Editorial : Extracellular Vesicles in Bone Oncology

Non peer reviewe

Perspectives for Clinical Translation of Adipose Stromal/Stem Cells

Adipose stromal/stem cells (ASCs) are an ideal cell type for regenerative medicine applications, as they can easily be harvested from adipose tissue in large quantities. ASCs have excellent proliferation, differentiation, and immunoregulatory capacities that have been demonstrated in numerous studies. Great interest and investment have been placed in efforts to exploit the allogeneic use and immunomodulatory and anti-inflammatory effects of ASCs. However, bridging the gap between in vitro and in vivo studies and moving into clinical practice remain a challenge. For the clinical translation of ASCs, several issues must be considered, including how to characterise such a heterogenic cell population and how to ensure their safety and efficacy. This review explores the different phases of in vitro and preclinical ASC characterisation and describes the development of appropriate potency assays. In addition, good manufacturing practice requirements are discussed, and cell-based medicinal products holding marketing authorisation in the European Union are reviewed. Moreover, the current status of clinical trials applying ASCs and the patent landscape in the field of ASC research are presented. Overall, this review highlights the applicability of ASCs for clinical cell therapies and discusses their potential.Peer reviewe

The burden of chronic rhinosinusitis with nasal polyps and its relation to asthma in Finland

Background Chronic rhinosinusitis with nasal polyps (CRSwNP) is commonly associated with asthma. Treatment of CRSwNP includes intranasal and systemic corticosteroids, with non-responsive patients commonly considered for endoscopic sinus surgery (ESS). This nationwide register-based study evaluated the incidence, prevalence, and treatment burden of CRSwNP in Finland, and their association with the presence and severity of comorbid asthma. Methods Electronic health records of patients diagnosed with CRSwNP between 1.1.2012 and 31.12.2018 in Finnish specialty and primary care were included in the study. The patients were divided into subgroups based on presence, severity, and control of asthma: no asthma, mild to moderate asthma, severe controlled asthma, and severe uncontrolled asthma. A mean cumulative count of ESS was calculated over time per subgroup. Results The prevalence of CRSwNP increased from 602.2 to 856.7 patients per 100,000 population between years 2012 and 2019 (p < 0.001). A total of 18,563 patients (59.9% male) had incident CRSwNP between 2012 and 2019, with 27% having asthma, 6% having severe asthma, and 1.5% having severe uncontrolled asthma. In the no asthma, severe controlled asthma, and severe uncontrolled asthma subgroups, systemic corticosteroids were used by 54.1%, 94.9% and 99.3% (p < 0.001), respectively, while the ESS count 3 years post diagnosis was 0.49, 0.68 and 0.80, respectively. Conclusions The prevalence of CRSwNP showed a significant increase in the recent decade in Finland. Comorbid asthma, and in particular severe asthma, increased the probability of receiving systemic corticosteroids and undergoing ESS. Thus, improved management of CRSwNP in patients with comorbid asthma is urgently needed.Peer reviewe

Effects of macromolecular crowding on human adipose stem cell culture in fetal bovine serum, human serum and defined xeno-free/serum-free conditions

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation

Extracellular small non-coding RNA contaminants in fetal bovine serum and serum-free media

In the research field of extracellular vesicles (EVs), the use of fetal bovine serum (FBS) depleted of EVs for in vitro studies is advocated to eliminate the confounding effects of media derived EVs. EV-depleted FBS may either be prepared by ultracentrifugation or purchased commercially. Nevertheless, these preparations do not guarantee an RNA-free FBS for in vitro use. In this study we address the RNA contamination issue, of small non-coding (nc)RNA in vesicular or non-vesicular fractions of FBS, ultracentrifugation EV-depleted FBS, commercial EV-depleted FBS, and in our recently developed filtration based EV-depleted FBS. Commercially available serum- and xeno-free defined media were also screened for small ncRNA contamination. Our small ncRNA sequencing data showed that all EV-depleted media and commercially available defined media contained small ncRNA contaminants. Out of the different FBS preparations studied, our ultrafiltration-based method for EV depletion performed the best in depleting miRNAs. Certain miRNAs such miR-122 and miR-203a proved difficult to remove completely and were found in all media. Compared to miRNAs, other small ncRNA (snRNA,Y RNA, snoRNA, and piRNA) were difficult to eliminate from all the studied media. Additionally, our tested defined media contained miRNAs and other small ncRNAs, albeit at a much lower level than in serum preparations. Our study showed that no media is free of small ncRNA contaminants. Therefore, in order to screen for baseline RNA contamination in culturing media, RNA sequencing data should be carefully controlled by adding a media sample as a control. This should be a mandatory step before performing cell culture experiments in order to eliminate the confounding effects of media.Peer reviewe

Proangiogenic Hypoxia-Mimicking Agents Attenuate Osteogenic Potential of Adipose Stem/Stromal Cells

BACKGROUND: Insufficient vascularization hampers bone tissue engineering strategies for reconstructing large bone defects. Delivery of prolyl-hydroxylase inhibitors (PHIs) is an interesting approach to upregulate vascular endothelial growth factor (VEGF) by mimicking hypoxic stabilization of hypoxia-inducible factor-1alpha (HIF-1α). This study assessed two PHIs: dimethyloxalylglycine (DMOG) and baicalein for their effects on human adipose tissue-derived mesenchymal stem/stromal cells (AT-MSCs). METHODS: Isolated AT-MSCs were characterized and treated with PHIs to assess the cellular proliferation response. Immunostaining and western-blots served to verify the HIF-1α stabilization response. The optimized concentrations for long-term treatment were tested for their effects on the cell cycle, apoptosis, cytokine secretion, and osteogenic differentiation of AT-MSCs. Gene expression levels were evaluated for alkaline phosphatase (ALPL), bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor A (VEGFA), secreted phosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). In addition, stemness-related genes Kruppel-like factor 4 (KLF4), Nanog homeobox (NANOG), and octamer-binding transcription factor 4 (OCT4) were assessed. RESULTS: PHIs stabilized HIF-1α in a dose-dependent manner and showed evident dose- and time dependent antiproliferative effects. With doses maintaining proliferation, DMOG and baicalein diminished the effect of osteogenic induction on the expression of RUNX2, ALPL, and COL1A1, and suppressed the formation of mineralized matrix. Suppressed osteogenic response of AT-MSCs was accompanied by an upregulation of stemness-related genes. CONCLUSION: PHIs significantly reduced the osteogenic differentiation of AT-MSCs and rather upregulated stemness-related genes. PHIs proangiogenic potential should be weighed against their longterm direct inhibitory effects on the osteogenic differentiation of AT-MSCs.Peer reviewe

Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation.Peer reviewe

Cranioplasty with Adipose-Derived Stem Cells, Beta-Tricalcium Phosphate Granules and Supporting Mesh : Six-Year Clinical Follow-Up Results

Several alternative techniques exist to reconstruct skull defects. The complication rate of the cranioplasty procedure is high and the search for optimal materials and techniques continues. To report long-term results of patients who have received a cranioplasty using autologous adipose-derived stem cells (ASCs) seeded on beta-tricalcium phosphate (betaTCP) granules. Between 10/2008 and 3/2010, five cranioplasties were performed (four females, one male; average age 62.0 years) using ASCs, betaTCP granules and titanium or resorbable meshes. The average defect size was 8.1 x 6.7 cm(2). Patients were followed both clinically and radiologically. The initial results were promising, with no serious complications. Nevertheless, in the long-term follow-up, three of the five patients were re-operated due to graft related problems. Two patients showed marked resorption of the graft, which led to revision surgery. One patient developed a late infection (7.3 years post-operative) that required revision surgery and removal of the graft. One patient had a successfully ossified graft, but was re-operated due to recurrence of the meningioma 2.2 years post-operatively. One patient had an uneventful clinical follow-up, and the cosmetic result is satisfactory, even though skull x-rays show hypodensity in the borders of the graft. Albeit no serious adverse events occurred, the 6-year follow-up results of the five cases are unsatisfactory. The clinical results are not superior to results achieved by conventional cranial repair methods. The use of stem cells in combination with betaTCP granules and supporting meshes in cranial defect reconstruction need to be studied further before continuing with clinical trials.Peer reviewe

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

Adipose-Derived Mesenchymal Stem Cells do not Affect the Invasion and Migration Potential of Oral Squamous Carcinoma Cells

Mesenchymal stem cells (MSCs) are commonly isolated from bone marrow and adipose tissue. Depending on the tissue of origin, MSCs have different characteristics and physiological effects. In various cancer studies, MSCs have been found to have either tumor-promoting or tumor-inhibiting action. This study investigated the effect of adipose tissue-MSCs (AT-MSCs) and bone marrow-MSCs (BM-MSCs) on global long interspersed nuclear element-1 (LINE-1) methylation, the expression level of microenvironment remodeling genes and cell proliferation, migration and invasion of oral tongue squamous cell carcinoma (OTSCC). Additionally, we studied the effect of human tongue squamous carcinoma (HSC-3)-conditioned media on LINE-1 methylation and the expression of microenvironment remodeling genes in AT-MSCs and BM-MSCs. Conditioned media from HSC-3 or MSCs did not affect LINE-1 methylation level in either cancer cells or MSCs, respectively. In HSC-3 cells, no effect of MSCs-conditioned media was detected on the expression of ICAM1, ITGA3 or MMP1. On the other hand, HSC-3-conditioned media upregulated ICAM1 and MMP1 expression in both types of MSCs. Co-cultures of AT-MSCs with HSC-3 did not induce proliferation, migration or invasion of the cancer cells. In conclusion, AT-MSCs, unlike BM-MSCs, seem not to participate in oral cancer progression