In vitro evaluation of therapeutic potential of exosomes delivered by human cardiac primitive cells in cardiac regeneration

Although, when injected, human Cardiac Primitive Cells (CPC) are not retained by host myocardium, they still improve cardiac function. Emerging evidence supports the hypothesis that exosomes may be responsible for beneficial effects induced by stem cells delivered in the infarcted myocardium. Exosomes are nano-sized vesicles naturally secreted by almost all cells and ubiquitously found in cell culture supernatants and biological fluids. Transporting and transferring peptides, lipids, and nucleic acids, exosomes have the potential to modulate signaling pathways, cell growth, migration, and proliferation of recipient cells. Accordingly, CPC may deliver chemoattractive, pro-survival and differentiating signals to resident cells through exosomes. To test our hypothesis, we isolated exosomes released in culture by CPC isolated from adult human myocardium (Exo-CPC) and analyzed the composition of their cargo and the effects elicited in vitro by their administration to resident population of CPC or fibroblasts. Specifically, we searched for the presence of specific factors known to regulate CPC migration, survival and differentiation. Additionally, we tested in vitro the potential of Exo-CPC of either regulating CPC proliferation and programmed cell death, and modulating interstitial fibrosis, extracellular-matrix (ECM) synthesis and deposition. Interestingly, on one hand, signals delivered by Exo-CPC affected proliferation and survival of CPC and, on the other hand, regulated ECM protein production. Therefore, we might speculate that Exo-CPC have potential effect on both resident CPC and fibroblasts when injected in cardiac wall

Non-modified RNA-Based Reprogramming of Human Dermal Fibroblasts into Induced Pluripotent Stem Cells

The generation of pluripotent stem cells from adult somatic cells by cell reprogramming has put a whole new perspective on stem cell biology and stem cell-based regenerative medicine. Cell reprogramming acts through the introduction of key genes that regulate and maintain the pluripotent cell state. In this chapter, we describe the optimized protocol for the efficient isolation of fibroblasts from a skin punch biopsy and the subsequent easy and effective generation of integration-free induced pluripotent stem cell (iPSC) colonies forcing the expression of specific factors by non-modified RNAs. © 2021, Springer Science+Business Media, LLC

Metabolic Reprogramming of Cancer Associated Fibroblasts: The Slavery of Stromal Fibroblasts

Cancer associated fibroblasts (CAFs) are the main stromal cell type of solid tumour microenvironment and undergo an activation process associated with secretion of growth factors, cytokines, and paracrine interactions. One of the important features of solid tumours is the metabolic reprogramming that leads to changes of bioenergetics and biosynthesis in both tumour cells and CAFs. In particular, CAFs follow the evolution of tumour disease and acquire a catabolic phenotype: in tumour tissues, cancer cells and tumour microenvironment form a network where the crosstalk between cancer cells and CAFs is associated with cell metabolic reprogramming that contributes to CAFs activation, cancer growth, and progression and evasion from cancer therapies. In this regard, the study of CAFs metabolic reprogramming could contribute to better understand their activation process, the interaction between stroma, and cancer cells and could offer innovative tools for the development of new therapeutic strategies able to eradicate the protumorigenic activity of CAFs. Therefore, this review focuses on CAFs metabolic reprogramming associated with both differentiation process and cancer and stromal cells crosstalk. Finally, therapeutic responses and potential anticancer strategies targeting CAFs metabolic reprogramming are reviewed

Non-integrating Methods to Produce Induced Pluripotent Stem Cells for Regenerative Medicine: An Overview

Induced Pluripotent Stem cells (iPSC) are adult somatic cells genetically reprogrammed to an embryonic stem cell-like state. Due to their autologous origin from adult somatic cells, iPSCs are considered a tremendously valuable tool for regenerative medicine, disease modeling, drug discovery and testing. iPSCs were first obtained by introducing specific transcription factors through retroviral transfection. However, cell reprogramming obtained by integrating methods prevent clinical application of iPSC because of potential risk for infection, teratomas and genomic instability. Therefore, several integration-free alternate methods have been developed and tested thus far to overcome safety issues. The present chapter provides an overview and a critical analysis of advantages and disadvantages of non-integrating methods used to generate iPSCs

Jump and balance test in judo athletes with or without visual impairments

The study was conducted for four months with 8 judo athletes: 4 sighted people (4 M) and 4 visual impairment people (3 M and 1 F), aged between 18 and 52 (30.75 ± 12.74). According to the IBSA Visual Classification, all visual impairment subjects participating in our study were covered in the B1 category of visual deficit. This is a group represented by patients with no light perception in either eye up to light perception, and with an inability to recognize the shape of a hand at any distance or in any direction. From our cohort it was excluded subjects who have had low extremities musculoskeletal, neurological, or orthopaedic disorders in the previous six months. The aim of the study was to evaluate their balance with both closed and opened eyes and to set their lower limbs’ strength: these are indispensable characteristics to carry out technical actions of judo. Anthropometric measures were compared between groups and data about jump protocol and balance protocol were analysed. Results of current research showed that postural stability is different in function of assessment with closed and open eyes. The result of the jump tests differs because the data do not show significant differences between long jump and high jump. The comparison between blinded and sighted judo athletes highlighted greater difficulties with eyes closed for sighted athletes than blinded ones

Human Cardiac Progenitor Cell-Derived Extracellular Vesicles Exhibit Promising Potential for Supporting Cardiac Repair in Vitro

Although human Cardiac Progenitor Cells (hCPCs) are not retained by host myocardium they still improve cardiac function when injected into schemic heart. Emerging evidence supports the hypothesis that hCPC beneficial effects are induced by paracrine action on resident cells. Extracellular vesicles (EVs) are an intriguing mechanism of cell communication based on the transport and transfer of peptides, lipids, and nucleic acids that have the potential to modulate signaling pathways, cell growth, migration, mand proliferation of recipient cells. We hypothesize that EVs are involved in the paracrine effects elicited by hCPCs and held accountable for the response of the infarcted myocardium to hCPC-based cell therapy. To test this theory, we collected EVs released by hCPCs isolated from healthy myocardium and evaluated the effects they elicited when administered to resident hCPC and cardiac fibroblasts (CFs) isolated from patients with post-ischemic end-stage heart failure. Evidence emerging from our study indicated that hCPC-derived EVs impacted upon proliferation and survival of hCPCs residing in the ischemic heart and regulated the synthesis and deposition of extracellular-matrix by CFs. These findings suggest that beneficial effects exerted by hCPC injection are, at least to some extent, ascribable to the delivery of signals conveyed by EVs

Compact and tunable stretch bioreactor advancing tissue engineering implementation. Application to engineered cardiac constructs

Physical stimuli are crucial for the structural and functional maturation of tissues both in vivo and in vitro . In tissue engineering applications, bioreactors have become fundamental and effective tools for provid- ing biomimetic culture conditions that recapitulate the native physical stimuli. In addition, bioreactors play a key role in assuring strict control, automation, and standardization in the production process of cell-based products for future clinical application. In this study, a compact, easy-to-use, tunable stretch bioreactor is proposed. Based on customizable and low-cost technological solutions, the bioreactor was designed for providing tunable mechanical stretch for biomimetic dynamic culture of different engineered tissues. In-house validation tests demonstrated the accuracy and repeatability of the imposed mechanical stimulation. Proof of concepts biological tests performed on engineered cardiac constructs, based on de- cellularized human skin scaffolds seeded with human cardiac progenitor cells, confirmed the bioreactor Good Laboratory Practice compliance and ease of use, and the effectiveness of the delivered cyclic stretch stimulation on the cardiac construct maturation

Limited diagnostic value of questionnaire-based pre-participation screening algorithms: A "risk-exposed" approach to sports activity

Background: Several pre-participation screening algorithms (PPSAs) have been proposed to assess sports eligibility in different populations. They are usually based on self-administered questionnaires, without further medical assessment if no risk factors are documented. The Med-Ex "Formula Benessere"worksite program includes a complete cardiovascular (CV) screening for all participants. The purpose of this study was to assess PPSAs accuracy in detecting medical and/or CV abnormalities in the general population, comparing the results with the date derived from Med-Ex program. Methods: The Med-Ex medical evaluation, consisting of medical history, physical examination (including body composition), resting electrocardiogram (ECG) and exercise stress test in 464 male subjects (38.4 aged) was analyzed and matched to several PPSAs - Physical Activity Readiness Questionnaire (PAR-Q) (2002-2020), American Heart Association (AHA)/American College of Sport Medicine (ACSM) (1998-2009-2014-2015), European Association of Cardiovascular Prevention and Rehabilitation (EACPR) (2011) - retrospectively simulated. Results: Five-hundred and 67 abnormalities were detected though Med-Ex medical evaluation, and one-fourth (24%) would have been undetected applying PPSA alone. In particular 28% of high blood pressure, 21% of impaired fasting glycaemia, 21% of high Body Mass Index (BMI) values and 19% of ECG abnormalities would have been missed, on average, by all PPSAs. Conclusions: The simulation analysis model performed in this study allowed to highlight the limits of PPSAs in granting sport eligibility, compared to a medical-guided CV screening. These findings emphasize the importance of a more balanced approach to pre-participation screening that includes a thorough evaluation of the cost/benefit ratio

A low-cost 3D-printed sample-holder for stirring-based decellularization of biological tissues

An innovative, low-cost, 3D-printed sample-holder is proposed for reproducible and effective stirring-based decellularization of biological tissues. The sample-holder was designed to be low-cost, easy to use with conventional laboratory equipment, and manufacturable through 3D printing. During stirring-based decellularization, the sample holder exposes the samples to convective flow, enhancing the reagent transport while protecting the samples from disruptive forces. Computational fluid dynamics analyses were carried out to elucidate the developing hydrodynamics. Explanatory tests, performed on human cardiac tissue samples, demonstrated the effectiveness of the presented device

A low-cost scalable 3D-printed sample-holder for agitation-based decellularization of biological tissues

Decellularized extracellular matrix is one of the most promising biological scaffold supporting in vitro tissue growth and in vivo tissue regeneration in both preclinical research and clinical practice. In case of thick tissues or even organs, conventional static decellularization methods based on chemical or enzymatic treatments are not effective in removing the native cellular material without affecting the extracellular matrix. To overcome this limitation, dynamic decellularization methods, mostly based on perfusion and agitation, have been proposed. In this study, we developed a low-cost scalable 3D-printed sample-holder for agitation-based decellularization purposes, designed for treating multiple specimens simultaneously and for improving efficiency, homogeneity and reproducibility of the decellularization treatment with respect to conventional agitation-based approaches. In detail, the proposed sample-holder is able to house up to four specimens and, immersed in the decellularizing solution within a beaker placed on a magnetic stirrer, to expose them to convective flow, enhancing the solution transport through the specimens while protecting them. Computational fluid dynamics analyses were performed to investigate the fluid phenomena establishing within the beaker and to support the sample-holder design. Exploratory biological tests performed on human skin specimens demonstrated that the sample-holder reduces process duration and increases treatment homogeneity and reproducibility