Multiobjective Optimization of Rotodynamic Blood Pumps: The Use Case of a Cavopulmonary Assist Device.

Comprehensive optimization of rotodynamic blood pumps (RBPs) requires the consideration of three partially conflicting objectives: size, hemocompatibility, and motor efficiency. Optimizing these individual objectives independently, the potential of multiobjective optimizations often remains untapped. This study aimed at the multiobjective optimization of an RBP for cavopulmonary support accounting for all three objectives simultaneously. Hydraulic and electromagnetic design spaces were characterized using computational fluid dynamics and computational electromagnetics, respectively. Design variables included secondary flow gap widths, impeller diameters, and stator heights. The size objective encompassed the RBP widths and heights, the hemocompatibility objective was a weighted composite measure of well-established metrics, and the motor objective was determined by motor losses. Multiobjective optimization was performed through Pareto analysis. 81 designs were considered, and 21 Pareto-optimal designs were identified. The Pareto analysis indicated that hemocompatibility performance could be improved by 72.4% with a concomitant 1.5% reduction in the baseline pump volume. This, however, entailed an increase in motor losses by 0.2 W, while still meeting design requirements, with maximum local temperature rises remaining below 0.4 K. The multiobjective optimization led to a Pareto front, demonstrating the feasibility to improve hemocompatibility at reduced pump volume, however, at the cost of a diminished yet still acceptable motor performance.

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

Microglia and glioblastoma heterocellular interplay sustains tumour growth and proliferation as an off-target effect of radiotherapy.

Glioblastoma (GBM), a WHO grade IV glioma, is a malignant primary brain tumour for which combination of surgery, chemotherapy and radiotherapy is the first-line approach despite adverse effects. Tumour microenvironment (TME) is characterized by an interplay of cells and soluble factors holding a critical role in neoplastic development. Significant pathophysiological changes have been found in GBM TME, such as glia activation and oxidative stress. Microglia play a crucial role in favouring GBM growth, representing target cells of immune escape mechanisms. Our study aims at analysing radiation-induced effects in modulating intercellular communication and identifying the basis of protective mechanisms in radiation-naïve GBM cells. Tumour cells were treated with conditioned media (CM) derived from 0, 2 or 15 Gy irradiated GBM cells or 0, 2 or 15 Gy irradiated human microglia. We demonstrated that irradiated microglia promote an increase of GBM cell lines proliferation through paracrine signalling. On the contrary, irradiated GBM-derived CM affect viability, triggering cell death mechanisms. In addition, we investigated whether these processes involve mitochondrial mass, fitness and oxidative phosphorylation and how GBM cells respond at these induced alterations. Our study suggests that off-target radiotherapy modulates microglia to support GBM proliferation and induce metabolic modifications.

Molecular Mechanisms and Therapeutic Implications of Human Pericyte-like Adipose-Derived Mesenchymal Stem Cells in an In Vitro Model of Diabetic Retinopathy.

The blood-retinal barrier (BRB) is strongly compromised in diabetic retinopathy (DR) due to the detachment of pericytes (PCs) from retinal microvessels, resulting in increased permeability and impairment of the BRB. Western blots, immunofluorescence and ELISA were performed on adipose mesenchymal stem cells (ASCs) and pericyte-like (P)-ASCs by co-cultured human retinal endothelial cells (HRECs) under hyperglycemic conditions (HG), as a model of DR. Our results demonstrated that: (a) platelet-derived growth factor receptor (PDGFR) and its activated form were more highly expressed in monocultured P-ASCs than in ASCs, and this expression increased when co-cultured with HRECs under high glucose conditions (HG); (b) the transcription factor Nrf2 was more expressed in the cytoplasmic fraction of ASCs and in the P-ASC nuclear fraction, under normal glucose and, even more, under HG conditions; (c) cytosolic phospholipase A2 activity and prostaglandin E2 release, stimulated by HG, were significantly reduced in P-ASCs co-cultured with HRECs; (d) HO-1 protein content was significantly higher in HG-P-ASCs/HRECs than P-ASCs/HRECs; and (e) VEGF-A levels in media from HG-co-cultures were reduced in P-ASCs/HRECs with respect to ASCs/HRECs. The data obtained highlighted the potential of autologous differentiated ASCs in future clinical applications based on cell therapy to counteract the damage induced by DR.

A Novel Pumping Principle for a Total Artificial Heart.

OBJECTIVE: Total artificial hearts (TAH) serve as a temporary treatment for severe biventricular heart failure. The limited durability and complication rates of current devices hamper long-term cardiac replacement. The aim of this study was to assess the feasibility of a novel valveless pumping principle for a durable pulsatile TAH (ShuttlePump). METHODS: The pump features a rotating and linearly shuttling piston within a cylindrical housing with two in- and outlets. With a single moving piston, the ShuttlePump delivers pulsatile flow to both systemic and pulmonary circulation. The pump and actuation system were designed iteratively based on analytical and in silico methods, utilizing finite element methods (FEM) and computational fluid dynamics (CFD). Pump characteristics were evaluated experimentally in a mock circulation loop mimicking the cardiovascular system, while hemocompatibility-related parameters were calculated numerically. RESULTS: Pump characteristics cover the entire required operating range for a TAH, providing 2.5-9 L/min of flow rate against 50-160 mmHg arterial pressures at stroke frequencies of 1.5-5 Hz while balancing left and right atrial pressures. FEM analysis showed mean overall copper losses of 8.84 W, resulting in a local maximum blood temperature rise of <2 K. The CFD results of the normalized index of hemolysis were 3.57 mg/100 L, and 95% of the pump's blood volume was exchanged after 1.42 s. CONCLUSION AND SIGNIFICANCE: This study indicates the feasibility of a novel pumping system for a TAH with numerical and experimental results substantiating further development of the ShuttlePump.

Adipose-Derived Mesenchymal Stromal Cells: A Tool for Bone and Cartilage Repair.

The osteogenic and chondrogenic differentiation ability of adipose-derived mesenchymal stromal cells (ASCs) and their potential therapeutic applications in bone and cartilage defects are reported in this review. This becomes particularly important when these disorders can only be poorly treated by conventional therapeutic approaches, and tissue engineering may represent a valuable alternative. Being of mesodermal origin, ASCs can be easily induced to differentiate into chondrocyte-like and osteocyte-like elements and used to repair damaged tissues. Moreover, they can be easily harvested and used for autologous implantation. A plethora of ASC-based strategies are being developed worldwide: they include the transplantation of freshly harvested cells, in vitro expanded cells or predifferentiated cells. Moreover, improving their positive effects, ASCs can be implanted in combination with several types of scaffolds that ensure the correct cell positioning; support cell viability, proliferation and migration; and may contribute to their osteogenic or chondrogenic differentiation. Examples of these strategies are described here, showing the enormous therapeutic potential of ASCs in this field. For safety and regulatory issues, most investigations are still at the experimental stage and carried out in vitro and in animal models. Clinical applications have, however, been reported with promising results and no serious adverse effects.

Mesenchymal stromal cells in tumor microenvironment remodeling of BCR-ABL negative myeloproliferative diseases.

Chronic myeloproliferative neoplasms encompass the BCR-ABL1-negative neoplasms polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These are characterized by calreticulin (CALR), myeloproliferative leukemia virus proto-oncogene (MPL) and the tyrosine kinase Janus kinase 2 (JAK2) mutations, eventually establishing a hyperinflammatory tumor microenvironment (TME). Several reports have come to describe how constitutive activation of JAK-STAT and NFκB signaling pathways lead to uncontrolled myeloproliferation and pro-inflammatory cytokines secretion. In such a highly oxidative TME, the balance between Hematopoietic Stem Cells (HSCs) and Mesenchymal Stromal Cells (MSCs) has a crucial role in MPN development. For this reason, we sought to review the current literature concerning the interplay between HSCs and MSCs. The latter have been reported to play an outstanding role in establishing of the typical bone marrow (BM) fibrotic TME as a consequence of the upregulation of different fibrosis-associated genes including PDGF- ß upon their exposure to the hyperoxidative TME characterizing MPNs. Therefore, MSCs might turn to be valuable candidates for niche-targeted targeting the synthesis of cytokines and oxidative stress in association with drugs eradicating the hematopoietic clone.

Epigenetics and Metabolism Reprogramming Interplay into Glioblastoma: Novel Insights on Immunosuppressive Mechanisms.

The central nervous system represents a complex environment in which glioblastoma adapts skillfully, unleashing a series of mechanisms suitable for its efficient development and diffusion. In particular, changes in gene expression and mutational events that fall within the domain of epigenetics interact complexly with metabolic reprogramming and stress responses enacted in the tumor microenvironment, which in turn fuel genomic instability by providing substrates for DNA modifications. The aim of this review is to analyze this complex interaction that consolidates several conditions that confer a state of immunosuppression and immunoevasion, making glioblastoma capable of escaping attack and elimination by immune cells and therefore invincible against current therapies. The progressive knowledge of the cellular mechanisms that underlie the resistance of the glioblastoma represents, in fact, the only weapon to unmask its weak points to be exploited to plan successful therapeutic strategies.

Functional Roles of Connexins and Gap Junctions in Osteo-Chondral Cellular Components.

Gap junctions (GJs) formed by connexins (Cxs) play an important role in the intercellular communication within most body tissues. In this paper, we focus on GJs and Cxs present in skeletal tissues. Cx43 is the most expressed connexin, participating in the formation of both GJs for intercellular communication and hemichannels (HCs) for communication with the external environment. Through GJs in long dendritic-like cytoplasmic processes, osteocytes embedded in deep lacunae are able to form a functional syncytium not only with neighboring osteocytes but also with bone cells located at the bone surface, despite the surrounding mineralized matrix. The functional syncytium allows a coordinated cell activity through the wide propagation of calcium waves, nutrients and anabolic and/or catabolic factors. Acting as mechanosensors, osteocytes are able to transduce mechanical stimuli into biological signals that spread through the syncytium to orchestrate bone remodeling. The fundamental role of Cxs and GJs is confirmed by a plethora of investigations that have highlighted how up- and downregulation of Cxs and GJs critically influence skeletal development and cartilage functions. A better knowledge of GJ and Cx mechanisms in physiological and pathological conditions might help in developing therapeutic approaches aimed at the treatment of human skeletal system disorders.

IGFBP-6 Alters Mesenchymal Stromal Cell Phenotype Driving Dasatinib Resistance in Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML), BCR-ABL1-positive, is classified as a myeloproliferative characterized by Philadelphia chromosome/translocation t(9;22) and proliferating granulocytes. Despite the clinical success of tyrosine kinase inhibitors (TKi) agents in the treatment of CML, most patients have minimal residual disease contained in the bone marrow microenvironment, within which stromal cells assume a pro-inflammatory phenotype that determines their transformation in cancer-associated fibroblasts (CAF) which, in turn can play a fundamental role in resistance to therapy. Insulin-like Growth Factor Binding Protein-6 (IGFBP-6) is expressed during tumor development, and is involved in immune-escape and inflammation as well, providing a potential additional target for CML therapy. Here, we aimed at investigating the role of IGFBP-6/SHH/TLR4 axis in TKi response. We used a CML cell line, LAMA84-s, and healthy bone marrow stromal cells, HS-5, in mono- or co-culture. The two cell lines were treated with Dasatinib and/or IGFBP-6, and the expression of inflammatory markers was tested by qRT-PCR; furthermore, expression of IGFBP-6, TLR4 and Gli1 were evaluated by Western blot analysis and immumocytochemistry. The results showed that both co-culture and Dasatinib exposure induce inflammation in stromal and cancer cells so that they modulate the expression of TLR4, and these effects were more marked following IGFBP-6 pre-treatment suggesting that this molecule may confer resistance through the inflammatory processes. This phenomenon was coupled with sonic hedgehog (SHH) signaling. Indeed, our data also demonstrate that HS-5 treatment with PMO (an inducer of SHH) induces significant modulation of TLR4 and overexpression of IGFPB-6 suggesting that the two pathways are interconnected with each other and with the TLR-4 pathway. Finally, we demonstrated that pretreatment with IGFBP-6 and/or PMO restored LAMA-84 cell viability after treatment with Dasatinib, suggesting that both IGFBP-6 and SHH are involved in the resistance mechanisms induced by the modulation of TLR-4, thus indicating that the two pathways may be considered as potential therapeutic targets.

Protective Effects of Human Pericyte-like Adipose-Derived Mesenchymal Stem Cells on Human Retinal Endothelial Cells in an In Vitro Model of Diabetic Retinopathy: Evidence for Autologous Cell Therapy.

Diabetic retinopathy (DR) is characterized by morphologic and metabolic alterations in endothelial cells (ECs) and pericytes (PCs) of the blood-retinal barrier (BRB). The loss of interendothelial junctions, increased vascular permeability, microaneurysms, and finally, EC detachment are the main features of DR. In this scenario, a pivotal role is played by the extensive loss of PCs. Based on previous results, the aim of this study was to assess possible beneficial effects exerted by adipose mesenchymal stem cells (ASCs) and their pericyte-like differentiated phenotype (P-ASCs) on human retinal endothelial cells (HRECs) in high glucose conditions (25 mM glucose, HG). P-ASCs were more able to preserve BRB integrity than ASCs in terms of (a) increased transendothelial electrical resistance (TEER); (b) increased expression of adherens junction and tight junction proteins (VE-cadherin and ZO-1); (c) reduction in mRNA levels of inflammatory cytokines TNF-α, IL-1ß, and MMP-9; (d) reduction in the angiogenic factor VEGF and in fibrotic TGF-ß1. Moreover, P-ASCs counteracted the HG-induced activation of the pro-inflammatory phospho-ERK1/2/phospho-cPLA2/COX-2 pathway. Finally, crosstalk between HRECs and ASCs or P-ASCs based on the PDGF-B/PDGFR-ß axis at the mRNA level is described herein. Thus, P-ASCs might be considered valuable candidates for therapeutic approaches aimed at countering BRB disruption in DR.

The Role of Epigenetics in Neuroinflammatory-Driven Diseases.

Neurodegenerative disorders are characterized by the progressive loss of central and/or peripheral nervous system neurons. Within this context, neuroinflammation comes up as one of the main factors linked to neurodegeneration progression. In fact, neuroinflammation has been recognized as an outstanding factor for Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and multiple sclerosis (MS). Interestingly, neuroinflammatory diseases are characterized by dramatic changes in the epigenetic profile, which might provide novel prognostic and therapeutic factors towards neuroinflammatory treatment. Deep changes in DNA and histone methylation, along with histone acetylation and altered non-coding RNA expression, have been reported at the onset of inflammatory diseases. The aim of this work is to review the current knowledge on this field.

Effects of Mangiferin on LPS-Induced Inflammation and SARS-CoV-2 Viral Adsorption in Human Lung Cells.

The growing interest in natural bioactive molecules, as an approach to many pathological contexts, is widely justified by the necessity to overcome the disadvantageous benefit-risk ratio related to traditional therapies. Among them, mangiferin (MGF) shows promising beneficial properties such as antioxidant, anti-inflammatory, and immunomodulatory effects. In this study, we aimed to investigate the antioxidant and anti-inflammatory properties of MGF on lipopolysaccharide (LPS)-induced lung NCI-H292 cells, focusing on its role against COVID-19 adsorption. In order to obtain this information, cells treated with LPS, with or without MGF, were analyzed performing wound healing, gene expression of inflammatory cytokines, GSH quantification, and JC-1 staining. Moreover, the inhibition of viral adsorption was evaluated microbiologically and the results were further confirmed by molecular docking analysis. In this regard, MGF downregulates the expression of several inflammatory factors, enhances GSH levels, promotes the wound healing rate, and restores the mitochondrial dysfunction caused by LPS. In addition, MGF significantly inhibits SARS-CoV-2 adsorption as shown by the gene expression of ACE2 and TMPRSS-2, and furtherly confirmed by microbiological and molecular modeling evaluation. Although more investigations are still needed, all data obtained constitute a solid background, demonstrating the cytoprotective role of MGF in inflammatory mechanisms including COVID-19 infection.

Postural Evaluation in Young Healthy Adults through a Digital and Reproducible Method.

Different tools for the assessment of posture exist, from the simplest and cheap plumb line to complex, expensive, 3D-marker-based systems. The aim of this study is to present digital postural normative data of young adults collected through a mobile app to expand the possibilities of digital postural evaluation. A sample of 100 healthy volunteers, 50 males and 50 females, was analyzed with the mobile app Apecs-AI Posture Evaluation and Correction System® (Apecs). The Student's t-test evaluated differences between gender to highlight if the digital posture evaluation may differ between groups. A significant difference was present in the anterior coronal plane for axillary alignment (p = 0.04), trunk inclination (p = 0.03), and knee alignment (p = 0.01). Head inclination (p = 0.04), tibia shift (p = 0.01), and foot angle (p < 0.001) presented significant differences in the sagittal plane, while there were no significant differences in the posterior coronal plane. The intraclass correlation coefficient (ICC) was considered to evaluate reproducibility. Thirteen parameters out of twenty-two provided an ICC > 0.90, three provided an ICC > 0.60, and six variables did not meet the cut-off criteria. The results highlight that digital posture analysis of healthy individuals may present slight differences related to gender. Additionally, the mobile app showed good reproducibility according to ICC. Digital postural assessment with Apecs could represent a quick method for preventing screening in the general population. Therefore, clinicians should consider this app's worth as an auxiliary posture evaluation tool.

GJA1/CX43 High Expression Levels in the Cervical Spinal Cord of ALS Patients Correlate to Microglia-Mediated Neuroinflammatory Profile.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motoneurons (MNs) with a fatal outcome. The typical degeneration of cortico-spinal, spinal, and bulbar MNs, observed in post-mortem biopsies, is associated with the activation of neuroimmune cells. GJA1, a member of the connexins (Cxs) gene family, encodes for connexin 43 (Cx43), a core gap junctions (GJs)- and hemichannels (HCs)-forming protein, involved in cell death, proliferation, and differentiation. Recently, Cx43 expression was found to play a role in ALS pathogenesis. Here, we used microarray and RNA-seq datasets from the NCBI of the spinal cord of control (NDC) and ALS patients, which were stratified according to the GJA1 gene expression. Genes that positively or negatively correlated to GJA1 expression were used to perform a genomic deconvolution analysis (GDA) using neuroimmune signatures. Expression analysis revealed a significantly higher GJA1 expression in the MNs of ALS patients as compared to NDC. Gene deconvolution analysis revealed that positively correlated genes were associated with microglia activation, whereas negatively correlated genes were associated with neuronal activation profiles. Moreover, gene ontology analysis, performed on genes characterizing either microglia or neuronal signature, indicated immune activation or neurogenesis as main biological processes. Finally, using a synthetic analysis of drugs able to revert the GJA1 transcriptomic signatures, we found a specific drug profile for ALS patients with high GJA1 expression levels, composed of amlodipine, sertraline, and prednisolone. In conclusion, our exploratory study suggests GJA1 as a new neuro-immunological gene correlated to microglial cellular profile in the spinal cord of ALS patients. Further studies are warranted to confirm these results and to evaluate the therapeutic potential of drugs able to revert typical GJA1/CX43 signature in ALS patients.

Lactate Rewrites the Metabolic Reprogramming of Uveal Melanoma Cells and Induces Quiescence Phenotype.

Uveal melanoma (UM), the most common primary intraocular cancer in adults, is among the tumors with poorer prognosis. Recently, the role of the oncometabolite lactate has become attractive due to its role as hydroxycarboxylic acid receptor 1 (HCAR1) activator, as an epigenetic modulator inducing lysine residues lactylation and, of course, as a glycolysis end-product, bridging the gap between glycolysis and oxidative phosphorylation. The aim of the present study was to dissect in UM cell line (92.1) the role of lactate as either a metabolite or a signaling molecule, using the known modulators of HCAR1 and of lactate transporters. Our results show that lactate (20 mM) resulted in a significant decrease in cell proliferation and migration, acting and switching cell metabolism toward oxidative phosphorylation. These results were coupled with increased euchromatin content and quiescence in UM cells. We further showed, in a clinical setting, that an increase in lactate transporters MCT4 and HCAR1 is associated with a spindle-shape histological type in UM. In conclusion, our results suggest that lactate metabolism may serve as a prognostic marker of UM progression and may be exploited as a potential therapeutic target.

Neurotrophic and immunomodulatory effects of olfactory ensheathing cells as a strategy for neuroprotection and regeneration.

Accumulating evidence sustains glial cells as critical players during central nervous system (CNS) development, homeostasis and disease. Olfactory ensheathing cells (OECs), a type of specialized glia cells sharing properties with both Schwann cells and astrocytes, are of critical importance in physiological condition during olfactory system development, supporting its regenerative potential throughout the adult life. These characteristics prompted research in the field of cell-based therapy to test OEC grafts in damaged CNS. Neuroprotective mechanisms exerted by OEC grafts are not limited to axonal regeneration and cell differentiation. Indeed, OEC immunomodulatory properties and their phagocytic potential encourage OEC-based approaches for tissue regeneration in case of CNS injury. Herein we reviewed recent advances on the immune role of OECs, their ability to modulate CNS microenvironment via bystander effects and the potential of OECs as a cell-based strategy for tissue regeneration.

Adult stem cell niches for tissue homeostasis.

Adult stem cells are fundamental to maintain tissue homeostasis, growth, and regeneration. They reside in specialized environments called niches. Following activating signals, they proliferate and differentiate into functional cells that are able to preserve tissue physiology, either to guarantee normal turnover or to counteract tissue damage caused by injury or disease. Multiple interactions occur within the niche between stem cell-intrinsic factors, supporting cells, the extracellular matrix, and signaling pathways. Altogether, these interactions govern cell fate, preserving the stem cell pool, and regulating stem cell proliferation and differentiation. Based on their response to body needs, tissues can be largely classified into three main categories: tissues that even in normal conditions are characterized by an impressive turnover to replace rapidly exhausting cells (blood, epidermis, or intestinal epithelium); tissues that normally require only a basal cell replacement, though able to efficiently respond to increased tissue needs, injury, or disease (skeletal muscle); tissues that are equipped with less powerful stem cell niches, whose repairing ability is not able to overcome severe damage (heart or nervous tissue). The purpose of this review is to describe the main characteristics of stem cell niches in these different tissues, highlighting the various components influencing stem cell activity. Although much has been done, more work is needed to further increase our knowledge of niche interactions. This would be important not only to shed light on this fundamental chapter of human physiology but also to help the development of cell-based strategies for clinical therapeutic applications, especially when other approaches fail.

ARPE-19 conditioned medium promotes neural differentiation of adipose-derived mesenchymal stem cells.

BACKGROUND: Adipose-derived stem cells (ASCs) have been increasingly explored for cell-based medicine because of their numerous advantages in terms of easy availability, high proliferation rate, multipotent differentiation ability and low immunogenicity. In this respect, they have been widely investigated in the last two decades to develop therapeutic strategies for a variety of human pathologies including eye disease. In ocular diseases involving the retina, various cell types may be affected, such as Müller cells, astrocytes, photoreceptors and retinal pigment epithelium (RPE), which plays a fundamental role in the homeostasis of retinal tissue, by secreting a variety of growth factors that support retinal cells. AIM: To test ASC neural differentiation using conditioned medium (CM) from an RPE cell line (ARPE-19). METHODS: ASCs were isolated from adipose tissue, harvested from the subcutaneous region of healthy donors undergoing liposuction procedures. Four ASC culture conditions were investigated: ASCs cultured in basal Dulbecco's Modified Eagle Medium (DMEM); ASCs cultured in serum-free DMEM; ASCs cultured in serum-free DMEM/F12; and ASCs cultured in a CM from ARPE-19, a spontaneously arising cell line with a normal karyotype derived from a human RPE. Cell proliferation rate and viability were assessed by crystal violet and MTT assays at 1, 4 and 8 d of culture. At the same time points, ASC neural differentiation was evaluated by immunocytochemistry and western blot analysis for typical neuronal and glial markers: Nestin, neuronal specific enolase (NSE), protein gene product (PGP) 9.5, and glial fibrillary acidic protein (GFAP). RESULTS: Depending on the culture medium, ASC proliferation rate and viability showed some significant differences. Overall, less dense populations were observed in serum-free cultures, except for ASCs cultured in ARPE-19 serum-free CM. Moreover, a different cell morphology was seen in these cultures after 8 d of treatment, with more elongated cells, often showing cytoplasmic ramifications. Immunofluorescence results and western blot analysis were indicative of ASC neural differentiation. In fact, basal levels of neural markers detected under control conditions significantly increased when cells were cultured in ARPE-19 CM. Specifically, neural marker overexpression was more marked at 8 d. The most evident increase was observed for NSE and GFAP, a modest increase was observed for nestin, and less relevant changes were observed for PGP9.5. CONCLUSION: The presence of growth factors produced by ARPE-19 cells in tissue culture induces ASCs to express neural differentiation markers typical of the neuronal and glial cells of the retina.

Potential therapeutic applications of mesenchymal stem cells for the treatment of eye diseases.

Stem cell-based treatments have been extensively explored in the last few decades to develop therapeutic strategies aimed at providing effective alternatives for those human pathologies in which surgical or pharmacological therapies produce limited effects. Among stem cells of different sources, mesenchymal stem cells (MSCs) offer several advantages, such as the absence of ethical concerns, easy harvesting, low immunogenicity and reduced tumorigenesis risks. Other than a multipotent differentiation ability, MSCs can release extracellular vesicles conveying proteins, mRNA and microRNA. Thanks to these properties, new therapeutic approaches have been designed for the treatment of various pathologies, including ocular diseases. In this review, the use of different MSCs and different administration strategies are described for the treatment of diabetic retinopathy, glaucoma, and retinitis pigmentosa. In a large number of investigations, positive results have been obtained by in vitro experiments and by MSC administration in animal models. Most authors agree that beneficial effects are likely related to MSC paracrine activity. Based on these considerations, many clinical trials have already been carried out. Overall, although some adverse effects have been described, promising outcomes are reported. It can be assumed that in the near future, safer and more effective protocols will be developed for more numerous clinical applications to improve the quality of life of patients affected by eye diseases.