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.

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.

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.

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.

RNA-seq dataset of subcutaneous adipose tissue: Transcriptional differences between obesity and healthy women.

In this data article, we present the dataset from the RNA-Seq analysis of subcutaneous adipose tissue collected from 5 healthy normal weight women (NW, age 37 ± 6.7 years, BMI 24.3 ± 0.9 kg/m2) and 5 obese women (OBF, age 41 ± 12.5 years, BMI 38.2 ± 4.6 kg/m2). Raw data obtained from Illumina NextSeq 500 sequencer were processed through BlueBee® Genomics Platform while differential expression analysis was performed with the DESeq2 R package and deposited in the GEO public repository with GSE166047 as accession number. Specifically, 20 samples divided between NW (control), OBF (obese women), OBM (obese male) and OBT2D (obese women with diabetes) are deposited in the GSE166047. We hereby describe only 10 samples (5 healthy normal weight women reported as NW and 5 obese women reported as OBF) because we refer to the data published in the article "Transcriptional characterization of Subcutaneous Adipose Tissue in obesity affected women highlights metabolic dysfunction and implications for lncRNAs" (DOI: 10.1016/j.ygeno.2021.09.014). Pathways analyses were performed on g:Profiler, Enrichr, ClueGO and GSEA to gain biological insights on gene expression. Raw data reported in GEO database along with detailed methods description reported in this data article could be reused for comparisons with other datasets on the topic to obtain transcriptional differences in a wider co-hort. Moreover, detailed pathways analysis along with cross-referenced data with other datasets will allow to identify novel dysregulated pathways and genes responsible for this regulation. The biological interpretation of this dataset, along with related in vitro experiments, is reported by Rey et al., in Genomics (DOI: 10.1016/j.ygeno.2021.09.014).

α-Synuclein antisense transcript SNCA-AS1 regulates synapses- and aging-related genes suggesting its implication in Parkinson's disease.

SNCA protein product, α-synuclein, is widely renowned for its role in synaptogenesis and implication in both aging and Parkinson's disease (PD), but research efforts are still needed to elucidate its physiological functions and mechanisms of regulation. In this work, we aim to characterize SNCA-AS1, antisense transcript to the SNCA gene, and its implications in cellular processes. The overexpression of SNCA-AS1 upregulates both SNCA and α-synuclein and, through RNA-sequencing analysis, we investigated the transcriptomic changes of which both genes are responsible. We highlight how they impact neurites' extension and synapses' biology, through specific molecular signatures. We report a reduced expression of markers associated with synaptic plasticity, and we specifically focus on GABAergic and dopaminergic synapses, for their relevance in aging processes and PD, respectively. A reduction in SNCA-AS1 expression leads to the opposite effect. As part of this signature is co-regulated by the two genes, we discriminate between functions elicited by genes specifically altered by SNCA-AS1 or SNCA's overexpression, observing a relevant role for SNCA-AS1 in synaptogenesis through a shared molecular signature with SNCA. We also highlight how numerous deregulated pathways are implicated in aging-related processes, suggesting that SNCA-AS1 could be a key player in cellular senescence, with implications for aging-related diseases. Indeed, the upregulation of SNCA-AS1 leads to alterations in numerous PD-specific genes, with an impact highly comparable to that of SNCA's upregulation. Our results show that SNCA-AS1 elicits its cellular functions through the regulation of SNCA, with a specific modulation of synaptogenesis and senescence, presenting implications in PD.

Neural Precursor Cells Expanded Inside the 3D Micro-Scaffold Nichoid Present Different Non-Coding RNAs Profiles and Transcript Isoforms Expression: Possible Epigenetic Modulation by 3D Growth.

Non-coding RNAs show relevant implications in various biological and pathological processes. Thus, understanding the biological implications of these molecules in stem cell biology still represents a major challenge. The aim of this work is to study the transcriptional dysregulation of 357 non-coding genes, found through RNA-Seq approach, in murine neural precursor cells expanded inside the 3D micro-scaffold Nichoid versus standard culture conditions. Through weighted co-expression network analysis and functional enrichment, we highlight the role of non-coding RNAs in altering the expression of coding genes involved in mechanotransduction, stemness, and neural differentiation. Moreover, as non-coding RNAs are poorly conserved between species, we focus on those with human homologue sequences, performing further computational characterization. Lastly, we looked for isoform switching as possible mechanism in altering coding and non-coding gene expression. Our results provide a comprehensive dissection of the 3D scaffold Nichoid's influence on the biological and genetic response of neural precursor cells. These findings shed light on the possible role of non-coding RNAs in 3D cell growth, indicating that also non-coding RNAs are implicated in cellular response to mechanical stimuli.

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.

RNA-seq Characterization of Sex-Differences in Adipose Tissue of Obesity Affected Patients: Computational Analysis of Differentially Expressed Coding and Non-Coding RNAs.

Obesity is a multifactorial disease presenting sex-related differences including adipocyte functions, sex hormone effects, genetics, and metabolic inflammation. These can influence individuals' risk for metabolic dysfunctions, with an urgent need to perform sex-based analysis to improve prevention, treatment, and rehabilitation programs. This research work is aimed at characterizing the transcriptional differences present in subcutaneous adipose tissue (SAT) of five obesity affected men versus five obesity affected women, with an additional focus on the role of long non-coding RNAs. Through RNA-sequencing, we highlighted the presence of both coding and non-coding differentially expressed RNAs, and with numerous computational analyses we identified the processes in which these genes are implicated, along with their role in co-morbidities development. We report 51 differentially expressed transcripts, 32 of which were coding genes and 19 were non-coding. Using the WGCNA R package (Weighted Correlation Network Analysis, version 1.70-3), we describe the interactions between coding and non-coding RNAs, and the non-coding RNAs association with the insurgence of specific diseases, such as cancer development, neurodegenerative diseases, and schizophrenia. In conclusion, our work highlights a specific gender sex-related transcriptional signature in the SAT of obesity affected patients.

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.

Neural precursors cells expanded in a 3D micro-engineered niche present enhanced therapeutic efficacy in vivo.

Rationale: Stem Cells (SCs) show a great potential in therapeutics for restoring and regenerating native tissues. The clinical translation of SCs therapies is currently hindered by the inability to expand SCs in vitro in large therapeutic dosages, while maintaining their safety and potency. The use of biomaterials allows for the generation of active biophysical signals for directing SCs fate through 3D micro-scaffolds, such as the one named "Nichoid", fabricated with two-photon laser polymerization with a spatial resolution of 100 nm. The aims of this study were: i) to investigate the proliferation, differentiation and stemness properties of neural precursor cells (NPCs) following their cultivation inside the Nichoid micro-scaffold; ii) to assess the therapeutic effect and safety in vivo of NPCs cultivated in the Nichoid in a preclinical experimental model of Parkinson's Disease (PD). Methods: Nichoids were fabricated by two photon laser polymerization onto circular glass coverslips using a home-made SZ2080 photoresist. NPCs were grown inside the Nichoid for 7 days, counted and characterized with RNA-Seq, Real Time PCR analysis, immunofluorescence and Western Blot. Then, NPCs were transplanted in a murine experimental model of PD, in which parkinsonism was induced by the intraperitoneal administration of the neurotoxin MPTP in C57/bl mice. The efficacy of engrafted Nichoid-expanded NPCs was evaluated by means of specific behavioral tests and, after animal sacrifice, with immunohistochemical studies in brain slices. Results: NPCs grown inside the Nichoid show a significantly higher cell viability and proliferation than in standard culture conditions in suspension. Furthermore, we report the mechanical conditioning of NPCs in 3D micro-scaffolds, showing a significant increase in the expression of pluripotency genes. We also report that such mechanical reprogramming of NPCs produces an enhanced therapeutic effect in the in vivo model of PD. Recovery of PD symptoms was significantly increased when animals were treated with Nichoid-grown NPCs, and this is accompanied by the recovery of dopaminergic markers expression in the striatum of PD affected mice. Conclusion: SCs demonstrated an increase in pluripotency potential when expanded inside the Nichoid, without the need of any genetic modification of cells, showing great promise for large-scale production of safe and functional cell therapies to be used in multiple clinical applications.

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.

Bioengineering tools to speed up the discovery and preclinical testing of vaccines for SARS-CoV-2 and therapeutic agents for COVID-19.

This review provides an update for the international research community on the cell modeling tools that could accelerate the understanding of SARS-CoV-2 infection mechanisms and could thus speed up the development of vaccines and therapeutic agents against COVID-19. Many bioengineering groups are actively developing frontier tools that are capable of providing realistic three-dimensional (3D) models for biological research, including cell culture scaffolds, microfluidic chambers for the culture of tissue equivalents and organoids, and implantable windows for intravital imaging. Here, we review the most innovative study models based on these bioengineering tools in the context of virology and vaccinology. To make it easier for scientists working on SARS-CoV-2 to identify and apply specific tools, we discuss how they could accelerate the discovery and preclinical development of antiviral drugs and vaccines, compared to conventional models.

Advances in Tissue Engineering and Innovative Fabrication Techniques for 3-D-Structures: Translational Applications in Neurodegenerative Diseases.

In the field of regenerative medicine applied to neurodegenerative diseases, one of the most important challenges is the obtainment of innovative scaffolds aimed at improving the development of new frontiers in stem-cell therapy. In recent years, additive manufacturing techniques have gained more and more relevance proving the great potential of the fabrication of precision 3-D scaffolds. In this review, recent advances in additive manufacturing techniques are presented and discussed, with an overview on stimulus-triggered approaches, such as 3-D Printing and laser-based techniques, and deposition-based approaches. Innovative 3-D bioprinting techniques, which allow the production of cell/molecule-laden scaffolds, are becoming a promising frontier in disease modelling and therapy. In this context, the specific biomaterial, stiffness, precise geometrical patterns, and structural properties are to be considered of great relevance for their subsequent translational applications. Moreover, this work reports numerous recent advances in neural diseases modelling and specifically focuses on pre-clinical and clinical translation for scaffolding technology in multiple neurodegenerative diseases.