Engineering synthetic signaling receptors to enable erythropoietin-free erythropoiesis
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ABSTRACT:
PROVIDER: PRJNA1205380 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR31866700_1.fastq.gz | Fastqsanger.gz | |||
| SRR31866700_2.fastq.gz | Fastqsanger.gz | |||
| SRR31866701_1.fastq.gz | Fastqsanger.gz | |||
| SRR31866701_2.fastq.gz | Fastqsanger.gz | |||
| SRR31866702_1.fastq.gz | Fastqsanger.gz |
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Project description:Blood transfusion plays a vital role in modern medicine. However, frequent shortages pose a significant healthcare challenge. Ex vivo manufacturing of red blood cells (RBCs) derived from universal donor pluripotent stem cells is limited by the high cost of recombinant cytokines required for ex vivo erythroid differentiation. Erythropoietin (EPO) signaling through the EPO receptor is indispensable to RBC development, and EPO is one of the most expensive components in erythroid-promoting media. Here, we use design-build-test cycles to develop highly optimized small molecule-inducible synthetic EPO receptors (synEPORs) which are integrated at a variety of genomic loci using homology-directed repair genome editing. We find that integration of synEPOR at the endogenous EPOR locus in an induced pluripotent stem cell producer line cultured with small molecule yields equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production compared to cells cultured with EPO. Due to the dramatically lower cost of small molecules vs. recombinant cytokines, these efforts eliminate one of the most expensive elements of ex vivo culture media—EPO cytokine. As cytokines dependence is a common barrier to ex vivo cell production, these strategies could improve scalable manufacturing of multiple clinically relevant cell types.
2025-01-09 | GSE285656 | GEO
Project description:Blood transfusion plays a vital role in modern medicine, but frequent shortages occur. Ex vivo manufacturing of red blood cells (RBCs) from universal donor cells offers a potential solution, yet the high cost of recombinant cytokines remains a barrier. Erythropoietin (EPO) signaling is crucial for RBC development, and EPO is among the most expensive media components. To address this challenge, we develop highly optimized small molecule-inducible synthetic EPO receptors (synEPORs) using design-build-test cycles and genome editing. By integrating synEPOR at the endogenous EPOR locus in O-negative induced pluripotent stem cells, we achieve equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production using small molecules compared to EPO-supplemented cultures. This approach dramatically reduces culture media costs. Our strategy not only addresses RBC production challenges but also demonstrates how protein and genome engineering can introduce precisely regulated cellular behaviors, potentially improving scalable manufacturing of a wide range of clinically relevant cell types.
| S-EPMC11779867 | biostudies-literature
Project description:Normal erythropoiesis requires a critical balance between proapoptotic and antipaoptotic pathways. Bcl-xl, an antiapoptotic protein is induced at end-stages of differentiation of erythroid precursors in response to erythropoietin. The details of the proapoptotic pathway and the critical proapoptotic proteins inhibited by Bcl-xl in erythropoiesis are not well understood. We employed gene targeting to ablate Nix, a proapoptotic BH3-domain only Bcl2 family protein, which is known to be transcriptionally induced during erythropoiesis. Nix null mice exhibited reticulocytosis and thrombocytosis in the peripheral blood; and profound splenomegaly with erythroblastosis in the spleen and bone marrow despite normal erythropoietin levels and blood oxygen tension. In vivo apoptosis was diminished in erythroblast precursors from Nix null spleens. To define the molecular consequences of Nix ablation on apoptosis and erythropoiesis, we conducted a detailed comparative analysis of gene expression in spleens from 8 week old Nix null mice and wild type controls. Of 45,101 genes analyzed, 514 were significantly upregulated and 386 down-regulated in Nix-/- splenocytes. Functional cluster analysis delineated the ten most highly regulated gene sets, revealing increased levels of cell cycle and erythroid genes, with decreased levels of cell death and B-cell genes. Keywords: strain comparison
2007-02-14 | GSE7020 | GEO
Project description:Erythropoietin (EPO) is the primary regulator of erythropoiesis in the mammalian fetus and the adult through interaction with erythropoietin receptor (EPOR). Deficiency of EPO induces anemia that is a major cause of death in patients with chronic kidney disease. Thus, development of safe treatment for anemia is an urgent issue. In this study, we investigated the effect of γ-aminobutyric acid (GABA) on serum EPO level and erythropoiesis, and its mechanism was elucidated in rat. GABA significantly (P < 0.05) increased EPO level in serum and expression levels of EPO and EPOR in a dose dependent manner. GABA increased the expression levels of HIF-1 and HIF-2 in a dose dependent manner compared with the negative control; while GABA did not affect the expression of prolyl-hydroxylase domain protein-2α (PHD-2α) gene, an oxygen sensor. GABA supplementation alters energy production pathway resulted in hypoxic condition, which increases EPO level in rat through overexpression of HIF-1 and HIF-2. This study shows a new physiological role of GABA in EPO production and thus GABA could contribute to the prevention of anemia by using alone or in combination with other anemia treating drugs.
2020-03-04 | GSE146299 | GEO
Project description:Hypoxia is associated with increased erythropoietin (EPO) release to drive erythropoiesis. However, a prolonged sojourn at high altitude results in an increase in EPO levels followed by a decrease, although erythropoiesis remains elevated at a stable level. The role of hypoxia and related EPO adjustments are not fully understood and contributed to the formulation of the theory of neocytolysis. In this study, we aimed to exclusively evaluate the role of oxygen on erythropoiesis comparing in vitro erythroid differentiation performed at atmospheric oxygen, with a lower oxygen concentration (3% O2) and with cultures of erythroid precursors isolated from peripheral blood after a 19-day sojourn at high altitude (3450 m). Results highlight an accelerated erythroid maturation at low oxygen and more concave morphology of reticulocytes. No differences in deformability were observed in the formed reticulocytes in the tested conditions. Moreover, hematopoietic stem and progenitor cells isolated from blood affected by hypoxia at high altitude did not result in a different erythroid development, suggesting no retention of high altitude signature but rather an immediate adaptation to oxygen concentration. This adaptation was observed during in vitro erythropoiesis at 3% oxygen, displaying a significantly increased glycolytic metabolic profile. These hypoxia-induced effects on in vitro erythropoiesis fail to provide an intrinsic explanation to the concept of neocytolysis.
2022-04-06 | PXD031776 | Pride
Project description:Normal erythropoiesis requires a critical balance between proapoptotic and antipaoptotic pathways. Bcl-xl, an antiapoptotic protein is induced at end-stages of differentiation of erythroid precursors in response to erythropoietin. The details of the proapoptotic pathway and the critical proapoptotic proteins inhibited by Bcl-xl in erythropoiesis are not well understood. We employed gene targeting to ablate Nix, a proapoptotic BH3-domain only Bcl2 family protein, which is known to be transcriptionally induced during erythropoiesis. Nix null mice exhibited reticulocytosis and thrombocytosis in the peripheral blood; and profound splenomegaly with erythroblastosis in the spleen and bone marrow despite normal erythropoietin levels and blood oxygen tension. In vivo apoptosis was diminished in erythroblast precursors from Nix null spleens. To define the molecular consequences of Nix ablation on apoptosis and erythropoiesis, we conducted a detailed comparative analysis of gene expression in spleens from 8 week old Nix null mice and wild type controls. Of 45,101 genes analyzed, 514 were significantly upregulated and 386 down-regulated in Nix-/- splenocytes. Functional cluster analysis delineated the ten most highly regulated gene sets, revealing increased levels of cell cycle and erythroid genes, with decreased levels of cell death and B-cell genes. Experiment Overall Design: In the study, we hybridized RNA from 8 week old spleens of wild type (WT) control and Nix null mice (Nix-/-) to Affymetrix MOE430A GeneChip® arrays containing 45,101 well characterized mouse genes/ESTs.
2007-11-07 | E-GEOD-7020 | biostudies-arrayexpress
Project description:Development of red blood cells from progenitors requires profound reshaping of both gene expression and metabolism. How these processes are coupled is unclear. Nprl3, an inhibitor of mTORC1, has remained in synteny with the -globin genes for >500 million years, and harbours most of the -globin enhancers. However, whether Nprl3 itself serves an erythroid role is unknown. While hematopoietic progenitors rely on tonic expression of baseline Nprl3, erythroblasts experience ‘boosted’ Nprl3 expression. Using Nprl3-deficient fetal liver and adult competitive bone marrow - fetal liver chimaeras, we show that NprI3 is required for sufficient erythropoiesis. Loss of Nprl3 elevates mTORC1 signalling, suppresses autophagy and disrupts erythroblast glycolysis. Human NPRL3-knockout erythroid progenitors produce fewer enucleated cells and demonstrate dysregulated mTORC1 signalling in response to nutrient availability and erythropoietin. Thus, Nprl3 is a key regulator of erythroid metabolism. Finally, we show that the alpha-globin enhancers upregulate erythoid NprI3 expression, and that this activity supports optimal erythropoiesis.
2025-02-12 | PXD060557 | Pride
Project description:High-dose erythropoiesis-stimulating agents (ESA) for anemia of chronic kidney disease (CKD) have been associated with adverse clinical outcomes and do not always improve erythropoiesis. We hypothesized that high-dose ESA requirement would be associated with elevated inflammatory biomarkers, decreased adipokines, and increased circulating, endogenous soluble erythropoietin receptors (sEpoR).A cross-sectional cohort of anemic 32 CKD participants receiving ESA were enrolled at a single center and cytokine profiles, adipokines, and sEpoR were compared between participants stratified by ESA dose requirement (usual-dose darbepoetin-? (< 1 ?g/kg/week) and high-dose (? 1 ?g/kg/week)).Baseline characteristics were similar between groups; however, hemoglobin was lower among participants on high-dose (1.4 ?g/kg/week) vs usual-dose (0.5 ?g/kg/week) ESA.In adjusted analyses, high-dose ESA was associated with an increased odds for elevations in c-reactive protein and interleukin-6 (p < 0.05 for both). There was no correlation between high-dose ESA and adipokines. Higher ESA dose correlated with higher levels of sEpoR (rs = 0.39, p = 0.03). In adjusted analyses, higher ESA dose (per ?cg/kg/week) was associated with a 53% greater odds of sEpoR being above the median (p < 0.05).High-dose ESA requirement among anemic CKD participants was associated with elevated inflammatory biomarkers and higher levels of circulating sEpoR, an inhibitor of erythropoiesis. Further research confirming these findings is warranted.Clinicaltrials.gov NCT00526747.
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