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

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

Majority of cerebrospinal fluid-contacting neurons in the spinal cord of C57Bl/6N mice is present in ectopic position unlike in other studied experimental mice strains and mammalian species.

Cerebrospinal fluid contacting neurons (CSF-cNs) represent a specific class of neurons located in close vicinity of brain ventricles and central canal. In contrast with knowledge gained from other vertebrate species, we found that vast majority of CSF-cNs in the spinal cord of C57Bl/6N mice is located in ectopic distal ventral position. However, we found that small number of ectopic CSF-cNs is present also in spinal cord of other investigated experimental mice strains (C57Bl/6J, Balb/C) and mammalian species (Wistar rats, New Zealand White rabbits). Similarly, as the proximal populations, ectopic CSF-cNs retain PKD2L1-immunoreactivity and synaptic contacts with other neurons. On the other side, they show rather multipolar morphology lacking thick dendrite contacting central canal lumen. Ectopic CSF-cNs in the spinal cord of C57Bl/6N mice emerge during whole period devoted to production of CSF-cNs and reach their ventral destinations during first postnatal weeks. In order to identify major gene, whose impairment could trigger translocation of CSF-cNs outside the central canal area, we took advantage of close consanguinity of C57Bl/6J substrain with normal CSF-cN distribution and C57Bl/6N substrain with majority of CSF-cNs in ectopic position. Employing in silico analyses, we ranked polymorphisms in C57Bl/6N substrain and selected genes Crb1, Cyfip2, Adamts12, Plk1, and Herpud2 as the most probable candidates, whose product dysfunction might be responsible for the ectopic distribution of CSF-cNs. Furthermore, segregation analysis of F2 progeny of parental C57Bl/6N and Balb/C mice revealed that polymorphic loci of Crb1 and Cyfip2 underlie the ectopic position of CSF-cNs in the spinal cord of C57Bl/6N mice.

Adipose-Derived Stem Cells from Fat Tissue of Breast Cancer Microenvironment Present Altered Adipogenic Differentiation Capabilities.

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.

Bioink Composition and Printing Parameters for 3D Modeling Neural Tissue.

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.

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

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

Neuroprotection, Recovery of Function and Endogenous Neurogenesis in Traumatic Spinal Cord Injury Following Transplantation of Activated Adipose Tissue.

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.

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

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

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

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

Transcriptional role of androgen receptor in the expression of long non-coding RNA Sox2OT in neurogenesis.

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.

Recovery from experimental parkinsonism by intrastriatal application of erythropoietin or EPO-releasing neural precursors.

An extensive literature has shown a powerful neuroprotective action of Erythropoietin (EPO) both in vivo and in vitro. This study shows that EPO, whether ectopically administered or released by neural precursors, does reverse MPTP-induced parkinsonism in mice. Unilateral stereotaxic injection of 2.5 × 105 erythropoietin-releasing neural precursor cells (Er-NPCs) rescued degenerating striatal dopaminergic neurons and promoted behavioral recovery as shown by three independent behavioral tests. These effects were replicated through direct intrastriatal administration of recombinant human EPO. At the end of the observational period, most of the transplanted Er-NPCs were vital and migrated via the striatum to reach Substantia Nigra. The restorative effects appear to be mediated by EPO since co-injection of anti-EPO or anti-EPOR antibodies antagonized the positive outcomes. Furthermore, this report supports the neuroprotective action of EPO, which may also be achieved via administration of EPO-releasing cells such as Er-NPCs.

Grafted Neural Precursors Integrate Into Mouse Striatum, Differentiate and Promote Recovery of Function Through Release of Erythropoietin in MPTP-Treated Mice.

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 × 105) 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.

The Ribonucleic Complex HuR-MALAT1 Represses CD133 Expression and Suppresses Epithelial-Mesenchymal Transition in Breast Cancer.

Epithelial-to-mesenchymal transition (EMT) is a core process underlying cell movement during embryonic development and morphogenesis. Cancer cells hijack this developmental program to execute a multi-step cascade, leading to tumorigenesis and metastasis. CD133 (PROM1), a marker of cancer stem cells, has been shown to facilitate EMT in various cancers, but the regulatory networks controlling CD133 gene expression and function in cancer remain incompletely delineated. In this study, we show that a ribonucleoprotein complex including the long noncoding RNA MALAT1 and the RNA-binding protein HuR (ELAVL1) binds the CD133 promoter region to regulate its expression. In luminal nonmetastatic MCF-7 breast cancer cells, HuR silencing was sufficient to upregulate N-cadherin (CDH2) and CD133 along with a migratory and mesenchymal-like phenotype. Furthermore, we found that in the basal-like metastatic cell line MDA-MB-231 and primary triple-negative breast cancer tumor cells, the repressor complex was absent from the CD133-regulatory region, but was present in the MCF-7 and primary ER+ tumor cells. The absence of the complex from basal-like cells was attributed to diminished expression of MALAT1, which, when overexpressed, dampened CD133 levels. In conclusion, our findings suggest that the failure of a repressive complex to form or stabilize in breast cancer promotes CD133 upregulation and an EMT-like program, providing new mechanistic insights underlying the control of prometastatic processes. Cancer Res; 76(9); 2626-36. ©2016 AACR.

Human Antigen R Binding and Regulation of SOX2 mRNA in Human Mesenchymal Stem Cells.

Since 2005, sex determining region y-box 2 (SOX2) has drawn the attention of the scientific community for being one of the key transcription factors responsible for pluripotency induction in somatic stem cells. Our research investigated the turnover regulation of SOX2 mRNA in human adipose-derived stem cells, considered one of the most valuable sources of somatic stem cells in regenerative medicine. Mitoxantrone is a drug that acts on nucleic acids primarily used to treat certain types of cancer and was recently shown to ameliorate the outcome of autoimmune diseases such as multiple sclerosis. In addition, mitoxantrone has been shown to inhibit the binding of human antigen R (HuR) RNA-binding protein to tumor necrosis factor-α mRNA. Our results show that HuR binds to the 3'-untranslated region of SOX2 mRNA together with the RNA-induced silencing complex miR145. The HuR binding works by stabilizing the interaction between the 3'-untranslated region and the RNA-induced silencing complex. Cell exposure to mitoxantrone leads to HuR detachment and the subsequent prolongation of the SOX2 mRNA half-life. The prolonged SOX2 half-life allows improvement of the spheroid-forming capability of the adipose-derived stem cells. The silencing of HuR confirmed the above observations and illustrates how the RNA-binding protein HuR may be a required molecule for regulation of SOX2 mRNA decay.

Enhanced brain release of erythropoietin, cytokines and NO during carotid clamping.

Although effective and safe, carotid endarterectomy (CEA) implies a reduced blood flow to the brain and likely an ischemia/reperfusion event. The high rate of uneventful outcomes associated with CEA suggests the activation of brain endogenous protection mechanisms aimed at limiting the possible ischemia/reperfusion damage. This study aims at assessing whether CEA triggers protective mechanisms such as brain release of erythropoietin and nitric oxide. CEA was performed in 12 patients; blood samples were withdrawn simultaneously from the surgically exposed ipsilateral jugular and leg veins before, during (2 and 40 min) and after clamp removal (2 min). Plasma antioxidant capacity, carbonylated proteins, erythropoietin, nitrates and nitrites (NOx) were determined. No changes in intraoperative EEG, peripheral and transcranial blood oxygen saturation were detectable, and no patients showed any neurologic sign after the intervention. Antioxidant capacity and protein carbonylation in plasma were unaffected. Differently, erythropoietin, VEGF, TNF-α and NOx increased during clamping in the jugular blood (2 and 40 min), while no changes were observed in the peripheral circulation. These results show that blood erythropoietin, VEGF, TNF-α, and NOx increased in the brain during uncomplicated CEA. This may represent an endogenous self-activated neuroprotective mechanism aimed at the prevention of ischemia/reperfusion damage.

Anti-EGFR antibody reduces lung nodules by inhibition of EGFR-pathway in a model of lymphangioleiomyomatosis.

EGFR belongs to the HER/ErbB family of tyrosine kinase receptors and its activation in cancer cells has been linked with increased proliferation, angiogenesis, and metastasis. Lymphangioleiomyomatosis (LAM) is a rare, low-grade neoplasm that occurs sporadically or in association with tuberous sclerosis complex (TSC), a genetic, multisystem disorder characterized by hamartomas in several organs. From chylous of a LAM/TSC patient, we previously isolated smooth muscle-like LAM/TSC cells whose proliferation depends on EGF and monoclonal anti-EGFR antibodies reduced proliferation and caused cell death. We demonstrated that the dependency from EGF was caused by the absence of tuberin. To study the role of EGFR pathway in vivo, we developed a mouse model by administration of LAM/TSC cells to female nude mice. LAM/TSC cells caused pulmonary airspace enlargement and, after 30 weeks, nodule formation which express EGFR. Anti-EGFR antibody decreased the number and dimension of lung nodules likely for the inhibition of Erk and S6 signaling, reversed the pulmonary alterations, and reduced lymphatic and blood vessels. Moreover, in pulmonary nodules anti-EGFR antibody reduced the positivity to estrogen and progesterone receptors which enhance survival of LAM cells and Snail expression. These results suggest that the inhibition of EGFR signalling has a potential in treatment of LAM/TSC lung alterations.

Exogenous adult postmortem neural precursors attenuate secondary degeneration and promote myelin sparing and functional recovery following experimental spinal cord injury.

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 ß-tubulin III-positive transplanted cells that are also mostly labeled by anti-ChAT antibodies.

Characteristics and Properties of Mesenchymal Stem Cells Derived From Microfragmented Adipose Tissue.

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 ß-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.

Neural stem cell transplantation in experimental contusive model of spinal cord injury.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

TSC2 epigenetic defect in primary LAM cells. Evidence of an anchorage-independent survival.

Tuberous sclerosis complex (TSC) is caused by mutations in TSC1 or TSC2 genes. Lymphangioleiomyomatosis (LAM) can be sporadic or associated with TSC and is characterized by widespread pulmonary proliferation of abnormal α-smooth muscle (ASM)-like cells. We investigated the features of ASM cells isolated from chylous thorax of a patient affected by LAM associated with TSC, named LAM/TSC cells, bearing a germline TSC2 mutation and an epigenetic defect causing the absence of tuberin. Proliferation of LAM/TSC cells is epidermal growth factor (EGF)-dependent and blockade of EGF receptor causes cell death as we previously showed in cells lacking tuberin. LAM/TSC cells spontaneously detach probably for the inactivation of the focal adhesion kinase (FAK)/Akt/mTOR pathway and display the ability to survive independently from adhesion. Non-adherent LAM/TSC cells show an extremely low proliferation rate consistent with tumour stem-cell characteristics. Moreover, LAM/TSC cells bear characteristics of stemness and secrete high amount of interleukin (IL)-6 and IL-8. Anti-EGF receptor antibodies and rapamycin affect proliferation and viability of non-adherent cells. In conclusion, the understanding of LAM/TSC cell features is important in the assessment of cell invasiveness in LAM and TSC and should provide a useful model to test therapeutic approaches aimed at controlling their migratory ability.

Third trimester NG2-positive amniotic fluid cells are effective in improving repair in spinal cord injury.

Spinal cord injury presents a significant therapeutic challenge since the treatments available are mostly vain. The use of stem cells to treat this condition represents a promising new therapeutic strategy; therefore, a variety of stem cell treatments have been recently examined in animal models of CNS trauma. In this work, we analyzed the effects of third trimester amniotic fluid cells in a mouse model of spinal cord injury. Among the different cultures used for transplantation, some were able to induce a significant improvement in motor recovery (cultures #3.5, #3.6 and #7.30), evaluated by means of open field free locomotion. All effective cell cultures expressed the surface marker nerve/glial antigen 2, ortholog of the human chondroitin sulfate proteoglycan 4, which is present on several types of immature progenitor cells. The improved motor functional recovery was correlated with higher myelin preservation in the ventral horn white matter and an increased vascularization in the peri-lesion area. Real-Time PCR analysis showed higher expression levels of vascular endothelial growth factor and hypoxia-inducible factor-1α mRNA two days after cells transplantation compared to PBS-treated animals, indicating that an angiogenic pathway might have been activated by these cells, possibly through the production of hepatocyte growth factor. This cytokine appears to be produced mostly in filtering organs, such as the lung, of the transplanted animals and is likely released in the blood suggesting an endocrine role of hepatocyte growth factor in targeting the injury site.