Cholesterol Deficiency Causes Impaired Osmotic Stability of Cultured Red Blood Cells.

Claudia Bernecker,Harald Köfeler,Slave Trajanoski,Peter Schlenke,Gerhard A. Holzapfel,Georg Pabst,Karl Strohmayer,Isabel Dorn,Dagmar Kolb,Martin Trötzmüller

doi:10.3389/fphys.2019.01529

Abstract

Ex vivo generation of red blood cells (cRBCs) is an attractive tool in basic research and for replacing blood components donated by volunteers. As a prerequisite for the survival of cRBCs during storage as well as in the circulation, the quality of the membrane is of utmost importance. Besides the cytoskeleton and embedded proteins, the lipid bilayer is critical for membrane integrity. Although cRBCs suffer from increased fragility, studies investigating the lipid content of their membrane are still lacking. We investigated the membrane lipid profile of cRBCs from CD34+ human stem and progenitor cells compared to native red blood cells (nRBCs) and native reticulocytes (nRETs). Ex vivo erythropoiesis was performed in a well-established liquid assay. cRBCs showed a maturation grade between nRETs and nRBCs. High-resolution mass spectrometry analysis for cholesterol and the major phospholipid classes, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin and lysophosphatidylcholin, demonstrated severe cholesterol deficiency in cRBCs. Although cRBCs showed normal deformability capacity, they suffered from increased hemolysis due to minimal changes in the osmotic conditions. After additional lipid supplementation, especially cholesterol during culturing, the cholesterol content of cRBCs increased to a subnormal amount. Concurrently, the osmotic resistance recovered completely and became comparable to that of nRETs. Minor differences in the amount of phospholipids in cRBCs compared to native cells could mainly be attributed to the ongoing membrane remodeling process from the reticulocyte to the erythrocyte stage. Obtained results demonstrate severe cholesterol deficiency as a reason for enhanced fragility of cRBCs. Therefore, the supplementation of lipids, especially cholesterol during ex vivo erythropoiesis may overcome this limitation and strengthens the survival of cRBCs ex vivo and in vivo.

Highlights

Ex vivo generation of red blood cells (RBCs) is a common tool in basic and translational research investigating RBC physiology, RBC affecting disorders and developmental biology
Our results demonstrate the importance of lipid, especially cholesterol supplementation during ex vivo erythropoiesis and its influence on the functionality of cultured red blood cells (cRBCs)
Already on day 8 >95% of cells from both sources expressed the early erythroid marker CD36, in cRBCcb, higher percentages of CD45+ erythroblasts and lower percentages of already glycophorin A+ (GPA+) cells were observed on days 8 and 11 (Supplementary Figures S1C,G)

Summary

INTRODUCTION

Ex vivo generation of red blood cells (RBCs) is a common tool in basic and translational research investigating RBC physiology, RBC affecting disorders and developmental biology. In addition to its lipid-ordering effect, cholesterol is considered to be involved in various processes, including raft formation (Simons and Ikonen, 2000) and specific lipid/protein interactions (Fantini and Barrantes, 2013). Another major membrane lipid is phosphatidylcholine (PC), a glycerophospholipid that creates fluid bilayers when unsaturated. After transendothelial migration into the blood stream, nRETs become functional erythrocytes within 3 days (Chasis et al, 1989) This maturation process includes extensive membrane remodeling, cytoskeletal rearrangement, loss of organelles and RNA and a reduction of the cell volume. Our results demonstrate the importance of lipid, especially cholesterol supplementation during ex vivo erythropoiesis and its influence on the functionality of cRBCs

MATERIALS AND METHODS

RESULTS

DISCUSSION

ETHICS STATEMENT