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Dexamethasone Predisposes Human Erythroblasts Toward Impaired Lipid Metabolism and Renders Their ex vivo Expansion Highly Dependent on Plasma Lipoproteins.

ABSTRACT: Cultures of stem cells from discarded sources supplemented with dexamethasone, a synthetic glucocorticoid receptor agonist, generate cultured red blood cells (cRBCs) in numbers sufficient for transfusion. According to the literature, however, erythroblasts generated with dexamethasone exhibit low enucleation rates giving rise to cRBCs that survive poorly in vivo. The knowledge that the glucocorticoid receptor regulates lipid metabolism and that lipid composition dictates the fragility of the plasma membrane suggests that insufficient lipid bioavailability restrains generation of cRBCs. To test this hypothesis, we first compared the expression profiling of erythroblasts generated with or without dexamethasone. This analysis revealed differences in expression of 55 genes, 6 of which encoding proteins involved in lipid metabolism. These were represented by genes encoding the mitochondrial proteins 3-Hydroxymethyl-3-Methylglutaryl-CoA lyase, upregulated, and 3-Oxoacid CoA-Transferase1 and glycerol-3-phosphate acyltransferase1, both downregulated, and the proteins ATP-binding cassette transporter1 and Hydroxysteroid-17-Beta-Dehydrogenase7, upregulated, and cAMP-dependent protein kinase catalytic subunit beta, downregulated. This profiling predicts that dexamethasone, possibly by interfering with mitochondrial functions, impairs the intrinsic lipid metabolism making the synthesis of the plasma membrane of erythroid cells depend on lipid-uptake from external sources. Optical and electron microscopy analyses confirmed that the mitochondria of erythroblasts generated with dexamethasone are abnormal and that their plasma membranes present pebbles associated with membrane ruptures releasing exosomes and micro-vesicles. These results indicate that the lipid supplements of media currently available are not adequate for cRBCs. To identify better lipid supplements, we determined the number of erythroblasts generated in synthetic media supplemented with either currently used liposomes or with lipoproteins purified from human plasma [the total lipoprotein fraction (TL) or its high (HDL), low (LDL) and very low (VLDL) density lipoprotein components]. Both LDL and VLDL generated numbers of erythroid cells 3-2-fold greater than that observed in controls. These greater numbers were associated with 2-3-fold greater amplification of erythroid cells due both to increased proliferation and to resistance to stress-induced death. In conclusion, dexamethasone impairs lipid metabolism making ex vivo expansion of erythroid cells highly dependent on lipid absorbed from external sources and the use of LDL and VLDL as lipid supplements improves the generation of cRBCs.

SUBMITTER: Zingariello M

PROVIDER: S-EPMC6458278 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Dexamethasone Predisposes Human Erythroblasts Toward Impaired Lipid Metabolism and Renders Their <i>ex vivo</i> Expansion Highly Dependent on Plasma Lipoproteins.

Zingariello Maria M Bardelli Claudio C Sancillo Laura L Ciaffoni Fiorella F Genova Maria Luisa ML Girelli Gabriella G Migliaccio Anna Rita AR

Frontiers in physiology 20190404

Cultures of stem cells from discarded sources supplemented with dexamethasone, a synthetic glucocorticoid receptor agonist, generate cultured red blood cells (cRBCs) in numbers sufficient for transfusion. According to the literature, however, erythroblasts generated with dexamethasone exhibit low enucleation rates giving rise to cRBCs that survive poorly <i>in vivo</i>. The knowledge that the glucocorticoid receptor regulates lipid metabolism and that lipid composition dictates the fragility of ...[more]

PMID: 31019464

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Project description:Hematopoietic stem and progenitor cells (HSPCs) have been cultured using a wide variety of cytokines to promote differentiation into megakaryocytic cells (Mks), the precursors to platelets. Greater Mk DNA content, or ploidy, has been correlated with increased platelet release. Gradients of pH, pO2, and signaling factors regulate megakaryopoiesis in the bone marrow niche. In this study, we demonstrate that a 3-phase culture process with increasing pH and pO2 and different cytokine cocktails greatly increases megakaryocyte production. CD34(+) HSPCs were first cultured at 5% O2 and pH 7.2 with a cytokine cocktail previously shown to promote Mk progenitor production. At day 5, cells were shifted to 20% O2 and pH 7.4 and maintained in 1 of 17 cytokine cocktails identified using a 2(4) factorial design of experiments method to evaluate the effects of interleukin (IL)-3, IL-6, IL-9, and high- or low-dose stem cell factor (SCF), in conjunction with thrombopoietin (Tpo) and IL-11, on expansion of mature Mks from progenitors. The combination of Tpo, high-dose SCF, IL-3, IL-9, and IL-11 best promoted Mk expansion. IL-3 greatly increased total cell fold expansion, but this was partially offset by lower Mk purity. IL-9 promoted CD41 and CD42b expression. High-dose (100?ng/mL) SCF increased Mk production and ploidy. Different commercial media and IL-3 sources substantially impacted differentiation, and X-VIVO 10 serum-free media best supported mature Mk expansion. Shifting from pH 7.4 to pH 7.6 at day 7 increased Mk production by 30%. Treatment with nicotinamide at day 7 or day 8 more than doubled the fraction of high-ploidy (>4N) Mks. Ultimately, the 3-phase culture system gave rise to 44.5±8.1?Mks and 8.5±3.1 high-ploidy Mks per input HSPC. Further optimization was required to improve platelet production. Using Iscove's modified Dulbecco's medium (IMDM)+20% BSA, insulin and transferin (BIT) 9500 Serum Substitute greatly improved the frequency and quality of Mk proplatelet extensions without affecting Mk expansion, commitment, or polyploidization in the 3-phase process. Mks cultured in IMDM+20% BIT 9500 gave rise to platelets with functional activity similar to that of fresh platelets from normal donors, as evidenced by basal tubulin distribution and the expression of surface markers and spreading in response to platelet agonists.

Project description:Aims/hypothesisRegulatory factor X 6 (RFX6) is crucial for pancreatic endocrine development and differentiation. The RFX6 variant p.His293LeufsTer7 is significantly enriched in the Finnish population, with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 and gestational diabetes. However, the precise mechanism of this predisposition remains unknown.MethodsTo understand the role of this variant in beta cell development and function, we used CRISPR technology to generate allelic series of pluripotent stem cells. We created two isogenic stem cell models: a human embryonic stem cell model; and a patient-derived stem cell model. Both were differentiated into pancreatic islet lineages (stem-cell-derived islets, SC-islets), followed by implantation in immunocompromised NOD-SCID-Gamma mice.ResultsStem cell models of the homozygous variant RFX6-/- predictably failed to generate insulin-secreting pancreatic beta cells, mirroring the phenotype observed in Mitchell-Riley syndrome. Notably, at the pancreatic endocrine stage, there was an upregulation of precursor markers NEUROG3 and SOX9, accompanied by increased apoptosis. Intriguingly, heterozygous RFX6+/- SC-islets exhibited RFX6 haploinsufficiency (54.2% reduction in protein expression), associated with reduced beta cell maturation markers, altered calcium signalling and impaired insulin secretion (62% and 54% reduction in basal and high glucose conditions, respectively). However, RFX6 haploinsufficiency did not have an impact on beta cell number or insulin content. The reduced insulin secretion persisted after in vivo implantation in mice, aligning with the increased risk of variant carriers to develop diabetes.Conclusions/interpretationOur allelic series isogenic SC-islet models represent a powerful tool to elucidate specific aetiologies of diabetes in humans, enabling the sensitive detection of aberrations in both beta cell development and function. We highlight the critical role of RFX6 in augmenting and maintaining the pancreatic progenitor pool, with an endocrine roadblock and increased cell death upon its loss. We demonstrate that RFX6 haploinsufficiency does not affect beta cell number or insulin content but does impair function, predisposing heterozygous carriers of loss-of-function variants to diabetes.Data availabilityUltra-deep bulk RNA-seq data for pancreatic differentiation stages 3, 5 and 7 of H1 RFX6 genotypes are deposited in the Gene Expression Omnibus database with accession code GSE234289. Original western blot images are deposited at Mendeley ( https://data.mendeley.com/datasets/g75drr3mgw/2 ).

Dataset Information

Dexamethasone Predisposes Human Erythroblasts Toward Impaired Lipid Metabolism and Renders Their ex vivo Expansion Highly Dependent on Plasma Lipoproteins.

Publications

Dexamethasone Predisposes Human Erythroblasts Toward Impaired Lipid Metabolism and Renders Their <i>ex vivo</i> Expansion Highly Dependent on Plasma Lipoproteins.

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