Cellulase-assisted platelet-rich plasma release from nanofibrillated cellulose hydrogel enhances wound healing.

Platelet-rich plasma (PRP) is a source of growth factors, which are implicated in active tissue regeneration. However, after transplantation the efficacy of these bioactive compounds is often diminished due to rapid degradation and untargeted localization. For this reason, we evaluated the potential of nanofibrillated cellulose (NFC) hydrogel as a PRP carrier. NFC hydrogel is an animal-free biomaterial that, when doped with cellulase, can assist the release of PRP in a wound site. In this study, we examined the effects of 0.5% (m/v) NFC hydrogel formulations, including PRP and cellulase, on the migration and proliferation of skin cells via an in vitro scratch wound model. The suitability of the 0.8% NFC hydrogel formulations for accelerated wound healing and PRP carrying was studied in vitro in diffusion studies and in vivo in a full-thickness excisional wound model in SKH1 mice. None of the NFC hydrogel formulations with or without PRP and cellulase disturbed the normal cell behavior in vitro, and cellulase was successfully used to degrade NFC. NFC hydrogel slowed fibroblast migration rate in vitro. In vivo, NFC hydrogel treatment showed significantly enhanced re-epithelialization compared to control and supported collagen deposition. In addition, angiogenesis was significantly induced via PRP release after degrading NFC hydrogel with cellulase without abnormal host reaction. This study demonstrates the potential of NFC hydrogel with cellulase as a carrier for PRP with controlled release in future skin tissue engineering applications.

Development of tandem cation exchange chromatography for high purity extracellular vesicle isolation: The effect of ligand steric availability.

Purification of extracellular vesicles for research and therapeutic applications requires updated methodology to address the limitations of traditional ultracentrifugation and other size-based separation techniques. Their downfalls include induced extracellular vesicle aggregation, low yields, poor scalability and one-dimensionality of the separation process, as the size or sedimentation speed of extracellular vesicles is often the only selection criterion. Ion exchange chromatography is a promising alternative or supplementary method candidate, as it offers a different approach for extracellular vesicle separation, which is surface charge. For now, mostly anion exchange chromatography has been evaluated for extracellular vesicle purification, as it successfully relies on the strongly negative surface charge of extracellular vesicles. However, as extracellular vesicles are very complex in their structure, also cation exchange chromatography could be applicable, due to individual cationic domains on the extracellular vesicle surface. Here, we compare anion exchange chromatography to different types of cation exchange chromatography for the purification of platelet extracellular vesicle samples also containing plasma-derived impurities. We found that the choice of resin structure used for cation exchange chromatography is critical for binding platelet extracellular vesicles, as a conventional-type cation exchanger was found to only capture and elute less than 20% of extracellular vesicles. With the tentacle-type resin, it was possible to obtain comparable platelet extracellular vesicle yields (over 90%) with cation exchange chromatography compared to anion exchange chromatography, as well as superior purity, especially when it was combined to conventional cation exchange resin.

OxLDL sensitizes platelets for increased formation of extracellular vesicles capable of finetuning macrophage gene expression.

Platelet extracellular vesicles (PEVs) generated upon platelet activation may play a role in inflammatory pathologies such as atherosclerosis. Oxidized low-density lipoprotein (oxLDL), a well-known contributor to atherogenesis, activates platelets and presensitizes them for activation by other agonists. We studied the effect of oxLDL on the secretion, composition, and inflammatory functions of PEVs using contemporary EV analytics. Platelets were activated by co-stimulation with thrombin (T) and collagen (C) ± oxLDL and characterized by high-resolution flow cytometry, nanoparticle tracking analysis, proximity extension assay, western blot, and electron microscopy. The effect of PEVs on macrophage differentiation and functionality was examined by analyzing macrophage surface markers, cytokine secretion, and transcriptome. OxLDL upregulated TC-induced formation of CD61+, P-selectin+ and phosphatidylserine+ PEVs. Blocking the scavenger receptor CD36 significantly suppressed the oxLDL+TC-induced PEV formation, and HDL caused a slight but detectable suppression. The inflammatory protein cargo differed between the PEVs from stimulated and unstimulated platelets. Both oxLDL+TC- and TC-induced PEVs enhanced macrophage HLA-DR and CD86 expression and decreased CD11c expression as well as secretion of several cytokines. Pathways related to cell cycle and regulation of gene expression, and immune system signaling were overrepresented in the differentially expressed genes between TC PEV -treated vs. control macrophages and oxLDL+TC PEV -treated vs. control macrophages, respectively. In conclusion, we speculate that oxLDL and activated platelets contribute to proatherogenic processes by increasing the number of PEVs that provide an adhesive and procoagulant surface, contain inflammatory mediators, and subtly finetune the macrophage gene expression.

Urinary extracellular vesicles: Assessment of pre-analytical variables and development of a quality control with focus on transcriptomic biomarker research.

Urinary extracellular vesicles (uEV) are a topical source of non-invasive biomarkers for health and diseases of the urogenital system. However, several challenges have become evident in the standardization of uEV pipelines from collection of urine to biomarker analysis. Here, we studied the effect of pre-analytical variables and developed means of quality control for uEV isolates to be used in transcriptomic biomarker research. We included urine samples from healthy controls and individuals with type 1 or type 2 diabetes and normo-, micro- or macroalbuminuria and isolated uEV by ultracentrifugation. We studied the effect of storage temperature (-20°C vs. -80°C), time (up to 4 years) and storage format (urine or isolated uEV) on quality of uEV by nanoparticle tracking analysis, electron microscopy, Western blotting and qPCR. Urinary EV RNA was compared in terms of quantity, quality, and by mRNA or miRNA sequencing. To study the stability of miRNA levels in samples isolated by different methods, we created and tested a list of miRNAs commonly enriched in uEV isolates. uEV and their transcriptome were preserved in urine or as isolated uEV even after long-term storage at -80°C. However, storage at -20°C degraded particularly the GC-rich part of the transcriptome and EV protein markers. Transcriptome was preserved in RNA samples extracted with and without DNAse, but read distributions still showed some differences in e.g. intergenic and intronic reads. MiRNAs commonly enriched in uEV isolates were stable and concordant between different EV isolation methods. Analysis of never frozen uEV helped to identify surface characteristics of particles by EM. In addition to uEV, qPCR assays demonstrated that uEV isolates commonly contained polyoma viruses. Based on our results, we present recommendations how to store and handle uEV isolates for transcriptomics studies that may help to expedite standardization of the EV biomarker field.

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.

Human Mesenchymal Stromal Cell Secretome Promotes the Immunoregulatory Phenotype and Phagocytosis Activity in Human Macrophages.

Human mesenchymal stromal/stem cells (hMSCs) show great promise in cell therapy due to their immunomodulatory properties. The overall immunomodulatory response of hMSCs resembles the resolution of inflammation, in which lipid mediators and regulatory macrophages (Mregs) play key roles. We investigated the effect of hMSC cell-cell contact and secretome on macrophages polarized and activated toward Mreg phenotype. Moreover, we studied the effect of supplemented polyunsaturated fatty acids (PUFAs): docosahexaenoic acid (DHA) and arachidonic acid, the precursors of lipid mediators, on hMSC immunomodulation. Our results show that unlike hMSC cell-cell contact, the hMSC secretome markedly increased the CD206 expression in both Mreg-polarized and Mreg-activated macrophages. Moreover, the secretome enhanced the expression of programmed death-ligand 1 on Mreg-polarized macrophages and Mer receptor tyrosine kinase on Mreg-activated macrophages. Remarkably, these changes were translated into improved Candida albicans phagocytosis activity of macrophages. Taken together, these results demonstrate that the hMSC secretome promotes the immunoregulatory and proresolving phenotype of Mregs. Intriguingly, DHA supplementation to hMSCs resulted in a more potentiated immunomodulation with increased CD163 expression and decreased gene expression of matrix metalloproteinase 2 in Mreg-polarized macrophages. These findings highlight the potential of PUFA supplementations as an easy and safe method to improve the hMSC therapeutic potential.

Assessment of Time-Dependent Platelet Activation Using Extracellular Vesicles, CD62P Exposure, and Soluble Glycoprotein V Content of Platelet Concentrates with Two Different Platelet Additive Solutions.

Novel analytical measures are needed to accurately monitor the properties of platelet concentrates (PCs). Since activated platelets produce platelet-derived extracellular vesicles (EVs), analyzing EVs of PCs may provide additional information about the condition of platelets. The prospect of using EVs as an auxiliary measure of platelet activation state was investigated by examining the effect of platelet additive solutions (PASs) on EV formation and platelet activation during PC storage. The time-dependent activation of platelets in PCs with PAS-B or with the further developed PAS-E was compared by measuring the exposure of CD62P by flow cytometry and the content of soluble glycoprotein V (sGPV) of PCs by an immunoassay. Changes in the concentration and size distribution of EVs were determined using nanoparticle tracking analysis. A time-dependent increase in platelet activation in PCs was demonstrated by increased CD62P ex-posure, sGPV content, and EV concentration. Using these strongly correlating parameters, PAS-B platelets were shown to be more activated compared to PAS-E platelets. Since the EV concentration correlated well with the established platelet activation markers CD62P and sGPV, it could potentially be used as a complementary parameter for platelet activation for PCs. More detailed characterization of the resulting EVs could help to understand how the PC components contribute the functional effects of transfused PCs.

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 E2 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.

Lipid mediators in platelet concentrate and extracellular vesicles: Molecular mechanisms from membrane glycerophospholipids to bioactive molecules.

Platelets are collected for transfusion to patients with different haematological disorders, and for logistical reasons, platelets are stored as concentrates. Despite carefully controlled conditions, platelets become activated during storage, and platelet concentrates (PlaCs) may cause adverse inflammatory reactions in recipients. The time-dependent changes in the lipidome of clinical PlaCs, platelets isolated from PlaCs, and extracellular vesicles (EVs) thereof were examined by mass spectrometry. The relative amount of arachidonic acid containing glycerophospholipids, especially those in the phosphatidylethanolamine and phosphatidylserine classes during storage, but the relative amount of other polyunsaturated fatty acid containing glycerophospholipids remained stable in all sample types. These changes were not directly translated to lipid mediator (LM) profile since the levels of arachidonic acid-derived proinflammatory LMs were not specifically elevated. Instead, several monohydroxy pathway markers and functionally relevant LMs, both proinflammatory and proresolving, were detected in the PlaCs and the EVs, and some representatives of both kind clearly accumulated during storage. By Western blot, the key enzymes of these pathways were shown to be present in platelets, and in many cases, EVs. Since the EVs were enriched in the fatty acid precursors of LMs in their (phospholipid) membranes, harboured LM-producing enzymes, contained the related monohydroxy pathway markers, and secreted the final LM products, PlaC-derived EVs could participate in the regulation of inflammation and healing, and thereby aid the platelets in exerting their essential physiological functions.

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 E2 (PGE2) 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 PGE2. These data suggest that both human MSC and MSC-EVs may potentiate tolerance-promoting proresolving phenotype of human Mregs.

Phospholipid composition of packed red blood cells and that of extracellular vesicles show a high resemblance and stability during storage.

Red blood cells (RBCs) are stored up to 35-42days at 2-6°C in blood banks. During storage, the RBC membrane is challenged by energy depletion, decreasing pH, altered cation homeostasis, and oxidative stress, leading to several biochemical and morphological changes in RBCs and to shedding of extracellular vesicles (EVs) into the storage medium. These changes are collectively known as RBC storage lesions. EVs accumulate in stored RBC concentrates and are, thus, transfused into patients. The potency of EVs as bioactive effectors is largely acknowledged, and EVs in RBC concentrates are suspected to mediate some adverse effects of transfusion. Several studies have shown accumulation of lipid raft-associated proteins in RBC EVs during storage, whereas a comprehensive phospholipidomic study on RBCs and corresponding EVs during the clinical storage period is lacking. Our mass spectrometric and chromatographic study shows that RBCs maintain their major phospholipid (PL) content well during storage despite abundant vesiculation. The phospholipidomes were largely similar between RBCs and EVs. No accumulation of raft lipids in EVs was seen, suggesting that the primary mechanism of RBC vesiculation during storage might not be raft -based. Nonetheless, a slight tendency of EV PLs for shorter acyl chains was observed.

Metabolomic Profiling of Extracellular Vesicles and Alternative Normalization Methods Reveal Enriched Metabolites and Strategies to Study Prostate Cancer-Related Changes.

Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the secreting cells. So far, biomarkers for pathological conditions have been mainly searched from their protein, (mi)RNA, DNA and lipid cargo. Here, we explored the small molecule metabolites from urinary and platelet EVs relative to their matched source samples. As a proof-of-concept study of intra-EV metabolites, we compared alternative normalization methods to profile urinary EVs from prostate cancer patients before and after prostatectomy and from healthy controls. METHODS: We employed targeted ultra-performance liquid chromatography-tandem mass spectrometry to profile over 100 metabolites in the isolated EVs, original urine samples and platelets. We determined the enrichment of the metabolites in the EVs and analyzed their subcellular origin, pathways and relevant enzymes or transporters through data base searches. EV- and urine-derived factors and ratios between metabolites were tested for normalization of the metabolomics data. RESULTS: Approximately 1 x 1010 EVs were sufficient for detection of metabolite profiles from EVs. The profiles of the urinary and platelet EVs overlapped with each other and with those of the source materials, but they also contained unique metabolites. The EVs enriched a selection of cytosolic metabolites including members from the nucleotide and spermidine pathways, which linked to a number of EV-resident enzymes or transporters. Analysis of the urinary EVs from the patients indicated that the levels of glucuronate, D-ribose 5-phosphate and isobutyryl-L-carnitine were 2-26-fold lower in all pre-prostatectomy samples compared to the healthy control and post-prostatectomy samples (p < 0.05). These changes were only detected from EVs by normalization to EV-derived factors or with metabolite ratios, and not from the original urine samples. CONCLUSIONS: Our results suggest that metabolite analysis of EVs from different samples is feasible using a high-throughput platform and relatively small amount of sample material. With the knowledge about the specific enrichment of metabolites and normalization methods, EV metabolomics could be used to gain novel biomarker data not revealed by the analysis of the original EV source materials.

Methylome Analysis of Human Bone Marrow MSCs Reveals Extensive Age- and Culture-Induced Changes at Distal Regulatory Elements.

Human bone marrow stromal cells, or mesenchymal stem cells (BM-MSCs), need expansion prior to use as cell-based therapies in immunological and tissue repair applications. Aging and expansion of BM-MSCs induce epigenetic changes that can impact therapeutic outcomes. By applying sequencing-based methods, we reveal that the breadth of DNA methylation dynamics associated with aging and expansion is greater than previously reported. Methylation changes are enriched at known distal transcription factor binding sites such as enhancer elements, instead of CpG-rich regions, and are associated with changes in gene expression. From this, we constructed hypo- and hypermethylation-specific regulatory networks, including a sub-network of BM-MSC master regulators and their predicted target genes, and identified putatively disrupted signaling pathways. Our genome-wide analyses provide a broader overview of age- and expansion-induced DNA methylation changes and a better understanding of the extent to which these changes alter gene expression and functionality of human BM-MSCs.

Tenascin-C and fibronectin expression divide early stage tongue cancer into low- and high-risk groups.

BACKGROUND: Oral tongue squamous cell carcinoma (OTSCC) metastasises early, especially to regional lymph nodes. There is an ongoing debate on which early stage (T1-T2N0) patients should be treated with elective neck dissection. We need prognosticators for early stage tongue cancer. METHODS: Mice immunisation with human mesenchymal stromal cells resulted in production of antibodies against tenascin-C (TNC) and fibronectin (FN), which were used to stain 178 (98 early stage), oral tongue squamous cell carcinoma samples. Tenascin-C and FN expression in the stroma (negative, moderate or abundant) and tumour cells (negative or positive) were assessed. Similar staining was obtained using corresponding commercial antibodies. RESULTS: Expression of TNC and FN in the stroma, but not in the tumour cells, proved to be excellent prognosticators both in all stages and in early stage cases. Among early stages, when stromal TNC was negative, the 5-year survival rate was 88%. Correspondingly, when FN was negative, no cancer deaths were observed. Five-year survival rates for abundant expression of TNC and FN were 43% and 25%, respectively. CONCLUSIONS: Stromal TNC and, especially, FN expressions differentiate patients into low- and high-risk groups. Surgery alone of early stage primary tumours might be adequate when stromal FN is negative. Aggressive treatments should be considered when both TNC and FN are abundant.

Low oxygen tension reveals distinct HOX codes in human cord blood-derived stromal cells associated with specific endochondral ossification capacities in vitro and in vivo.

Effects of oxygen tension on the generation, expansion, proliferation and differentiation of stromal cell types is widely described in the literature. However, data on the internal heterogeneity of applied cell populations at different O2 levels and possible impacts on differentiation potentials are controversial. Here, the expression of 39 human HOX genes was determined in neonatal cord blood stromal cells and linked to differentiation-associated signatures. In cord blood, unrestricted somatic stromal cells (USSCs), lacking HOX gene expression, and cord blood-derived multipotent stromal cells (CB-MSCs), expressing about 20 HOX genes, are distinguished by their specific HOX code. Interestingly, 74% of the clones generated at 21% O2 were HOX-negative USSCs, whereas 73% of upcoming clones at 3% O2 were HOX-positive CB-MSCs. In order to better categorize distinct cell lines generated at 3% O2 , the expression of all 39 HOX genes within HOX clusters A, B, C and D were tested and new subtypes defined: cells negative in all four HOX clusters (USSCs); cells positive in all four clusters (CB-MSCsABCD ); and subpopulations missing a single cluster (CB-MSCsACD and CB-MSCsBCD ). Comprehensive qPCR analyses of established chondro-osteomarkers revealed subtype-specific signatures verifiably associated with in vitro and in vivo differentiation capacity. The data presented here underline the necessity of better characterizing distinct cell populations at a clonal level, taking advantage of the inherent specific HOX code as a distinguishing feature between individual subtypes. Moreover, the correlation of subtype-specific molecular signatures with in vitro and in vivo bone formation is discussed. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolism and phospholipid assembly of polyunsaturated fatty acids in human bone marrow mesenchymal stromal cells.

High arachidonic acid (20:4n-6) and low n-3 PUFA levels impair the capacity of cultured human bone marrow mesenchymal stromal cells (hBMSCs) to modulate immune functions. The capacity of the hBMSCs to modify PUFA structures was found to be limited. Therefore, different PUFA supplements given to the cells resulted in very different glycerophospholipid (GPL) species profiles and substrate availability for phospholipases, which have preferences for polar head group and acyl chains when liberating PUFA precursors for production of lipid mediators. When supplemented with 20:4n-6, the cells increased prostaglandin E2 secretion. However, they elongated 20:4n-6 to the less active precursor, 22:4n-6, and also incorporated it into triacylglycerols, which may have limited the proinflammatory signaling. The n-3 PUFA precursor, 18:3n-3, had little potency to reduce the GPL 20:4n-6 content, while the eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acid supplements efficiently displaced the 20:4n-6 acyls, and created diverse GPL species substrate pools allowing attenuation of inflammatory signaling. The results emphasize the importance of choosing appropriate PUFA supplements for in vitro hBMSC expansion and suggests that for optimal function they require an exogenous fatty acid source providing 20:5n-3 and 22:6n-3 sufficiently, but 20:4n-6 moderately, which calls for specifically designed optimal PUFA supplements for the cultures.

Expansion induced microRNA changes in bone marrow mesenchymal stromal cells reveals interplay between immune regulation and cell cycle.

Mesenchymal stromal cells (MSC) are currently used in many cell based therapies. Prior to use in therapy, extensive expansion is required. We used microarray profiling to investigate expansion induced miRNA and mRNA expression changes of bone marrow MSCs (BM-MSCs) derived from old and young donors. The expression levels of 36 miRNAs were altered in cells derived from the old and respectively 39 miRNAs were altered in cells derived from young donors. Of these, only 12 were differentially expressed in both young and old donor BM-MSCs, and their predicted target mRNAs, were mainly linked to cell proliferation and senescence. Further qPCR verification showed that the expression of miR-1915-3p, miR-1207, miR-3665, and miR-762 correlated with the expansion time at passage 8. Previously described BM-MSC-specific miRNA fingerprints were also detected but these remained unchanged during expansion. Interestingly, members of well-studied miR-17/92 cluster, involved in cell cycle regulation, aging and also development of immune system, were down-regulated specifically in cells from old donors. The role of this cluster in MSC functionality is worth future studies since it links expansion, aging and immune system together.

Real-Time Label-Free Monitoring of Nanoparticle Cell Uptake.

The surface plasmon resonance technique in combination with whole cell sensing is used for the first time for real-time label-free monitoring of nanoparticle cell uptake. The uptake kinetics of several types of nanoparticles relevant to drug delivery applications into HeLa cells is determined. The cell uptake of the nanoparticles is confirmed by confocal microscopy. The cell uptake of silica nanoparticles and polyethylenimine-plasmid DNA polyplexes is studied as a function of temperature, and the uptake energies are determined by Arrhenius plots. The phase transition temperature of the HeLa cell membrane is detected when monitoring cell uptake of silica nanoparticles at different temperatures. The HeLa cell uptake of the mesoporous silica nanoparticles is energy-independent at temperatures slightly higher than the phase transition temperature of the HeLa cell membrane, while the uptake of polyethylenimine-DNA polyplexes is energy-dependent and linear as a function of temperature with an activation energy of Ea = 62 ± 7 kJ mol-1 = 15 ± 2 kcal mol-1 . The HeLa cell uptake of red blood cell derived extracellular vesicles is also studied as a function of the extracellular vesicle concentration. The results show a concentration dependent behavior reaching a saturation level of the extracellular vesicle uptake by HeLa cells.

The effects of culture conditions on the functionality of efficiently obtained mesenchymal stromal cells from human cord blood.

BACKGROUND AIMS: Cord blood (CB) is an attractive source of mesenchymal stromal cells (MSCs) because of its abundant availability and ease of collection. However, the success rate of generating CB-MSCs is low. In this study, our aim was to demonstrate the efficiency of our previously described method to obtain MSCs from CB and further characterize them and to study the effects of different culture conditions on MSCs. METHODS: CB-MSC cultures were established in low oxygen (3%) conditions on fibronectin in 10% fetal bovine serum containing culture medium supplemented with combinations of growth factors. Cells were characterized for their adipogenic, osteogenic and chondrogenic differentiation capacity; phenotype; and HOX gene expression profile. The functionality of the cells cultured in different media was tested in vitro with angiogenesis and T-cell proliferation assays. RESULTS: We demonstrate 87% efficacy in generating MSCs from CB. The established cells had typical MSC characteristics with reduced adipogenic differentiation potential and a unique HOX gene fingerprint. Growth factor-rich medium and a 3% oxygen condition enhanced cell proliferation; however, the growth factor-rich medium had a negative effect on the expression of CD90. Dexamethasone-containing medium improved the capacity of the cells to suppress T-cell proliferation, whereas the cells grown without dexamethasone were more able to support angiogenesis. CONCLUSIONS: Our results demonstrate that the composition of expansion medium is critical for the functionality of MSCs and should always be appropriately defined for each purpose.

Adenosinergic Immunosuppression by Human Mesenchymal Stromal Cells Requires Co-Operation with T cells.

Mesenchymal stem/stromal cells (MSCs) have the capacity to counteract excessive inflammatory responses. MSCs possess a range of immunomodulatory mechanisms, which can be deployed in response to signals in a particular environment and in concert with other immune cells. One immunosuppressive mechanism, not so well-known in MSCs, is mediated via adenosinergic pathway by ectonucleotidases CD73 and CD39. In this study, we demonstrate that adenosine is actively produced from adenosine 5'-monophosphate (AMP) by CD73 on MSCs and MSC-derived extracellular vesicles (EVs). Our results indicate that although MSCs express CD39 at low level and it colocalizes with CD73 in bulge areas of membranes, the most efficient adenosine production from adenosine 5'-triphosphate (ATP) requires co-operation of MSCs and activated T cells. Highly CD39 expressing activated T cells produce AMP from ATP and MSCs produce adenosine from AMP via CD73 activity. Furthermore, adenosinergic signaling plays a role in suppression of T cell proliferation in vitro. In conclusion, this study shows that adenosinergic signaling is an important immunoregulatory mechanism of MSCs, especially in situations where ATP is present in the extracellular environment, like in tissue injury. An efficient production of immunosuppressive adenosine is dependent on the concerted action of CD39-positive immune cells with CD73-positive cells such as MSCs or their EVs.