Medulloblastoma (MB) is the most common malignant brain tumor in children and among the subtypes, Group 3 MB has the worst outcome. Here, we perform an in vivo, patient-specific screen leading to the ...identification of Otx2 and c-MYC as strong Group 3 MB inducers. We validated our findings in human cerebellar organoids where Otx2/c-MYC give rise to MB-like organoids harboring a DNA methylation signature that clusters with human Group 3 tumors. Furthermore, we show that SMARCA4 is able to reduce Otx2/c-MYC tumorigenic activity in vivo and in human cerebellar organoids while SMARCA4 T910M, a mutant form found in human MB patients, inhibits the wild-type protein function. Finally, treatment with Tazemetostat, a EZH2-specific inhibitor, reduces Otx2/c-MYC tumorigenesis in ex vivo culture and human cerebellar organoids. In conclusion, human cerebellar organoids can be efficiently used to understand the role of genes found altered in cancer patients and represent a reliable tool for developing personalized therapies.
The transition of neural progenitors to differentiated postmitotic neurons is mainly considered irreversible in physiological conditions. In the present work, we show that Shh pathway activation ...through SmoM2 expression promotes postmitotic neurons dedifferentiation, re-entering in the cell cycle and originating medulloblastoma in vivo. Notably, human adult patients present inactivating mutations of the chromatin reader BRPF1 that are associated with SMO mutations and absent in pediatric and adolescent patients. Here, we found that truncated BRPF1 protein, as found in human adult patients, is able to induce medulloblastoma in adult mice upon SmoM2 activation. Indeed, postmitotic neurons re-entered the cell cycle and proliferated as a result of chromatin remodeling of neurons by BRPF1. Our model of brain cancer explains the onset of a subset of human medulloblastoma in adult individuals where granule neuron progenitors are no longer present.
Display omitted
•SmoM2 overexpression promotes cerebellar granule neurons dedifferentiation in vivo•SmoM2 and mutant BRPF1 cooperation in vivo mimics human adult SHH MBs•Granule neurons are putative cells of origin of adult SHH MBs•Truncated BRPF1 increases the accessibility of a subset of super-enhancers
Medulloblastoma is a brain tumor affecting the cerebellum of infants and adults. Aiello et al. establish a mouse model for adult onset, which allows investigation of the pathogenesis of the disease and identifies neurons as putative cells of origin.
Medulloblastoma and high-grade glioma represent the most aggressive and frequent lethal solid tumors affecting individuals during pediatric age. During the past years, several models have been ...established for studying these types of cancers. Human organoids have recently been shown to be a valid alternative model to study several aspects of brain cancer biology, genetics and test therapies. Notably, brain cancer organoids can be generated using genetically modified cerebral organoids differentiated from human induced pluripotent stem cells (hiPSCs). However, the protocols to generate them and their downstream applications are very rare. Here, we describe the protocols to generate cerebellum and forebrain organoids from hiPSCs, and the workflow to genetically modify them by overexpressing genes found altered in patients to finally produce cancer organoids. We also show detailed protocols to use medulloblastoma and high-grade glioma organoids for orthotopic transplantation and co-culture experiments aimed to study cell biology in vivo and in vitro, for lineage tracing to investigate the cell of origin and for drug screening. The protocol takes 60-65 d for cancer organoids generation and from 1-4 weeks for downstream applications. The protocol requires at least 3-6 months to become proficient in culturing hiPSCs, generating organoids and performing procedures on immunodeficient mice.
Abstract
Brain cancer is now the deadliest form of childhood cancer in the United States. In particular, Group3 Medulloblastoma (MB) is the pediatric brain tumor with the highest morbidity and ...mortality. Patients with Group3 MB currently have the worst outcome and nearly 50% are metastatic at the time of diagnosis. However, the cellular and molecular mechanisms underlying Group3 MB are still unknown. What is still lacking in the field is the possibility to obtain tumors by direct genetic modification of mice and to be able to recapitulate the growth and metastasis formation of Group3 MB. Exploiting in-vivo transfection of mouse cerebellar cells with CRISPR-Cas9 and PiggyBac transposase systems, we tested different combinations of putative oncosuppressors and putative oncogenes, derived from human Medulloblastoma NGS data, for their ability to induce Group3 MB in mice. Surprisingly, concomitant overexpression of c-Myc and other transcription factors in mouse cerebellum is able to induce MB in a few months. The newly generated mouse model is able to fully recapitulate human Group 3 MB. Using this proposed patient-specific model, we were able to unravel the molecular aspects of Group 3 medulloblastoma tumorigenesis and identify molecules inhibiting tumor growth in a targeted manner.
Abstract
Three-dimensional (3D) cell culture systems have gained increasing interest in drug discovery and tissue engineering due to their evident advantages in providing more relevant information ...and more predictive data for in vivo tests. Willing to increase the knowledge about Medulloblastoma Brain tumor we are developing Human cerebellar organoids derived from human iPSC: three-dimensional cultures that include multiple stem cell types, cell organization and features of human cerebellum. Notably, Medulloblastoma is the most common brain tumor affecting infants and stands as a cause for a high percentage of morbidity and mortality among cancer patients. To identify new Medulloblastoma driver genes we set up an innovative way to modify human cerebellar organoid, developing the first in-vitro 3D model of Medulloblastoma. We are mimicking human pathological conditions modifying this organoids with different putative oncogenes. Notably thanks to this type of model, we are able to study the functions of new Medulloblastoma driver genes in modulating stem cells differentiation and commitment in a three-dimensional structure, during the development of the organoid and the tumor. We speculate that our new Medulloblastoma 3D culture systems hold great promise for applications in infant tumor research, cancer cell biology and drug discovery, being the first human 3D in-vitro model that resembles human pathological conditions.
Abstract
In the last years significant progress has been made to identify the cells of origin of many cancers and the majority of data points towards stem cells and progenitors. Notably, it is common ...opinion that these cells should possess a proliferative capacity, therefore postmitotic neurons have not been considered as suitable cell of origin of human and mouse cancer. Indeed, the transition of neural progenitor to differentiated postmitotic neurons is thought to be irreversible in physiological and pathological conditions. Here we show that neurons reprograming may occur upon oncogenes activation and it leads to brain cancer formation in vivo. Using a conditional Cre-recombination system we manage to recapitulate human cancer model in mice ad to demonstrate that the postmigratory mature granule neurons (MGNs) can be reprogrammed in-vivo. Interestingly, MGNs seem to possess defined characteristic that allow the reprogramming and the cancer formation, since activation of oncogenic pathways leads to cancer formation only in specific area of brain. For the first time we demonstrate the chance to reprogram postmitotic neurons into cancer cells shedding new light on the mechanisms of cancer formation. Postmitotic cells of other tissues may also possess tumor initianting cell features and our findings can be considered as a starting point to a new field in cancer biology.
In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an ...antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the genomic DNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientations and frames compatible with expression. These results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against infected erythrocytes and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.
Brain neurons arise from relatively few progenitors generating an enormous diversity of neuronal types. Nonetheless, a cardinal feature of mammalian brain neurogenesis is thought to be that ...excitatory and inhibitory neurons derive from separate, spatially segregated progenitors. Whether bi-potential progenitors with an intrinsic capacity to generate both lineages exist and how such a fate decision may be regulated are unknown. Using cerebellar development as a model, we discover that individual progenitors can give rise to both inhibitory and excitatory lineages. Gradations of Notch activity determine the fates of the progenitors and their daughters. Daughters with the highest levels of Notch activity retain the progenitor fate, while intermediate levels of Notch activity generate inhibitory neurons, and daughters with very low levels of Notch signaling adopt the excitatory fate. Therefore, Notch-mediated binary cell fate choice is a mechanism for regulating the ratio of excitatory to inhibitory neurons from common progenitors.
Display omitted
•GABAergic and glutamatergic cerebellar neurons are generated from Sox2+ progenitors•Single Sox2+ ECPs can give rise to both excitatory and inhibitory cerebellar neurons•Notch activity mediates GABAergic versus glutamatergic cell fates in Sox2+ ECPs
Zhang et al. report that excitatory and inhibitory neurons of the cerebellum can arise from individual embryonic cerebellar progenitors (ECPs). Notch activity controls the fate of bi-potential ECP daughters whereby cells with higher Notch activity adopt an inhibitory cell fate, whereas cells with lower Notch activity adopt an excitatory fate.
•We present here the first IonTorrent PGM profiling of the human saliva microbiome.•The saliva microbiome complexity increases after short-term probiotic intake.•Streptococcus, Actinomyces and Rothia ...characterize the post-probiotic microbiome.•The computational pipelines need to be adapted to the IonTorrent characteristics.•We suggest IonTorrent PGM as an overall good compromise for 16S rRNA sequencing.
Microbial communities populating several human body habitats are important determinants of human health. Cultivation-free community-wide approaches like bacterial 16S rRNA sequencing recently revolutionized the study of such human-associated microbial diversity, and the continuously decreasing cost/throughput ratio of current sequencing platforms is further enhancing the availability and effectiveness of microbiome research. The IonTorrent PGM platform is among the latest available commercial high-throughput sequencing tools, but it is just starting to be used for 16S rRNA surveys with only episodic assessments of its performance. We present here the first IonTorrent profiling of the human saliva microbiome collected from 12 healthy individuals. In this cohort, a subset of the volunteers was asked to assume a probiotic product, in order to investigate its impact on the composition and the structure of the saliva microbiome. Analysis of the generated dataset suggests the suitability of the IonTorrent platform for 16S rRNA surveys, even though some platform-specific choices are required to optimize the consistency of the obtained bacterial profiles. Interestingly, we found a marked and statistically significant increase of the overall bacterial diversity in the saliva of individuals who received the probiotic product compared to the control group, suggesting a short-term effect of probiotic product administration on oral microbiome composition.
PDF
