Analysis of the stability of 70 housekeeping genes during iPS reprogramming

Studies on induced pluripotent stem (iPS) cells highly rely on the investigation of their gene expression which requires normalization by housekeeping genes. Whether the housekeeping genes are stable during the iPS reprogramming, a transition of cell state known to be associated with profound changes, has been overlooked. In this study we analyzed the expression patterns of the most comprehensive list to date of housekeeping genes during iPS reprogramming of a mouse neural stem cell line N31. Our results show that housekeeping genes’ expression fluctuates significantly during the iPS reprogramming. Clustering analysis shows that ribosomal genes’ expression is rising, while the expression of cell-specific genes, such as vimentin (Vim) or elastin (Eln), is decreasing. To ensure the robustness of the obtained data, we performed a correlative analysis of the genes. Overall, all 70 genes analyzed changed the expression more than two-fold during the reprogramming. The scale of this analysis, that takes into account 70 previously known and newly suggested genes, allowed us to choose the most stable of all genes. We highlight the fact of fluctuation of housekeeping genes during iPS reprogramming, and propose that, to ensure robustness of qPCR experiments in iPS cells, housekeeping genes should be used together in combination, and with a prior testing in a specific line used in each study. We suggest that the longest splice variants of Rpl13a, Rplp1 and Rps18 can be used as a starting point for such initial testing as the most stable candidates.

CBIO-17. IDENTIFYING A NETWORK OF ESSENTIAL & TUMORIGENIC GENES IN GLIOBLASTOMA USING WHOLE-GENOME CRISPR Cas9 SCREENING

GBM is the most common primary malignant brain tumor in adults, with a 100% recurrence rate and a median survival of 21 months. As such, advances in therapy are desperately needed. Genomic approaches have shown that GBM has high intra-tumoral heterogeneity. However, a comprehensive understanding of determinants of growth is required to identify new therapeutic targets. CRISPR-Cas9 screening technology has enabled whole-genome screens that allow objective identification of genes governing specific phenotypes. Here, we performed a genome-wide CRISPR knockout screen in H4 human glioma cells to identify genes that drive proliferation. Our screen identified ~150 novel essential growth genes. From this list, we identified 5 genes that were previously unstudied, show significant elevations in expression at the RNA and protein levels (p< 0.05), and show significant survival benefit in patient datasets (p< 0.05) – PSMB3, CHCHD4, THOC1, SPDYE5, HSPA1. Our validation experiments showed that knocking out these genes resulted in cell death in multiple GBM patient-derived xenograft (PDX) lines. In addition, animals with knockout cells implanted demonstrated extended survival (p< 0.01). Furthermore, overexpression of these genes in a normal neural stem cell line resulted in transformation to a cancer phenotype, as evidenced by sphere formation in a soft agar assay (p< 0.01). Further investigation of one of these genes, PSMB3, which is a subunit of the proteasome, showed that ubiquitinated proteins were significantly increased after PSMB3 knockdown, suggesting that disruption of the proteasome system was the likely cause of cell death. We performed a ubiquitin immunoprecipitation (IP) to identify which genes were being uniquely ubiquitinated in GBM. Results showed pathways involving retinoblastoma genes, DNA repair genes, and DNA replication. Together, this data suggests that both our CRISPR screens and our ubiquitin IP have yielded promising and novel therapeutic targets for GBM, a disease desperately in need of new strategies.

Establishment and Characterization of Immortalized Minipig Neural Stem Cell Line

Despite the increasing importance of minipigs in biomedical research, there has been relatively little research concerning minipig-derived adult stem cells as a promising research tool that could be used to develop stem cell-based therapies. We first generated immortalized neural stem cells (iNSCs) from primary minipig olfactory bulb cells (pmpOBCs) and defined the characteristics of the cell line. Primary neural cells were prepared from minipig neonate olfactory bulbs and immortalized by infection with retrovirus carrying the v-myc gene. The minipig iNSCs (mpiNSCs) had normal karyotypes and expressed NSC-specific markers, including nestin, vimentin, Musashi1, and SOX2, suggesting a similarity to human NSCs. On the basis of the global gene expression profiles from the microarray analysis, neurogenesis-associated transcript levels were predominantly altered in mpiNSCs compared with pmpOBCs. These findings increase our understanding of minipig stem cells and contribute to the utility of mpiNSCs as resources for immortalized stem cell experiments.