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Metabolomics-based elucidation of active metabolic pathways in erythrocytes and HSC-derived reticulocytes Anubhav Srivastava, Krystal J Evans, Anna Elizabeth Sexton, Louis Schofield, and Darren John Creek J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00902 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 8, 2017
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Metabolomics-based elucidation of active metabolic pathways in erythrocytes and HSC-derived reticulocytes Anubhav Srivastava1†, Krystal J. Evans2†, Anna E. Sexton1, Louis Schofield2,
3
and
Darren J. Creek1 * 1. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia 2. Walter and Eliza Hall Institute of Medical Research, Division of Infection and Immunity, Parkville, Victoria, Australia 3. Australian Institute of Tropical Health and Medicine, James Cook University, Douglas, Queensland, Australia †
These authors contributed equally.
*
Corresponding author:
[email protected] ; Phone: +61399039249
Abstract A detailed analysis of the metabolic state of human stem cell derived erythrocytes allowed us to characterize the existence of active metabolic pathways in younger 1 ACS Paragon Plus Environment
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reticulocytes and compare them to mature erythrocytes. Using high resolution LCMS based, untargeted metabolomics, we found that reticulocytes had a comparatively much richer repertoire of metabolites, which spanned a range of metabolite classes. An untargeted metabolomics analysis using stable isotopelabelled glucose showed that only glycolysis and the pentose phosphate pathway actively contributed to the biosynthesis of metabolites in erythrocytes, and these pathways were upregulated in reticulocytes. Most metabolite species found to be enriched in reticulocytes were residual pools of metabolites produced by earlier erythropoietic processes, and their systematic depletion in mature erythrocytes aligns with the simplification process which is also seen at the cellular and the structural level. Our work shows that high resolution LC-MS based, untargeted metabolomics provides a global coverage of the biochemical species which are present in erythrocytes. However, the incorporation of stable isotope labelling provides a more accurate description of the active metabolic processes that occur in each developmental stage. To our knowledge, this is the first detailed characterization of the active metabolic pathways of the erythroid lineage and it provides a rich database for understanding the physiology of the maturation of reticulocytes in to mature erythrocytes.
Keywords Metabolomics, stable-isotope labelling, erythrocytes, reticulocytes, metabolism. Introduction Human stem cell (HSC)-derived erythrocytes offer a highly useful model to study red blood cell biology throughout their development, and present an exciting prospect for therapeutic production of ex vivo erythrocytes. A number of factors responsible for the differentiation and growth of CD34+ hematopoietic progenitor cells have been 2 ACS Paragon Plus Environment
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identified and successfully applied to generate erythrocytes under laboratory conditions1-4. Apart from the exciting prospect of generating red blood cells for clinical transfusion5, the ability to generate human erythrocytes in vitro has also allowed the scientific community to obtain sufficient numbers of younger red blood cells (reticulocytes) to study. Stem cell derived reticulocytes can aid the study of infectious diseases by the development of in vitro culture methodologies6 for reticulocyte-invading species of malaria parasites, such as Plasmodium vivax7, 8. Traditional in vitro methods to study these species have previously relied on the limited number of reticulocytes obtained from peripheral blood (naturally 1-2% of red blood cells)9, 10. Ready access to large numbers of reticulocytes facilitates investigations of basic erythrocyte physiology and metabolism using modern molecular and systems biology techniques. Mature erythrocytes, which comprise of almost 98% of circulating red blood cells, were shown to be metabolically active in some of the earliest biochemical studies, with the major metabolic pathways being glycolysis11 and the pentose phosphate pathway12. Studies on erythrocytes involving radioisotope labelling and metabolic inhibitors linked phosphate permeability to glycolysis, and established the existence of an active ion transport mechanism13. Abnormalities in erythrocytic enzymes involved in glycolysis and the Luebering-Rapoport shunt, which is unique to mammalian erythrocytes, have been studied in detail by biochemical and genetic methods14. Other pathways have also been described in erythrocytes, including fatty acid synthesis (FASI pathway)15 and nucleotide salvage pathways16. Furthermore, erythrocyte metabolism and oxygen carrying capacity have been simulated in an in silico model17,
18
, and we have recently applied untargeted
metabolomics to study rodent erythrocyte metabolism19. 3 ACS Paragon Plus Environment
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Development and maturation of red blood cells is a multistep process, which begins in the bone marrow and continues in the peripheral circulation20,
21
. There exists a
hierarchy in structural complexity in the erythrocyte stages involved in the process of erythropoiesis, as the bone marrow precursors of erythroid lineage are much more complex than the enucleated young red blood cells (reticulocytes)20 which in turn are more complex than their mature counterpart erythrocytes21-24. The simplification of red blood cell biology from reticulocyte to mature erythrocyte in peripheral circulation has been documented at the transcriptome25 and proteome levels26-28. Recently, attempts have also been made to study the metabolism of cord blood reticulocytes29 30
and that of sickle cell disease erythrocytes31,
32
. We19 and others33 have also
reflected on the metabolic complexity of reticulocytes and their role in supporting species specific growth of malaria parasites. However, to date, reticulocyte metabolomics studies have looked at the steady state levels of metabolites in reticulocytes to draw conclusions about their biochemistry or infer their role in malaria biology. The active metabolic pathways in HSC derived reticulocytes have not been investigated by stable isotope tracing methodologies. Metabolomics is a powerful and unbiased approach to study complex biological systems in depth and provides a functional readout of biological processes at the cellular level. Metabolomics typically involves measurement of the abundance of small molecule metabolites from diverse metabolic pathways, and bridges the gaps between the genome, transcriptome, proteome and the ultimate phenotype of the organism studied. A notable limitation of untargeted metabolomics is the inability to directly determine the activity of metabolic pathways based on steady state metabolite levels. Tracer fate studies using stable isotope labelled metabolic precursors allow direct measurement of intracellular metabolite turnover, and 4 ACS Paragon Plus Environment
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indicate the level of activity in pre-defined metabolic pathways. These targeted labelling approaches have led to important biological discoveries ranging from parasite34, 35 and cancer biology36, 37 to identification of novel disease biomarkers38. Recent advances in high resolution mass spectrometry, coupled with the availability of genome scale metabolic networks, has enabled the combination of stable isotope tracing with untargeted metabolomics, allowing detection of the active metabolic pathways throughout the metabolic network39-41. This
study
applied
untargeted
metabolomics
and
stable
isotope
labelled
metabolomics to establish the metabolic differences between HSC derived reticulocytes and mature erythrocytes. The stable isotope labelling approach demonstrated the limited extent of active metabolism in reticulocytes, which is primarily associated with glycolysis and sugar phosphate pathways. Experimental Procedures Production of Human CD34+ stem cell derived cultured reticulocytes Peripheral blood mononucleated cells were obtained from blood by Percoll density purification and CD34+ hemopoietic progenitor cells were isolated by magnetic bead separation according to the manufacturer's instructions (Miltenyi Biotec). CD34+ cells were cultured in a three-stage protocol based on methods previously described24. Initially cells were cultured at 37 °C in a humid atmosphere of 5% CO2 at a density of 1 x 104 cells/mL and then maintained in the range of 2-10 × 105 cells/mL in IMDM (LifeTech) containing 5% (v/v) AB Serum (Interstate Companies Laboratories), 10 µg/mL Insulin (Sigma), 3 U/mL heparin (Pfizer), 200 µg/mL Transferrin (Prospec), 3 U/mL EPO (Eprex). During stage one (days 0-8) this was supplemented with 10 ng/mL SCF (GenScript) and 1 ng/mL IL-3 (R&D systems);
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during stage two (days 8-11) with 10 ng/mL SCF and additional 800 µg/mL transferrin and stage 3 (days 11-18) with 3 U/mL EPO and additional 800 µg/mL transferrin. Cultured reticulocytes were filtered at day 18 using a PALL WBF leukocyte filter to select for enucleated reticulocytes. Isogenic control red blood cells (RBCs) were retained from donor blood, washed in IMDM and stored in salineadenine-glucose-mannitol solution (SAG-M) at 4°C prior to use. Before analysis, cells were washed and cultured overnight in stage 3-supplemented IMDM (as outlined above). Metabolic labelling and sample extraction Metabolic labelling was carried out by incubating reticulocytes and mature erythrocytes in the presence of 50% U-13C-glucose labelledfor 1 or 20 hours and harvested for metabolite extraction. To achieve 50% labelled glucose, 10 mM U-13Cglucose was added to the stage 3-IMDM media (which contains 10 mM U-12Cglucose). The stage 3-IMDM media was replaced with the 50% labelled media either 20 hours, or 1 hour, before sample extraction. All samples were extracted at the same time to avoid time-dependent variability, including the unlabelled control samples, which were exposed to stage 3-IMDM media (with unlabelled glucose) throughout incubation. Metabolism was quenched by immersion of cultures in an ethanol/dry ice bath to 04 °C. Cells were pelleted by centrifugation (10,000 rpm for 1 min at 4°C) and washed in cold PBS (1 mL). Metabolites were extracted from 1 x 108 RBCs and 0.5 x 108 reticulocytes by addition of 300 µL chloroform/methanol/water (1:3:1 v/v) containing internal standards (CHAPS, CAPS, PIPES and TRIS; 1 µM) and left for 30 mins at 4 °C with periodic mixing and sonication. The number of cells used for RBCs and reticulocytes was based on the mean cell volume of each cell type measured using a 6 ACS Paragon Plus Environment
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Coulter counter. This ensured that the total cell pellet volume was equivalent for each sample, allowing direct comparison of metabolite concentrations from these samples. After mixing, cellular debris was removed by centrifugation (16,000 rpm for 10 mins at 4°C) and the supernatant was kept at -80 °C prior to analysis. Three biological replicates were prepared for each cell line, and the whole experiment was repeated with blood from a different donor, which revealed consistent results (all data in Files S1 and S2). LCMS analysis Samples were analyzed by hydrophilic interaction liquid chromatography coupled to high resolution-mass spectrometry (LC-MS) according to a previously published method42. Briefly, the chromatography utilized a ZIC-p(HILIC) column (column temperature 25 °C) with a gradient elution of 20 mM ammonium carbonate (A) and acetonitrile (B) (linear gradient time- %B as follows: 0min- 80%, 15min- 50%, 18min5%, 21min- 5%, 24min- 80%, 32min- 80%) on a Dionex RSLC3000 UHPLC (Thermo). The flow rate was maintained at 300 µl/min. Samples were kept at 4 °C in the autosampler and 10 µL injected for analysis. The mass spectrometry was performed at 35,000 resolution (accuracy calibrated to
