Identification of a novel pathway in sporadic Amyotrophic Lateral Sclerosis mediated by the long non-coding RNA ZEB1-AS1.

BACKGROUND: Deregulation of transcription in the pathogenesis of sporadic Amyotrophic Lateral Sclerosis (sALS) is taking central stage with RNA-sequencing analyses from sALS patients tissues highlighting numerous deregulated long non-coding RNAs (lncRNAs). The oncogenic lncRNA ZEB1-AS1 is strongly downregulated in peripheral blood mononuclear cells of sALS patients. In addition, in cancer-derived cell lines, ZEB1-AS1 belongs to a negative feedback loop regulation with hsa-miR-200c, acting as a molecular sponge for this miRNA. The role of the lncRNA ZEB1-AS1 in sALS pathogenesis has not been characterized yet, and its study could help identifying a possible disease-modifying target. METHODS: the implication of the ZEB1-AS1/ZEB1/hsa-miR-200c/BMI1 pathway was investigated in multiple patients-derived cellular models (patients-derived peripheral blood mononuclear cells and induced pluripotent stem cells-derived neural stem cells) and in the neuroblastoma cell line SH-SY5Y, where its function was inhibited via RNA interference. Molecular techniques such as Real Time PCR, Western Blot and Immunofluorescence were used to assess the pathway dysregulation. RESULTS: Our results show a dysregulation of a signaling pathway involving ZEB1-AS1/hsa-miR-200c/ß-Catenin in peripheral blood mononuclear cells and in induced pluripotent stem cells-derived neural stem cells from sALS patients. These results were validated in vitro on the cell line SH-SY5Y with silenced expression of ZEB1-AS1. Moreover, we found an increase for ZEB1-AS1 during neural differentiation with an aberrant expression of ß-Catenin, highlighting also its aggregation and possible impact on neurite length. CONCLUSIONS: Our results support and describe the role of ZEB1-AS1 pathway in sALS and specifically in neuronal differentiation, suggesting that an impairment of ß-Catenin signaling and an alteration of the neuronal phenotype are taking place.

Ferritin nanoconjugates guide trastuzumab brain delivery to promote an antitumor response in murine HER2 + breast cancer brain metastasis.

Brain metastasis (BM) represents a clinical challenge for patients with advanced HER2 + breast cancer (BC). The monoclonal anti-HER2 antibody trastuzumab (TZ) improves survival of BC patients, but it has low central nervous system penetrance, being ineffective in treating BM. Previous studies showed that ferritin nanoparticles (HFn) may cross the blood brain barrier (BBB) through binding to the transferrin receptor 1 (TfR1). However, whether this has efficacy in promoting the trans-BBB delivery of TZ and combating BC BM was not studied yet. Here, we investigated the potential of HFn to drive TZ brain delivery and promote a targeted antitumor response in a murine model of BC BM established by stereotaxic injection of engineered BC cells overexpressing human HER2. HFn were covalently conjugated with TZ to obtain a nanoconjugate endowed with HER2 and TfR1 targeting specificity (H-TZ). H-TZ efficiently achieved TZ brain delivery upon intraperitoneal injection and triggered stable targeting of cancer cells. Treatment with H-TZ plus docetaxel significantly reduced tumor growth and shaped a protective brain microenvironment by engaging macrophage activation toward cancer cells. H-TZ-based treatment also avoided TZ-associated cardiotoxicity by preventing drug accumulation in the heart and did not induce any other major side effects when combined with docetaxel. These results provided in vivo demonstration of the pharmacological potential of H-TZ, able to tackle BC BM in combination with docetaxel. Indeed, upon systemic administration, the nanoconjugate guides TZ brain accumulation, reduces BM growth and limits side effects in off-target organs, thus showing promise for the management of HER2 + BC metastatic to the brain.

MINCR: A long non-coding RNA shared between cancer and neurodegeneration.

The multitasking nature of lncRNAs allows them to play a central role in both physiological and pathological conditions. Often the same lncRNA can participate in different diseases. Specifically, the MYC-induced Long non-Coding RNA MINCR is upregulated in various cancer types, while downregulated in Amyotrophic Lateral Sclerosis patients. Therefore, this work aims to investigate MINCR potential mechanisms of action and its implications in cancer and neurodegeneration in relation to its expression levels in SH-SY5Y cells through RNA-sequencing approach. Our results show that MINCR overexpression causes massive alterations in cancer-related genes, leading to disruption in many fundamental processes, such as cell cycle and growth factor signaling. On the contrary, MINCR downregulation influences a small number of genes involved in different neurodegenerative disorders, mostly concerning RNA metabolism and inflammation. Thus, understanding the cause and functional consequences of MINCR deregulation gives important insights on potential pathogenetic mechanisms both in cancer and in neurodegeneration.

Transcriptional characterization of subcutaneous adipose tissue in obesity affected women highlights metabolic dysfunction and implications for lncRNAs.

Obesity is a complex disease with multifactorial causes, and its prevalence is becoming a serious health crisis. For this reason, there is a crucial need to identify novel targets and players. With this aim in mind, we analyzed via RNA-sequencing the subcutaneous adipose tissue of normal weight and obesity-affected women, highlighting the differential expression in the two tissues. We specifically focused on long non-coding RNAs, as 6 of these emerged as dysregulated in the diseased-tissue (COL4A2-AS2, RPS21-AS, PELATON, ITGB2-AS1, ACER2-AS and CTEPHA1). For each of them, we performed both a thorough in silico dissection and in vitro validation, to predict their function during adipogenesis. We report the lncRNAs expression during adipose derived stem cells differentiation to adipocytes as model of adipogenesis and their potential modulation by adipogenesis-related transcription factors (C/EBPs and PPARγ). Moreover, inhibiting CTEPHA1 expression we investigated its impact on adipogenesis-related transcription factors, showing its significative dysregulation of C/EBPα expression. Lastly, we dissected the subcellular localization, pathway involvement and disease-correlation for coding differentially expressed genes. Together, these findings highlight a transcriptional deregulation at the basis of obesity, impacted by both coding and long non-coding RNAs.

Transcriptome Analysis of Subcutaneous Adipose Tissue from Severely Obese Patients Highlights Deregulation Profiles in Coding and Non-Coding Oncogenes.

Obesity is a major risk factor for a large number of secondary diseases, including cancer. Specific insights into the role of gender differences and secondary comorbidities, such as type 2 diabetes (T2D) and cancer risk, are yet to be fully identified. The aim of this study is thus to find a correlation between the transcriptional deregulation present in the subcutaneous adipose tissue of obese patients and the oncogenic signature present in multiple cancers, in the presence of T2D, and considering gender differences. The subcutaneous adipose tissue (SAT) of five healthy, normal-weight women, five obese women, five obese women with T2D and five obese men were subjected to RNA-sequencing, leading to the identification of deregulated coding and non-coding RNAs, classified for their oncogenic score. A panel of DE RNAs was validated via Real-Time PCR and oncogene expression levels correlated the oncogenes with anthropometrical parameters, highlighting significant trends. For each analyzed condition, we identified the deregulated pathways associated with cancer, the prediction of possible prognosis for different cancer types and the lncRNAs involved in oncogenic networks and tissues. Our results provided a comprehensive characterization of oncogenesis correlation in SAT, providing specific insights into the possible molecular targets implicated in this process. Indeed, the identification of deregulated oncogenes also in SAT highlights hypothetical targets implicated in the increased oncogenic risk in highly obese subjects. These results could shed light on new molecular targets to be specifically modulated in obesity and highlight which cancers should receive the most attention in terms of better prevention in obesity-affected patients.

Biomaterials in Neurodegenerative Disorders: A Promising Therapeutic Approach.

Neurodegenerative disorders (i.e., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and spinal cord injury) represent a great problem worldwide and are becoming prevalent because of the increasing average age of the population. Despite many studies having focused on their etiopathology, the exact cause of these diseases is still unknown and until now, there are only symptomatic treatments. Biomaterials have become important not only for the study of disease pathogenesis, but also for their application in regenerative medicine. The great advantages provided by biomaterials are their ability to mimic the environment of the extracellular matrix and to allow the growth of different types of cells. Biomaterials can be used as supporting material for cell proliferation to be transplanted and as vectors to deliver many active molecules for the treatments of neurodegenerative disorders. In this review, we aim to report the potentiality of biomaterials (i.e., hydrogels, nanoparticles, self-assembling peptides, nanofibers and carbon-based nanomaterials) by analyzing their use in the regeneration of neural and glial cells their role in axon outgrowth. Although further studies are needed for their use in humans, the promising results obtained by several groups leads us to suppose that biomaterials represent a potential therapeutic approach for the treatments of neurodegenerative disorders.

Dissecting the Effect of a 3D Microscaffold on the Transcriptome of Neural Stem Cells with Computational Approaches: A Focus on Mechanotransduction.

3D cell cultures are becoming more and more important in the field of regenerative medicine due to their ability to mimic the cellular physiological microenvironment. Among the different types of 3D scaffolds, we focus on the Nichoid, a miniaturized scaffold with a structure inspired by the natural staminal niche. The Nichoid can activate cellular responses simply by subjecting the cells to mechanical stimuli. This kind of influence results in different cellular morphology and organization, but the molecular bases of these changes remain largely unknown. Through RNA-Seq approach on murine neural precursors stem cells expanded inside the Nichoid, we investigated the deregulated genes and pathways showing that the Nichoid causes alteration in genes strongly connected to mechanobiological functions. Moreover, we fully dissected this mechanism highlighting how the changes start at a membrane level, with subsequent alterations in the cytoskeleton, signaling pathways, and metabolism, all leading to a final alteration in gene expression. The results shown here demonstrate that the Nichoid influences the biological and genetic response of stem cells thorough specific alterations of cellular signaling. The characterization of these pathways elucidates the role of mechanical manipulation on stem cells, with possible implications in regenerative medicine applications.

Alzheimer's, Parkinson's Disease and Amyotrophic Lateral Sclerosis Gene Expression Patterns Divergence Reveals Different Grade of RNA Metabolism Involvement.

Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) are neurodegenerative disorders characterized by a progressive degeneration of the central or peripheral nervous systems. A central role of the RNA metabolism has emerged in these diseases, concerning mRNAs processing and non-coding RNAs biogenesis. We aimed to identify possible common grounds or differences in the dysregulated pathways of AD, PD, and ALS. To do so, we performed RNA-seq analysis to investigate the deregulation of both coding and long non-coding RNAs (lncRNAs) in ALS, AD, and PD patients and controls (CTRL) in peripheral blood mononuclear cells (PBMCs). A total of 293 differentially expressed (DE) lncRNAs and 87 mRNAs were found in ALS patients. In AD patients a total of 23 DE genes emerged, 19 protein coding genes and four lncRNAs. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses, we found common affected pathways and biological processes in ALS and AD. In PD patients only five genes were found to be DE. Our data brought to light the importance of lncRNAs and mRNAs regulation in three principal neurodegenerative disorders, offering starting points for new investigations on deregulated pathogenic mechanisms.

A New Selective PPARγ Modulator Inhibits Triglycerides Accumulation during Murine Adipocytes' and Human Adipose-Derived Mesenchymal Stem Cells Differentiation.

Understanding the molecular basis of adipogenesis is vital to identify new therapeutic targets to improve anti-obesity drugs. The adipogenic process could be a new target in the management of this disease. Our aim was to evaluate the effect of GMG-43AC, a selective peroxisome proliferator-activated receptor γ (PPARγ) modulator, during adipose differentiation of murine pre-adipocytes and human Adipose Derived Stem Cells (hADSCs). We differentiated 3T3-L1 cells and primary hADSCs in the presence of various doses of GMG-43AC and evaluated the differentiation efficiency measuring lipid accumulation, the expression of specific differentiation markers and the quantification of accumulated triglycerides. The treatment with GMG-43AC is not toxic as shown by cell viability assessments after the treatments. Our findings demonstrate the inhibition of lipid accumulation and the significant decrease in the expression of adipocyte-specific genes, such as PPARγ, FABP-4, and leptin. This effect was long lasting, as the removal of GMG-43AC from culture medium did not allow the restoration of adipogenic process. The above actions were confirmed in hADSCs exposed to adipogenic stimuli. Together, these results indicate that GMG-43AC efficiently inhibits adipocytes differentiation in murine and human cells, suggesting its possible function in the reversal of adipogenesis and modulation of lipolysis.

HuR interacts with lincBRN1a and lincBRN1b during neuronal stem cells differentiation.

LncRNAs play crucial roles in cellular processes and their regulatory effects in the adult brain and neural stem cells (NSCs) remain to be entirely characterized. We report that 10 lncRNAs (LincENC1, FABL, lincp21, HAUNT, PERIL, lincBRN1a, lincBRN1b, HOTTIP, TUG1 and FENDRR) are expressed during murine NSCs differentiation and interact with the RNA-binding protein ELAVL1/HuR. Furthermore, we characterize the function of two of the deregulated lncRNAs, lincBRN1a and lincBRN1b, during NSCs' differentiation. Their inhibition leads to the induction of differentiation, with a concomitant decrease in stemness and an increase in neuronal markers, indicating that they exert key functions in neuronal cells differentiation. Furthermore, we describe here that HuR regulates their half-life, suggesting their synergic role in the differentiation process. We also identify six human homologs (PANTR1, TUG1, HOTTIP, TP53COR, ELDRR and FENDRR) of the mentioned 10 lncRNAs and we report their deregulation during human iPSCs differentiation into neurons. In conclusion, our results strongly indicate a key synergic role for lncRNAs and HuR in neuronal stem cells fate.

Counteracting neuroinflammation in experimental Parkinson's disease favors recovery of function: effects of Er-NPCs administration.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease, presenting with midbrain dopaminergic neurons degeneration. A number of studies suggest that microglial activation may have a role in PD. It has emerged that inflammation-derived oxidative stress and cytokine-dependent toxicity may contribute to nigrostriatal pathway degeneration and exacerbate the progression of the disease in patients with idiopathic PD. Cell therapies have long been considered a feasible regenerative approach to compensate for the loss of specific cell populations such as the one that occurs in PD. We recently demonstrated that erythropoietin-releasing neural precursors cells (Er-NPCs) administered to MPTP-intoxicated animals survive after transplantation in the recipient's damaged brain, differentiate, and rescue degenerating striatal dopaminergic neurons. Here, we aimed to investigate the potential anti-inflammatory actions of Er-NPCs infused in an MPTP experimental model of PD. METHODS: The degeneration of dopaminergic neurons was caused by MPTP administration in C57BL/6 male mice. 2.5 × 105 GFP-labeled Er-NPCs were administered by stereotaxic injection unilaterally in the left striatum. Functional recovery was assessed by two independent behavioral tests. Neuroinflammation was investigated measuring the mRNAs levels of pro-inflammatory and anti-inflammatory cytokines, and immunohistochemistry studies were performed to evaluate markers of inflammation and the potential rescue of tyrosine hydroxylase (TH) projections in the striatum of recipient mice. RESULTS: Er-NPC administration promoted a rapid anti-inflammatory effect that was already evident 24 h after transplant with a decrease of pro-inflammatory and increase of anti-inflammatory cytokines mRNA expression levels. This effect was maintained until the end of the observational period, 2 weeks post-transplant. Here, we show that Er-NPCs transplant reduces macrophage infiltration, directly counteracting the M1-like pro-inflammatory response of murine-activated microglia, which corresponds to the decrease of CD68 and CD86 markers, and induces M2-like pro-regeneration traits, as indicated by the increase of CD206 and IL-10 expression. Moreover, we also show that this activity is mediated by Er-NPCs-derived erythropoietin (EPO) since the co-injection of cells with anti-EPO antibodies neutralizes the anti-inflammatory effect of the Er-NPCs treatment. CONCLUSION: This study shows the anti-inflammatory actions exerted by Er-NPCs, and we suggest that these cells may represent good candidates for cellular therapy to counteract neuroinflammation in neurodegenerative disorders.

From Neuronal Differentiation of iPSCs to 3D Neuro-Organoids: Modelling and Therapy of Neurodegenerative Diseases.

In the last decade, the advances made into the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) led to great improvements towards their use as models of diseases. In particular, in the field of neurodegenerative diseases, iPSCs technology allowed to culture in vitro all types of patient-specific neural cells, facilitating not only the investigation of diseases' etiopathology, but also the testing of new drugs and cell therapies, leading to the innovative concept of personalized medicine. Moreover, iPSCs can be differentiated and organized into 3D organoids, providing a tool which mimics the complexity of the brain's architecture. Furthermore, recent developments in 3D bioprinting allowed the study of physiological cell-to-cell interactions, given by a combination of several biomaterials, scaffolds, and cells. This technology combines bio-plotter and biomaterials in which several types of cells, such as iPSCs or differentiated neurons, can be encapsulated in order to develop an innovative cellular model. IPSCs and 3D cell cultures technologies represent the first step towards the obtainment of a more reliable model, such as organoids, to facilitate neurodegenerative diseases' investigation. The combination of iPSCs, 3D organoids and bioprinting will also allow the development of new therapeutic approaches. Indeed, on the one hand they will lead to the development of safer and patient-specific drugs testing but, also, they could be developed as cell-therapy for curing neurodegenerative diseases with a regenerative medicine approach.

Role of epigenetics and alterations in RNA metabolism in leukodystrophies.

Leukodystrophies are a class of rare heterogeneous disorders which affect the white matter of the brain, ultimately leading to a disruption in brain development and a damaging effect on cognitive, motor and social-communicative development. These disorders present a great clinical heterogeneity, along with a phenotypic overlap and this could be partially due to contributions from environmental stimuli. It is in this context that there is a great need to investigate what other factors may contribute to both disease insurgence and phenotypical heterogeneity, and novel evidence are raising the attention toward the study of epigenetics and transcription mechanisms that can influence the disease phenotype beyond genetics. Modulation in the epigenetics machinery including histone modifications, DNA methylation and non-coding RNAs dysregulation, could be crucial players in the development of these disorders, and moreover an aberrant RNA maturation process has been linked to leukodystrophies. Here, we provide an overview of these mechanisms hoping to supply a closer step toward the analysis of leukodystrophies not only as genetically determined but also with an added level of complexity where epigenetic dysregulation is of key relevance. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNA RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.

Towards genomic-Newborn Screening: Technical feasibility of Exome Sequencing starting from dried blood spots.

Each year thousands of babies are born with rare genetic disorders not identified by current NBS panels, due to programs which are not yet optimal. Next-generation sequencing technologies have the potential to overcome many NBS drawbacks and provide large amounts of molecular data, broadening the number of diseases investigated. Here, we design and set up an NGS-based approach to evaluate the feasibility of NGS from dried blood spot starting from 34 DBSs. After assessing gDNA yield and integrity, libraries were performed using three target enrichment approaches, sequenced on NS500 platform, and analyzed on commercial platform. Specifically, we focus on virtual gene panels related to highly actionable neonatal/pediatric disorders. WES show that amount and quality of DBS-extracted gDNA are suitable for high-throughput sequencing. We obtain 500-1500 ng for each specimen, 1.7-1.8 260/280 wavelength, and DIN of 7 resulting DNA integrity, on par with traditional venous blood collection. A high read depth with 94.3% coverage uniformity is achieved for all samples. Data results on mean coverage are comparable among the different workflows tested and demonstrate that DBS from newborn collected at birth is a suitable material for the developing of gNBS programs.

Study of lncRNAs in Pediatric Neurological Diseases: Methods, Analysis of the State-of-Art and Possible Therapeutic Implications.

Long non-coding RNAs (lncRNAs) have emerged as crucial regulators in various cellular processes, and their roles in pediatric neurological diseases are increasingly being explored. This review provides an overview of lncRNA implications in the central nervous system, both in its physiological state and when a pathological condition is present. We describe the role of lncRNAs in neural development, highlighting their significance in processes such as neural stem cell proliferation, differentiation, and synaptogenesis. Dysregulation of specific lncRNAs is associated with multiple pediatric neurological diseases, such as neurodevelopmental or neurodegenerative disorders and brain tumors. The collected evidence indicates that there is a need for further research to uncover the full spectrum of lncRNA involvement in pediatric neurological diseases and brain tumors. While challenges exist, ongoing advancements in technology and our understanding of lncRNA biology offer hope for future breakthroughs in the field of pediatric neurology, leveraging lncRNAs as potential therapeutic targets and biomarkers.

Subcutaneous Adipose Tissue Transcriptome Highlights Specific Expression Profiles in Severe Pediatric Obesity: A Pilot Study.

The prevalence of pediatric obesity is rising rapidly worldwide, and "omic" approaches are helpful in investigating the molecular pathophysiology of obesity. This work aims to identify transcriptional differences in the subcutaneous adipose tissue (scAT) of children with overweight (OW), obesity (OB), or severe obesity (SV) compared with those of normal weight (NW). Periumbilical scAT biopsies were collected from 20 male children aged 1-12 years. The children were stratified into the following four groups according to their BMI z-scores: SV, OB, OW, and NW. scAT RNA-Seq analyses were performed, and a differential expression analysis was conducted using the DESeq2 R package. A pathways analysis was performed to gain biological insights into gene expression. Our data highlight the significant deregulation in both coding and non-coding transcripts in the SV group when compared with the NW, OW, and OB groups. A KEGG pathway analysis showed that coding transcripts were mainly involved in lipid metabolism. A GSEA analysis revealed the upregulation of lipid degradation and metabolism in SV vs. OB and SV vs. OW. Bioenergetic processes and the catabolism of branched-chain amino acids were upregulated in SV compared with OB, OW, and NW. In conclusion, we report for the first time that a significant transcriptional deregulation occurs in the periumbilical scAT of children with severe obesity compared with those of normal weight or those with overweight or mild obesity.

Oxygen Sensing in Neurodegenerative Diseases: Current Mechanisms, Implication of Transcriptional Response, and Pharmacological Modulation.

Significance: Oxygen (O2) sensing is the fundamental process through which organisms respond to changes in O2 levels. Complex networks exist allowing the maintenance of O2 levels through the perception, capture, binding, transport, and delivery of molecular O2. The brain extreme sensitivity to O2 balance makes the dysregulation of related processes crucial players in the pathogenesis of neurodegenerative diseases (NDs). In this study, we wish to review the most relevant advances in O2 sensing in relation to Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Recent Advances: Over the years, it has been clarified that most NDs share common pathways, a great number of which are in relation to O2 imbalance. These include hypoxia, hyperoxia, reactive oxygen species production, metabolism of metals, protein misfolding, and neuroinflammation. Critical Issues: There is still a gap in knowledge concerning how O2 sensing plays a role in the above indicated neurodegenerations. Specifically, O2 concentrations are perceived in body sites that are not limited to the brain, but primarily reside in other organs. Moreover, the mechanisms of O2 sensing, gene expression, and signal transduction seem to correlate with neurodegeneration, but many aspects are mechanistically still unexplained. Future Directions: Future studies should focus on the precise characterization of O2 level disruption and O2 sensing mechanisms in NDs. Moreover, advances need to be made also concerning the techniques used to assess O2 sensing dysfunctions in these diseases. There is also the need to develop innovative therapies targeting this precise mechanism rather than its secondary effects, as early intervention is necessary. Antioxid. Redox Signal. 38, 160-182.

Redox Imbalance in Neurological Disorders in Adults and Children.

Oxygen is a central molecule for numerous metabolic and cytophysiological processes, and, indeed, its imbalance can lead to numerous pathological consequences. In the human body, the brain is an aerobic organ and for this reason, it is very sensitive to oxygen equilibrium. The consequences of oxygen imbalance are especially devastating when occurring in this organ. Indeed, oxygen imbalance can lead to hypoxia, hyperoxia, protein misfolding, mitochondria dysfunction, alterations in heme metabolism and neuroinflammation. Consequently, these dysfunctions can cause numerous neurological alterations, both in the pediatric life and in the adult ages. These disorders share numerous common pathways, most of which are consequent to redox imbalance. In this review, we will focus on the dysfunctions present in neurodegenerative disorders (specifically Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis) and pediatric neurological disorders (X-adrenoleukodystrophies, spinal muscular atrophy, mucopolysaccharidoses and Pelizaeus-Merzbacher Disease), highlighting their underlining dysfunction in redox and identifying potential therapeutic strategies.

Bone Marrow Mesenchymal Stem Cells Expanded Inside the Nichoid Micro-Scaffold: a Focus on Anti-Inflammatory Response.

Purpose: Mesenchymal stem cells (MSCs) represent a promising source for stem cell therapies in numerous diseases, including pediatric respiratory system diseases. Characterized by low immunogenicity, high anti-inflammatory, and immunoregulatory features, MSCs demonstrated an excellent therapeutic profile in numerous in vitro and preclinical models. MSCs reside in a specialized physiologic microenvironment, characterized by a unique combination of biophysical, biochemical, and cellular properties. The exploitation of the 3D micro-scaffold Nichoid, which simulates the native niche, enhanced the anti-inflammatory potential of stem cells through mechanical stimulation only, overcoming the limitation of biochemical and xenogenic growth factors application. Materials and Methods: In this work, we expanded pediatric bone marrow MSCs (BM-MSCs) inside the Nichoid and performed a complete cellular characterization with different approaches including viability assays, immunofluorescence analyses, RNA sequencing, and gene expression analysis. Results: We demonstrated that BM-MSCs inside the scaffold remain in a stem cell quiescent state mimicking the condition of the in vivo environment. Moreover, the gene expression profile of these cells shows a significant up-regulation of genes involved in immune response when compared with the flat control. Conclusion: The significant changes in the expression profile of anti-inflammatory genes could potentiate the therapeutic effect of BM-MSCs, encouraging the possible clinical translation for the treatment of pediatric congenital and acquired pulmonary disorders, including post-COVID lung manifestations. Lay Summary: Regenerative medicine is the research field integrating medicine, biology, and biomedical engineering. In this context, stem cells, which are a fundamental cell source able to regenerate tissues and restore damage in the body, are the key component for a regenerative therapeutic approach. When expanded outside the body, stem cells tend to differentiate spontaneously and lose regenerative potential due to external stimuli. For this reason, we exploit the scaffold named Nichoid, which mimics the in vivo cell niche architecture. In this scaffold, mesenchymal stem cells "feel at home" due to the three-dimensional mechanical stimuli, and our findings could be considered as an innovative culture system for the in vitro expansion of stem cells for clinical translation. Future Perspective: The increasing demand of safe and effective cell therapies projects our findings toward the possibility of improving cell therapies based on the use of BM-MSCs, particularly for their clinical translation in lung diseases.

3D photopolymerized microstructured scaffolds influence nuclear deformation, nucleo/cytoskeletal protein organization, and gene regulation in mesenchymal stem cells.

Mechanical stimuli from the extracellular environment affect cell morphology and functionality. Recently, we reported that mesenchymal stem cells (MSCs) grown in a custom-made 3D microscaffold, the Nichoid, are able to express higher levels of stemness markers. In fact, the Nichoid is an interesting device for autologous MSC expansion in clinical translation and would appear to regulate gene activity by altering intracellular force transmission. To corroborate this hypothesis, we investigated mechanotransduction-related nuclear mechanisms, and we also treated spread cells with a drug that destroys the actin cytoskeleton. We observed a roundish nuclear shape in MSCs cultured in the Nichoid and correlated the nuclear curvature with the import of transcription factors. We observed a more homogeneous euchromatin distribution in cells cultured in the Nichoid with respect to the Flat sample, corresponding to a standard glass coverslip. These results suggest a different gene regulation, which we confirmed by an RNA-seq analysis that revealed the dysregulation of 1843 genes. We also observed a low structured lamina mesh, which, according to the implemented molecular dynamic simulations, indicates reduced damping activity, thus supporting the hypothesis of low intracellular force transmission. Also, our investigations regarding lamin expression and spatial organization support the hypothesis that the gene dysregulation induced by the Nichoid is mainly related to a reduction in force transmission. In conclusion, our findings revealing the Nichoid's effects on MSC behavior is a step forward in the control of stem cells via mechanical manipulation, thus paving the way to new strategies for MSC translation to clinical applications.