroblasts adapt to turbulent flow by accelerating maturation and activating cholesterol biosynthesis.
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ABSTRACT:
PROVIDER: PRJNA1036247 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR26684825_1.fastq.gz | Fastqsanger.gz | |||
| SRR26684825_2.fastq.gz | Fastqsanger.gz | |||
| SRR26684826_1.fastq.gz | Fastqsanger.gz | |||
| SRR26684826_2.fastq.gz | Fastqsanger.gz | |||
| SRR26684827_1.fastq.gz | Fastqsanger.gz |
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Project description:In vitro culture of erythroblasts and production of mature erythrocytes for transfusion purposes requires upscaling in bioreactors with turbulent fluidic environments, resulting in membrane shear stress. For the implementation of erythroid cultures in bioreactors, further understanding of the effects of mechanical stress on terminal erythroblast differentiation are required. To this end, we investigated the effect of orbital shaking-induced shear stress on differentiation of CD49d+CD235low primary human erythroblast (EBL) towards enucleated reticulocytes at the molecular, cellular, and functional level. Orbital shaking at the onset of erythroblast differentiation enhanced cell maturation and increased enucleation percentage compared to static cultures, without loss of cell viability. Transcriptome analysis uncovered 505 genes to be differentially expressed between static and non-static cultures, where genes involved in lipids and cholesterol biosynthesis pathways where upregulated in non-static conditions. In line with this finding, cells differentiated in orbital shakers showed an increased cholesterol concentration and higher osmotic resistance compared to cells differentiated in static condition. HMGCR (3-Hydroxy-3-Methylglutaryl-CoA-Reductase), a rate limiting enzyme of the cholesterol biosynthesis pathway, is significantly increased and showed earlier induction during differentiation in shear stress environments. Inhibition of HMGCR using lovastatin led to severe loss of EBL differentiation in shear environments but not in static conditions. In conclusion, differentiating EBL display a dynamic capability to adapt to shear stress environments, by modulating their transcriptional program and consequently upregulating cholesterol biosynthesis, which is critical for their survival. This work sheds light into specific mechanisms which will assist on the successful upscaling of erythroid differentiation in turbulent bioreactors.
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Project description:Human erythroblasts purified from cord blood were cultured in vitro and FACS-sorted into five highly purified populations representing distinct differentiation stages: proerythroblasts, early basophilic erythroblasts, late basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromatophilic erythroblasts. The methods for culture and sorting experiments are given in Hu et al. 2013. For each RNA-seq library, RNA was isolated from 1x 106 sorted human erythroblasts using RNeasy Plus Mini kits (Qiagen). Libraries were then prepared using Illumina TruSeqTM RNA kits to obtain 50 nt reads. Collaborators at the New Your Blood Center were responsible for erythroblast culture, FACS purification of erythroblast populations, and acquisition of RNA-seq data. Collaborators at U.C. Berkeley and Lawrence Berkeley National Laboratory performed data analysis and experimental validation of alternative splicing in erythroblasts. Results: Differentiating erythroblasts execute a dynamic alternative splicing program that is enriched in genes affecting cell cycle, organelle organization, chromatin function, and RNA processing. Alternative splicing plays a major role in regulating gene expression to ensure synthesis of appropriate proteome at each stage as the cells remodel in preparation for production of mature red cells.
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Project description:Human erythroblasts purified from cord blood were cultured in vitro and FACS-sorted into five highly purified populations representing distinct differentiation stages: proerythroblasts, early basophilic erythroblasts, late basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromatophilic erythroblasts. The methods for culture and sorting experiments are given in Hu et al. 2013. For each RNA-seq library, RNA was isolated from 1x 106 sorted human erythroblasts using RNeasy Plus Mini kits (Qiagen). Libraries were then prepared using Illumina TruSeqTM RNA kits to obtain 50 nt reads. Collaborators at the New Your Blood Center were responsible for erythroblast culture, FACS purification of erythroblast populations, and acquisition of RNA-seq data. Collaborators at U.C. Berkeley and Lawrence Berkeley National Laboratory performed data analysis and experimental validation of alternative splicing in erythroblasts. Results: Differentiating erythroblasts execute a dynamic alternative splicing program that is enriched in genes affecting cell cycle, organelle organization, chromatin function, and RNA processing. Alternative splicing plays a major role in regulating gene expression to ensure synthesis of appropriate proteome at each stage as the cells remodel in preparation for production of mature red cells. Erythroid differentiation stage-specific transcriptome analysis was performed by RNA-seq analysis of highly purified erythroblast populations
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Project description:Transcriptome analysis of total RNA samples from FACS sorted erythroblasts To further verify the role of cytokine triggered inflammation signaling in erythropoiesis, we decided to follow the profile of gene expression in erythroblasts using DNA microarrays. To elucidated the gene expression pattern in cytokine, especially IL-6, challenged erythroblasts, in vitro IL-6 treated erythroblasts in EPO containing medium both at day 1 and day 2 were collected. We also sorted the erythroblasts at different stages (Proerythroblasts(I),Basophilic(II) and polychromatic(III) erythroblasts,Orthochromatic(IV) erythroblasts) from both DWT and DKO bone marrow cells through fluorescence activated cell sorting. Total RNA were extracted and processed to hybridize with Affymetrix mouse Clariom S microarrays.
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Project description:This study analyzes gene expression in beta-thalassemic fetal liver erythroblasts in the Th3 murine model. FACS-purified wild-type, heterozygous, and homozygous stage-matched erythroblasts from E14.5 fetal livers are compared.
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Project description:Erythroblasts from human cord blood
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Project description:Erythroblasts-rich fraction expanded from three healthy commercial available cord blood mononuclear cells were analyzed.
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Project description:This study analyzes gene expression in beta-thalassemic fetal liver erythroblasts in the Th3 murine model. FACS-purified wild-type, heterozygous, and homozygous stage-matched erythroblasts from E14.5 fetal livers are compared. We used FACS to purify CD71+Ter119+FSChigh matched populations from E14.5 fetal livers of wild-type, Th3/+, and Th3/Th3 embryos
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