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Supplementary Table S1

Supplementary Fig. S3. Differentiation of ML NK cells and Preactivated-Expanded CB-NK cells.

Supplementary Materials and Methods

Purpose:Natural killer (NK)-cell recognition and function against NK-resistant cancers remain substantial barriers to the broad application of NK-cell immunotherapy. Potential solutions include bispecific engagers that target NK-cell activity via an NK-activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL12/15/18 cytokine pre-activation.Experimental Design:We assessed single-cell NK-cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL12/15/18 pre-activation of blood and cord blood–derived NK cells was investigated in vitro and in vivo.Results:We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK-cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from patients with Hodgkin lymphoma. IL12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared with cNK cells. Cord-blood NK cells preactivated with IL12/15/18 and ex vivo expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK-cell effector function. AFM13–NK complex cells exhibited enhanced responses to CD30+ lymphomas in vitro and in vivo.Conclusions:We identify AFM13 as a promising combination with cytokine-activated adult blood or cord-blood NK cells to treat CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.

Amyotrophic lateral sclerosis type 6 (ALS6) is a familial subtype of ALS linked to Fused in Sarcoma (FUS) gene mutation. FUS mutations lead to decreased global protein synthesis, but the mechanism that drives this has not been established. Here, we used ALS6 patient-derived induced pluripotent stem cells (hIPSCs) to study the effect of the ALS6 FUSR521Hmutation on the translation machinery in motor neurons (MNs). We find, in agreement with findings of others, that protein synthesis is decreased in ALS6 MNs. Furthermore, ALS6 MNs are more sensitive to oxidative stress and display reduced expression of TGF-β and mTORC gene pathways when stressed. Finally, we show that IFNγtreatment reduces apoptosis of ALS6 MNs exposed to oxidative stress and partially restores the translation rates in ALS6 MNs. Overall, these findings suggest that a functional IFNγresponse is important for FUS-mediated protein synthesis, possibly by FUS nuclear translocation in ALS6.Highlights and eTOC blurbALS6 patient-derived motor neurons show decreased viability and reduced production of innate immune cytokines following oxidative stressFUS cytoplasmic localization coincides with decreased protein synthesis ratesIFNγtreatment of ALS6 patient-derived motor neurons reduces apoptosis and ameliorates translation rates resulting from oxidative stress

It is estimated that 5–10% of breast cancer cases are hereditary. The identification of pathogenic germline variants allows individualized preventive health care, improvement of clinical management and genetic counseling. Studies in ethnically admixed Latin American populations have identified regions with increased frequency of deleterious variants in breast cancer predisposing genes. In this context, the Brazilian population exhibits great genetic heterogeneity, and is not well represented in international databases, which makes it difficult to interpret the clinical relevance of germline variants. We evaluated the frequency of pathogenic/likely pathogenic (P/LP) germline variants in up to 37 breast cancer predisposing genes, in a cohort of 105 breast and/or ovarian cancer Brazilian women referred to two research centers between 2014 and 2019. A total of 22 patients (21%) were found to carry P/LP variants, and 16 VUS were detected in 15 patients (14.3%). Additionally, a novel pathogenic ATM intragenic deletion was identified in an early-onset breast cancer. We also detected a BRCA1 pathogenic variant (c.5074+2T>C) in higher frequency (10×) than in other studies with similar cohorts. Our findings contribute to the characterization of the genetic background of breast cancer predisposition in the Brazilian population as a useful resource to discriminate between deleterious variants and VUS, thus enabling improvement in the preventive health care and clinical management of carriers.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with no cure. The reports showed the role of nearby astrocytes around the motor neurons as one among the causes of the disease. However, the exact mechanistic insights are not explored so far. Thus, in the present investigations, we employed the induced pluripotent stem cells (iPSCs) of Cu/Zn-SOD1L39R linked ALS patient to convert them into the motor neurons (MNs) and astrocytes. We report that the higher expression of stress granule (SG) marker protein G3BP1, and its co-localization with the mutated Cu/Zn-SOD1L39R protein in patient's MNs and astrocytes are linked with AIF1-mediated upregulation of caspase 3/7 and hyper activated autophagy. We also observe the astrocyte-mediated non-cell autonomous neurotoxicity on MNs in ALS. The secretome of the patient's iPSC-derived astrocytes exerts significant oxidative stress in MNs. The findings suggest the hyperactive status of autophagy in MNs, as witnessed by the co-distribution of LAMP1, P62 and LC3 I/II with the autolysosomes. Conversely, the secretome of normal astrocytes has shown neuroprotection in patient's iPSC-derived MNs. The whole-cell patch-clamp assay confirms our findings at a physiological functional level in MNs. Perhaps for the first time, we are reporting that the MN degeneration in ALS triggered by the hyper-activation of autophagy and induced apoptosis in both cell-autonomous and non-cell autonomous conditions.

Amyotrophic lateral sclerosis type 8 (ALS8) is an autosomal dominant form of ALS, which is caused by pathogenic variants in the VAPB gene. Here we investigated five ALS8 patients, classified as ‘severe’ and ‘mild’ from a gigantic Brazilian kindred, carrying the same VAPB mutation but displaying different clinical courses. Copy number variation and whole exome sequencing analyses in such individuals ruled out previously described genetic modifiers of pathogenicity. After deriving induced pluripotent stem cells (iPSCs) for each patient (N = 5) and controls (N = 3), motor neurons were differentiated, and high-throughput RNA-Seq gene expression measurements were performed. Functional cell death and oxidative metabolism assays were also carried out in patients’ iPSC-derived motor neurons. The degree of cell death and mitochondrial oxidative metabolism were similar in iPSC-derived motor neurons from mild patients and controls and were distinct from those of severe patients. Similar findings were obtained when RNA-Seq from such cells was performed. Overall, 43 genes were upregulated and 66 downregulated in the two mild ALS8 patients when compared with severe ALS8 individuals and controls. Interestingly, significantly enriched pathways found among differentially expressed genes, such as protein translation and protein targeting to the endoplasmic reticulum (ER), are known to be associated with neurodegenerative processes. Taken together, the mitigating mechanisms here presented appear to maintain motor neuron survival by keeping translational activity and protein targeting to the ER in such cells. As ALS8 physiopathology has been associated with proteostasis mechanisms in ER–mitochondria contact sites, such differentially expressed genes appear to relate to the bypass of VAPB deficiency.

The Zika virus (ZIKV) has shown a promising oncolytic effect against embryonal CNS tumors. However, studies on the effect of different administration routes and the ideal viral load in preclinical models are highly relevant aiming for treatment safety and efficiency. Here, we investigated the effect and effectiveness of different routes of administration, and the number of ZIKVBR injections on tumor tropism, destruction, and side effects. Furthermore, we designed an early-stage human brain organoid co-cultured with embryonal CNS tumors to analyze the ZIKVBR oncolytic effect. We showed that in the mice bearing subcutaneous tumors, the ZIKVBR systemically presented a tropism to the brain. When the tumor was located in the mice’s brain, serial systemic injections presented efficient tumor destruction, with no neurological or other organ injury and increased mice survival. In the human cerebral organoid model co-cultured with embryonal CNS tumor cells, ZIKVBR impaired tumor progression. The gene expression of cytokines and chemokines in both models suggested an enhancement of immune cells recruitment and tumor inflammation after the treatment. These results open new perspectives for virotherapy using the ZIKVBR systemic administration route and multiple doses of low virus load for safe and effective treatment of embryonal CNS tumors, an orphan disease that urges new effective therapies.

Cell therapy with T cells expressing chimeric antigen receptor (CAR-T) is a type of immunotherapy that involves the manipulation and reprogramming of immune cells (T lymphocytes) in order to recognize and kill tumor cells. For use in patients, CAR-T cells must be manufcatured inside a GMP facility according to a established procedure.

Manufacturing of customized gene or cell therapy products such as CAR-T cells is complex and depends on release tests and exams that can attest to a consistent quality standard for each product. The quality of CAR-T cell products is subject to donor variation, but also includes the manufacturing environment, as well as the quality and availability of materials and reagents. Quality must be carefully monitored and integrated into the manufacturing process.

Transplantation is essential and crucial for individuals suffering from end-stage organ failure diseases. However, there are still many challenges regarding these procedures, such as high rates of organ rejection, shortage of organ donors, and long waiting lines. Thus, investments and efforts to develop laboratory-grown organs have increased over the past years, and with the recent progress in regenerative medicine, growing organs in vitro might be a reality within the next decades. One of the many different strategies to address this issue relies on organoid technology, a miniaturized and simplified version of an organ. Here, we address recent progress on organoid research, focusing on transplantation of intestine, retina, kidney, liver, pancreas, brain, lung, and heart organoids. Also, we discuss the main outcomes after organoid transplantation, common challenges faced by these promising regenerative medicine approaches, and future perspectives on the field.

Background Natural killer (NK) cells are a nascent cellular immunotherapy for hematologic malignancies. Target recognition of NK cell-resistant cancers remains a substantial barrier to broad application of NK cell therapy. One solution are bispecific engagers that trigger NK cells via an NK activating receptor when simultaneously engaging a tumor-specific antigen. Methods Here, we investigated single NK cell responses stimulated by the tetravalent bispecific innate cell engager (ICE®) AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on several types of NK cells. Results Multidimensional mass cytometry revealed heterogeneity within AFM13-directed conventional (c)NK cell responses, as well as consistent polyfunctional activation of mature terminally differentiated NK cells across donors. The source of NK cells also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from Hodgkin lymphoma patients. IL-12, IL-15, and IL-18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared to cNK cells. Cord-blood expanded NK cells that were pre-activated with IL-12, IL-15 and IL-18 also exhibited enhanced killing with AFM13 stimulation, via upregulation of signaling pathways related to NK cell effector function. These cells were stably pre-loaded with AFM13 enhancing responses to CD30+ lymphomas in vitro and in vivo in immunodeficient NSG mouse models. Conclusions Collectively, these data identify promising combinations of AFM13 with cytokine-activated adult blood or cord blood NK cells against CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with no cure. The reports showed the role of nearby astrocytes around the motor neurons as one among the causes of the disease. However, the exact mechanistic insights are not explored so far. Thus, in the present investigations, we employed the induced pluripotent stem cells (iPSCs) of Cu/Zn-SOD1

Patients with SARS-CoV-2 may be affected by the acute respiratory distress syndrome (ARDS), which has been associated with high mortality rate. As no specific drugs are available for ARDS, mesenchymal stem cells (MSC) seems to be a promising cell therapy due to immunomodulatory effects on reducing and healing inflammation-induced lung and other tissue injuries. The goal of this Phase I clinical trial was to explore the safety and efficacy of bone marrow-derived MSC (BM-MSC) infusions in patients with COVID-19 ARDS. The inclusions criteria were age between 18 to 70 years and PaO2/FiO2≤200mmHg. The BM-MSC infusions were as follow: one to 3 infusions intravenous doses of BM-MSC of 1x10 6 cells/kg; each dose could be administered with an interval between 3 to 7 days. The primary endpoint was safety (adverse events) within 6 hours; cardiac arrest or death within 24 hours post-infusion. The secondary endpoint includes patient survival at 30 days after the first infusion. Six patients were included in the trial and treated with at least one infusion of BM-MSC. The median age was 60,3 years (54 to 69), 5 were male. The median time between the worsening of respiratory distress and the BM-MSC infusion was 10 days (3 to 31 days). The median of PaO2/FiO2 before infusion was 151.86 (127.80-164.44) and median PaCO2 was 63,85 (39 to 117). One patient was treated with 3 MSC doses, two patients with 2 doses and 3 patients one dose. No serious adverse effects were observed within 24 hours post-infusion; only one death was observed following 30 days of cell administration. None of them showed adverse events during BM-MSC infusion. Only one patient showed signs of pulmonary infection one week after first BM-MSC infusion. This patient was at increased risk for infection due to prolonged intubation and a high dose of corticosteroid. Therefore, it was not possible to conclude its association with BM-MSC treatment. Only two patients showed clinical improvement after BM-MSC infusion. Patient 1 had BM-MSC infusion 72 hours after worsening of respiratory parameters, and thorax CT suggested the hypothesis of cryptogenic organizing pneumonia, which led to decision of using methylprednisolone 125mg I.V. for 3 days and carry on with MSC infusion. We observed a decrease in CRP levels from 126 to 67 mg/dL on day 1 after the first infusion, and PaO2/FiO2 ratio improved from 155 to 297 mmHg on day 5. He received the second dose within 7 days interval and by day 11 of the first infusion a new thorax TC showed complete resolution of alveolar consolidation areas in both lungs (Figure 1A and 1B). Patient 2 had BM-MSC administration 11 days after respiratory worsening and also presented improvement of PaO2/FiO2 ratio (148 to 215 mmHg after 2 days of infusion) and had thorax CT images suggesting cryptogenic organizing pneumonia with administration of methylprednisolone 250mg I.V. Nevertheless, the second dose was not administered due to ventilator-associated pneumonia and urinary infection. Four patients showed a non-sustained increase of PaO2/FiO2 ratio, with higher median PCO2 levels of 69,3 mmHg (range, 61,2 to 117) comparing to 39 and 47,3 mmHg of patients 1 and 2, respectively. PCO2 parameter could be a marker to indicate a worse response to MSC treatment, since it could point out chronic phases of COVID-19 disease. The patients died due to COVID-19 complications. No difference in inflammatory markers, such as interleukin 6, C-protein reactive test, procalcitonin, ferritin was observed before and after treatment. Inclusion criteria did not defined interval between respiratory worsening and first BM-MSC infusion. Four patients had chronic phase of COVID-19 without inflammatory markers and hypercapnia. It could be related to severity of pulmonary disease, such as reported in chronic obstructive pulmonary disease. Two patients were discharged after MSC treatment and they received methylprednisolone to treat cryptogenic organizing pneumonia. There are only a few clinical trials and observational studies evaluating the use of high-dose of glucocorticoid for severe COVID-19 pneumonia. Therefore, it is not possible to conclude that use of glucocorticoid has contributed to favorable outcomes. In conclusion, BM-MSC showed to be a secure therapeutic option for severe COVID-19 pneumonia, possibly with superior benefit in acute phases and lower PCO2 levels. Further studies involving a large cohort or randomized controlled trials are warranted. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

// Patricia Benites Goncalves da Silva 1 , Marcia Cristina Teixeira dos Santos 1 , Carolina Oliveira Rodini 1 , Carolini Kaid 1 , Marcia Cristina Leite Pereira 1 , Gabriela Furukawa 1 , Daniel Sanzio Gimenes da Cruz 2 , Mauricio Barbugiani Goldfeder 3 , Clarissa Ribeiro Reily Rocha 4 , Carla Rosenberg 1 , Oswaldo Keith Okamoto 1 1 Centro de Pesquisa sobre o Genoma Humano e Celulas-Tronco, Departamento de Genetica e Biologia Evolutiva, Instituto de Biociencias, Universidade de Sao Paulo, Sao Paulo, SP, Brazil 2 Departamento de Patologia, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Sao Paulo, SP, Brazil 3 Laboratorio de Bioquimica e Biofisica, Instituto Butantan, Sao Paulo, SP, Brazil 4 Departmento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo, SP, Brazil Correspondence to: Oswaldo Keith Okamoto, email: keith.okamoto@usp.br Keywords: OCT4A, POU5F1, LIN28A, medulloblastoma, aggressiveness Received: May 26, 2016 Accepted: January 22, 2017 Published: February 07, 2017 ABSTRACT Medulloblastoma is a highly aggressive pediatric brain tumor, in which sporadic expression of the pluripotency factor OCT4 has been recently correlated with poor patient survival. However the contribution of specific OCT4 isoforms to tumor aggressiveness is still poorly understood. Here, we report that medulloblastoma cells stably overexpressing the OCT4A isoform displayed enhanced clonogenic, tumorsphere generation, and invasion capabilities. Moreover, in an orthotopic metastatic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive and infiltrative tumors, with tumor-bearing mice attaining advanced metastatic disease and shorter survival rates. Pro-oncogenic OCT4A effects were expression-level dependent and accompanied by distinct chromosomal aberrations. OCT4A overexpression in medulloblastoma cells also induced a marked differential expression of non-coding RNAs, including poorly characterized long non-coding RNAs and small nucleolar RNAs. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer.

Primary central nervous system (CNS) tumors are the most common deadly childhood cancer. Several patients with medulloblastoma experience local or metastatic recurrences after standard treatment, a condition associated with very poor prognosis. Current neuroimaging techniques do not accurately detect residual stem-like medulloblastoma cells promoting tumor relapses. In attempt to identify candidate tumor markers that could be circulating in blood or cerebrospinal (CSF) fluid of patients, we evaluated the proteome and miRNome content of extracellular microvesicles (MVs) released by highly-aggressive stem-like medulloblastoma cells overexpressing the pluripotent factor OCT4A. These cells display enhanced tumor initiating capability and resistance to chemotherapeutic agents. A common set of 464 proteins and 10 microRNAs were exclusively detected in MVs of OCT4A-overexpressing cells from four distinct medulloblastoma cell lines, DAOY, CHLA-01-MED, D283-MED, and USP13-MED. The interactome mapping of these exclusive proteins and miRNAs revealed ERK, PI3K/AKT/mTOR, EGF/EGFR, and stem cell self-renewal as the main oncogenic signaling pathways altered in these aggressive medulloblastoma cells. Of these MV cargos, four proteins (UBE2M, HNRNPCL2, HNRNPCL3, HNRNPCL4) and five miRNAs (miR-4449, miR-500b, miR-3648, miR-1291, miR-3607) have not been previously reported in MVs from normal tissues and in CSF. These proteins and miRNAs carried within MVs might serve as biomarkers of aggressive stem-like medulloblastoma cells to improve clinical benefit by helping refining diagnosis, patient stratification, and early detection of relapsed disease.

Medulloblastoma is a highly aggressive brain tumor and one of the leading causes of morbidity and mortality related to childhood cancer. These tumors display differential ability to metastasize and respond to treatment, which reflects their high degree of heterogeneity at the genetic and molecular levels. Such heterogeneity of medulloblastoma brings an additional challenge to the understanding of its physiopathology and impacts the development of new therapeutic strategies. This translational effort has been the focus of most pre-clinical studies which invariably employ experimental models using human tumor cell lines. Nonetheless, compared to other cancers, relatively few cell lines of human medulloblastoma are available in central repositories, partly due to the rarity of these tumors and to the intrinsic difficulties in establishing continuous cell lines from pediatric brain tumors. Here, we report the establishment of a new human medulloblastoma cell line which, in comparison with the commonly used and well-established cell line Daoy, is characterized by enhanced proliferation and invasion capabilities, stem cell properties, increased chemoresistance, tumorigenicity in an orthotopic metastatic model, replication of original medulloblastoma behavior in vivo, strong chromosome structural instability and deregulation of genes involved in neural development. These features are advantageous for designing biologically relevant experimental models in clinically oriented studies, making this novel cell line, named USP-13-Med, instrumental for the study of medulloblastoma biology and treatment.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease whose pathophysiology is poorly understood. Aiming to better understand the cause of motor neuron death, the use of experimental cell-based models increased significantly over the past years. In this scenario, much knowledge has been generated from the study of motor neurons derived from embryonic stem cells and induced pluripotent stem cells. These methods, however, have advantages and disadvantages, which must be balanced on experimental design. Preclinical studies provide valuable information, making it possible to combine diverse methods to build an expanded knowledge of ALS pathophysiology. In addition to using stem cells as experimental models for understanding disease mechanism, these cells had been quoted for therapy in ALS. Despite ethical issues involved in its use, cell therapy with neural stem cells stands out. A phase I clinical trial was recently completed and a phase II is on its way, attesting the method's safety. In another approach, mesenchymal stromal cells capable of releasing neuroregulatory and anti-inflammatory factors have also been listed as candidates for cell therapy for ALS, and have been admitted as safe in a phase I trial. Despite recent advances, application of stem cells as an actual therapy for ALS patients is still in debate. Here, we discuss how stem cells have been useful in modeling ALS and address critical topics concerning their therapeutic use, such as administration protocols, injection site, cell type to be administered, type of transplantation (autologous vs. allogeneic) among other issues with particular implications for ALS therapy.

// Carolina Oliveira Rodini 1 , Patricia Benites Goncalves da Silva 1 , Amanda Faria Assoni 1, 2 , Valdemir Melechco Carvalho 2 and Oswaldo Keith Okamoto 1, 3 1 Centro de Pesquisa sobre o Genoma Humano e Celulas-Tronco, Departamento de Genetica e Biologia Evolutiva, Instituto de Biociencias, Universidade de Sao Paulo, Cidade Universitaria, Sao Paulo, SP, Brazil 2 Fleury Group, Sao Paulo, Jabaquara, Sao Paulo, SP, Brazil 3 Departamento de Hemoterapia e Terapia Celular, Hospital Israelita Albert Einstein, Sao Paulo, SP, Brazil Correspondence to: Oswaldo Keith Okamoto, email: keith.okamoto@usp.br Keywords: glioblastoma; tumor microenvironment; mesenchymal stem cells; aggressiveness; secretome Received: November 03, 2017 Accepted: April 23, 2018 Published: May 15, 2018 ABSTRACT Mesenchymal stem cells (MSC) display tumor tropism and have been addressed as vehicles for delivery of anti-cancer agents. As cellular components of the tumor microenvironment, MSC also influence tumor progression. However, the contribution of MSC in brain cancer is not well understood since either oncogenic or tumor suppressor effects have been reported for these cells. Here, MSC were found capable of stimulating human Glioblastoma (GBM) cell proliferation through a paracrine effect mediated by TGFB1. Moreover, when in direct cell-cell contact with GBM cells, MSC elicited an increased proliferative and invasive tumor cell behavior under 3D conditions, as well as accelerated tumor development in nude mice, independently of paracrine TGFB1. A secretome profiling of MSC-GBM co-cultures identified 126 differentially expressed proteins and 10 proteins exclusively detected under direct cell-cell contact conditions. Most of these proteins are exosome cargos and are involved in cell motility and tissue development. These results indicate a dynamic interaction between MSC and GBM cells, favoring aggressive tumor cell traits through alternative and independent mechanisms. Overall, these findings indicate that MSC may exert pro-tumorigenic effects when in close contact with tumor cells, which must be carefully considered when employing MSC in targeted cell therapy protocols against 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.

Several methods have been used to study the neuropathogenesis of Down syndrome (DS), such as mouse aneuploidies, post mortem human brains, and in vitro cell culture of neural progenitor cells. More recently, induced pluripotent stem cell (iPSC) technology has offered new approaches in investigation, providing a valuable tool for studying specific cell types affected by DS, especially neurons and astrocytes. Here, we investigated the role of astrocytes in DS developmental disease and the impact of the astrocyte secretome in neuron mTOR signaling and synapse formation using iPSC derived from DS and wild-type (WT) subjects. We demonstrated for the first time that DS neurons derived from hiPSC recapitulate the hyperactivation of the Akt/mTOR axis observed in DS brains and that DS astrocytes may play a key role in this dysfunction. Our results bear out that 21 trisomy in astrocytes contributes to neuronal abnormalities in addition to cell autonomous dysfunctions caused by 21 trisomy in neurons. Further research in this direction will likely yield additional insights, thereby improving our understanding of DS and potentially facilitating the development of new therapeutic approaches.

Pericytes are important cellular components of the tumor microenviroment with established roles in angiogenesis and metastasis. These two cancer hallmarks are modulated by enzymes of the LOX family, but thus far, information about LOX relevance in tumor-associated pericytes is lacking. Here, we performed a comparative characterization of normal and tumoral pericytes and report for the first time the modulatory effects of LOX enzymes on activated pericyte properties. Tumoral pericytes isolated from childhood ependymoma and neuroblastoma specimens displayed angiogenic properties in vitro and expressed typical markers, including CD146, NG2, and PDGFRβ. Expression of all LOX family members could be detected in both normal and tumor-associated pericytes. In most pericyte samples, LOXL3 was the family member displaying the highest transcript levels. Inhibition of LOX/LOXL activity with the inhibitor β-aminopropionitrile (βAPN) significantly reduced migration of pericytes, while proliferation rates were kept unaltered. Formation of tube-like structures in vitro by pericytes was also significantly impaired upon inhibition of LOX/LOXL activity with βAPN, which induced more prominent effects in tumor-associated pericytes. These findings reveal a novel involvement of the LOX family of enzymes in migration and angiogenic properties of pericytes, with implications in tumor development and in therapeutic targeting tumor microenvironment constituents.

National Institute of Science and Technology-Stem Cells in Human Genetic Diseases Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) In a previous genome-wide expression profiling study, we identified E2F2 as a hyperexpressed gene in stem-like cells of distinct glioblastoma multiforme (GBM) specimens. Since the encoded E2F2 transcription factor has been implicated in both tumor suppression and tumor development, we conducted a functional study to investigate the pertinence of E2F2 to human gliomagenesis. E2F2 expression was knocked down by transfecting U87MG cells with plasmids carrying a specific silencing shRNA. Upon E2F2 silencing, in vitro cell proliferation was significantly reduced, as indicated by a time-course analysis of viable tumor cells. Anchorage-independent cell growth was also significantly inhibited after E2F2 silencing, based on cell colony formation in soft agar. Subcutaneous and orthotopic xenograft models of GBM in nude mice also indicated inhibition of tumor development in vivo, following E2F2 silencing. As expression of the E2F2 gene is associated with glioblastoma stem cells and is involved in the transformation of human astrocytes, the present findings suggest that E2F2 is involved in gliomagenesis and could be explored as a potential therapeutic target in malignant gliomas. Universidade Federal de São Paulo, Dept Neurol & Neurosurg, Expt Neurol Unit, BR-04023900 São Paulo, Brazil Univ São Paulo, Human Genome & Stem Cell Res Ctr, Inst Biosci, Dept Genet & Evolutionary Biol, BR-05508090 São Paulo, Brazil Universidade Federal de São Paulo, Dept Neurol & Neurosurg, Expt Neurol Unit, BR-04023900 São Paulo, Brazil Web of Science

Long-term structural and functional changes in the hippocampus have been identified as the primary physiopathological basis for temporal lobe epilepsy. These changes include reactive gliosis and granule cell axonal sprouting within the dentate gyrus. The intimate mechanisms of these changes are beginning to be revealed. Here, we show the possibility of using laser capture microdissection (LCM) to isolate the dentate granular cell layer of Wistar rats submitted to the pilocarpine model of epilepsy. Using two-dimensional gel electrophoresis (2-D PAGE) and mass spectrometry for laser-captured cells, we identified molecular events that could be altered as part of the epileptic pathogenic process. According to our results, eight proteins related to energy metabolism were differentially expressed between both the control and pilocarpine-treated animals. These results provide, for the first time, new molecular insights into the altered protein profile of the epileptogenic dentate gyrus and can contribute to a better understanding of the phenomena involved in the genesis and maintenance of the epileptic state.