A promoter-dependent upstream activator augments CFTR expression in diverse epithelial cell types.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an anion-selective channel found in epithelial cell membranes. Mutations in CFTR cause cystic fibrosis (CF), an inherited disorder that impairs epithelial function in multiple organs. Most men with CF are infertile due to loss of intact genital ducts. Here we investigated a novel epididymis-selective cis-regulatory element (CRE), located within a peak of open chromatin at -9.5 kb 5' to the CFTR gene promoter. Activation of the -9.5 kb CRE alone by CRISPRa had no impact on CFTR gene expression. However, CRISPRa co-activation of the -9.5 kb CRE and the CFTR gene promoter in epididymis cells significantly augmented CFTR mRNA and protein expression when compared to promoter activation alone. This increase was accompanied by enhanced chromatin accessibility at both sites. Furthermore, the combined CRISPRa strategy activated CFTR expression in other epithelial cells that lack open chromatin at the -9.5 kb site and in which the locus is normally inactive. However, the -9.5 kb CRE does not function as a classical enhancer of the CFTR promoter in transient reporter gene assays. These data provide a novel mechanism for activating/augmenting CFTR expression, which may have therapeutic utility for mutations that perturb CFTR transcription.

The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35 kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.

Piperlongumine inhibits antioxidant enzymes, increases ROS levels, induces DNA damage and G2/M cell cycle arrest in breast cell lines.

Piperlongumine (PLN) is a biologically active alkaloid/amide derived from Piper longum, with known promising anticancer activity. The aim of this study was to compare the antiproliferative activity of PLN in human breast MCF-7 adenocarcinoma cell line with effects in HB4a normal mammary epithelial non-tumor cell line. The parameters examined were cell growth, viability, reactive oxygen species (ROS) levels and DNA damage, as well as the effects on the modulating targets responsible through regulation of these pathways. PLN increased ROS levels and expression of the SOD1 antioxidant enzyme. PLN inhibited the expression of the antioxidant enzymes catalase, TRx1, and PRx2. The ability of PLN to inhibit antioxidant enzyme expression was associated with the oxidative stress response. PLN induced genotoxicity in both cell lines and upregulated the levels of GADD45A mRNA and p21 protein. The DNA damage response ATR protein was downregulated in both cell lines and contributed to an enhanced PLN genotoxicity. In HB4a cells, Chk1 protein, and mRNA levels were also decreased. In response to elevated ROS levels and DNA damage induction, the cells were arrested at the G2/M phase, probably in an attempt to promote cell survival. Although cell viability was reduced in both cell lines, only HB4a cells underwent apoptotic cell death, whereas other types of cellular death may be involved in MCF-7 cells. Taken together, these data provide insight into the anticancer mechanisms attributed to PLN effects, which acts as an inhibitor of DNA damage response (DDR) proteins and antioxidant enzymes.

Dual SMAD inhibition enhances the longevity of human epididymis epithelial cells.

Primary human epididymis epithelial (HEE) cells are valuable reagents for functional studies on the human epididymis. We used them previously to determine the transcriptional networks that establish cell identity along the length of the epididymis from caput, corpus, and cauda. These studies on HEE cells and organoids derived from them revealed important cellular properties. However, similar to other primary cells, HEE cells undergo replicative senescence and de-differentiation in culture. A cocktail of small molecules was shown elsewhere to extend longevity of epithelial basal cells. The components included transforming growth factor ß (TGF-ß)/bone morphogenetic protein (BMP) antagonists, WNT agonist, and Rho-associated and coiled-coil containing protein kinase (ROCK) inhibitor (ROCKi), which together prevented the senescence-related upregulation of TGF-ß signaling pathway members. Here, we treat HEE cells with the same cocktail and observed enhanced replicative potential and prolonged expression of markers of HEE differentiation. This treatment expands the differentiated HEE cell population available from individual epididymis tissue samples that can be used for molecular, cellular, and functional studies.

Differential mRNA expression in the induction of DNA damage, G2/M arrest, and cell death by zerumbone in HepG2/C3A cells.

Zerumbone (ZER) is a phytochemical with antioxidant and antiproliferative properties. This study evaluated the cytoxicity of ZER combined with chemotherapeutic agents and the expression of mRNA genes related to cell cycle, cell death, xenobiotic metabolism, DNA damage, and endoplasmic reticulum (ER) stress in HepG2/C3A cells. ZER was cytotoxic (IC50, 44.31 µM). ZER-induced apoptosis was related to BBC3 and ERN1 upregulation (ER stress), and its antiproliferative effects were attributable to MYC, IGF1, and NF-kB mRNA inhibition. ZER-induced G2/M arrest and DNA damage was associated with mRNA expression of cell cycle (CDKN1A) and DNA damage (GADD45A) genes. Increased CYP1A2 and CYP2C19 mRNA expression suggested ZER metabolization, and reduced CYP1A1 and CYP2D6 expression indicated a longer time of action of ZER in the cell, enhancing its pharmacological effect. ZER downregulated TP53, PARP1, BIRC5 (apoptosis), and MAP1LC3A (autophagy). In apoptosis assay, the data of the association treatments with ZER suggested antagonism. In cytotoxicity assay, the data of the association treatments with ZER suggested synergism action to cisplatin and antagonism action to doxorubicin and 5-fluorouracil. Thus, ZER has potential for application in chemotherapy as it modulates mRNA targets; however, it may not have the desired efficiency when combined with other chemotherapeutic agents.

DNA damage and reticular stress in cytotoxicity and oncotic cell death of MCF-7 cells treated with fluopsin C.

Fluopsin C is an antibiotic compound derived from secondary metabolism of different microorganisms, which possesses antitumor, antibacterial, and antifungal activity. Related to fluopsin C antiproliferative activity, the aim of this study was to examine the following parameters: cytotoxicity, genotoxicity, cell cycle arrest, cell death induction (apoptosis), mitochondrial membrane potential (MMP), colony formation, and mRNA expression of genes involved in adaptive stress responses and cellular death utilizing a monolayer. In addition, a three-dimensional cell culture was used to evaluate the effects on growth of tumor spheroids. Fluopsin C was cytotoxic (1) producing cell division arrest in the G1 phase, (2) elevating expression of mRNA of the CDKN1A gene and (3) decrease in expression of mRNA H2AFX gene. Further, fluopsin C enhanced DNA damage as evidenced by increased expression of mRNA of GADD45A and GPX1 genes, indicating that reactive oxygen species (ROS) may be involved in the observed genotoxic response. Reticulum stress was also detected as noted from activation of the ribonuclease inositol-requiring protein 1 (IRE1) pathway, since a rise in mRNA expression of the ERN1 and TRAF2 genes was observed. During the cell death process, an increase in mRNA expression of the BBC3 gene was noted, indicating participation of this antibiotic in oncotic (ischemic) cell death. Data thus demonstrated for the first time that fluopsin C interferes with the volume of tumor spheroids, in order to attenuate their growth. Our findings show that fluopsin C modulates essential molecular processes in response to stress and cell death.

Dimethoxycurcumin reduces proliferation and induces apoptosis in renal tumor cells more efficiently than demethoxycurcumin and curcumin.

Curcumin (Cur), is a pigment with antiproliferative activity but has some pharmacokinetic limitations, which led researchers to look for more effective structure analogs. This work investigated the effects of Cur and compared them with the two analogs, demethoxycurcumin (DeMC) and dimethoxycurcumin (DiMC), to elucidate their mechanisms of action. The cytotoxic, antiproliferative, and genotoxic effects these compounds were correlated based on gene expression analysis in the human renal adenocarcinoma cells (786-O). Cur decreased CYP2D6 expression and exhibited cytotoxic effects, such as inducing monopolar spindle formation and mitotic arrest mediated by the increase in CDKN1A (p21) mRNA. This dysregulation induced cell death through a caspase-independent pathway but was mediated by decrease in MTOR and BCL2 mRNA expression, suggesting that apoptosis occurred by autophagy. DeMC and DiMC had similar effects in that they induced monopolar spindle and mitotic arrest, were genotoxic, and activated GADD45A, an important molecule in repair mechanisms, and CDKN1A. However, the induction of apoptosis by DeMC was delayed and regulated by the decrease of antiapoptotic mRNA BCL.XL and subsequent activation of caspase 9 and caspase 3/7. DiMC treatment increased the expression of CYP1A2, CYP2C19, and CYP3A4 and exhibited higher cytotoxicity compared with other compounds. It induced apoptosis by increasing mRNA expression of BBC3, MYC, and CASP7 and activation of caspase 9 and caspase 3/7. These data revealed that different gene regulation processes are involved in cell death induced by Cur, DeMC, and DiMC. All three can be considered as promising chemotherapy candidates, with DiMC showing the greatest potency.

Random changepoint segmented regression with smooth transition.

We consider random changepoint segmented regression models to analyse data from a study conducted to verify whether treatment with stem cells may delay the onset of a symptom of amyotrophic lateral sclerosis in genetically modified mice. The proposed models capture the biological aspects of the data, accommodating a smooth transition between the periods with and without symptoms. An additional changepoint is considered to avoid negative predicted responses. Given the nonlinear nature of the model, we propose an algorithm to estimate the fixed parameters and to predict the random effects by fitting linear mixed models iteratively via standard software. We compare the variances obtained in the final step with bootstrapped and robust ones.

An epilepsy-associated ACTL6B variant captures neuronal hyperexcitability in a human induced pluripotent stem cell model.

ACTL6B is a component of the neuronal BRG1/brm-associated factor (nBAF) complex, which is required for chromatin remodeling in postmitotic neurons. We recently reported biallelic pathogenic variants in ACTL6B in patients diagnosed with early infantile epileptic encephalopathy, subtype 76 (EIEE-76), presenting with severe, global developmental delay, epileptic encephalopathy, cerebral atrophy, and abnormal central nervous system myelination. However, the pathophysiological mechanisms underlying their phenotype is unknown. Here, we investigate the molecular pathogenesis of ACTL6B p.(Val421_Cys425del) using in silico 3D protein modeling predictions and patient-specific induced pluripotent stem cell-derived neurons. We found neurons derived from EIEE-76 patients showed impaired accumulation of ACTL6B compared to unaffected relatives, caused by reduced protein stability. Furthermore, EIEE-76 patient-derived neurons had dysregulated nBAF target gene expression, including genes important for neuronal development and disease. Multielectrode array system analysis unveiled elevated electrophysiological activity of EIEE-76 patients-derived neurons, consistent with the patient phenotype. Taken together, our findings validate a new model for EIEE-76 and reveal how reduced ACTL6B expression affects neuronal function.

mRNAs biomarker related to the control of proliferation and cell death in HepG2/C3A spheroid and monolayer cultures treated with piperlongumine

Background: Cell culture (spheroid and 2D monolayer cultures) is an essential tool in drug discovery. Piperlongumine (PLN), a naturally occurring alkaloid present in the long pepper (Piper longum), has been implicated in the regulation of GSTP1 activity. In vitro treatment of cancer cells with PLN increases ROS (reactive oxygen species) levels and induces cell death, but its molecular mode of action has not been entirely elucidated. Methods: In this study, we correlated the antiproliferative effects (2D and 3D cultures) of PLN (CAS 20069­09-4, Sigma-Aldrich) with morphological and molecular analyses in HepG2/C3A cell line. We performed assays for cytotoxicity (MTT), comet assays for genotoxicity, induction of apoptosis, analysis of the cell cycle phase, and analysis of the membrane integrity by flow cytometry. Relative expression of mRNA of genes related to proliferation, apoptosis, cell cycle control, metabolism of xenobiotics, and reticulum endoplasmic stress. Results: PLN reduced the cell proliferation by the cell cycle arrest in G2/M. Changes in the mRNA expression for CDKN1A (4.9x) and CCNA2 (0.5x) of cell cycle control genes were observed. Cell death occurred due to apoptosis, which may have been induced by increased expression of proapoptotic mRNAs (BAK1, 3.1x; BBC3, 2.4x), and by an increase in 9 and 3/7 active caspases. PLN induced cellular injury by ROS generation and DNA damage. DNA damage induced MDM2 signaling (3.0x) associated with the appearance of the monastral spindle in mitosis. Genes associated with ROS degradation also showed increased mRNA expression (GSR, 2.0x; SOD1, 2.1x). PLN induce endoplasmic reticulum stress with the increase in the mRNA expression of ERN1 (4.5x) and HSPA14 (2.2x). The xenobiotic metabolism showed increased mRNA expression for CYP1A2 (2.2x) and CYP3A4 (3.4x). In addition to 2D culture, PLN treatment also inhibited the growth of 3D culture (spheroids). Conclusion: Thus, the findings of our study show that several gene expression biomarkers (mRNAs) and monastral spindle formation indicated the many pathways of damage induced by PLN treatment that contributes to its antiproliferative effects

Up and down-regulation of mRNA in the cytotoxicity and genotoxicity of Plumbagin in HepG2/C3A.

Studies that evaluated the mechanisms of action of Plumbagin (PLB) and its toxicity may contribute to future therapeutic applications of this compound. We investigate biomarker important in the mechanisms of action correlate the expression of mRNA with the cytotoxic and genotoxic effects of PLB on HepG2/C3A. In the analysis of cytotoxicity, PLB decreased cell viability and membrane integrity at concentrations ≥ 15µM. Xenobiotic-metabolizing system showed strong mRNA induction of CYP1A1, CYP1A2, and CYP3A4, suggesting extensive metabolization. PLB induced apoptosis and an increase in the mRNA expression of genes BBC3, CASP3, and CASP8. At a concentration of 15µM, there was a reduction in the expression of PARP1 mRNA and an increase in the expression of BECN1 mRNA, suggesting that PLB may also induce cell death by autophagy. PLB induced an arrest at the G2/M phase due to DNA damage, as observed in the comet assay. This damage is associated with the increased mRNA expression of genes p21, GADD45A, and H2AFX and with changes in the expression of proteins H2AX, p21, p53, Chk1, and Chk2. These results allow a better understanding of the cellular action of PLB and of its toxicity, thereby contributing to the development of PLB-based drugs, with markers of mRNA expression possibly playing a role as indicators for monitoring toxicity in human cells.

Mitotic spindle defects and DNA damage induced by dimethoxycurcumin lead to an intrinsic apoptosis pathway in HepG2/C3A cells.

Dimethoxycurcumin (DiMC), a synthetic analog of curcumin, was shown to have antiproliferative activity in human tumor cell lines. Therefore, we investigated its cytotoxic, antiproliferative, genotoxic, and apoptotic effect and correlated these evaluations with the expression of transcripts and proteins in the human hepatocellular carcinoma cell line (HepG2/C3A). Treatment with DiMC resulted in increased CYP2E1, CYP2C19 and CYP1A2 transcripts levels and was cytotoxic (≥10 µM). DiMC caused mitotic arrest by inducing monopolar spindle formation and was genotoxic increasing expression of the CDKN1A, GADD45A and PARP1 gene, key effectors in the cell cycle arrest and DNA repair pathways, respectively. This genotoxicity was caused by generation of reactive oxygen species and reduction of antioxidant proteins levels. Furthermore, we observed a decrease in important proteins involved in DNA repair. In addition to the observed apoptotic morphology and the presence of annexin labeling, we observed increased expression of BAK1 and CASP7 genes and caspase 3/7 protein activity, showing that these effects caused apoptosis through the intrinsic pathway in HepG2/C3A cells. Our results indicate that DiMC modulates important molecular targets leading to cell death even in metabolic competent cells models has considerable potential in anticancer therapy.

Molecular pathways related to the control of proliferation and cell death in 786-O cells treated with plumbagin.

Plumbagin (PLB) is a phytochemical being used for centuries in traditional medicines. Recently, its capacity to inhibit the development of human tumors has been observed, through the induction of apoptosis, cell cycle arrest, and inhibition of angiogenesis and metastasis. Here we evaluated the mechanism of action of PLB in the kidney adenocarcinoma 786-O cell line, which are metabolizing cells important for toxicology studies. After the treatment with PLB, we observed increased apoptosis and cell cycle arrest in S and G2/M phases, starting at 5 µM. In addition, PLB was cytotoxic, genotoxic and induced loss of cell membrane integrity. Regarding gene expression, treatment with 7.5 µM PLB reduced the amount of MTOR, BCL2 and ATM transcripts, and increased CDKN1A (p21) transcripts. Phosphorylation levels of yH2AX was increased and MDM2 protein level was reduced following the treatment with PLB, demonstrating its genotoxic effect. Our results suggest that PLB acts in molecular pathways related to the control of proliferation and cell death in 786-O cells.

The Role of Pericytes in Amyotrophic Lateral Sclerosis.

In amyotrophic lateral sclerosis (ALS), motor neurons die selectively. Therefore, initial symptoms that include fasciculation, spasticity, muscle atrophy, and weakness emerge following axons retraction and consequent muscles' denervation. Patients lose the ability to talk and swallow and rely on parenteral nutrition and assisted ventilation to survive. The degeneration caused by ALS is progressive and irreversible. In addition to the autonomous mechanism of neuronal cell death, non-autonomous mechanisms have been proved to be toxic for motor neurons, such as the activation of astrocytes and microglia. Among the cells being studied to unveil these toxic mechanisms are pericytes, cells that help keep the integrity of the blood-brain barrier and blood-spinal cord barrier. In this chapter, we aim to discuss the role of pericytes in ALS.

Response of HepG2/C3A cells supplemented with sodium selenite to hydrogen peroxide-induced oxidative stress.

Oxidative stress (OS) is involved in the onset of various pathological processes, and sodium selenite (Na2SeO3) is known to have antioxidant activity. This study evaluated the cellular response of human HepG2/C3A cells supplemented with Na2SeO3 when exposed to hydrogen peroxide (H2O2)-induced OS. We analyzed cytotoxicity, cell proliferation, and genotoxicity in comparison with molecular data of mRNA and protein expression. The MTT and comet assays revealed that Na2SeO3 conferred cytoprotective and anti-genotoxic effects. In contrast, RTCA (Real-Time Cell Analysis) and flow cytometry analysis revealed that Na2SeO3 did not inhibit H2O2-induced anti-proliferative effects or cell cycle arrest (G2/M). Cells exposed simultaneously to Na2SeO3 and H2O2 showed overexpression of GPX1 mRNA, indicating that Na2SeO3 influenced the cellular antioxidant system. Furthermore, downregulation of CAT mRNA and SOD1 and PRX2 proteins induced by H2O2, was minimal after the Na2SeO3+H2O2 treatment. Although normalization of CCN2B mRNA expression by Na2SeO3 was observed after the Na2SeO3+H2O2 treatment, this was not observed for other genes such as CDKN1A, CDKN1C, and CDKN2B, which are related to cell cycle control, nor for GADD45A, which is involved in the cellular response to DNA damage. Furthermore, both CDKN1B and CDKN1C expression were downregulated in HepG2/C3A cells treated with Na2SeO3 only. Our results indicate that cellular response to Na2SeO3 involved the modulation of the antioxidant system. Na2SeO3 was unable completely recover HepG2/C3A cells from H2O2-induced oxidative damage, as evidenced by analysis of cell proliferation kinetics, cell cycle assay, and expression of key genes involved in cell cycle progression and response to DNA damage.

Genistein Affects Expression of Cytochrome P450 (CYP450) Genes in Hepatocellular Carcinoma (HEPG2/C3A) Cell Line.

BACKGROUND: Genistein (5,7-Dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is the most abundant isoflavone in soybean, which has been associated with a lower risk of development of cancer and cardiovascular diseases. Of particular interest regarding cancer preventive properties of flavonoids is their interaction with cytochrome P450 enzymes (CYPs). However, contradictory data report the effect of genistein on expression of СYPs enzymes. OBJECTIVE: The aim of this study was to investigate the effects of genistein on cytochrome P450 (CYP) gene expression levels in human hepatocellular carcinoma (HepG2/C3A) and colon adenocarcinoma (HT29) cells. METHODS: Real-time RT-PCR was used to examine the expression of genes families involved in xenobiotic metabolism, such as CYP1 (CYP1A1, CYP1B1), CYP2 (CYP2E1, CYP2D6), CYP3 (CYP3A4); and of a family involved in the catabolism of the all-trans-retinoic acid (ATRA), CYP26 (CYP26A1, CYP26B1). RESULTS: RT-qPCR data analysis showed that after 12 h of exposure of HepG2/C3A cells to genistein (5 and 50 µM) there was an upregulation of CYP1A1 and CYP1B1 and downregulation of CYP2D6, CYP26A1 and CYP26B1 mRNA levels. There was no change in the mRNA levels of CYP P450 genes in HT29 cells. CONCLUSION: Our results suggest that treatment with genistein in non-toxic concentrations may impact the expression level of CYPs involved in the biotransformation of xenobiotics and drug metabolizing enzymes. Moreover, the downregulation of ATRA metabolism-related genes opens a new research path for the study of genistein as retinoic acid metabolism blocking agent for treating cancer and other pathologies.

Human Adipose-Derived CD146+ Stem Cells Increase Life Span of a Muscular Dystrophy Mouse Model More Efficiently than Mesenchymal Stromal Cells.

Duchenne muscular dystrophy is the most common and severe form of progressive muscular dystrophy. Previous results showed an increased survival in double knockout mice (dko) when treated with adipose-derived CD146+ cells. In this study, we analyzed the effect of CD146+ cells compared to mesenchymal stem/stromal cells (MSCs) derived from the same human adipose sample when injected in the dko mouse model without immunosuppression. Both CD146+ cells and MSCs increased the survival of treated mice when compared to vehicle-injected mice, with a more prominent effect of CD146+ cells than MSCs. Both CD146+ cells and MSCs suppressed peripheral blood mononuclear cell proliferation, indicating immunomodulatory properties. Co-culture experiments showed that MSCs have a more inflammatory profile expression, and angiogenesis assay showed that CD146+ cells can improve blood vessel formation. CD146+ cells can extend survival of muscular dystrophy mice more efficiently than MSCs, possibly due to immunomodulatory and angiogenic properties. Further investigations focusing on exogenous CD146+ cell role in vivo will improve cell therapy understanding and effectiveness.

Immunoglobulin therapy ameliorates the phenotype and increases lifespan in the severely affected dystrophin-utrophin double knockout mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, caused by mutations in the dystrophin gene, affecting 1:3500-5000 boys worldwide. The lack of dystrophin induces degeneration of muscle cells and elicits an immune response characterized by an intensive secretion of pro-inflammatory cytokines. Immunoglobulins modulate the inflammatory response through several mechanisms and have been widely used as an adjuvant therapy for autoimmune diseases. Here we evaluated the effect of immunoglobulin G (IG) injected intraperitoneally in a severely affected double knockout (dko) mouse model for Duchenne muscular dystrophy. The IG dko treated mice were compared regarding activity rates, survival and histopathology with a control untreated group. Additionally, dendritic cells and naïve lymphocytes from these two groups and WT mice were obtained to study in vitro the role of the immune system associated to DMD pathophysiology. We show that IG therapy significantly enhances activity rate and lifespan of dko mice. It diminishes muscle tissue inflammation by decreasing the expression of costimulatory molecules MHC, CD86 and CD40 and reducing Th1-related cytokines IFN-γ, IL-1ß and TNF-α release. IG therapy dampens the effector immune responses supporting the hypothesis according to which the immune response accelerates DMD progression. As IG therapy is already approved by FDA for treating autoimmune disorders, with less side-effects than currently used glucocorticoids, our results may open a new therapeutic option aiming to improve life quality and lifespan of DMD patients.

Deepening a Simple Question: Can MSCs Be Used to Treat Cancer?

In cancer, mesenchymal stem/stromal cells (MSCs) have been considered as vehicles for targeted delivery of drugs due to their inherent tropism toward primary and metastatic tumors. However, it is still unclear whether MSCs could be therapeutically explored without significant harm, since a great amound of evidence indicates that MSCs are able to exert both tumor-suppressive and pro-oncogenic effects. Here, we discuss how MSCs might adopt a pro- or anti-inflammatory profile in response to changes within the tumor microenvironment and how these features may lead to opposite outcomes in tumor development. Additionally, we address how differences in experimental design might impact interpretation and consistency of the current literature in this specific field. Finally, we point-out critical issues to be addressed at a pre-clinical stage, regarding safety and therapeutic effectiveness of MSCs application in cancer treatment.

Pericytes Extend Survival of ALS SOD1 Mice and Induce the Expression of Antioxidant Enzymes in the Murine Model and in IPSCs Derived Neuronal Cells from an ALS Patient.

Amyotrophic Lateral Sclerosis (ALS) is one of the most common adult-onset motor neuron disease causing a progressive, rapid and irreversible degeneration of motor neurons in the cortex, brain stem and spinal cord. No effective treatment is available and cell therapy clinical trials are currently being tested in ALS affected patients. It is well known that in ALS patients, approximately 50% of pericytes from the spinal cord barrier are lost. In the central nervous system, pericytes act in the formation and maintenance of the blood-brain barrier, a natural defense that slows the progression of symptoms in neurodegenerative diseases. Here we evaluated, for the first time, the therapeutic effect of human pericytes in vivo in SOD1 mice and in vitro in motor neurons and other neuronal cells derived from one ALS patient. Pericytes and mesenchymal stromal cells (MSCs) were derived from the same adipose tissue sample and were administered to SOD1 mice intraperitoneally. The effect of the two treatments was compared. Treatment with pericytes extended significantly animals survival in SOD1 males, but not in females that usually have a milder phenotype with higher survival rates. No significant differences were observed in the survival of mice treated with MSCs. Gene expression analysis in brain and spinal cord of end-stage animals showed that treatment with pericytes can stimulate the host antioxidant system. Additionally, pericytes induced the expression of SOD1 and CAT in motor neurons and other neuronal cells derived from one ALS patient carrying a mutation in FUS. Overall, treatment with pericytes was more effective than treatment with MSCs. Our results encourage further investigations and suggest that pericytes may be a good option for ALS treatment in the future. Graphical Abstract ᅟ.