Monocyte-derived extracellular vesicles stimulate cytokinesecretion and gene expression of matrixmetalloproteinases by mesenchymal stem/stromal cells

Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. DatabaseGene expression data are available in the GEO database under the accession number .Peer reviewe

Efficient ultrafiltration based protocol to deplete extracellular vesicles from fetal bovine serum

Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EVdepleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories.Peer reviewe

Epigenetic alterations in mesenchymal stem cells by osteosarcoma-derived extracellular vesicles

Extracellular vesicles (EVs) are central to intercellular communication and play an important role in cancer progression and development. Osteosarcoma (OS) is an aggressive bone tumour, characterized by the presence of malignant mesenchymal cells. The specific tumour-driving genetic alterations that are associated with OS development are currently poorly understood. Mesenchymal stem cells (MSCs) of osteogenic lineage have been postulated as likely candidates as the cells of origin for OS, thus indicating that MSCs and OS stroma cells may be related cell types. Therefore, this study set out to examine the EV-mediated intercellular crosstalk of MSCs and OS. MSCs and pre-osteoblasts were treated with OS-EVs at different time points, and the epigenetic signature of OS-EVs was assessed by methylation analysis of LINE-1 (long interspersed element) and tumour suppressor genes. In addition, surface markers and expression of specific genes were also evaluated. Our data indicated that OS-EVs mediated LINE-1 hypomethylation in MSCs, whereas an opposite effect was seen in pre-osteoblasts, indicating that MSCs but not pre-osteoblasts were susceptible to epigenetic transformation. Thus, OS-EVs modulated the fate of MSCs by modulating the epigenetic status, and also influenced the expression of genes related to bone microenvironment remodelling. Overall, this study provided evidence that epigenetic regulation appears to be an early event in the transformation of MSCs during the development of OS. Elucidating the mechanisms of EV-mediated communication may lead to new avenues for therapeutic exploitation.Peer reviewe

Patient-Specific Bioimplants and Reconstruction Plates for Mandibular Defects: Production Workflow and In Vivo Large Animal Model Study

A major challenge with extensive craniomaxillofacial bone reconstruction is the limited donor-site availability to reconstruct defects predictably and accurately according to the anatomical shape of the patient. Here, patient-specific composite bioimplants, consisting of cross-linked poly(trimethylene carbonate) (PTMC) networks and beta-tricalcium phosphate (beta-TCP), are tested in vivo in twelve Gottingen minipigs in a large mandibular continuity defect model. The 25 mm defects are supported by patient-specific titanium reconstruction plates and receive either osteoconductive composite bioimplants (PTMC+TCP), neat polymer network bioimplants (PTMC), autologous bone segments (positive control), or are left empty (negative control). Postoperatively, defects treated with bioimplants show evident ossification at 24 weeks. Histopathologic evaluation reveals that neat PTMC bioimplant surfaces are largely covered with fibrous tissue, while in the PTMC+TCP bioimplants, bone attached directly to the implant surface shows good osteoconduction and histological signs of osteoinductivity. However, PTMC+TCP bioimplants are associated with high incidence of necrosis and infection, possibly due to rapid resorption and/or particle size of the used beta-TCP. The study highlights the importance of testing bone regeneration implants in a clinically relevant large animal model and at the in situ reconstruction site, since results on small animal models and studies in nonloadbearing areas do not translate directly.Peer reviewe

ECMO for COVID-19 patients in Europe and Israel

Since March 15th, 2020, 177 centres from Europe and Israel have joined the study, routinely reporting on the ECMO support they provide to COVID-19 patients. The mean annual number of cases treated with ECMO in the participating centres before the pandemic (2019) was 55. The number of COVID-19 patients has increased rapidly each week reaching 1531 treated patients as of September 14th. The greatest number of cases has been reported from France (n = 385), UK (n = 193), Germany (n = 176), Spain (n = 166), and Italy (n = 136) .The mean age of treated patients was 52.6 years (range 16–80), 79% were male. The ECMO configuration used was VV in 91% of cases, VA in 5% and other in 4%. The mean PaO2 before ECMO implantation was 65 mmHg. The mean duration of ECMO support thus far has been 18 days and the mean ICU length of stay of these patients was 33 days. As of the 14th September, overall 841 patients have been weaned from ECMO support, 601 died during ECMO support, 71 died after withdrawal of ECMO, 79 are still receiving ECMO support and for 10 patients status n.a. . Our preliminary data suggest that patients placed on ECMO with severe refractory respiratory or cardiac failure secondary to COVID-19 have a reasonable (55%) chance of survival. Further extensive data analysis is expected to provide invaluable information on the demographics, severity of illness, indications and different ECMO management strategies in these patients

Insights of Bone-Remodeling : Limitations and Alternatives in Bone Tissue Engineering

Defects in bone structures are extensive problems, faced by millions of people worldwide. Still, current methods do not always provide a satisfactory result in restoring functions after trauma or disease treatment. Bone tissue engineering methods require refinement; therefore, cell-derived agents could be used to enhance treatment outcomes. The extracellular vesicles (EVs) are potential new agents that could improve bone formation in tissue engineering. EVs are released from the cellular membrane and contain mRNA, miRNA and other tissue factors, affecting recipient cells. As EVs can initiate cell differentiation, it is important to understand the mechanisms behind their operation, since they have shown applicability as biomimetic tools for guided lineage-specific differentiation and cell-free therapies. In this thesis, a novel cost-effective and easily standardizable method for EV isolation was developed, based on ultrafiltration of cell culture media. The method may be used in a wide range of cell culture applications increasing reproducibility of EV research results between laboratories. As EVs are proposed to be central in intercellular communication, the potency of EVs from activated monocytes (MC) and osteoclasts was evaluated on adipose tissue mesenchymal stromal cells (MSCs). The EVs from activated MCs promoted secretion of cytokines, potentially representing an immunomodulatory mechanism, while also upregulating gene expression of matrix metalloproteinases in MSCs, affecting tissue remodeling. Further, to investigate the role of EVs in malignant bone conditions, MSCs and pre-osteoblasts were treated with EVs released from osteosarcoma cells (OS-EVs) and the epigenetic signature of OS-EVs was assessed. Overall, this study provided evidence that epigenetic regulation appears to be an early event in the transformation of MSCs during the development of OS. To develop new methods for tissue engineering, we conducted an in vivo study on minipigs. The minipig was selected as the large animal model of choice due to physiological and anatomical similarities of the jaws and teeth with humans. To reconstruct a large continuous defect in the mandible, a composite consisting of PTMC (poly[trimethylene carbonate]) and calcium phosphate ceramic was tested in a mechanically loaded area. The bioimplant scaffolds were manufactured individually based on CT imaging data from each minipig using computer-aided design and manufacturing (CAD-CAM). The mandibular defect was stabilized with custom-made 3D printed titanium reconstruction plates as the bioimplant itself was not load-bearing. The results of this thesis work investigated different strategies in bone remodeling, e.g., the effect and mechanisms of EVs on the immune system and bone remodeling as well as in malignant transformation. Further studies are necessary to confirm the proof-of-concept in bone remodeling to implement these strategies in patient treatments.Luupuutokset leukojen ja kasvojen alueella ovat laaja ongelma, joka koskettaa miljoonia ihmisiä maailmassa. Tällä hetkellä käytössä olevat menetelmät eivät aina tarjoa toiminnallisesti ja esteettisesti tyydyttäviä tuloksia. Soluviljelykokeissa käytetään usein kasvatusliuoksessa naudan seerumia lisäravinteena. Tässä väitöskirjassa kehitettiin uusi kustannustehokas ja helposti standardisoitava menetelmä EV-eristykseen, joka perustuu seerumin ultrasuodatukseen ja tarjoaa merkittävää parannusta nykyisiin käytössä oleviin menetelmiin. Koska EV:t ovat keskeisiä tekijöitä solujen välisessä viestinnässä, selvitimme aktivoituneista monosyyteistä ja osteoklasteista peräisin olevien EV:den vaikutus rasvakudosperäisten kantasolujen geenien ilmentymiseen. Aktivoituneiden monosyyttien EV:t edistivät kantasolujen sytokiinien eritystä, kuvastaen EV:den immunomoduloivaa mekanismia, sekä myös matriisimetalloproteinaasien geenien ilmentymiseen, vaikuttamalla luun mikroympäristön uudistumiseen. EV:en merkitystä tutkittiin lisäksi myös pahanlaatuisen kasvaimen, osteosarkooman (OS), kehittymisessä, altistamalla kudosperäisiä kantasoluja ja pre-osteoblasteja OS-soluista peräisin olevilla EV:llä (OS-EV). Tutkimuksemme osoitti, että OS-EV:t vaikuttivat muokkaamalla sekä kudosperäisten kantasolujen epigeneettistä tilaa, että luun mikroympäristön uudistumiseen liittyvien geenien ilmentymistä. Näin ollen osoitimme, että epigeneettinen säätely voisi olla varhaisimpia tapahtumia kudosperäisten kantasolujen transformaatiossa OS:n muodostuksessa. Kehittääksemme uusia kudosteknologisia menetelmiä luukudoksen tuottamiseksi, suoritimme in vivo-tutkimuksen minisialla. Ihmisen ja minisian hampaiden ja leukojen samankaltaisuuksien vuoksi minisika valikoitui tutkimuksemme eläinmalliksi. Jokainen bioimplantti valmistettiin yksilöllisesti minisian leuan tietokonetomografian kuvantamisdatan pohjalta, tietokoneavusteisen suunnittelun ja valmistuksen (CAD-CAM) avulla. Alaleuka tuettiin yksilöidyillä mittatilaustyönä tehdyillä 3D-tulostetuilla titaanirekonstruktiolevyillä, koska itse bioimplantti ei yksin olisi kestänyt pureskelusta ja muusta purentaelimen toiminnasta syntyviä voimia. Tämän väitöskirjan tulokset osoittivat EV:en vaikuttavan solujen immuunijärjestelmään ja mikroympäristöön, sekä luun uudistumisessa että luusyövän muodostumisessa. Kuitenkin, lisätutkimukset ovat välttämättömiä luun uudistamisen konseptien vahvistamiseksi kudosteknologisten strategioiden toteuttamiseksi potilashoidoissa

Genome-Wide Association and Exome Sequencing Study of Language Disorder in an Isolated Population

WOS: 000373197500020PubMed ID: 27016271BACKGROUND AND OBJECTIVE: Developmental language disorder (DLD) is a highly prevalent neurodevelopmental disorder associated with negative outcomes in different domains; the etiology of DLD is unknown. To investigate the genetic underpinnings of DLD, we performed genome-wide association and whole exome sequencing studies in a geographically isolated population with a substantially elevated prevalence of the disorder (ie, the AZ sample). METHODS: DNA samples were collected from 359 individuals for the genome-wide association study and from 12 severely affected individuals for whole exome sequencing. Multifaceted phenotypes, representing major domains of expressive language functioning, were derived from collected speech samples. RESULTS: Gene-based analyses revealed a significant association between SETBP1 and complexity of linguistic output (P = 5.47 x 10(-7)). The analysis of exome variants revealed coding sequence variants in 14 genes, most of which play a role in neural development. Targeted enrichment analysis implicated myocyte enhancer factor-2 (MEF2)-regulated genes in DLD in the AZ population. The main findings were successfully replicated in an independent cohort of children at risk for related disorders (n = 37). CONCLUSIONS: MEF2-regulated pathways were identified as potential candidate pathways in the etiology of DLD. Several genes (including the candidate SETBP1 and other MEF2-related genes) seem to jointly influence certain, but not all, facets of the DLD phenotype. Even when genetic and environmental diversity is reduced, DLD is best conceptualized as etiologically complex. Future research should establish whether the signals detected in the AZ population can be replicated in other samples and languages and provide further characterization of the identified pathway.National Institute of Health [R01 DC007665, P50 HD052120]; NIH Centers for Mendelian Genomics [5U54HG006504]; National Science Foundation Integrative Graduate Education and Research Traineeship grant [114399]; Government of the Russian Federation [14.Z50.31.0027]; National Institutes of Health (NIH)Supported by National Institute of Health grants R01 DC007665 (Dr Grigorenko, Principal Investigator) and P50 HD052120 (Richard Wagner, Principal Investigator), NIH Centers for Mendelian Genomics (5U54HG006504), National Science Foundation Integrative Graduate Education and Research Traineeship grant 114399 (Dr Magnuson, Principal Investigator), and grant 14.Z50.31.0027 from the Government of the Russian Federation (Dr Grigorenko, Principal Investigator). Funded by the National Institutes of Health (NIH)

Genome-Wide Association and Exome Sequencing Study of Language Disorder in an Isolated Population

BACKGROUND AND OBJECTIVE: Developmental language disorder (DLD) is a highly prevalent neurodevelopmental disorder associated with negative outcomes in different domains; the etiology of DLD is unknown. To investigate the genetic underpinnings of DLD, we performed genome-wide association and whole exome sequencing studies in a geographically isolated population with a substantially elevated prevalence of the disorder (ie, the AZ sample)

Genome-Wide Association and Exome Sequencing Study of Language Disorder in an Isolated Population

BACKGROUND AND OBJECTIVE: Developmental language disorder (DLD) is a highly prevalent neurodevelopmental disorder associated with negative outcomes in different domains; the etiology of DLD is unknown. To investigate the genetic underpinnings of DLD, we performed genome-wide association and whole exome sequencing studies in a geographically isolated population with a substantially elevated prevalence of the disorder (ie, the AZ sample). METHODS: DNA samples were collected from 359 individuals for the genome-wide association study and from 12 severely affected individuals for whole exome sequencing. Multifaceted phenotypes, representing major domains of expressive language functioning, were derived from collected speech samples. RESULTS: Gene-based analyses revealed a significant association between SETBP1 and complexity of linguistic output (P = 5.47 × 10(−7)). The analysis of exome variants revealed coding sequence variants in 14 genes, most of which play a role in neural development. Targeted enrichment analysis implicated myocyte enhancer factor–2 (MEF2)-regulated genes in DLD in the AZ population. The main findings were successfully replicated in an independent cohort of children at risk for related disorders (n = 372). CONCLUSIONS: MEF2-regulated pathways were identified as potential candidate pathways in the etiology of DLD. Several genes (including the candidate SETBP1 and other MEF2-related genes) seem to jointly influence certain, but not all, facets of the DLD phenotype. Even when genetic and environmental diversity is reduced, DLD is best conceptualized as etiologically complex. Future research should establish whether the signals detected in the AZ population can be replicated in other samples and languages and provide further characterization of the identified pathway