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In the last years, mesenchymal stem cells (MSCs) have been identified as an attractive cell population in regenerative medicine. In view of future therapeutic applications, the study of specific differentiation‐related gene expression is a pivotal prerequisite to define the most appropriate MSC source for clinical translation. In this context, it is crucial to use stable housekeeping genes (HGs) for normalization of qRT‐PCR to obtain validated and comparable results. By our knowledge, an exhaustive validation study of HGs comparing MSCs from different sources under various differentiation conditions is still missing. In this pivotal study, we compared the expression levels of 12 genes (ACTB, Β2M, EF1alpha, GAPDH, GUSB, PPIA, RPL13A, RPLP0, TBP, UBC, YWHAZ and 18S rRNA) to assess their suitability as HGs in MSCs during adipogenic, osteogenic and chondrogenic differentiation. We demonstrated that many of the most popular HGs including 18S rRNA, B2M and ACTB were inadequate for normalization, whereas TBP/YWHAZ/GUSB were frequently identified among the best performers. Moreover, we showed the dramatic effects of suboptimal HGs choice on the quantification of cell differentiation markers, thus interfering with a reliable comparison of the lineage potential properties among various MSCs. Thus, in the emerging field of regenerative medicine, the identification of the most appropriate MSC source and cell line is so crucial for the treatment of patients that being inaccurate in the first step of the stem cell characterization can bring important consequences for the patients and for the promising potential of stem cell therapy.

Bone marrow mesenchymal stem/stromal cells (BMSCs) show great promise for bone repair, however they are isolated by an invasive bone marrow harvest and their regenerative potential decreases with age. Conversely, cord blood can be collected non-invasively after birth and contains MSCs (CBMSCs) that can be stored for future use. However, whether CBMSCs can replace BMSCs targeting bone repair is unknown. This study evaluates the in vitro osteogenic potential of unprimed, osteogenically primed, or chondrogenically primed CBMSCs and BMSCs and their in vivo bone forming capacity following ectopic implantation on biphasic calcium phosphate ceramics in nude mice. In vitro, alkaline phosphatase (intracellular, extracellular, and gene expression), and secretion of osteogenic cytokines (osteoprotegerin and osteocalcin) was significantly higher in BMSCs compared with CBMSCs, while CBMSCs demonstrated superior chondrogenic differentiation and secretion of interleukins IL-6 and IL-8. BMSCs yielded significantly more cell engraftment and ectopic bone formation compared to CBMSCs. However, priming of CBMSCs with either chondrogenic or BMP-4 supplements led to bone formation by CBMSCs. This study is the first direct quantification of the bone forming abilities of BMSCs and CBMSCs in vivo and, while revealing the innate superiority of BMSCs for bone repair, it provides avenues to induce osteogenesis by CBMSCs.

Background: The COVID-19 pandemic and control measures may have had an impact on unpleasant emotions experimented during the lockdown (LD). This may have increased the number of hours spent online and could have impacted the quality of the enacted behavior, in terms of loss of control of Internet use. In this online survey, we were interested in measure how much loss of control was perceived regarding online gambling, online shopping, the fruition of online pornographic content and web navigation.Design and methods: The online survey was carried out during the COVID-19 pandemic in the post-lockdown and 1232 subjects participated in the survey. In the participating sample, healthcare workers (HW) were 43.1% of the sample, of which 18.7% were directly involved in the Coronavirus emergency, and 52.3% of the sample is not a HW. Only 0.6% of the sample gambled online and 37.5% of those reported losing control of their gambling mode. Most of the sample shopped online during the LD (70.1%), but only 7.2% of those lost control by buying and/or spending more than what they had set themselves.Results: Significant data emerged showing that those who lost control while online shopping also lost control regarding the amount of time spent online (p<0.001); 21.6% of the sample, reported making use of online pornographic material during LD, 4.7% of them stated that the frequency increased and 5.1% reported losing control by having spent more money or more time than what was intended. Finally, 44.7% of the sample have experienced loss of control during the web navigation. Furthermore, during the LD 67.8% of the sample reports having experienced unpleasant emotions. Of these, 8.4% state that they enacted behaviors such as online gambling, online shopping, online pornographic material viewing and web navigation to counter their negative emotions. Interestingly, we found a correlation between loss of control during web navigation and online shopping and the emotional states “upset”, “scared” and “restless” (p<0.05).Conclusion: To conclude, there was no significant increase in potentially addictive behaviors, nor an increase in loss of control of these behaviors when enacted online. However, the loss of control in online shopping and web navigation was significantly correlated to the unpleasant emotional states of nervousness, fear and restlessness, whereas those who reported feeling strong and able to handle the situation experienced a lower loss of control in their web navigation. These correlations may suggest that these online behaviors may act as modulators of unpleasant emotional states.

Background The COVID-19 pandemic and the lockdown period lasted from March to May 2020, resulted in a highly stressful situation yielding different negative health consequences, including the worsening of smoking habit. Methods A web-based cross-sectional study on a convenient sample of 1013 Italian ever smokers aged 18 years or more was conducted. Data were derived from surveys compiled by three different groups of people: subjects belonging to Smoking Cessation Services, Healthcare Providers and Nursing Sciences’ students. All institutions were from Northern Italy. The primary outcome self-reported worsening (relapse or increase) or improvement (quit or reduce) of smoking habit during lockdown period. Multiple unconditional (for worsening) and multinomial (for improving) logistic regressions were carried out. Results Among 962 participants, 56.0% were ex-smokers. Overall, 13.2% of ex-smokers before lockdown reported relapsing and 32.7% of current smokers increasing cigarette intake. Among current smokers before lockdown, 10.1% quit smoking and 13.5% decreased cigarette intake. Out of 7 selected stressors related to COVID-19, four were significantly related to relapse (OR for the highest vs. the lowest tertile ranging between 2.24 and 3.62): fear of being infected and getting sick; fear of dying due to the virus; anxiety in listening to news of the epidemic; sense of powerlessness in protecting oneself from contagion. In addition to these stressors, even the other 3 stressors were related with increasing cigarette intensity (OR ranging between 1.90 and 4.18): sense of powerlessness in protecting loved ones from contagion; fear of losing loved ones due to virus; fear of infecting other. Conclusion The lockdown during the COVID-19 pandemic was associated with both self-reported relapse or increase smoking habit and also quitting or reduction of it.

Investigators observed the healing of a broncholpeural fistula soon after the injection of mesenchymal stem cells into the area surrounding the fistula. To the Editor: Large-airway defects and tracheobronchial dehiscence after lung resection present a problem for clinicians because there are few effective methods of treatment. 1 Bronchopleural fistula is a pathologic connection between the airway and the pleural space that may develop after lung resection. For many patients with empyema, the presence or absence of a fistula makes the difference between recovery, chronic illness, and death. 2 , 3 In our previous preclinical experiments, we found that bronchoscopic transplantation of mesenchymal stem cells derived from bone marrow could close a bronchopleural fistula with the extraluminal proliferation of fibroblasts and the development of collagenous matrix. . . .

Background A growing number of clinical trials have shown that regulatory T (T reg ) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional T reg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for T reg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of T reg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype). Results The GMP-compliant protocol defined in this work allows at least 4.11 × 10 9 T reg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit T reg cell immunomodulatory function. Conclusions These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the T reg cell number and function, which may slow the progression of certain diseases.

Background and objectives Children with multi-drug resistant idiopathic nephrotic syndrome (MDR-INS) usually progress to end-stage kidney disease with a consistent risk of disease recurrence after transplantation. New therapeutic options are needed for these patients. Mesenchymal stromal cells (MSCs) are multipotential non-hematopoietic cells with several immunomodulatory properties and growing clinical applications. Cord blood-derived MSC have peculiar anti-inflammatory and immunosuppressive properties. We aimed at assessing safety and efficacy of cord-blood-derived MSCs (CB-MSCs) in children with MDR-INS. Design, setting, participants Prospective, open-label, single arm phase I–II pilot study. Pediatric patients with MDR-INS, resistant to at least two lines of therapy, were enrolled. Allogenic CB-MSCs were administered intravenously on days 0, 14, and 21 at a dose of 1.5 × 10 6 cells/kg. Patients were followed for at least 12 months. The primary outcomes were safety and toxicity. The secondary outcome was remission at 12 months evaluated by urinary protein/urinary creatinine ratio (uPr/uCr). Circulating regulatory T cells (Tregs) were monitored. Results Eleven pediatric patients with MDR-INS (10 females, median age 13 years) resistant to a median of 3 previous lines of therapy were enrolled. All patients completed the CB-MSC infusion schedule. No patient experienced any infusion-related adverse event or toxicity. Nine patients were assessable for efficacy. At the 12 months follow-up after the treatment, the median uPr/uCr did not change significantly from baseline (8.13 vs. 9.07; p  = 0.98), while 3 patients were in partial or complete remission. A lower baseline uPr/uCr was a predictor of remission (2.55 vs. 8.74; p  = 0.0238). Tregs count was not associated with CB-MSCs therapy. Conclusions CB-MSCs are safe and may have a role in the immunosuppressive therapy of pediatric patients with MDR-INS. This preliminary experience paves the way toward further phase II studies addressing MSC efficacy in immune-mediated kidney diseases.

During the last decade it has been demonstrated that mesenchymal progenitors are present and can be isolated also from cord blood (CB). Recently, we managed to set up a standard protocol allowing the isolation of mesenchymal stromal cells (MSCs) with high proliferative potential and multiple differentiation capabilities, whereas the generation rate of MSC-initiating colonies could still be further improved. Herein, we strikingly succeeded in defining some simple and basic culture conditions based on the use of a chemically defined medium that increased the colony isolation efficiency up to almost 80% of processed CB units. Importantly, this result was achieved irrespective of CB unit white blood cell content and time elapsed from delivery, two limiting parameters involved with processing CB units. Thus, this high efficiency is guaranteed without strict selection of the starting material. In addition, since we are profoundly concerned about how different culture conditions can influence cell behavior, we devoted part of this study to in-depth characterization of the established CB-MSC populations to confirm their stemness features in this novel isolation and culture system. Therefore, an extended study of their immunophenotype, including classical pericytic markers, and a detailed molecular analysis addressing telomere length and also stemness-related microRNA contribution were performed. In summary, we propose a straightforward, extremely efficient, and reliable approach to isolate and expand thoroughly characterized CB-MSCs, even when poor-quality CB units are the only available source, or there is no space for an isolation to fail.

Background Recently, mesenchymal stromal cells (MSCs) have gained recognition for their clinical utility in transplantation to induce tolerance and to improve/replace pharmacological immunosuppression. Cord blood (CB)-derived MSCs are particularly attractive for their immunological naivety and peculiar anti-inflammatory and anti-apoptotic properties. Objectives: The objective of this study was to obtain an inventory of CB MSCs able to support large-scale advanced therapy medicinal product (ATMP)-based clinical trials. Study design: We isolated MSCs by plastic adherence in a GMP-compliant culture system. We established a well-characterized master cell bank and expanded a working cell bank to generate batches of finished MSC(CB) products certified for clinical use. The MSC(CB) produced by our facility was used in approved clinical trials or for therapeutic use, following single-patient authorization as an immune-suppressant agent. Results: We show the feasibility of a well-defined MSC manufacturing process and describe the main indications for which the MSCs were employed. We delve into a regulatory framework governing advanced therapy medicinal products (ATMPs), emphasizing the need of stringent quality control and safety assessments. From March 2012 to June 2023, 263 of our Good Manufacturing Practice (GMP)-certified MSC(CB) preparations were administered as ATMPs in 40 subjects affected by Graft-vs.-Host Disease, nephrotic syndrome, or bronco-pulmonary dysplasia of the newborn. There was no infusion-related adverse event. No patient experienced any grade toxicity. Encouraging preliminary outcome results were reported. Clinical response was registered in the majority of patients treated under therapeutic use authorization. Conclusions: Our 10 years of experience with MSC(CB) described here provides valuable insights into the use of this innovative cell product in immune-mediated diseases.

Mesenchymal stromal cells (MSC) for cellular therapy in European Union are classified as advanced therapy medicinal products (ATMPs), and their production must fulfill the requirements of Good Manufacturing Practice (GMP) rules. Despite their classification as medicinal products is already well recognized, there is still a lack of information and indications to validate methods and to adapt the noncompendial and compendial methods to these peculiar biological products with intrinsic characteristics that differentiate them from classic synthetic or biologic drugs. In the present paper, we present the results of the validation studies performed in the context of MSC development as ATMPs for clinical experimental use. Specifically, we describe the validation policies followed for sterility testing, endotoxins, adventitious viruses, cell count, and immunophenotyping. Our work demonstrates that it is possible to fully validate analytical methods also for ATMPs and that a risk-based approach can fill the gap between the prescription of the available guidelines shaped on traditional medicinal products and the peculiar characteristics of these novel and extremely promising new drugs.