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Abstract Statins, widely used cardiovascular drugs that lower cholesterol by inhibiting HMG-CoA reductase, have been increasingly recognized for their potential anticancer properties. This study elucidates the underlying mechanism, revealing that statins exploit Synthetic Lethality, a principle where the co-occurrence of two non-lethal events leads to cell death. Our computational analysis of approximately 37,000 SL pairs identified statins as potential drugs targeting genes involved in SL pairs with metastatic genes. In vitro validation on various cancer cell lines confirmed the anticancer efficacy of statins. This data-driven drug repurposing strategy provides a molecular basis for the anticancer effects of statins, offering translational opportunities in oncology.

Abstract Background A reliable preclinical model of patient-derived organoids (PDOs) was developed in a case study of a 69-year-old woman diagnosed with breast cancer (BC) to investigate the tumour evolution before and after neoadjuvant chemotherapy and surgery. The results were achieved due to the development of PDOs from tissues collected before (O-PRE) and after (O-POST) treatment. Methods PDO cultures were characterized by histology, immunohistochemistry (IHC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), confocal microscopy, flow cytometry, real-time PCR, bulk RNA-seq, single-cell RNA sequencing (scRNA-seq) and drug screening. Results Both PDO cultures recapitulated the histological and molecular profiles of the original tissues, and they showed typical mammary gland organization, confirming their reliability as a personalized in vitro model. Compared with O-PRE, O-POST had a greater proliferation rate with a significant increase in the Ki67 proliferation index. Moreover O-POST exhibited a more stem-like and aggressive phenotype, with increases in the CD24low/CD44low and EPCAMlow/CD49fhigh cell populations characterized by increased tumour initiation potential and multipotency and metastatic potential in invasive lobular carcinoma. Analysis of ErbB receptor expression indicated a decrease in HER-2 expression coupled with an increase in EGFR expression in O-POST. In this context, deregulation of the PI3K/Akt signalling pathway was assessed by transcriptomic analysis, confirming the altered transcriptional profile. Finally, transcriptomic single-cell analysis identified 11 cell type clusters, highlighting the selection of the luminal component and the decrease in the number of Epithelial–mesenchymal transition cell types in O-POST. Conclusion Neoadjuvant treatment contributed to the enrichment of cell populations with luminal phenotypes that were more resistant to chemotherapy in O-POST. PDOs represent an excellent 3D cell model for assessing disease evolution.

Hypomyelinating leukodystrophies (HLD) are a group of heterogeneous genetic disorders characterized by a deficit in myelin deposition during brain development. Specifically, 4H-Leukodystrophy is a recessive disease due to biallelic mutations in the POLR3A gene, which encodes one of the subunits forming the catalytic core of RNA polymerase III (PolIII). The disease also presents non-neurological signs such as hypodontia and hypogonadotropic hypogonadism. Here, we report the generation of a human induced pluripotent stem cell (hiPSC) line from fibroblasts of the first identified carrier of the biallelic POLR3A variants c.1802 T > A and c.4072G > A.

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.

The vast corpus of heterogeneous biomedical data stored in databases, ontologies, and terminologies presents a unique opportunity for drug design. Integrating and fusing these sources is essential to develop data representations that can be analyzed using artificial intelligence methods to generate novel drug candidates or hypotheses. Here, we propose Non-Negative Matrix Tri-Factorization as an invaluable tool for integrating and fusing data, as well as for representation learning. Additionally, we demonstrate how representations learned by Non-Negative Matrix Tri-Factorization can effectively be utilized by traditional artificial intelligence methods. While this approach is domain-agnostic and applicable to any field with vast amounts of structured and semi-structured data, we apply it specifically to computational pharmacology and drug repurposing. This field is poised to benefit significantly from artificial intelligence, particularly in personalized medicine. We conducted extensive experiments to evaluate the performance of the proposed method, yielding exciting results, particularly compared to traditional methods. Novel drug–target predictions have also been validated in the literature, further confirming their validity. Additionally, we tested our method to predict drug synergism, where constructing a classical matrix dataset is challenging. The method demonstrated great flexibility, suggesting its applicability to a wide range of tasks in drug design and discovery.

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.

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.

Traumatic spinal cord injuries (SCIs) often result in sensory, motor, and vegetative function loss below the injury site. Although preclinical results have been promising, significant solutions for SCI patients have not been achieved through translating repair strategies to clinical trials. In this study, we investigated the effective potential of mechanically activated lipoaspirated adipose tissue when transplanted into the epicenter of a thoracic spinal contusion. Male Sprague Dawley rats were divided into three experimental groups: SHAM (uninjured and untreated), NaCl (spinal cord contusion with NaCl application), and AF (spinal cord contusion with transplanted activated human fat). Pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) were measured to assess endogenous inflammation levels 14 days after injury. Sensorimotor recovery was monitored weekly for 12 weeks, and gait and electrophysiological analyses were performed at the end of this observational period. The results indicated that AF reduced endogenous inflammation post-SCI and there was a significant improvement in sensorimotor recovery. Moreover, activated adipose tissue also reinstated the segmental sensorimotor loop and the communication between supra- and sub-lesional spinal cord regions. This investigation highlights the efficacy of activated adipose tissue grafting in acute SCI, suggesting it is a promising therapeutic approach for spinal cord repair after traumatic contusion in humans.

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/β-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 β-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 β-Catenin signaling and an alteration of the neuronal phenotype are taking place.

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.

Fundamental organelles that occur in every cell type with the exception of mammal erythrocytes, the mitochondria are required for multiple pivotal processes that include the production of biological energy, the biosynthesis of reactive oxygen species, the control of calcium homeostasis, and the triggering of cell death. The disruption of anyone of these processes has been shown to impact strongly the function of all cells, but especially of neurons. In this review, we discuss the role of the mitochondria impairment in the development of the neurodegenerative diseases Amyotrophic Lateral Sclerosis, Parkinson’s disease and Alzheimer’s disease. We highlight how mitochondria disruption revolves around the processes that underlie the mitochondria’s life cycle: fusion, fission, production of reactive oxygen species and energy failure. Both genetic and sporadic forms of neurodegenerative diseases are unavoidably accompanied with and often caused by the dysfunction in one or more of the key mitochondrial processes. Therefore, in order to get in depth insights into their health status in neurodegenerative diseases, we need to focus into innovative strategies aimed at characterizing the various mitochondrial processes. Current techniques include Mitostress, Mitotracker, transmission electron microscopy, oxidative stress assays along with expression measurement of the proteins that maintain the mitochondrial health. We will also discuss a panel of approaches aimed at mitigating the mitochondrial dysfunction. These include canonical drugs, natural compounds, supplements, lifestyle interventions and innovative approaches as mitochondria transplantation and gene therapy. In conclusion, because mitochondria are fundamental organelles necessary for virtually all the cell functions and are severely impaired in neurodegenerative diseases, it is critical to develop novel methods to measure the mitochondrial state, and novel therapeutic strategies aimed at improving their health.

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.

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.

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).

Background: Telomere length (TL) and mitochondrial DNA (mtDNA) copy number shifts are linked to metabolic abnormalities, and possible modifications by diet-induced weight loss are poorly explored. We investigated the variations before (T0) and after a 1-year (T12) lifestyle intervention (diet + physical activity) in a group of outpatients with obesity. Methods: Patients aged 25–70 years with BMI ≥ 30 kg/m2 were enrolled. Clinical and biochemical assessments (including a blood sample for TL, mtDNA copy number and total antioxidant capacity, and TAC determinations) were performed at T0 and T12. Results: The change in TL and the mtDNA copy number was heterogeneous and not significantly different at T12. Patients were then divided by baseline TL values into lower than median TL (L-TL) and higher than median TL (H-TL) groups. The two groups did not differ at baseline for anthropometric, clinical, and laboratory characteristics. At T12, the L-TL group when compared to H-TL showed TL elongation (respectively, +0.57 ± 1.23 vs. −2.15 ± 1.13 kbp, p = 0.04), higher mtDNA copy number (+111.5 ± 478.5 vs. −2314.8 ± 724.2, respectively, p < 0.001), greater weight loss (−8.1 ± 2.7 vs. −6.1 ± 4.6 Kg, respectively, p = 0.03), fat mass reduction (−1.42 ± 1.3 vs. −1.22 ± 1.5%, respectively, p = 0.04), and increased fat-free mass (+57.8 ± 6.5 vs. +54.9 ± 5.3%, respectively, p = 0.04) and TAC levels (+58.5 ± 18.6 vs. +36.4 ± 24.1 µM/L, respectively, p = 0.04). Conclusions: TL and the mtDNA copy number significantly increased in patients with obesity and with lower baseline TL values after a 1-year lifestyle intervention. Larger longitudinal studies are needed to confirm the results of this pilot study.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease (NDD) that affects motor neurons, causing weakness, muscle atrophy and spasticity. Unfortunately, there are only symptomatic treatments available. Two important innovations in recent years are three-dimensional (3D) bioprinting and induced pluripotent stem cells (iPSCs). The aim of this work was to demonstrate the robustness of 3D cultures for the differentiation of stem cells for the study of ALS. We reprogrammed healthy and sALS peripheral blood mononuclear cells (PBMCs) in iPSCs and differentiated them in neural stem cells (NSCs) in 2D. NSCs were printed in 3D hydrogel-based constructs and subsequently differentiated first in motor neuron progenitors and finally in motor neurons. Every step of differentiation was tested for cell viability and characterized by confocal microscopy and RT-qPCR. Finally, we tested the electrophysiological characteristics of included NSC34. We found that NSCs maintained good viability during the 3D differentiation. Our results suggest that the hydrogel does not interfere with the correct differentiation process or with the electrophysiological features of the included cells. Such evidence confirmed that 3D bioprinting can be considered a good model for the study of ALS pathogenesis.

Mitochondria alterations are present in tissues derived from patients and animal models, but no data are available for peripheral blood mononuclear cells (PBMCs) of ALS patients. This work aims to investigate mitophagy in PBMCs of sporadic (sALS) patients and how this pathway can be tuned by using small molecules. We found the presence of morphologically atypical mitochondria by TEM and morphological abnormalities by MitoTracker™. We found a decreased number of healthy mitochondria in sALS PBMCs and an impairment of mitophagy with western blot and immunofluorescence. After rapamycin treatment, we found a higher increase in the LC3 marker in sALS PBMCs, while after NH4Cl treatment, we found a lower increase in the LC3 marker. Finally, mTOR-independent autophagy induction with trehalose resulted in a significant decrease in the lysosomes level sALS PBMCs. Our data suggest that the presence of morphologically altered mitochondria and an inefficient turnover of damaged mitochondria in PBMCs of sALS patients rely on the impairment of the mitophagy pathway. We also found that the induction of the mTOR-independent autophagy pathway leads to a decrease in lysosomes level, suggesting a more sensitivity of sALS PBMCs to trehalose. Such evidence suggests that trehalose could represent an effective treatment for ALS patients.

Superoxide dismutase 1 (SOD1) is one of the causative genes associated with amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder. SOD1 aggregation contributes to ALS pathogenesis. A fraction of the protein is localized in the nucleus (nSOD1), where it seems to be involved in the regulation of genes participating in the oxidative stress response and DNA repair. Peripheral blood mononuclear cells (PBMCs) were collected from sporadic ALS (sALS) patients (n = 18) and healthy controls (n = 12) to perform RNA-sequencing experiments and differential expression analysis. Patients were stratified into groups with “high” and “low” levels of nSOD1. We obtained different gene expression patterns for high- and low-nSOD1 patients. Differentially expressed genes in high nSOD1 form a cluster similar to controls compared to the low-nSOD1 group. The pathways activated in high-nSOD1 patients are related to the upregulation of HSP70 molecular chaperones. We demonstrated that, in this condition, the DNA damage is reduced, even under oxidative stress conditions. Our findings highlight the importance of the nuclear localization of SOD1 as a protective mechanism in sALS patients.

Abstract 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.

The inflammatory response plays a central role in the complications of congenital pulmonary airway malformations (CPAM) and severe coronavirus disease 2019 (COVID-19). The aim of this study was to evaluate the transcriptional changes induced by SARS-CoV-2 exposure in pediatric MSCs derived from pediatric lung (MSCs-lung) and CPAM tissues (MSCs-CPAM) in order to elucidate potential pathways involved in SARS-CoV-2 infection in a condition of exacerbated inflammatory response. MSCs-lung and MSCs-CPAM do not express angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TRMPSS2). SARS-CoV-2 appears to be unable to replicate in MSCs-CPAM and MSCs-lung. MSCs-lung and MSCs-CPAM maintained the expression of stemness markers MSCs-lung show an inflammatory response (IL6, IL1B, CXCL8, and CXCL10), and the activation of Notch3 non-canonical pathway; this route appears silent in MSCs-CPAM, and cytokine genes expression is reduced. Decreased value of p21 in MSCs-lung suggested no cell cycle block, and cells did not undergo apoptosis. MSCs-lung appears to increase genes associated with immunomodulatory function but could contribute to inflammation, while MSCs-CPAM keeps stable or reduce the immunomodulatory receptors expression, but they also reduce their cytokines expression. These data indicated that, independently from their perilesional or cystic origin, the MSCs populations already present in a patient affected with CPAM are not permissive for SARS-CoV-2 entry, and they will not spread the disease in case of infection. Moreover, these MSCs will not undergo apoptosis when they come in contact with SARS-CoV-2; on the contrary, they maintain their staminality profile.

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.

According to the neurological symptoms of SARS-CoV-2 infection, it is known that the nervous system is influenced by the virus. We used pediatric human cerebral cortical cell line HCN-2 as a neuronal model of SARS-CoV-2 infection, and, through transcriptomic analysis, our aim was to evaluate the effect of SARS-CoV-2 in this type of cells. Transcriptome analyses revealed impairment in TXN gene, resulting in deregulation of its antioxidant functions, as well as a decrease in the DNA-repairing mechanism, as indicated by the decrease in KAT5. Western blot analyses of SOD1 and iNOS confirmed the impairment of reduction mechanisms and an increase in oxidative stress. Upregulation of CDKN2A and a decrease in CDK4 and CDK6 point to the blocking of the cell cycle that, according to the deregulation of repairing mechanism, has apoptosis as the outcome. A high level of proapoptotic gene PMAIP1 is indeed coherent with neuronal death, as also supported by increased levels of caspase 3. The upregulation of cell-cycle-blocking genes and apoptosis suggests a sufferance state of neurons after SARS-CoV-2 infection, followed by their inevitable death, which can explain the neurological symptoms reported. Further analyses are required to deeply explain the mechanisms and find potential treatments to protect neurons from oxidative stress and prevent their death.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease caused in 10% of cases by inherited mutations considered “familial”. An ever-increasing amount of evidence is showing a fundamental role for RNA metabolism in ALS pathogenesis, and long non-coding RNAs (lncRNAs) appear to play a role in ALS development. Here, we aim to investigate the expression of a panel of lncRNAs (linc-Enc1, linc–Brn1a, linc–Brn1b, linc-p21, Hottip, Tug1, Eldrr, and Fendrr) which could be implicated in early phases of ALS. Via Real-Time PCR, we assessed their expression in a murine familial model of ALS (SOD1-G93A mouse) in brain and spinal cord areas of SOD1-G93A mice in comparison with that of B6.SJL control mice, in asymptomatic (week 8) and late-stage disease (week 18). We highlighted a specific area and pathogenetic-stage deregulation in each lncRNA, with linc-p21 being deregulated in all analyzed tissues. Moreover, we analyzed the expression of their human homologues in SH-SY5Y-SOD1-WT and SH-SY5Y-SOD1-G93A, observing a profound alteration in their expression. Interestingly, the lncRNAs expression in our ALS models often resulted opposite to that observed for the lncRNAs in cancer. These evidences suggest that lncRNAs could be novel disease-modifying agents, biomarkers, or pathways affected by ALS neurodegeneration.

Different mechanisms were proposed as responsible for COVID-19 neurological symptoms but a clear one has not been established yet. In this work we aimed to study SARS-CoV-2 capacity to infect pediatric human cortical neuronal HCN-2 cells, studying the changes in the transcriptomic profile by next generation sequencing. SARS-CoV-2 was able to replicate in HCN-2 cells, that did not express ACE2, confirmed also with Western blot, and TMPRSS2. Looking for pattern recognition receptor expression, we found the deregulation of scavenger receptors, such as SR-B1, and the downregulation of genes encoding for Nod-like receptors. On the other hand, TLR1, TLR4 and TLR6 encoding for Toll-like receptors (TLRs) were upregulated. We also found the upregulation of genes encoding for ERK, JNK, NF-κB and Caspase 8 in our transcriptomic analysis. Regarding the expression of known receptors for viral RNA, only RIG-1 showed an increased expression; downstream RIG-1, the genes encoding for TRAF3, IKKε and IRF3 were downregulated. We also found the upregulation of genes encoding for chemokines and accordingly we found an increase in cytokine/chemokine levels in the medium. According to our results, it is possible to speculate that additionally to ACE2 and TMPRSS2, also other receptors may interact with SARS-CoV-2 proteins and mediate its entry or pathogenesis in pediatric cortical neurons infected with SARS-CoV-2. In particular, TLRs signaling could be crucial for the neurological involvement related to SARS-CoV-2 infection.

Aims: Hyperoxic breathing might lead to redox imbalance and signaling changes that affect cerebral function. Paradoxically, hypoxic breathing is also believed to cause oxidative stress. Our aim is to dissect the cerebral tissue responses to altered O2 fractions in breathed air by assessing the redox imbalance and the recruitment of the hypoxia signaling pathways. Results: Mice were exposed to mild hypoxia (10%O2), normoxia (21%O2) or mild hyperoxia (30%O2) for 28 days, sacrificed and brain tissue excised and analyzed. Although one might expect linear responses to %O2, only few of the examined variables exhibited this pattern, including neuroprotective phospho- protein kinase B and the erythropoietin receptor. The major reactive oxygen species (ROS) source in brain, NADPH oxidase subunit 4 increased in hypoxia but not in hyperoxia, whereas neither affected nuclear factor (erythroid-derived 2)-like 2, a transcription factor that regulates the expression of antioxidant proteins. As a result of the delicate equilibrium between ROS generation and antioxidant defense, neuron apoptosis and cerebral tissue hydroperoxides increased in both 10%O2 and 30%O2, as compared with 21%O2. Remarkably, the expression level of hypoxia-inducible factor (HIF)−2α (but not HIF-1α) was higher in both 10%O2 and 30%O2 with respect to 21%O2 Innovation: Comparing the in vivo effects driven by mild hypoxia with those driven by mild hyperoxia helps addressing whether clinically relevant situations of O2 excess and scarcity are toxic for the organism. Conclusion: Prolonged mild hyperoxia leads to persistent cerebral damage, comparable to that inferred by prolonged mild hypoxia. The underlying mechanism appears related to a model whereby the imbalance between ROS generation and anti-ROS defense is similar, but occurs at higher levels in hypoxia than in hyperoxia. Keywords: In vivo hypoxia, In vivo hyperoxia, Neurons, Hypoxia-inducible factor, Oxidative injury

Erythropoietin (EPO) is a cytokine mainly induced in hypoxia conditions. Its major production site is the kidney. EPO primarily acts on the erythroid progenitor cells in the bone marrow. More and more studies are highlighting its secondary functions, with a crucial focus on its role in the central nervous system. Here, EPO may interact with up to four distinct isoforms of its receptor (erythropoietin receptor [EPOR]), activating different signaling cascades with roles in neuroprotection and neurogenesis. Indeed, the EPO/EPOR axis has been widely studied in the neurodegenerative diseases field. Its potential therapeutic effects have been evaluated in multiple disorders, such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, spinal cord injury, as well as brain ischemia, hypoxia, and hyperoxia. EPO is showing great promise by counteracting secondary neuroinflammatory processes, reactive oxygen species imbalance, and cell death in these diseases. Multiple studies have been performed both in vitro and in vivo , characterizing the mechanisms through which EPO exerts its neurotrophic action. In some cases, clinical trials involving EPO have been performed, highlighting its therapeutic potential. Together, all these works indicate the potential beneficial effects of EPO.

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into multiple cell types, including adipocytes, osteoblasts, and chondrocytes. The role of adipose-derived stem cells (ADSCs) in cancers is significantly relevant. They seem to be involved in the promotion of tumour development and progression and relapse processes. For this reason, investigating the effects of breast cancer microenvironment on ADSCs is of high importance in order to understand the relationship between tumour cells and the surrounding stromal cells. With the current study, we aimed to investigate the specific characteristics of human ADSCs isolated from the adipose tissue of breast tumour patients. We compared ADSCs obtained from periumbilical fat (PF) of controls with ADSCs obtained from adipose tissue of breast cancer- (BC-) bearing patients. We analysed the surface antigens and the adipogenic differentiation ability of both ADSC populations. C/EBPδ expression was increased in PF and BC ADSCs induced to differentiate compared to the control while PPARγ and FABP4 expressions were enhanced only in PF ADSCs. Conversely, adiponectin expression was reduced in PF-differentiated ADSCs while it was slightly increased in differentiated BC ADSCs. By means of Oil Red O staining, we further observed an impaired differentiation capability of BC ADSCs. To investigate this aspect more in depth, we evaluated the effect of selective PPARγ activation and nutritional supplementation on the differentiation efficiency of BC ADSCs, noting that it was only with a strong differentiation stimuli that the process took place. Furthermore, we observed no response in BC ADSCs to the PPARγ inhibitor T0070907, showing an impaired activation of this receptor in adipose cells surrounding the breast cancer microenvironment. In conclusion, our study shows an impaired adipogenic differentiation capability in BC ADSCs. This suggests that the tumour microenvironment plays a key role in the modulation of the adipose microenvironment located in the surrounding tissue.

Pediatric obesity remains a challenge in modern society. Recently, research has focused on the role of the brown adipose tissue (BAT) as a potential target of intervention. In this review, we revised preclinical and clinical works on factors that may promote BAT or browning of white adipose tissue (WAT) from fetal age to adolescence. Maternal lifestyle, type of breastfeeding and healthy microbiota can affect the thermogenic activity of BAT. Environmental factors such as exposure to cold or physical activity also play a role in promoting and activating BAT. Most of the evidence is preclinical, although in clinic there is some evidence on the role of omega-3 PUFAs (EPA and DHA) supplementation on BAT activation. Clinical studies are needed to dissect the early factors and their modulation to allow proper BAT development and functions and to prevent onset of childhood obesity.

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.

Existing therapies for Parkinson’s disease (PD) are only symptomatic. As erythropoietin (EPO) is emerging for its benefits in neurodegenerative diseases, here, we test the protective effect driven by EPO in in vitro (SH-SY5Y cells challenged by MPP+) and in vivo (C57BL/6J mice administered with MPTP) PD models. EPO restores cell viability in both protective and restorative layouts, enhancing the dopaminergic recovery. Specifically, EPO rescues the PD-induced damage to mitochondria, as shown by transmission electron microscopy, Mitotracker assay and PINK1 expression. Moreover, EPO promotes a rescue of mitochondrial respiration while markedly enhancing the glycolytic rate, as shown by the augmented extracellular acidification rate, contributing to elevated ATP levels in MPP+-challenged cells. In PD mice, EPO intrastriatal infusion markedly improves the outcome of behavioral tests. This is associated with the rescue of dopaminergic markers and decreased neuroinflammation. This study demonstrates cellular and functional recovery following EPO treatment, likely mediated by the 37 Kda isoform of the EPO-receptor. We report for the first time, that EPO-neuroprotection is exerted through restoring ATP levels by accelerating the glycolytic rate. In conclusion, the redox imbalance and neuroinflammation associated with PD may be successfully treated by EPO.

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.

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.

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.

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.

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.

The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT.

Neurodegenerative diseases (NDs) are a broad class of pathologies characterized by the progressive loss of neurons in the central nervous system. The main problem in the study of NDs is the lack of an adequate realistic experimental model to study the pathogenic mechanisms. Induced pluripotent stem cells (iPSCs) partially overcome the problem, with their capability to differentiate into almost every cell types; even so, these cells alone are not sufficient to unveil the mechanisms underlying NDs. 3D bioprinting allows to control the distribution of cells such as neurons, leading to the creation of a realistic in vitro model. In this work, we analyzed two biomaterials: sodium alginate and gelatin, and three different cell types: a neuroblastoma cell line (SH-SY5Y), iPSCs, and neural stem cells. All cells were encapsulated inside the bioink, printed and cultivated for at least seven days; they all presented good viability. We also evaluated the maintenance of the printed shape, opening the possibility to obtain a reliable in vitro neural tissue combining 3D bioprinting and iPSCs technology, optimizing the study of the degenerative processes that are still widely unknown.

Spinal cord injury (SCI) is a devastating disease, which leads to paralysis and is associated to substantially high costs for the individual and society. At present, no effective therapies are available. Here, the use of mechanically-activated lipoaspirate adipose tissue (MALS) in a murine experimental model of SCI is presented. Our results show that, following acute intraspinal MALS transplantation, there is an engraftment at injury site with the acute powerful inhibition of the posttraumatic inflammatory response, followed by a significant progressive improvement in recovery of function. This is accompanied by spinal cord tissue preservation at the lesion site with the promotion of endogenous neurogenesis as indicated by the significant increase of Nestin-positive cells in perilesional areas. Cells originated from MALS infiltrate profoundly the recipient cord, while the extra-dural fat transplant is gradually impoverished in stromal cells. Altogether, these novel results suggest the potential of MALS application in the promotion of recovery in SCI.

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.

Erythropoietin-releasing neural precursor cells (Er-NPCs) are a subclass of subventricular zone-derived neural progenitors, capable of surviving for 6 hr after death of donor. They present higher neural differentiation. Here, Er-NPCs were studied in animal model of Parkinson’s disease. Dopaminergic degeneration was caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intraperitoneal administration in C57BL/6 mice. The loss of function was evaluated by specific behavioral tests. Er-NPCs (2.5 × 10 5 ) expressing the green fluorescent protein were administered by stereotaxic injection unilaterally in the left striatum. At the end of observational research period (2 weeks), most of the transplanted Er-NPCs were located in the striatum, while several had migrated ventrally and caudally from the injection site, up to ipsilateral and contralateral substantia nigra. Most of transplanted cells had differentiated into dopaminergic, cholinergic, or GABAergic neurons. Er-NPCs administration also promoted a rapid functional improvement that was already evident at the third day after cells administration. This was accompanied by enhanced survival of nigral neurons. These effects were likely promoted by Er-NPCs-released erythropoietin (EPO), since the injection of Er-NPCs in association with anti-EPO or anti-EPOR antibodies had completely neutralized the recovery of function. In addition, intrastriatal administration of recombinant EPO mimics the effects of Er-NPCs. We suggest that Er-NPCs, and cells with similar properties, may represent good candidates for cellular therapy in neurodegenerative disorders of this kind.

This study was aimed at the isolation of neural precursor cells (NPCs) capable of resisting to a prolonged ischemic insult as this may occur at the site of traumatic and ischemic CNS injuries. Adult mice were anesthetized and then killed by cervical dislocation. The cadavers were maintained at room temperature or at 4 °C for different time periods. Post mortem neural precursors (PM-NPCs) were isolated, grown in vitro and their differentiation capability was investigated by evaluating the expression of different neuronal markers. PM-NPCs differentiate mostly in neurons, show activation of hypoxia-inducible factor-1 and MAPK, and express both erythropoietin (EPO) and its receptor (EPO-R). The exposure of PM-NPCs to neutralizing antibodies to EPO or EPO-R dramatically reduced the extent of neuronal differentiation to about 11% of total PM-NPCs. The functionality of mTOR and MAPK is also required for the expression of the neuronal phenotype by PM-NPCs. These results suggest that PM-NPCs can be isolated from animal cadaver even several hours after death and their self-renewable capability is comparable to normal neural precursors. Differently, their ability to achieve a neural phenotype is superior to that of NPCs, and this is mediated by the activation of hypoxia-induced factor 1 and EPO signaling. PM-NPCs may represent good candidates for transplantation studies in animal models of neurodegenerative diseases.

The adipose tissue is a source of inflammatory proteins, such as TNF, IL-6, and CXCL8. Most of their production occurs in macrophages that act as scavengers of dying adipocytes. The application of an orbital mechanical force for 6–10 min at 97 g to the adipose tissue, lipoaspirated and treated according to Coleman procedures, abolishes the expression of TNF-α and stimulates the expression of the anti-inflammatory protein TNF-stimulated gene-6 (TSG-6). This protein had protective and anti-inflammatory effects when applied to animal models of rheumatic diseases. We examined biopsy, lipoaspirate, and mechanically activated fat and observed that in addition to the increased TSG-6, Sox2, Nanog, and Oct4 were also strongly augmented by mechanical activation, suggesting an effect on stromal cell stemness. Human adipose tissue-derived mesenchymal stem cells (hADSCs), produced from activated fat, grow and differentiate normally with proper cell surface markers and chromosomal integrity, but their anti-inflammatory action is far superior compared to those mesenchymal stem cells (MSCs) obtained from lipoaspirate. The expression and release of inflammatory cytokines from THP-1 cells was totally abolished in mechanically activated adipose tissue-derived hADSCs. In conclusion, we report that the orbital shaking of adipose tissue enhances its anti-inflammatory properties, and derived MSCs maintain such enhanced activity.

The subcutaneous adipose tissue provides a clear advantage over other mesenchymal stem cell sources due to the ease with which it can be accessed, as well as the ease of isolating the residing stem cells. Human adipose-derived stem cells (hADSCs), localized in the stromal–vascular portion, can be isolated ex vivo using a combination of washing steps and enzymatic digestion. In this study, we report that microfragmented human lipoaspirated adipose tissue is a better stem cell source compared to normal lipoaspirated tissue. The structural composition of microfragments is comparable to the original tissue. Differently, however, this procedure activates the expression of antigens, such as β-tubulin III. The hADSCs derived from microfragmented lipoaspirate tissue were systematically characterized for growth features, phenotype, and multipotent differentiation potential. They fulfill the definition of mesenchymal stem cells, although with a higher neural phenotype profile. These cells also express genes that constitute the core circuitry of self-renewal such as OCT4, SOX2, and NANOG, and neurogenic lineage genes such as NEUROD1, PAX6, and SOX3. Such findings suggest further studies by evaluating Microfrag-AT hADSC action in animal models of neurodegenerative conditions.

Spinal cord injury (SCI) is a debilitating clinical condition, characterized by a complex of neurological dysfunctions. Neural stem cells from the subventricular zone of the forebrain have been considered a potential tool for cell replacement therapies. We recently isolated a subclass of neural progenitors from the cadaver of mouse donors. These cells, named postmortem neural precursor cells (PM-NPCs), express both erythropoietin (EPO) and its receptor. Their EPO-dependent differentiation abilities produce a significantly higher percentage of neurons than regular NSCs. The cholinergic yield is also higher. The aim of the present study was to evaluate the potential repair properties of PM-NPCs in a mouse model of traumatic SCI. Labeled PM-NPCs were administered intravenously; then the functional recovery and the fate of transplanted cells were studied. Animals transplanted with PM-NPCs showed a remarkable improved recovery of hindlimb function that was evaluated up to 90 days after lesion. This was accompanied by reduced myelin loss, counteraction of the invasion of the lesion site by the inflammatory cells, and an attenuation of secondary degeneration. PM-NPCs migrate mostly at the injury site, where they survive at a significantly higher extent than classical NSCs. These cells accumulate at the edges of the lesion, where a reach neuropile is formed by MAP2- and β-tubulin III-positive transplanted cells that are also mostly labeled by anti-ChAT antibodies.

There have been considerable interests in attempting to reverse the deficit because of an SCI (spinal cord injury) by restoring neural pathways through the lesion and by rebuilding the tissue network. In order to provide an appropriate micro-environment for regrowing axotomized neurons and proliferating and migrating cells, we have implanted a small block of pHPMA [poly N-(2-hydroxypropyl)-methacrylamide] hydrogel into the hemisected T10 rat spinal cord. Locomotor activity was evaluated once a week during 14 weeks with the BBB rating scale in an open field. At the 14th week after SCI, the reflexivity of the sub-lesional region was measured. We also monitored the ventilatory frequency during an electrically induced muscle fatigue known to elicit the muscle metaboreflex and increase the respiratory rate. Spinal cords were then collected, fixed and stained with anti-ED-1 and anti-NF-H antibodies and FluoroMyelin. We show in this study that hydrogel-implanted animals exhibit: (i) an improved locomotor BBB score, (ii) an improved breathing adjustment to electrically evoked isometric contractions and (iii) an H-reflex recovery close to control animals. Qualitative histological results put in evidence higher accumulation of ED-1 positive cells (macrophages/monocytes) at the lesion border, a large number of NF-H positive axons penetrating the applied matrix, and myelin preservation both rostrally and caudally to the lesion. Our data confirm that pHPMA hydrogel is a potent biomaterial that can be used for improving neuromuscular adaptive mechanisms and H-reflex responses after SCI.

Erythropoietin (EPO), originally identified for its critical function in regulating production and survival of erythrocytes, is a member of the type 1 cytokine superfamily. Recent studies have shown that EPO has cytoprotective effects in a wide variety of cells and tissues. Here is presented the analysis of EPO effects on spinal cord injury (SCI), considering both animal experiments concerning to mechanisms of neurodegeneration in SCI and EPO as a neuroprotective agent, and some evidences coming from ongoing clinical trials. The evidences underling that EPO could be a promising therapeutic agent in a variety of neurological insults, including trauma, are mounting. In particular, it is highlighted that administration of EPO or other recently generated EPO analogues such as asialo-EPO and carbamylated-EPO demonstrate interesting preclinical and clinical characteristics, rendering the evaluation of these tissue-protective agents imperative in human clinical trials. Moreover the demonstration of rhEPO and its analogues’ broad neuroprotective effects in animal models of cord lesion and in human trial like stroke, should encourage scientists and clinicians to design clinical trials assessing the efficacy of these pharmacological compounds on SCI.

BACKGROUND: Tuberous sclerosis complex (TSC), a tumor syndrome caused by mutations in TSC1 or TSC2 genes, is characterized by the development of hamartomas. We previously isolated, from an angiomyolipoma of a TSC2 patient, a homogenous population of smooth muscle-like cells (TSC2(-/-) ASM cells) that have a mutation in the TSC2 gene as well as TSC2 loss of heterozygosity (LOH) and consequently, do not produce the TSC2 gene product, tuberin. TSC2(-/-) ASM cell proliferation is EGF-dependent. METHODS AND FINDINGS: Effects of EGF on proliferation of TSC2(-/-) ASM cells and TSC2(-/-) ASM cells transfected with TSC2 gene were determined. In contrast to TSC2(-/-) ASM cells, growth of TSC2-transfected cells was not dependent on EGF. Moreover, phosphorylation of Akt, PTEN, Erk and S6 was significantly decreased. EGF is a proliferative factor of TSC2(-/-) ASM cells. Exposure of TSC2(-/-) ASM cells to anti-EGFR antibodies significantly inhibited their proliferation, reverted reactivity to HMB45 antibody, a marker of TSC2(-/-) cell phenotype, and inhibited constitutive phosphorylation of S6 and ERK. Exposure of TSC2(-/-) ASM cells to rapamycin reduced the proliferation rate, but only when added at plating time. Although rapamycin efficiently inhibited S6 phosphorylation, it was less efficient than anti-EGFR antibody in reverting HMB45 reactivity and blocking ERK phosphorylation. In TSC2(-/-) ASM cells specific PI3K inhibitors (e.g. LY294002, wortmannin) and Akt1 siRNA had little effect on S6 and ERK phosphorylation. Following TSC2-gene transfection, Akt inhibitor sensitivity was observed. CONCLUSION: Our results show that an EGF independent pathway is more important than that involving IGF-I for growth and survival of TSC(-/-) ASM cells, and such EGF-dependency is the result of the lack of tuberin.