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.

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.

Human adipose-derived mesenchymal stem cells as a new model of spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) or Kennedy's disease is an X-linked CAG/polyglutamine expansion motoneuron disease, in which an elongated polyglutamine tract (polyQ) in the N-terminal androgen receptor (ARpolyQ) confers toxicity to this protein. Typical markers of SBMA disease are ARpolyQ intranuclear inclusions. These are generated after the ARpolyQ binds to its endogenous ligands, which promotes AR release from chaperones, activation and nuclear translocation, but also cell toxicity. The SBMA mouse models developed so far, and used in preclinical studies, all contain an expanded CAG repeat significantly longer than that of SBMA patients. Here, we propose the use of SBMA patients adipose-derived mesenchymal stem cells (MSCs) as a new human in vitro model to study ARpolyQ toxicity. These cells have the advantage to express only ARpolyQ, and not the wild type AR allele. Therefore, we isolated and characterized adipose-derived MSCs from three SBMA patients (ADSC from Kennedy's patients, ADSCK) and three control volunteers (ADSCs). We found that both ADSCs and ADSCKs express mesenchymal antigens, even if only ADSCs can differentiate into the three typical cell lineages (adipocytes, chondrocytes and osteocytes), whereas ADSCKs, from SBMA patients, showed a lower growth potential and differentiated only into adipocyte. Moreover, analysing AR expression on our mesenchymal cultures we found lower levels in all ADSCKs than ADSCs, possibly related to negative pressures exerted by toxic ARpolyQ in ADSCKs. In addition, with proteasome inhibition the ARpolyQ levels increased specifically in ADSCKs, inducing the formation of HSP70 and ubiquitin positive nuclear ARpolyQ inclusions. Considering all of this evidence, SBMA patients adipose-derived MSCs cultures should be considered an innovative in vitro human model to understand the molecular mechanisms of ARpolyQ toxicity and to test novel therapeutic approaches in SBMA.

Experimental models of brain ischemia: a review of techniques, magnetic resonance imaging, and investigational cell-based therapies.

Stroke continues to be a significant cause of death and disability worldwide. Although major advances have been made in the past decades in prevention, treatment, and rehabilitation, enormous challenges remain in the way of translating new therapeutic approaches from bench to bedside. Thrombolysis, while routinely used for ischemic stroke, is only a viable option within a narrow time window. Recently, progress in stem cell biology has opened up avenues to therapeutic strategies aimed at supporting and replacing neural cells in infarcted areas. Realistic experimental animal models are crucial to understand the mechanisms of neuronal survival following ischemic brain injury and to develop therapeutic interventions. Current studies on experimental stroke therapies evaluate the efficiency of neuroprotective agents and cell-based approaches using primarily rodent models of permanent or transient focal cerebral ischemia. In parallel, advancements in imaging techniques permit better mapping of the spatial-temporal evolution of the lesioned cortex and its functional responses. This review provides a condensed conceptual review of the state of the art of this field, from models and magnetic resonance imaging techniques through to stem cell therapies.

Circulating T regulatory cells migration and phenotype in glioblastoma patients: an in vitro study.

Glioblastoma multiforme (GBM) is the most aggressive primary human brain tumor. The relatively high amount of T regulatory lymphocytes present in the tumor, contributes to the establishment of an immunosuppressive microenvironment. Samples of peripheral blood were collected from GBM patients and healthy controls and a purified population of Treg (CD4(+)/CD25(bright)) was isolated using flow cytometric cell sorting. Treg migrating capacities toward human glioma cell line conditioned medium were evaluated through an in vitro migration test. Our data show that supernatants collected from GBM cell lines were more attractant to Treg when compared to complete standard medium. The addition of an anti-CCL2 antibody to conditioned medium decreased conditioned medium-depending Treg migration, suggesting that CCL2 (also known as Monocyte Chemoattractant Protein, MCP-1) is implicated in the process. The number of circulating CD4(+)/µL or Treg/µL was similar in GBM patients and controls. Specific Treg markers (FOXP3; CD127; Helios; GITR; CTLA4; CD95; CCR2, CCR4; CCR7) were screened in peripheral blood and no differences could be detected between the two populations. These data confirm that the tumor microenvironment is attractive to Treg, which tend to migrate toward the tumor region changing the immunological response. Though we provide evidence that CCL2 is implicated in Treg migration, other factors are needed as well to provide such effect.

Transforming growth factor-beta1 induces microvascular abnormalities through a down-modulation of neural cell adhesion molecule in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a very angiogenic and malignant cancer. Conventional chemotherapy is poorly effective because of the abnormal structural organization of HCC-infiltrating vessels. In previous work, we demonstrated that HCC angiogenesis is driven by transforming growth factor beta-1(TGF-ß1)/CD105 axis, stimulating liver-derived microvascular endothelial cells (Ld-MECs) migration. As TGF-ß1 also affects mural cells (MCs) recruitment and maturation, we asked whether it may contribute to HCC-induced vascular abnormalities. HCC and adjacent non-neoplastic liver (nNL) biopsies obtained from 12 patients were analyzed by immunohistochemistry for angiogenic markers CD105, TGF-ß1, CD44 and vascular endothelial growth factor-a (VEGFa) and for MC markers NG2, α-smooth muscle actin (αSMA) and neural cell adhesion molecule (NCAM). The same markers were also investigated by immunocytochemistry on cultured HCC-derived stromal cells (HCC-StCs) and nNL-derived StCs (nNL-StCs) isolated from the same liver biopsies. Angiogenic factors released by StCs were analyzed by ELISA and the interaction between StCs and Ld-MECs by adhesion assay. Compared with nNL, HCC biopsies showed increased angiogenic markers and αSMA that was localized in vessels. By contrast, NG2 and NCAM were substantially localized in tumor cells but absent in vessels and stroma. Cultured HCC-StCs showed less expression of NG2, αSMA and NCAM. They also demonstrated a lower capacity to release angiogenic factors and adhered on Ld-MECs. HCC-StCs and nNL-StCs treated with TGF-ß1 or with of HepG2 (a human hepatoma cell line) derived conditioned medium (CM), down-modulated NCAM expression, whereas anti-NCAM antibodies significantly reduced the adhesion of StCs to Ld-MECs. By further blocking TGF-ß1 with anti-TGF-ß1 antibodies or with Ly-364947 (a specific inhibitor TGF-ß1-receptor) adhesion to Ld-MECs and NCAM expression respectively was partially restored. TGF-ß1 contributes to HCC-induced vascular alterations by affecting the interaction between HCC-StCs and Ld-MECs through a down-modulation of NCAM expression.

Expression of neural and neurotrophic markers in nucleus pulposus cells isolated from degenerated intervertebral disc.

Intervertebral disc (IVD) degeneration is a common disorder of the lower spine. Since it is caused by loss of cellularity, there is interest in the comprehension of the cellular phenotypes. This study aimed to verify if stem cells isolated from nucleus pulposus of intervertebral discs (NPs-IVD), which may express neurogenic properties, may be implicated in IVD disease. NPs-IVD isolated from 14 human pathological discs were cultured under mesenchymal and neural differentiation. An induction of the neural markers GFAP, NF, MAP2, O4, and a decrement of the expression of the immature neural markers ß-tubulin III, Nestin, NG2, occurred within the neural differentiation. The expression of TrkA and p75NGFR, the receptors of NGF, was not correlated with neural induction; in contrast, TrkB, the BDNF receptor, increased and was co-expressed with acid sensing ion channel 3 (ASIC3). In the same condition, neuroinflammatory markers were over-expressed. We confirm our hypothesis that stem cells within IVD degeneration acquire neurogenic phenotype, causing the induction of markers related to inflammatory condition. These cells could promote the enrolment of neurotrophines in adaptation to the acidic microenvironment in degenerative conditions. These data could improve our knowledge about IVD cellularity and eventually lead to the development of pharmacological therapies.

Decrease in circulating endothelial progenitor cells in treated glioma patients.

High-grade gliomas are highly vascularized tumors, in which the amount of new blood vessels is closely related with the degree of malignancy. The role of endothelial progenitor cells (EPCs) in the neoangiogenesis of gliomas and the effects of post-surgical therapies (i.e., radiotherapy (RT) and chemotherapy) have not yet been fully elucidated. The aim of the present study was to evaluate the effect of surgery and post-surgical treatment on the levels of circulating EPCs in glioma patients and their correlation with vascular endothelial growth factor (VEGF). In this study, we assessed by flow cytometry the number of EPCs in the peripheral blood of 78 high-grade glioma patients (both untreated and treated with RT and chemotherapy) and 34 age- and sex-matched healthy controls. EPCs were markedly decreased in all treated glioma patients as compared to untreated ones. VEGF levels were significantly higher in patients as compared to controls, and surgery, but not chemotherapy, significantly decreased VEGF concentrations. We found no relationship between VEGF plasma levels and EPCs. In conclusion, the reliability of EPCs as a biomarker for monitoring angiogenesis in glioma patients needs further studies of correlations of this parameter with other markers of tumor-related vasculature.

Increased migration of a human glioma cell line after in vitro CyberKnife irradiation.

A human glioblastoma multiforme cell line (U87) and its derived-spheroids were irradiated either using a conventional irradiation (CIR) or a CK-like irradiation (IIR) in which the 8 Gy was delivered intermittently over a period of 40 minutes. The ability of glioma cells to migrate into a matrigel matrix was evaluated on days 1-8 from irradiation. Irradiation with CK-driven IIR significantly increased the invasion potential of U87 cells in a matrigel-based assay. In contrast to CIR, IIR was associated with increased levels of TGF-ß at four days (Real time PCR), ß1-integrin at 4-5 days (real-time PCR and western blot) and no elevation in phosphorylated AKT at days 4 and 5 (western blot). Our data suggests that glioma cell invasion as well as elevations of TGF-ß and ß1-integrin are associated with IIR and not CIR.

In vitro effects of Cyberknife-driven intermittent irradiation on glioblastoma cell lines.

Radiosurgery is used increasingly upon recurrence of high-grade gliomas to deliver a high dose of focused radiation to a defined target. The purpose of our study was to compare intermittent irradiation (IIR) by using a CyberKnife (CK) with continuous irradiation (CIR) by using a conventional linear accelerator (LINAC). A significant decrease in surviving fraction was observed after IIR irradiation compared with after CIR at a dose of 8 Gy. Three hours after irradiation, most of the DNA damage was repaired in U87. Slightly higher basal levels of Ku70/80 mRNA were found in U87 compared with A172, while radiation treatment induced only minor regulation of Ku70/80 and Rad51 transcription in either cell lines. IIR treatment using CK significantly decreased the survival in U87 and A172 compared with CIR. Although the two cell lines differed in DNA repair capability, the role of Ku70/80 and Rad51 in the cell line radiosensitivity seemed marginal.

Expression of the new CXCL12 receptor, CXCR7, in gliomas.

Gliomas are very invasive brain tumors with poor prognosis and therefore any attempt to limit tumor cell dissemination in the brain is expected to improve glioma treatment. The recent deorphanization of CXCR7 as additional receptor for CXCL12 and CXCL11 has raised key issues on its interaction with the CXCL12/CXCR4 axis as a mechanism to modulate glioma cell migration. In this work we investigated protein and mRNA expression of the two chemokines CXCL12 and CXCL11, together with their receptors CXCR4 and CXCR7 in human glioma specimens and cell lines by immunohistochemistry, flow cytometry and quantitative real-time PCR. The main purpose of this study was to find out whether and at what extent CXCR4 and CXCR7 are differentially expressed in glioma cells. In human glioma specimens the levels of CXCL11 and CXCR4 mRNA were significantly higher in glioblastomas compared to non-tumor controls or low grade gliomas, whilst no difference was found for CXCL12 and CXCR7 mRNA expression. In cell lines, flow cytometry and immunocytochemical experiments showed CXCR4 was mainly expressed irrespective of its membrane or intracellular localization. In contrast, a predominant intracellular localization together with a negligible membrane expression of CXCR7 was found in all cells examined. In in vitro experiments CXCR4 and CXCR7 antagonists and the silencing of CXCR4 showed complete inhibition of glioma proliferation. Our findings, in agreement with previous data, suggest that in human glioma cells the prevalent intracellular localization of CXCR7 might modulate the functionality of CXCL11/12 either acting as a scavenger for these chemokines or interfering with the signaling pathways activated by the stimulation of CXCR4.

CXCL12, CXCR4 and CXCR7 expression in brain metastases.

Brain metastases occur in about 25% of patients who die of cancer. The most common sources of brain metastases in adults are lung, breast, kidney, colorectal cancer and melanoma. The chemokine/receptor system CXCL12/CXCR4 plays a key role in multiple biological functions; among these, homing of neoplastic cells from the primary site to the target and metastasis progression. Recently, an alternative CXCL12 receptor CXCR7 has been discovered. The aim of our study was to investigate the expression of CXCL12 and its receptors CXCR4 and CXCR7 by immunohistochemistry in 56 patients with metastatic brain disease from different non-CNS primary tumors and evaluate their prognostic relevance as well as that of other patient/treatment-related features on patient survival. CXCL12 showed an expression in tumor cells and in tumor vessels; CXCR7 was expressed by tumor and endothelial cells (both within the tumor and in the adjacent brain tissue), while CXCR4 showed a positivity in all samples with a nuclear pattern. Among the investigated immunohistochemical parameters, only CXCL12 expression in tumor endothelial cells showed a statistically significant correlation with shorter survival (p = 0.04 log-rank), perhaps identifying more aggressive tumors. Thus, this is the first study evaluating at the same time the expression of CXCL12 and its two receptors in a cohort of brain metastases.