Maarit Takatalo

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

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. Stem Cells 2016;34:781–790

Noggin null allele mice exhibit a microform of holoprosencephaly

Holoprosencephaly (HPE) is a heterogeneous craniofacial and neural developmental anomaly characterized in its most severe form by the failure of the forebrain to divide. In humans, HPE is associated with disruption of Sonic hedgehog and Nodal signaling pathways, but the role of other signaling pathways has not yet been determined. In this study, we analyzed mice which, due to the lack of the Bmp antagonist Noggin, exhibit elevated Bmp signaling. Noggin(-/-) mice exhibited a solitary median maxillary incisor that developed from a single dental placode, early midfacial narrowing as well as abnormalities in the developing hyoid bone, pituitary gland and vomeronasal organ. In Noggin(-/-) mice, the expression domains of Shh, as well as the Shh target genes Ptch1 and Gli1, were reduced in the frontonasal region at key stages of early facial development. Using E10.5 facial cultures, we show that excessive BMP4 results in reduced Fgf8 and Ptch1 expression. These data suggest that increased Bmp signaling in Noggin(-/-) mice results in downregulation of the hedgehog pathway at a critical stage when the midline craniofacial structures are developing, which leads to a phenotype consistent with a microform of HPE.

Loss-of-Function of Gli3 in Mice Causes Abnormal Frontal Bone Morphology and Premature Synostosis of the Interfrontal Suture

Greig cephalopolysyndactyly syndrome (GCPS) is an autosomal dominant disorder with polydactyly and syndactyly of the limbs and a broad spectrum of craniofacial abnormalities. Craniosynostosis of the metopic suture (interfrontal suture in mice) is an important but rare feature associated with GCPS. GCPS is caused by mutations in the transcription factor GLI3, which regulates Hedgehog signaling. The Gli3 loss-of-function (Gli3Xt-J/Xt-J) mouse largely phenocopies the human syndrome with the mice exhibiting polydactyly and several craniofacial abnormalities. Here we show that Gli3Xt-J/Xt-J mice exhibit ectopic ossification in the interfrontal suture and in the most severe cases the suture fuses already prior to birth. We show that abnormalities in frontal bones occur early in calvarial development, before the establishment of the interfrontal suture. It provides a model for the metopic suture pathology that can occur in GCPS.

Expression of the novel Golgi protein GoPro49 is developmentally regulated during mesenchymal differentiation.

The Golgi complex is the major cell organelle responsible for protein glycosylation and secretion. In this article, we show that GoPro49 is a new gene expressed specifically in mesenchymal and cartilaginous tissues during development. The corresponding human homologue was identified in our previous Golgi proteomics study and was shown to localize to the Golgi complex as an EGFP‐fusion protein. Furthermore, we show using in situ hybridization that GoPro49 expression pattern is both restricted and developmentally regulated. It is specific in vertebrae, ribs, and limbs, and in the craniofacial area in nasal septum and dental follicle. In the trunk, GoPro49 expression decreases before final chondrocyte differentiation, while in the craniofacial area expression is still observed in postnatal tissues. This is the first time a Golgi membrane protein is shown to be expressed in a developmentally regulated manner during mesenchymal and cartilage development in mammals. Developmental Dynamics 237:2243–2255, 2008.

Prevention of Premature Fusion of Calvarial Suture in GLI-Kruppel Family Member 3 (Gli3)-deficient Mice by Removing One Allele of Runt-related Transcription Factor 2 (Runx2)

Background: Gli3-deficient mice (Gli3Xt-J/Xt-J) show premature suture closure (craniosynostosis). Results: Gli3Xt-J/Xt-J mice have aberrant cell proliferation and osteogenic differentiation in the sutures. Reducing the dosage of Runx2 (Gli3Xt-J/Xt-J;Runx2+/− mice) rescues the abnormality through canonical Bmp-Smad signaling. Conclusion: Gli3 represses bone formation Bmp-Smad signaling, which integrates Dlx5/Runx2-II cascade. Significance: Targeting Runx2 might provide an attractive way of preventing craniosynostosis in patients. Mutations in the gene encoding the zinc finger transcription factor GLI3 (GLI-Kruppel family member 3) have been identified in patients with Grieg cephalopolysyndactyly syndrome in which premature fusion of calvarial suture (craniosynostosis) is an infrequent but important feature. Here, we show that Gli3 acts as a repressor in the developing murine calvaria and that Dlx5, Runx2 type II isoform (Runx2-II), and Bmp2 are expressed ectopically in the calvarial mesenchyme, which results in aberrant osteoblastic differentiation in Gli3-deficient mouse (Gli3Xt-J/Xt-J) and resulted in craniosynostosis. At the same time, enhanced activation of phospho-Smad1/5/8 (pSmad1/5/8), which is a downstream mediator of canonical Bmp signaling, was observed in Gli3Xt-J/Xt-J embryonic calvaria. Therefore, we generated Gli3;Runx2 compound mutant mice to study the effects of decreasing Runx2 dosage in a Gli3Xt-J/Xt-J background. Gli3Xt-J/Xt-J Runx2+/− mice have neither craniosynostosis nor additional ossification centers in interfrontal suture and displayed a normalization of Dlx5, Runx2-II, and pSmad1/5/8 expression as well as sutural mesenchymal cell proliferation. These findings suggest a novel role for Gli3 in regulating calvarial suture development by controlling canonical Bmp-Smad signaling, which integrates a Dlx5/Runx2-II cascade. We propose that targeting Runx2 might provide an attractive way of preventing craniosynostosis in patients.

Novel Golgi protein, GoPro49, is a specific dental follicle marker.

GoPro49 is a recently identified, novel Golgi protein that is expressed in embryonic mesenchymal tissues, including dental follicle. In the present study, we have tested the hypothesis that the gene is a specific marker for the dental follicle, and examined its expression during the development of mouse incisors and molars. In situ hybridization showed that GoPro49 is expressed in dental follicles from bud to post-eruption stages. The expression is intense throughout the dental follicle during crown development, and persists in the root follicle during root development. In the forming periodontal ligament, GoPro49 expression is down-regulated upon differentiation of the follicle cells to cementoblasts and osteoblasts marked by Bsp1. In cultured dental follicle cells, the GoPro49 protein co-localizes with β-COP, suggesting that GoPro49 may function in the secretory pathway. We conclude that GoPro49 is a novel, specific marker for the dental follicle and can be used to identify this tissue. Abbreviations: Bsp1, bone sialoprotein 1; GoPro49, Golgi protein 49 kDa; E16, embryonic day 16; HERS, Hertwig’s epithelial root sheath; PDL, periodontal ligament; dpn, day post-natal.

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.

Modeling tumor predisposing FH mutations in yeast: Effects on fumarase activity, growth phenotype and gene expression profile

Heterozygous mutations in the fumarase (FH) gene cause the tumor predisposition syndrome hereditary leiomyomatosis and renal cell cancer (MIM 605839). While most families segregate a benign phenotype of multiple leiomyomas, others display a phenotype with early‐onset renal cancer and leiomyosarcoma. Modifier genes may play a role in this, but an alternative explanation is simple genotype–phenotype association. FH mutations predisposing to cancer appear to be truncating or in fully conserved amino acids, suggesting that mutations severely affecting FH activity might predispose to malignancy. In the present study, we analyzed 2 conserved fumarase mutations in yeast. H153R has been described in 3 cancer predisposition families; whereas all 3 reported K187R families have displayed the benign phenotype. Examining H153R and K187R should clarify whether cancer‐related FH mutations differ from their benign phenotype‐associated counterparts. Yeast strains containing the 2 mutations, and knockout and wild type (WT) references, were created and the growth phenotypes studied on selected carbon sources to assess mitochondrial function. Additionally, Fum1 protein production and activity were measured, and the strains were subjected to transcriptional profiling. On nonfermentable lactate medium, the fumarase knockout strains did not grow, whereas the mutants showed no differences, as compared to WT yeast. Although both mutant strains produced fumarase, a considerable decrease in enzyme activity was seen in mutants with respect to WT. Transcription of the majority of Krebs cycle enzymes was downregulated in response to mutations in fumarase. In conclusion, both mutants displayed some, albeit greatly reduced, fumarase activity. This activity was sufficient to support normal growth on nonfermentable carbon source, unlike the deletion phenotype, demonstrating the significance of the residual activity. The findings support the hypothesis that modifier gene(s), rather than phenotype–genotype effects, display a major role in determining tumor phenotypes in families segregating FH mutations.

Metastatic state of parent cells influences the uptake and functionality of prostate cancer cell-derived extracellular vesicles

ABSTRACT Extracellular vesicles (EVs), including microvesicles and exosomes, mediate intercellular signalling which has a profound role in cancer progression and in the development of metastasis. Internalisation of EVs can prompt functional changes in the recipient cells, the nature of which depends on the molecular composition and the cargo of the EVs. We hypothesised that the metastatic stage of cancerous parent cells would determine the uptake efficacy and the subsequent functional effects of the respective cancer cell-derived EVs. To address this question, we compared the internalisation of EVs derived from two metastatic site-derived prostate cancer cell lines (PC-3 and LNCaP), human telomerase reverse transcriptase immortalised primary malignant prostate epithelial cells (RC92a/hTERT), and a benign epithelial prostate cell line (PNT2). EVs isolated from the metastatic site-derived PC-3 and LNCaP cells were more efficiently internalised by the PC-3 and PNT2 cells compared to the EVs from the primary malignant RC92a/hTERT cells or the benign PNT2 cells, as determined by high content microscopy, confocal microscopy, and flow cytometry. EV uptake was also influenced by the phase of the cell cycle, so that an increased EV-derived fluorescence signal was observed in the cells at the G2/M phase compared to the G0/G1 or S phases. Finally, differences were also observed in the functions of the recipient cells based on the EV source. Proliferation of PNT2 cells and to a lesser extent also PC-3 cells was enhanced particularly by the EVs from the metastatic-site-derived prostate cancer cells in comparison to the EVs from the benign cells or primary cancer cells, whereas migration of PC-3 cells was enhanced by all cancerous EVs.