A Novel Three-Dimensional Culture Device Favors a Myelinating Morphology of Neural Stem Cell-Derived Oligodendrocytes

The complexity of the central nervous system (CNS) requires researchers to consider all the variables linked to the interaction between the different cell inhabitants. On this basis, any in vitro study of the physiological and pathological processes regarding the CNS should consider the balance between the standardization of the assay and the complexity of the cellular system which mimics the in vivo microenvironment. One of the main structural and functional components of the CNS is the oligodendrocyte precursor cell (OPC), responsible for developmental myelination and myelin turnover and repair during adulthood following differentiation into mature oligodendrocytes. In the present brief research report, we describe a 3D culture tool (VITVO) based on an inert and biocompatible synthetic polymer material scaffold, functionalized with laminin coating, and tested as a new culture microenvironment for neural stem/precursor cell (NSPC) differentiation compared to standard 2D cultures. NSPCs spontaneously differentiate in the three neural lineages (neurons, astrocytes and OPCs), identified by specific markers, along the fibers in the 3D structure. Analysis of the mRNA levels for lineage differentiation markers reveals a higher expression compared to those seeded on a 2D surface, suggesting an acceleration of the differentiation process. We then focused on the oligodendroglial lineage, showing that in VITVO, mature oligodendrocytes exhibit a myelinating morphology, proven by 3D image elaboration, linked to a higher expression of mature oligodendrocyte markers. This preliminary study on an innovative 3D culture system is the first robust step in producing new microenvironment-based strategies to investigate in vitro OPC and oligodendrocyte biology

A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors

We here investigated the dynamic cell-to-cell interactions between tumor and mesenchymal stromal/stem cells (MSCs) by the novel VITVOⓇ 3D bioreactor that was customized to develop in vivo-like metastatic nodules of Ewing’s sarcoma (ES). MSCs are known to contribute to tumor microenvironment as cancer associated fibroblast (CAF) precursors and, for this reason, they have also been used as anti-cancer tools. Using dynamic conditions, the process of tissue colonization and formation of metastatic niches was recreated through tumor cell migration aiming to mimic ES development in patients. ES is an aggressive tumor representing the second most common malignant bone cancer in children and young adults. An urgent and unmet need exists for the development of novel treatment strategies to improve the outcomes of metastatic ES. The tumor-tropic ability of MSCs offers an alternative approach, in which these cells can be used as vehicles for the delivery of antitumor molecules, such as the proapoptotic TNF-related apoptosis inducing ligand (TRAIL). However, the therapeutic targeting of metastases remains challenging and the interaction occurring between tumor cells and MSCs has not yet been deeply investigated. Setting up in vitro and in vivo models to study this interaction is a prerequisite for novel approaches where MSCs affinity for tumor is optimized to ultimately increase their therapeutic efficacy. Here, VITVOⓇ integrating a customized scaffold with an increased inter-fiber distance (VITVO50) was used to develop a dynamic model where MSCs and tumor nodules were evaluated under flow conditions. Colonization and interaction between cell populations were explored by droplet digital PCR (ddPCR). VITVO50 findings were then applied in vivo. An ES metastatic model was established in NSG mice and biodistribution of TRAIL-expressing MSCs in mice organs affected by metastases was investigated using a 4-plex ddPCR assay. VITVOⓇ proved to be an easy handling and versatile bioreactor to develop in vivo-like tumor nodules and investigate dynamic cell-to-cell interactions with MSCs. The proposed fluidic system promises to facilitate the understanding of tumor-stroma interaction for the development of novel tumor targeting strategies, simplifying the analysis of in vivo data, and ultimately accelerating the progress towards the early clinical phase

Challenges in Clinical Development of Mesenchymal Stromal/Stem Cells: Concise Review

Identified 50 years ago, mesenchymal stromal/stem cells (MSCs) immediately generated a substantial interest among the scientific community because of their differentiation plasticity and hematopoietic supportive function. Early investigations provided evidence of a relatively low engraftment rate and a transient benefit for challenging congenital and acquired diseases. The reasons for these poor therapeutic benefits forced the entire field to reconsider MSC mechanisms of action together with their ex vivo manipulation procedures. This phase resulted in advances in MSCs processing and the hypothesis that MSC-tissue supportive functions may be prevailing their differentiation plasticity, broadening the spectrum of MSCs therapeutic potential far beyond their lineage-restricted commitments. Consequently, an increasing number of studies have been conducted for a variety of clinical indications, revealing additional challenges and suggesting that MSCs are still lagging behind for a solid clinical translation. For this reason, our aim was to dissect the current challenges in the development of still promising cell types that, after more than half a century, still need to reach their maturity. Stem Cells Translational Medicine 2019;8:1135–1148

Spin dynamics of molecular nanomagnets fully unraveled by four-dimensional inelastic neutron scattering

Molecular nanomagnets are among the first examples of spin systems of finite size and have been test-beds for addressing a range of elusive but important phenomena in quantum dynamics. In fact, for short-enough timescales the spin wavefunctions evolve coherently according to the an appropriate cluster spin-Hamiltonian, whose structure can be tailored at the synthetic level to meet specific requirements. Unfortunately, to this point it has been impossible to determine the spin dynamics directly. If the molecule is sufficiently simple, the spin motion can be indirectly assessed by an approximate model Hamiltonian fitted to experimental measurements of various types. Here we show that recently-developed instrumentation yields the four-dimensional inelastic-neutron scattering function S(Q,E) in vast portions of reciprocal space and enables the spin dynamics to be determined with no need of any model Hamiltonian. We exploit the Cr8 antiferromagnetic ring as a benchmark to demonstrate the potential of this new approach. For the first time we extract a model-free picture of the quantum dynamics of a molecular nanomagnet. This allows us, for example, to examine how a quantum fluctuation propagates along the ring and to directly test the degree of validity of the N\'{e}el-vector-tunneling description of the spin dynamics

Field-regulated switching of the magnetization of Co-porphyrin on graphene

Differentmagnetic couplingmechanisms have been identified for a fewmonolayers of Co-porphyrin molecules deposited on a graphene-covered Ni(111) single crystal. A relatively strong antiferromagnetic coupling of the first molecular layer via graphene to the Ni crystal in comparison to a weaker intermolecular coupling gives rise to a complex field-dependent response of this hybrid system. By continuously increasing the magnetic field strength, the net magnetization of the molecular system switches from antiparallel to parallel to the field direction at 2.5 T. Utilizing x-ray absorption spectroscopy and x-ray magnetic circular dichroism, the element-specific magnetization and field dependence was probed. The nature of the magnetic couplings is identified by means of density functional theory and orbital-dependent susceptibilities

A Novel 3D In Vitro Platform for Pre-Clinical Investigations in Drug Testing, Gene Therapy, and Immuno-oncology.

Tumors develop within complex cell-to-cell interactions, with accessory cells playing a relevant role starting in the early phases of cancer progression. This event occurs in a three-dimensional (3D) environment, which to date, has been difficult to reproduce in vitro due to its complexity. While bi-dimensional cultures have generated substantial data, there is a progressive awareness that 3D culture strategies may rapidly increase the understanding of tumor development and be used in anti-cancer compound screening and for predicting response to new drugs utilizing personalized approaches. However, simple systems capable of rapidly rebuilding cancer tissues ex-vivo in 3D are needed and could be used for a variety of applications. Therefore, we developed a flat, handheld and versatile 3D cell culture bioreactor that can be loaded with tumor and/or normal cells in combination which can be monitored using a variety of read-outs. This biocompatible device sustained 3D growth of tumor cell lines representative of various cancers, such as pancreatic and breast adenocarcinoma, sarcoma, and glioblastoma. The cells repopulated the thin matrix which was completely separated from the outer space by two gas-permeable membranes and was monitored in real-time using both microscopy and luminometry, even after transportation. The device was tested in 3D cytotoxicity assays to investigate the anti-cancer potential of chemotherapy, biologic agents, and cell-based therapy in co-cultures. The addition of luciferase in target cancer cells is suitable for comparative studies that may also involve parallel in vivo investigations. Notably, the system was challenged using primary tumor cells harvested from lung cancer patients as an innovative predictive functional assay for cancer responsiveness to checkpoint inhibitors, such as nivolumab. This bioreactor has several novel features in the 3D-culture field of research, representing a valid tool useful for cancer investigations, drug screenings, and other toxicology approaches

Resistance to neoplastic transformation of ex-vivo expanded human mesenchymal stromal cells after exposure to supramaximal physical and chemical stress

The risk of malignant transformation of ex-vivo expanded human mesenchymal stromal cells (huMSCs) has been debated in the last years; however, the biosafety of these cells after exposure to supramaximal physical and chemical stress has never been systematically investigated. We established an experimental in vitro model to induce supramaximal physical (ionizing radiation, IR) and chemical (starvation) stress on ex-vivo expanded bone marrow (BM)-derived huMSCs and investigated their propensity to undergo malignant transformation. To this aim, we examined MSC morphology, proliferative capacity, immune-phenotype, differentiation potential, immunomodulatory properties and genetic profile before and after stressor exposure. Furthermore, we investigated the cellular mechanisms underlying MSC response to stress. MSCs were isolated from 20 healthy BM donors and expanded in culture medium supplemented with 5% platelet lysate (PL) up to passage 2 (P2). At this stage, MSCs were exposed first to escalating doses of IR (30, 100, 200 Gy) and then to starvation culture conditions (1% PL). With escalating doses of radiation, MSCs lost their typical spindle-shaped morphology, their growth rate markedly decreased and eventually stopped (at P4-P6) by reaching early senescence. Irradiated and starved MSCs maintained their typical immune-phenotype, ability to differentiate into adipocytes/osteoblasts and to inhibit mitogen-induced T-cell proliferation. The study of the genetic profile of irradiated/ starved MSCs did not show any alteration. While the induction of supramaximal stress triggered production of ROS and activation of DNA damage response pathway via multiple mechanisms, our data indicate that irradiated/starved MSCs, although presenting altered morphology/growth rate, do not display increased propensity for malignant transformation

Impact of HOXB7 overexpression on human adipose-derived mesenchymal progenitors

Background: The ex vivo expansion potential of mesenchymal stromal/stem cells (MSC) together with their differentiation and secretion properties makes these cells an attractive tool for transplantation and tissue engineering. Although the use of MSC is currently being tested in a growing number of clinical trials, it is still desirable to identify molecular markers that may help improve their performance both in vitro and after transplantation. Methods: Recently, HOXB7 was identified as a master player driving the proliferation and differentiation of bone marrow mesenchymal progenitors. In this study, we investigated the effect of HOXB7 overexpression on the ex vivo features of adipose mesenchymal progenitors (AD-MSC). Results: HOXB7 increased AD-MSC proliferation potential, reduced senescence, and improved chondrogenesis together with a significant increase of basic fibroblast growth factor (bFGF) secretion. Conclusion: While further investigations and in vivo models shall be applied for better understanding, these data suggest that modulation of HOXB7 may be a strategy for innovative tissue regeneration applications

Social distance during the covid-19 pandemic reflects perceived rather than actual risk

Interpersonal space (IPS) is the area surrounding our own bodies in which we interact comfortably with other individuals. During the COVID-19 pandemic, keeping larger IPS than usual, along with wearing a face mask, is one of the most effective measures to slow down the COVID-19 outbreak. Here, we explore the contribution of actual and perceived risk of contagion and anxiety levels in regulating our preferred social distance from other people during the first wave of the COVID-19 pandemic in Italy. In this study, 1293 individuals from six Italian regions with different levels of actual risk of infection participated in an online survey assessing their perceived risk to be infected, level of anxiety and IPS. Two tasks were adopted as measures of interpersonal distance: the Interpersonal Visual Analogue Scale and a questionnaire evaluating interpersonal distance with and without face mask. The results showed that the IPS regulation was affected by how people subjectively perceived COVID-19 risk and the related level of anxiety, not by actual objective risk. This clarifies that the role of threat in prompting avoidant behaviors expressed in increased IPS does not merely reflect environmental events but rather how they are subjectively experienced and represented