Exploring Transcriptomic Landscapes in Red Blood Cells, in Their Extracellular Vesicles and on A Single-Cell Level

Being enucleated, RBCs lack typical transcriptomes, but are known to contain small amounts of diverse long transcripts and microRNAs. However, the exact role and importance of these RNAs are lacking. Shedding of extracellular vesicles (EVs) from the plasma membrane constitutes an integral mechanism of RBC homeostasis, by which RBCs remove unnecessary cytoplasmic content and cell membrane. To study this further, we explored the transcriptomes of RBCs and extracellular vesicles (EVs) of RBCs using next-generation sequencing. Furthermore, we performed single-cell RNA sequencing on RBCs, which revealed that approximately 10% of the cells contained detectable levels of mRNA and cells formed three subpopulations based on their transcriptomes. A decrease in the mRNA quantity was observed across the populations. Qualitative changes included the differences in the globin transcripts and changes in the expression of ribosomal genes. A specific splice form of a long non-coding RNA, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), was the most enriched marker in one subpopulation of RBCs, co-expressing with ribosomal structural transcripts. MALAT1 expression was confirmed by qPCR in CD71-enriched reticulocytes, which were also characterized with imaging flow cytometry at the single cell level. Analysis of the RBC transcriptome shows enrichment of pathways and functional categories required for the maturation of reticulocytes and erythrocyte functions. The RBC transcriptome was detected in their EVs, making these transcripts available for intercellular communication in blood

Verisolujen solunulkoiset vesikkelit

Peer reviewe

Mesenchymal Stromal Cells and Their Extracellular Vesicles Enhance the Anti-Inflammatory Phenotype of Regulatory Macrophages by Downregulating the Production of Interleukin (IL)-23 and IL-22

Resolution-phase macrophage population orchestrates active dampening of the inflammation by secreting anti-inflammatory and proresolving products including interleukin (IL)-10 and lipid mediators (LMs). We investigated the effects of both human bone marrow-derived mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (MSC-EVs) on mature human regulatory macrophages (Mregs). The cytokines and LMs were determined from cell culture media of Mregs cultivated with MSCs and MSC-EVs. In addition, the alterations in the expression of cell surface markers and the phagocytic ability of Mregs were investigated. Our novel findings indicate that both MSC coculture and MSC-EVs downregulated the production of IL-23 and IL-22 enhancing the anti-inflammatory phenotype of Mregs and amplifying proresolving properties. The levels of prostaglandin E-2 (PGE(2)) were substantially upregulated in MSC coculture media, which may endorse proresolving LM class switching. In addition, our results manifest, for the first time, that MSC-EVs mediate the Mreg phenotype change via PGE(2). These data suggest that both human MSC and MSC-EVs may potentiate tolerance-promoting proresolving phenotype of human Mregs.Peer reviewe

Label-free characterization and real-time monitoring of cell uptake of extracellular vesicles

Extracellular vesicles (EVs) have the ability to function as molecular vehicles and could therefore be harnessed to deliver drugs to target cells in diseases such as cancer. The composition of EVs determines their function as well as their interactions with cells, which consequently affects the cell uptake efficacy of EVs. In this study, we present two novel label-free approaches for studying EVs; characterization of EV composition by time-gated surface-enhanced Raman spectroscopy (TG-SERS) and monitoring the kinetics and amount of cellular uptake of EVs by surface plasmon resonance (SPR) in real-time. Using these methods, we characterized the most abundant EVs of human blood, red blood cell (RBC)- and platelet (PLT)-derived EVs and studied their interactions with prostate cancer cells. Complementary studies were performed with nanoparticle tracking analysis for concentration and size determinations of EVs, zeta potential measurements for surface charge analysis, and fluorophore-based confocal imaging and flow cytometry to confirm EV uptake. Our results revealed distinct biochemical features between the studied EVs and demonstrated that PLT-derived EVs were more efficiently internalized by PC-3 cells than RBC-derived EVs. The two novel label-free techniques introduced in this study were found to efficiently complement conventional techniques and paves the way for further use of TG-SERS and SPR in EV studies.Peer reviewe

Polyunsaturated fatty acids modify the extracellular vesicle membranes and increase the production of proresolving lipid mediators of human mesenchymal stromal cells

Human mesenchymal stromal/stem cells (hMSCs) are used in experimental cell therapy to treat various immunological disorders, and the extracellular vesicles (hMSC-EVs) they produce have emerged as an option for cell-free therapeutics. The immunomodulatory function of hMSCs resembles the resolution of inflammation, in which proresolving lipid mediators (LMs) play key roles. Multiple mechanisms underlying the hMSC immunosuppressive effect has been elucidated; however, the impact of LMs and EVs in the resolution is poorly understood. In this study, we supplemented hMSCs with polyunsaturated fatty acids (PUFAs); arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, which serve as precursors for multiple LMs. We then determined the consequent compositional modifications in the fatty acid, phospholipid, and LM profiles. Mass spectrometric analyses revealed that the supplemented PUFAs were incorporated into the main membrane phospholipid classes with different dynamics, with phosphatidylcholine serving as the first acceptor. Most importantly, the PUFA modifications were transferred into hMSC-EVs, which are known to mediate hMSC immunomodulation. Furthermore, the membrane-incorporated PUFAs influenced the LM profile by increasing the production of downstream prostaglandin E-2 and proresolving LMs, including Resolvin E2 and Resolvin D6. The production of LMs was further enhanced by a highly proinflammatory stimulus, which resulted in an increase in a number of mediators, most notably prostaglandins, while other stimulatory conditions had less a pronounced impact after a 48-h incubation. The current findings suggest that PUFA manipulations of hMSCs exert significant immunomodulatory effects via EVs and proresolving LMs, the composition of which can be modified to potentiate the therapeutic impact of hMSCs.Peer reviewe

Transcriptome Profiling of Human Pre-Implantation Development

BACKGROUND: Preimplantation development is a crucial step in early human development. However, the molecular basis of human preimplantation development is not well known. METHODOLOGY: By applying microarray on 397 human oocytes and embryos at six developmental stages, we studied the transcription dynamics during human preimplantation development. PRINCIPAL FINDINGS: We found that the preimplantation development consisted of two main transitions: from metaphase-II oocyte to 4-cell embryo where mainly the maternal genes were expressed, and from 8-cell embryo to blastocyst with down-regulation of the maternal genes and up-regulation of embryonic genes. Human preimplantation development proved relatively autonomous. Genes predominantly expressed in oocytes and embryos are well conserved during evolution. SIGNIFICANCE: Our database and findings provide fundamental resources for understandin

Immunomodulatory Properties of Packed Red Blood Cells during Storage

Introduction: Red blood cell (RBC) transfusion may affect the recipient immune system. During RBC storage in an unphysiological environment, RBC quality and function are impaired, the cells bleb extracellular vesicles (EVs), and other bioactive substances accumulate in the storage medium. EVs can carry reactive biomolecules and mediate cell-cell interactions. Thus, EVs could explain RBC transfusion related immunomodulation, particularly after prolonged storage. Methods: We exposed peripheral blood mononuclear cells (PBMCs) to allogeneic RBC supernatant (SN) and EVs from fresh and longer-stored RBC units, diluted plasma, and storage solution SAGM, and studied activation and proliferation of T-cells by flow cytometry, and cytokine secretion of LPS-stimulated PBMCs by enzyme-linked immunosorbent assay (ELISA). Results: Both fresh and longer-stored RBC SN but not EVs induced immunomodulation in recipient cells. RBC SN and diluted plasma augmented the proliferation of particularly CD8(+) T-cells in a 4-day proliferation assay. T-cell activation by SN was evident already after 5 h as shown by upregulation of CD69. SN suppressed monocyte TNF-alpha and increased IL-10 secretion while diluted plasma increased secretion of both cytokines. Conclusion: This in vitro study demonstrates that stored RBC SN will have mixed immunomodulatory effects depending on responder cells and conditions, independent of RBC storage age. Fresh RBCs containing relatively few EVs can induce immune responses. Residual plasma in the products may contribute to these effects.Peer reviewe