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PDF file - 213K, Gene signatures distinguishing the different MB CSC populations segregate distinct human MB molecular subgroups, with MB1_EXCLUSIVE gene signature distinguishing the WNT molecular subgroup

PDF file - 183K, Long term-cultured hindbrain- and forebrain-derived NSCs are Sonic Hedgehog (Shh) pathway-independent, as cyclopamine treatment negatively affects the survival/proliferation of short-term cultured but not of long term-cultured NSCs

PDF file - 155K, Enforced expression of Ebf3 in CB WM and SVZ NSCs results in premature neuronal differentiation, under proliferative and differentiative conditions

PDF file - 157K, MB CSC lines are Sonic Hedgehog (Shh) pathway-independent, as their survival/proliferation is not affected by cyclopamine treatment and Smo silencing

PDF file - 104K, Details on experimental and bioinformatics procedures.

PDF file - 134K, Table S1 A. Lineage-specific differentiation of region-specific clonally derived NSCs is summarized as frequency of neurons, astrocytes and oligodendrocytes. B. Electrophysiological characteristics of neurons derived from the differentiation of region-specific NSCs. C. Analysis of the engraftment of region-specific clonally derived NSCs after transplantation in the early postnatal mouse cerebellum. Table S2 - Statistical analysis of GSEA for gene signatures generated by DEGs up regulated in hindbrain (CB/IVv) or forebrain (SVZ) NSCs. Table S3 - Lineage-specific differentiation of clonally derived MB CSCs is summarized as frequency of neuron-, astrocyte- and oligodendrocyte-like cells. Table S4 - Statistical analysis of GSEA for gene signatures generated by DEGs up regulated in tumorigenic Ptch+/- p53-/- MB CSCs vs. hindbrain NSCs (MB1_EXCLUSIVE), in non-tumorigenic Ptch+/- p53+/+ MB CSCs vs. hindbrain NSCs (MB2_EXCLUSIVE), and commonly expressed in both tumorigenic and non-tumorigenic CSCs vs. hindbrain NSCs (MB1_MB2 COMMON). Table S5 - Expression of EBF proteins in a collection of 33 human MB specimens as determined by immunohistochemistry.

PDF file - 130K, EBF3 expression can be retrieved in silico in human MBs by exploiting publicly available human data sets

PDF file - 132K, The gene signature containing the 297 genes differentially expressed between hindbrain- vs. forebrain-derived NSCs distinguishes the SHH molecular subgroup of human MBs

Cancer stem–like cells (CSC) could be a novel target for cancer therapy, including dendritic cell (DC) immunotherapy. To address this, we developed experiments aimed at DC targeting of neurospheres (NS) from GL261 glioma cells because neurospheres can be enriched in CSC. We obtained murine neurospheres by growing GL261 cells in epidermal growth factor/basic fibroblast growth factor without serum. GL261-NS recapitulated important features of glioblastoma CSC and expressed higher levels of radial glia stem cell markers than GL261 cells growing under standard conditions (GL261 adherent cells, GL261-AC), as assessed by DNA microarray and real-time PCR. GL261-NS brain gliomas were highly infiltrating and more rapidly lethal than GL261-AC, as evidenced by survival analysis (P < 0.0001), magnetic resonance imaging and histology. DC from the bone marrow of syngeneic mice were then used for immunotherapy of GL261-NS and GL261-AC tumors. Strikingly, DC loaded with GL261-NS (DC-NS) cured 80% and 60% of GL261-AC and GL261-NS tumors, respectively (P < 0.0001), whereas DC-AC cured only 50% of GL261-AC tumors (P = 0.0022) and none of the GL261-NS tumors. GL261-NS expressed higher levels of MHC and costimulatory molecules (CD80 and CD86) than GL261-AC; the JAM assay indicated that DC-NS splenocytes had higher lytic activity than DC-AC splenocytes on both GL261-NS and GL261-AC, and immunohistochemistry showed that DC-NS vaccination was associated with robust tumor infiltration by CD8+ and CD4+ T lymphocytes. These findings suggest that DC targeting of CSC provides a higher level of protection against GL261 gliomas, a finding with potential implications for the design of clinical trials based on DC vaccination. (Cancer Res 2006; 66(21): 10247-52)

Supplementary Methods and Figures 1-8 from Neurospheres Enriched in Cancer Stem–Like Cells Are Highly Effective in Eliciting a Dendritic Cell–Mediated Immune Response against Malignant Gliomas

Supplementary Table S2 from Neurospheres Enriched in Cancer Stem–Like Cells Are Highly Effective in Eliciting a Dendritic Cell–Mediated Immune Response against Malignant Gliomas

Background: Patient-oriented research (POR) incorporates patient partners as members of a research team. With the strengths and challenges of POR starting to emerge, one challenge we explore here is the research ethics application and review stages. Methods: We report the findings of a self-administered web-based survey in English and French for Canadian researchers or research ethics board (REB) perspectives (i.e., REB members or administrators) about the preparation and review of research ethics applications when patient partners are team members. The results are based on 165 survey responses and the team includes two patient partners who contributed fully to the conception, design, execution, interpretation of the findings, and in writing this manuscript.Results: The large majority of both groups correctly defined patient partner and had experience either reviewing or writing an application that included patient partners. REB perspective respondents indicated seeking more information to clarify patient partners’: roles, privacy training, experience/expertise, conflicts of interest, training, compensation; and at what stage the patient partner was recruited. Researchers indicated receiving questions about patient partners’: roles, training, compensation, access to data and participant information, affiliation, and curriculum vitae. Ideas related to adding questions to ethics applications about patient partners followed similar themes. Interpretation: This work reveals the knowledge base of REB perspectives and researchers in Canada with respect to ethics applications that include patient partners on research teams. We provide ideas for future work to help make the research ethics review and submission more efficient and clearer when patient partners are on research teams.

Middle-aged and older adults with diabetes are at increased risk for loneliness and functional limitations. Cross-sectional and longitudinal associations between loneliness and functional limitations have been demonstrated among the general population, but have not been established among those with diabetes. The purpose of this study was to directly compare the following models describing the direction of the association between loneliness and functional limitations among people with diabetes: (1) loneliness leads to functional limitations, (2) functional limitations lead to loneliness, and (3) a bidirectional association between loneliness and functional limitations.Data came from the Health and Retirement Study. Participants were middle-aged and older individuals with diabetes in the United States (n = 2934). Loneliness and functional limitations were measured at baseline, 4-year follow-up, and 8-year follow-up. Path models for each of the three models, as well as a stability model, were created. Model fit was compared using Akaike's Information Criteria (AIC).Participants were 54.6% female, 74.98% White, had a mean age of 69.66 years, had an average of 1.48 comorbid chronic conditions, and had diabetes for an average of 10.40 years. The bidirectional model best fit the data as evidenced by the lowest AIC value (AIC = 171,162.81). ∆AIC between the bidirectional model and the next best fitting model was 16.19, indicating strong support for selecting the bidirectional model. Higher levels of loneliness were associated with subsequent higher levels of functional limitations at some time points (βs = 0.07, 0.02) and higher levels of functional limitations were associated with subsequent higher levels of loneliness (βs = 0.13, 0.06) at all time points.Results suggest that the association between loneliness and functional limitations among individuals with diabetes is bidirectional. This study demonstrates the value of directly comparing directional models.

The pursuit of hedonic and eudaimonic activities is related to well-being. The current study examined whether hedonic and eudaimonic motives are associated with the strategies people choose to regulate their positive and negative emotions, which, in turn, predict well-being. Participants completed measures assessing hedonic and eudaimonic motives, down-regulation of positive emotions and up-regulation of negative emotions, and well-being. Eudaimonic motives correlated positively with down-regulation of negative emotions whereas hedonic motives correlated positively, but weakly, with up-regulation of positive emotions. Both types of motives and emotion regulation were associated with well-being, but eudaimonic motives and down-regulation of negative emotions were more strongly predictive of well-being than hedonic motives and up-regulation of positive emotions were. In addition, these associations appeared to be driven by people's choices for dysfunctional, rather than functional, strategies. In sum, the findings suggest that hedonic and eudaimonic motives may support different pathways to well-being, with eudaimonic motives and down-regulation of negative emotions playing a stronger role in well-being.

Recent models of emotion regulation suggest that the cognitive costs of reappraisal depend on stimulus intensity and habitual reappraisal. In the current experiment, we tested these hypotheses by manipulating the intensity of unpleasant and pleasant images, which participants reappraised, viewed, or suppressed their emotions to. To assess cognitive costs, we measured participants’ performance on a concurrent simple reaction time task. Participants also reported on their everyday use of reappraisal and suppression. Higher intensity stimuli were associated with greater cognitive costs of reappraisal, for unpleasant, but not pleasant pictures. Also, greater habitual reappraisal predicted lower cognitive costs of reappraisal and greater reductions in subjective feelings. Results support the role of stimulus intensity and habitual use of reappraisal in predicting the cognitive costs of reappraisal.

Several microRNAs (miRNAs) that are either specifically enriched or highly expressed in neurons and glia have been described, but the identification of miRNAs modulating neural stem cell (NSC) biology remains elusive. In this study, we exploited high throughput miRNA expression profiling to identify candidate miRNAs enriched in NSC/early progenitors derived from the murine subventricular zone (SVZ). Then, we used lentiviral miRNA sensor vectors (LV.miRT) to monitor the activity of shortlisted miRNAs with cellular and temporal resolution during NSC differentiation, taking advantage of in vitro and in vivo models that recapitulate physiological neurogenesis and gliogenesis and using known neuronal- and glial-specific miRNAs as reference. The LV.miRT platform allowed us monitoring endogenous miRNA activity in low represented cell populations within a bulk culture or within the complexity of CNS tissue, with high sensitivity and specificity. In this way we validated and extended previous results on the neuronal-specific miR-124 and the astroglial-specific miR-23a. Importantly, we describe for the first time a cell type- and differentiation stage-specific modulation of miR-93 and miR-125b in SVZ-derived NSC cultures and in the SVZ neurogenic niche in vivo, suggesting key roles of these miRNAs in regulating NSC function.

Pilot data showed that adding intratumoral (IT) injection of dendritic cells (DCs) prolongs survival of patients affected by glioblastoma multiforme (GBM) treated by subcutaneous (SC) delivery of DCs. Using a murine model resembling GBM, we investigated the immunological mechanisms underlying this effect. C57BL6/N mice received brain injections of GL261 glioma cells. Seven days later, mice were treated by 3 SC injections of DCs with or without 1 IT injection of DCs. DC maturation, induced by pulsing with GL261 lysates, was necessary to develop effective immune responses. IT injection of pulsed (pDC), but not unpulsed DCs (uDC), increased significantly the survival, either per se or in combination with SC-pDC (P.001 vs controls). Mice treated by IT-pDC plus SC-pDC survived longer than mice treated by SC-pDC only (P = .03). Injected pDC were detectable in tumor parenchyma, but not in cervical lymph nodes. In gliomas injected with IT-pDC, CD8+ cells were significantly more abundant and Foxp3+ cells were significantly less abundant than in other groups. Using real-time polymerase chain reaction, we also found enhanced expression of IFN-gamma and TNF-alpha and decreased expression of transforming growth factor-beta (TGF-beta) and Foxp3 in mice treated with SC-pDC and IT-pDC. In vitro, pDC produced more TNF-alpha than uDC: addition of TNF-alpha to the medium decreased the proliferation of glioma cells. Overall, the results suggest that IT-pDC potentiates the anti-tumor immune response elicited by SC-pDC by pro-immune modulation of cytokines in the tumor microenvironment, decrease of Treg cells, and direct inhibition of tumor proliferation by TNF-alpha.

Cancer stem–like cells (CSC) could be a novel target for cancer therapy, including dendritic cell (DC) immunotherapy. To address this, we developed experiments aimed at DC targeting of neurospheres (NS) from GL261 glioma cells because neurospheres can be enriched in CSC. We obtained murine neurospheres by growing GL261 cells in epidermal growth factor/basic fibroblast growth factor without serum. GL261-NS recapitulated important features of glioblastoma CSC and expressed higher levels of radial glia stem cell markers than GL261 cells growing under standard conditions (GL261 adherent cells, GL261-AC), as assessed by DNA microarray and real-time PCR. GL261-NS brain gliomas were highly infiltrating and more rapidly lethal than GL261-AC, as evidenced by survival analysis (P < 0.0001), magnetic resonance imaging and histology. DC from the bone marrow of syngeneic mice were then used for immunotherapy of GL261-NS and GL261-AC tumors. Strikingly, DC loaded with GL261-NS (DC-NS) cured 80% and 60% of GL261-AC and GL261-NS tumors, respectively (P < 0.0001), whereas DC-AC cured only 50% of GL261-AC tumors (P = 0.0022) and none of the GL261-NS tumors. GL261-NS expressed higher levels of MHC and costimulatory molecules (CD80 and CD86) than GL261-AC; the JAM assay indicated that DC-NS splenocytes had higher lytic activity than DC-AC splenocytes on both GL261-NS and GL261-AC, and immunohistochemistry showed that DC-NS vaccination was associated with robust tumor infiltration by CD8+ and CD4+ T lymphocytes. These findings suggest that DC targeting of CSC provides a higher level of protection against GL261 gliomas, a finding with potential implications for the design of clinical trials based on DC vaccination. (Cancer Res 2006; 66(21): 10247-52)

In recent years, the use of dendritic cells (DC), the most powerful antigen presenting cells, has been proposed for the creation of vaccines against gliomas. This approach has been demonstrated to be safe and non-toxic in phase I or I-II trials (2, 3). Immunotherapy plays a central role in the search for new treatments for glioblastoma multiforme (GBM). In particular, several phase I studies have been performed using DC pulsed by GBM proteins as a vaccine for patients with relapsing GBM. The studies demonstrated that DC vaccination is safe and may produce a significant increase in overall survival. As the first step in the preparation of appropriate conditions for a clinical evaluation in Italy, we have performed pre-clinical experiments on immune-competent mice injected intra-cerebrally with syngeneic GL261GBM cells and treated subcutaneously and intra-tumorally with DC loaded with a GL261 homogenate. These results show that vaccination with DC pulsed with a tumor lysate increases considerably survival in mice bearing intracranial glioblastomas and supports the development of DC-based clinical trials for patients with glioblastomas that do not respond to standard therapies.