Generation of a human induced pluripotent stem cell line from a patient with GM3 synthase deficiency using self-replicating RNA vector.

GM3 synthase deficiency (GM3SD) is caused by biallelic variants in the ST3GAL5 gene. Early clinical features of GM3SD include infantile onset of severe irritability and feeding difficulties, early intractable seizures, growth failure, hypotonia, sensorineural hearing impairment. We describe the generation and characterization the human induced pluripotent stem cell (hiPSC) line derived from fibroblasts of a 13-year-old girl with GM3 synthase deficiency resulted compound heterozygous for two new variants in the ST3GAL5 gene, c.1166A > G (p.His389Arg) and the c.1024G > A (p.Gly342Ser). The generated hiPSC line shows a normal karyotype, expresses pluripotency markers, and is able to differentiate into the three germ layers.

Polymorphisms in transcription factor binding sites and enhancer regions and pancreatic ductal adenocarcinoma risk.

Genome-wide association studies (GWAS) are a powerful tool for detecting variants associated with complex traits and can help risk stratification and prevention strategies against pancreatic ductal adenocarcinoma (PDAC). However, the strict significance threshold commonly used makes it likely that many true risk loci are missed. Functional annotation of GWAS polymorphisms is a proven strategy to identify additional risk loci. We aimed to investigate single-nucleotide polymorphisms (SNP) in regulatory regions [transcription factor binding sites (TFBSs) and enhancers] that could change the expression profile of multiple genes they act upon and thereby modify PDAC risk. We analyzed a total of 12,636 PDAC cases and 43,443 controls from PanScan/PanC4 and the East Asian GWAS (discovery populations), and the PANDoRA consortium (replication population). We identified four associations that reached study-wide statistical significance in the overall meta-analysis: rs2472632(A) (enhancer variant, OR 1.10, 95%CI 1.06,1.13, p = 5.5 × 10-8), rs17358295(G) (enhancer variant, OR 1.16, 95%CI 1.10,1.22, p = 6.1 × 10-7), rs2232079(T) (TFBS variant, OR 0.88, 95%CI 0.83,0.93, p = 6.4 × 10-6) and rs10025845(A) (TFBS variant, OR 1.88, 95%CI 1.50,1.12, p = 1.32 × 10-5). The SNP with the most significant association, rs2472632, is located in an enhancer predicted to target the coiled-coil domain containing 34 oncogene. Our results provide new insights into genetic risk factors for PDAC by a focused analysis of polymorphisms in regulatory regions and demonstrating the usefulness of functional prioritization to identify loci associated with PDAC risk.

The New Era of Therapeutic Strategies for the Treatment of Retinitis Pigmentosa: A Narrative Review of Pathomolecular Mechanisms for the Development of Cell-Based Therapies.

Retinitis pigmentosa, defined more properly as cone-rod dystrophy, is a paradigm of inherited diffuse retinal dystrophies, one of the rare diseases with the highest prevalence in the worldwide population and one of the main causes of low vision in the pediatric and elderly age groups. Advancements in and the understanding of molecular biology and gene-editing technologies have raised interest in laying the foundation for new therapeutic strategies for rare diseases. As a consequence, new possibilities for clinicians and patients are arising due to the feasibility of treating such a devastating disorder, reducing its complications. The scope of this review focuses on the pathomolecular mechanisms underlying RP better to understand the prospects of its treatment using innovative approaches.

Generation of human induced pluripotent stem cell line (AOUMEYi001-A) from a patient affected by Congenital disorders of glycosylation (ALG8-CDG) using self-replicating RNA vector.

Congenital Disorders of Glycosylation (CDG) are rare inherited metabolic diseases caused by genetic defects in the glycosylation of proteins and lipids. In this study, we describe the generation and characterization of one human induced pluripotent stem cell (hiPSC) line from a 15-year-old male patient with CDG. The patient carried three variants, one (c.122G > A; p.Arg41Gln) inherited from his father and two (c.445 T > G; p.Leu149Arg and the novel c.980C > G; p.Thr327Arg) inherited from his mother in the ALG8 gene (OMIM #608103). The generated hiPSC line shows a normal karyotype, expresses pluripotency markers, and is able to differentiate into the three germ layers.

Treatment of Inherited Retinal Dystrophies with Somatic Cell Therapy Medicinal Product: A Review.

Inherited retinal dystrophies and retinal degenerations related to more common diseases (i.e., age-related macular dystrophy) are a major issue and one of the main causes of low vision in pediatric and elderly age groups. Advancement and understanding in molecular biology and the possibilities raised by gene-editing techniques opened a new era for clinicians and patients due to feasible possibilities of treating disabling diseases and the reduction in their complications burden. The scope of this review is to focus on the state-of-the-art in somatic cell therapy medicinal products as the basis of new insights and possibilities to use this approach to treat rare eye diseases.

Extracellular vesicles miR-210 as a potential biomarker for diagnosis and survival prediction of oral squamous cell carcinoma patients.

BACKGROUND: The identification of early diagnostic and prognostic biomarkers in oral squamous cell carcinoma (OSCC) is an unmet clinical need. We hypothesized that extracellular vesicles miR-210 expression (EV-miR-210) could be a potential biomarker for OSCC diagnosis and follow-up. METHODS: The expression of EV-miR-210 was measured in the plasma of OSCC patients (n = 30) and compared to that of controls (n = 14). RESULTS: The median EV-miR-210 expression was significantly higher in OSCC patients compared to controls who had often, undetectable levels (p < 0.0001). We performed receiver operating characteristic (ROC) analysis for discriminating OSCC cases from controls. EV-miR-210 yielded an area under the curve (AUC) of 0.9513 with sensitivity 92.3% and specificity 86.6%. Kaplan-Meier curves indicated that high EV-miR-210 expression was associated with worse 3 years' survival (p < 0.05). Cox regression hazard model indicated that high EV-miR-210, G2, and G3 grading and pathological nodal status (pN)>1 were independent predictors of worse survival in OSCC patients. CONCLUSION: These preliminary data suggest that EV-mir-210 may be a novel diagnostic and prognostic biomarker in OSCC.

Platelet-rich plasma affects gap junctional features in myofibroblasts in vitro via vascular endothelial growth factor (VEGF)-A/VEGF receptor.

NEW FINDINGS: What is the central question of this study? It is a challenge to discover effective therapies for fibrosis. Increasing evidence supports the antifibrotic potential of platelet-rich plasma (PRP) as a source of bioactive molecules, such as vascular endothelial growth factor (VEGF)-A. However, the effects and mechanisms of action of PRP need to be clarified. What is the main finding and its importance? This report clarifies the mechanisms mediating the antifibrotic action of PRP, strengthening the role of VEGF-A/VEGF receptor, and identifies gap junction currents and connexin 43 as novel targets of this pathway in the fibroblast-to-myofibroblast transition induced by the transforming growth factor-ß1. ABSTRACT: Despite increasing experimental evidence, the antifibrotic potential of platelet-rich plasma (PRP) remains controversial, and its mechanisms of action are not fully clarified. This short report extends our previous research on the capability of PRP to prevent the in vitro differentiation of fibroblasts toward myofibroblasts, the key effectors of fibrosis, induced by the profibrotic agent transforming growth factor-ß1 (TGF-ß1). In particular, we focused on the involvement of signalling mediated by vascular endothelial growth factor (VEGF)-A/VEGF receptor (VEGFR) in the PRP-induced fibroblast response, highlighting gap junction features. Electrophysiological and morphological analyses revealed that PRP hindered morphofunctional differentiation of both murine NIH/3T3 and human primary adult skin fibroblasts toward myofibroblasts as judged by the analysis of membrane phenomena, α-smooth muscle actin and vinculin expression and cell morphology. Neutralization of VEGF-A by blocking antibodies or pharmacological inhibition of VEGFR by KRN633 in TGF-ß1-treated fibroblasts prevented the PRP-promoted effects, such as the reduction of voltage-dependent transjunctional currents in cell pairs and a decreased expression of connexin 43, the typical connexin isoform forming voltage-dependent connexons. The role of VEGF-A in inhibiting these events was confirmed by treating TGF-ß1-stimulated fibroblasts with soluble VEGF-A. The results obtained when cells were differentiated using KRN633 alone suggest an antagonistic cross-talk between TGF-ß1 and VEGFR. In conclusion, this study identifies, for the first time, gap junction currents as crucial targets in the VEGF-A/VEGFR-mediated antifibrotic pathway and provides new insights into mechanisms behind the action of PRP in preventing differentiation of fibroblasts to myofibroblasts.

Association of Genetic Variants Affecting microRNAs and Pancreatic Cancer Risk.

Genetic factors play an important role in the susceptibility to pancreatic cancer (PC). However, established loci explain a small proportion of genetic heritability for PC; therefore, more progress is needed to find the missing ones. We aimed at identifying single nucleotide polymorphisms (SNPs) affecting PC risk through effects on micro-RNA (miRNA) function. We searched in silico the genome for SNPs in miRNA seed sequences or 3 prime untranslated regions (3'UTRs) of miRNA target genes. Genome-wide association data of PC cases and controls from the Pancreatic Cancer Cohort (PanScan) Consortium and the Pancreatic Cancer Case-Control (PanC4) Consortium were re-analyzed for discovery, and genotyping data from two additional consortia (PanGenEU and PANDoRA) were used for replication, for a total of 14,062 cases and 11,261 controls. None of the SNPs reached genome-wide significance in the meta-analysis, but for three of them the associations were in the same direction in all the study populations and showed lower value of p in the meta-analyses than in the discovery phase. Specifically, rs7985480 was consistently associated with PC risk (OR = 1.12, 95% CI 1.07-1.17, p = 3.03 × 10-6 in the meta-analysis). This SNP is in linkage disequilibrium (LD) with rs2274048, which modulates binding of various miRNAs to the 3'UTR of UCHL3, a gene involved in PC progression. In conclusion, our results expand the knowledge of the genetic PC risk through miRNA-related SNPs and show the usefulness of functional prioritization to identify genetic polymorphisms associated with PC risk.

Comparison of CryoMACS Freezing Bags with Maco Biotech Freezing-Ethinyl Vinyl Acetate Bags for Hematopoietic Progenitor Cells Cryopreservation Using a CD34+-Enriched Product.

Background: Hematopoietic progenitor cells (HPCs) cryopreservation have applications, especially in the autologous setting, allowing therapeutic use several years after collection. Cryopreservation aims to preserve the therapeutic properties of HPCs, and successful cryopreservation depends on several factors such as preservation procedures, biopreservation media, freezing rates, and thawing procedures. In this context, the choice of the freezing bag is critical as it provides mechanical protection during the freezing process. Since Maco Biotech Freezing-ethinyl vinyl acetate (EVA) Bags® are no longer available in our country, a comparative study was developed to verify bioequivalence with the Miltenyi CryoMACS® freezing bag. Methods: In this study, a CD34+-enriched product was used to better reproduce HPC apheresis. Freezing bags were filled with the same volume, cryopreserved with controlled rate freezing, and stored in the vapor phase of liquid nitrogen for at least 6 months. After thawing, all bags were tested for integrity and sterility using a microbial challenge. In addition, a comparison was developed by evaluating recovery of white blood cells, mononuclear cells, lymphocytes, and CD34+ cells. Results: No significant differences between the two manufacturers' bags have been observed in terms of the evaluated parameters. Data were confirmed, even comparing bags according to filling volume. Data presented in this study support the conclusion that CryoMACS freezing bags are bioequivalent to Maco Biotech Freezing-EVA Bags for HPC cryopreservation.

An alternative procedure to leukapheresis for peripheral hematopoietic progenitor cell collection in very-low-weight children: A single pediatric center experience.

BACKGROUND: PBSC collection using a blood cell separator in very low weight patients can be frequently complicated by severe adverse effects and technical difficulties. MATERIAL AND METHODS: From March 2013 to January 2017, 14 PBSC collections were performed in 12 children weighing less than 10 kg, affected by different solid tumours. PBSC collection was performed with a "homemade" aseptically assembled circuit. The circuit is composed by a 150 mL collection bag connected with a 4 stopcock ramp, perfused with ACD. This circuit allows collection of a specific total blood amount from CVC, depending on CD34+ /kg target. RESULTS: Mean CD34+ cell performance per collection was 9.3 × 106 /kg. Tolerance to the procedure was very good as none of the patients experienced complications, with the exception of a patient who showed mild cyanosis and pallor after collection. Moreover, no bleeding or thrombotic complications have been observed. To date, 16 PBSC reinfusions have been performed in 7 children with a mean CD34+ cells viability of 98.1% ± 2.7 and mean WBC viability of 57% ± 10. Cell recovery after thawing was 87% ± 10.8. A rapid graft intake for both neutrophils and platelets, between day 7 and 20 after reinfusion was observed. DISCUSSION: The procedure of total blood collection without the use of a cell separator is feasible and allows a good PBSC collection without significant side effects in very low-weight children. Moreover, this method could represent a valid and safe alternative to leukapheresis in patients where classic procedure could be difficult to apply.

Platelet-Rich Plasma Modulates Gap Junction Functionality and Connexin 43 and 26 Expression During TGF-ß1-Induced Fibroblast to Myofibroblast Transition: Clues for Counteracting Fibrosis.

Skeletal muscle repair/regeneration may benefit by Platelet-Rich Plasma (PRP) treatment owing to PRP pro-myogenic and anti-fibrotic effects. However, PRP anti-fibrotic action remains controversial. Here, we extended our previous researches on the inhibitory effects of PRP on in vitro transforming growth factor (TGF)-ß1-induced differentiation of fibroblasts into myofibroblasts, the effector cells of fibrosis, focusing on gap junction (GJ) intercellular communication. The myofibroblastic phenotype was evaluated by cell shape analysis, confocal fluorescence microscopy and Western blotting analyses of α-smooth muscle actin and type-1 collagen expression, and electrophysiological recordings of resting membrane potential, resistance, and capacitance. PRP negatively regulated myofibroblast differentiation by modifying all the assessed parameters. Notably, myofibroblast pairs showed an increase of voltage-dependent GJ functionality paralleled by connexin (Cx) 43 expression increase. TGF-ß1-treated cells, when exposed to a GJ blocker, or silenced for Cx43 expression, failed to differentiate towards myofibroblasts. Although a minority, myofibroblast pairs also showed not-voltage-dependent GJ currents and coherently Cx26 expression. PRP abolished the TGF-ß1-induced voltage-dependent GJ current appearance while preventing Cx43 increase and promoting Cx26 expression. This study adds insights into molecular and functional mechanisms regulating fibroblast-myofibroblast transition and supports the anti-fibrotic potential of PRP, demonstrating the ability of this product to hamper myofibroblast generation targeting GJs.

Genome-wide association study identifies an early onset pancreatic cancer risk locus.

Early onset pancreatic cancer (EOPC) is a rare disease with a very high mortality rate. Almost nothing is known on the genetic susceptibility of EOPC, therefore, we performed a genome-wide association study (GWAS) to identify novel genetic variants specific for patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) at younger ages. In the first phase, conducted on 821 cases with age of onset ≤60 years, of whom 198 with age of onset ≤50, and 3227 controls from PanScan I-II, we observed four SNPs (rs7155613, rs2328991, rs4891017 and rs12610094) showing an association with EOPC risk (P < 1 × 10-4 ). We replicated these SNPs in the PANcreatic Disease ReseArch (PANDoRA) consortium and used additional in silico data from PanScan III and PanC4. Among these four variants rs2328991 was significant in an independent set of 855 cases with age of onset ≤60 years, of whom 265 with age of onset ≤50, and 4142 controls from the PANDoRA consortium while in the in silico data, we observed no statistically significant association. However, the resulting meta-analysis supported the association (P = 1.15 × 10-4 ). In conclusion, we propose a novel variant rs2328991 to be involved in EOPC risk. Even though it was not possible to find a mechanistic link between the variant and the function, the association is supported by a solid statistical significance obtained in the largest study on EOPC genetics present so far in the literature.

Targeting GD2-positive glioblastoma by chimeric antigen receptor empowered mesenchymal progenitors.

Tumor targeting by genetically modified mesenchymal stromal/stem cells (MSCs) carrying anti-cancer molecules represents a promising cell-based strategy. We previously showed that the pro-apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be successfully delivered by MSCs to cancer sites. While the interaction between TRAIL and its receptors is clear, more obscure is the way in which MSCs can selectively target tumors and their antigens. Several neuroectoderm-derived neoplasms, including glioblastoma (GBM), sarcomas, and neuroblastoma, express high levels of the tumor-associated antigen GD2. We have already challenged this cell surface disialoganglioside by a chimeric antigen receptor (CAR)-T cell approach against neuroblastoma. With the intent to maximize the therapeutic profile of MSCs delivering TRAIL, we here originally developed a bi-functional strategy where TRAIL is delivered by MSCs that are also gene modified with the truncated form of the anti-GD2 CAR (GD2 tCAR) to mediate an immunoselective recognition of GD2-positive tumors. These bi-functional MSCs expressed high levels of TRAIL and GD2 tCAR associated with a robust anti-tumor activity against GD2-positive GBM cells. Most importantly, the anti-cancer action was reinforced by the enhanced targeting potential of such bi-functional cells. Collectively, our results suggest that a truncated anti-GD2 CAR might be a powerful new tool to redirect MSCs carrying TRAIL against GD2-expressing tumors. This affinity-based dual targeting holds the promise to combine site-specific and prolonged retention of MSCs in GD2-expressing tumors, thereby providing a more effective delivery of TRAIL for still incurable cancers.

Genetic determinants of telomere length and risk of pancreatic cancer: A PANDoRA study.

Telomere deregulation is a hallmark of cancer. Telomere length measured in lymphocytes (LTL) has been shown to be a risk marker for several cancers. For pancreatic ductal adenocarcinoma (PDAC) consensus is lacking whether risk is associated with long or short telomeres. Mendelian randomization approaches have shown that a score built from SNPs associated with LTL could be used as a robust risk marker. We explored this approach in a large scale study within the PANcreatic Disease ReseArch (PANDoRA) consortium. We analyzed 10 SNPs (ZNF676-rs409627, TERT-rs2736100, CTC1-rs3027234, DHX35-rs6028466, PXK-rs6772228, NAF1-rs7675998, ZNF208-rs8105767, OBFC1-rs9420907, ACYP2-rs11125529 and TERC-rs10936599) alone and combined in a LTL genetic score ("teloscore", which explains 2.2% of the telomere variability) in relation to PDAC risk in 2,374 cases and 4,326 controls. We identified several associations with PDAC risk, among which the strongest were with the TERT-rs2736100 SNP (OR = 1.54; 95%CI 1.35-1.76; p = 1.54 × 10-10 ) and a novel one with the NAF1-rs7675998 SNP (OR = 0.80; 95%CI 0.73-0.88; p = 1.87 × 10-6 , ptrend = 3.27 × 10-7 ). The association of short LTL, measured by the teloscore, with PDAC risk reached genome-wide significance (p = 2.98 × 10-9 for highest vs. lowest quintile; p = 1.82 × 10-10 as a continuous variable). In conclusion, we present a novel genome-wide candidate SNP for PDAC risk (TERT-rs2736100), a completely new signal (NAF1-rs7675998) approaching genome-wide significance and we report a strong association between the teloscore and risk of pancreatic cancer, suggesting that telomeres are a potential risk factor for pancreatic cancer.

Platelet-Rich Plasma Prevents In Vitro Transforming Growth Factor-ß1-Induced Fibroblast to Myofibroblast Transition: Involvement of Vascular Endothelial Growth Factor (VEGF)-A/VEGF Receptor-1-Mediated Signaling †.

The antifibrotic potential of platelet-rich plasma (PRP) is controversial. This study examined the effects of PRP on in vitro transforming growth factor (TGF)-ß1-induced differentiation of fibroblasts into myofibroblasts, the main drivers of fibrosis, and the involvement of vascular endothelial growth factor (VEGF)-A in mediating PRP-induced responses. The impact of PRP alone on fibroblast differentiation was also assessed. Myofibroblastic phenotype was evaluated by confocal fluorescence microscopy and western blotting analyses of α-smooth muscle actin (sma) and type-1 collagen expression, vinculin-rich focal adhesion clustering, and stress fiber assembly. Notch-1, connexin 43, and VEGF-A expression were also analyzed by RT-PCR. PRP negatively regulated fibroblast-myofibroblast transition via VEGF-A/VEGF receptor (VEGFR)-1-mediated inhibition of TGF-ß1/Smad3 signaling. Indeed TGF-ß1/PRP co-treated fibroblasts showed a robust attenuation of the myofibroblastic phenotype concomitant with a decrease of Smad3 expression levels. The VEGFR-1 inhibition by KRN633 or blocking antibodies, or VEGF-A neutralization in these cells prevented the PRP-promoted effects. Moreover PRP abrogated the TGF-ß1-induced reduction of VEGF-A and VEGFR-1 cell expression. The role of VEGF-A signaling in counteracting myofibroblast generation was confirmed by cell treatment with soluble VEGF-A. PRP as single treatment did not induce fibroblast myodifferentiation. This study provides new insights into cellular and molecular mechanisms underpinning PRP antifibrotic action.

Correction: Inherited variants in genes somatically mutated in thyroid cancer.

[This corrects the article DOI: 10.1371/journal.pone.0174995.].

Human platelet lysate in mesenchymal stromal cell expansion according to a GMP grade protocol: a cell factory experience.

BACKGROUND: The use of platelet lysate (PL) for the ex-vivo expansion of mesenchymal stromal/stem cells (MSCs) was initially proposed by Doucet et al. in 2005, as an alternative to animal serum. Moreover, regulatory authorities discourage the use of fetal bovine serum (FBS) or other animal derivatives, to avoid risk of zoonoses and xenogeneic immune reactions. Even if many studies investigated PL composition, there still are some open issues related to its use in ex-vivo MSC expansion, especially according to good manufacturing practice (GMP) grade protocols. METHODS: As an authorized cell factory, we report our experience using standardized PL produced by Azienda Ospedaliero Universitaria Meyer Transfusion Service for MSC expansion according to a GMP grade clinical protocol. As suggested by other authors, we performed an in-vitro test on MSCs versus MSCs cultured with FBS that still represents the best way to test PL batches. We compared 12 MSC batches cultured with DMEM 5% PL with similar batches cultured with DMEM 10% FBS, focusing on the MSC proliferation rate, MSC surface marker expression, MSC immunomodulatory and differentiation potential, and finally MSC relative telomere length. RESULTS: Results confirmed the literature data as PL increases cell proliferation without affecting the MSC immunophenotype, immunomodulatory potential, differentiation potential and relative telomere length. CONCLUSIONS: PL can be considered a safe alternative to FBS for ex-vivo expansion of MSC according to a GMP grade protocol. Our experience confirms the literature data: a large number of MSCs for clinical applications can be obtained by expansion with PL, without affecting the MSC main features. Our experience underlines the benefits of a close collaboration between the PL producers (transfusion service) and the end users (cell factory) in a synergy of skills and experiences that can lead to standardized PL production.