Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from diploid and aneuoploid human pluripotent stem cells-derived teratomas

ABSTRACT: Teratoma formation is the gold standard assay for testing the capacity of human stem cells to differentiate into all embryonic germ layers. Although widely used, little effort has been made to transform this qualitative assay into a quantitative one. Using gene expression data from a wide variety of cells, we created a gene scorecard representing tissues from all three germ layers as well as an extraembryonic tissue. A calculated grade using this gene list successfully distinguishes pluripotent stem cell-initiated teratomas from malignant tumors, thereby translating cell potency into a quantitative measure. This new methodology, named TeratoScore, thus assesses the pluripotency of human cells, and is easily performed using an open-source code. The new teratoma database also allowed us to examine the gene expression differences between tumors with a diploid karyotype and those initiated by aneuploid cells. We found that while teratomas originating from aneuploid cells pass the TeratoScore benchmark for pluripotency, they exhibit aberrant gene expression congruent with human chromosomal syndromes (such as Down syndrome). This gene expression signature is significantly different from that of teratomas originating from diploid cells, particularly in central nervous system-specific genes, suggesting aberrant teratomas may be beneficial for in vivo disease modeling. Teratoma formation followed by TeratoScore analysis can rapidly assess cell potency and allows comparison between different pluripotent cell lines. Total RNA was extracted from hPSC (diploid or aneuploid)-derived teratomas using RNeasy mini kit (Qiagen), according to the manufacture instruction. RNA was subjected to Human Genome U133 Plus 2.0 microarray platform (Affymetrix); washing and scanning were performed according to the manufacturer’s protocol

ORGANISM(S): Homo sapiens

SUBMITTER: Yishai Avior

PROVIDER: E-GEOD-64669 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Studying early human developmental processes requires access to a model system that is easy to perturb, either genetically or chemically, genetically tractable and recapitulates key features of organogenesis. We propose that the teratoma, a recognized standardused for for validating pluripotency in stem cells, could be a promising platform for modeling human development. Teratomas differentiate to all germ layers, have regions of complex tissue-like organization with internal vasculature, and are relatively easy to generate and thus accessible, especially in comparison to human fetal tissue. Teratomas, however, have not been characterized rigorously to determine their suitability to model human developmental processes the diversity and identities of the cell types present, and their level of maturity in comparison to human developmental cell types. We thus systematically analyzed, perturbed, and engineered human pluripotent stem cell (PSC)-derived teratomas. Usingused histology, RNA in situ hybridization, and single cell RNA-seq (scRNA-seq) of 179,632 cells across 23 teratomas generated from 4 cell lines, we observed to find that teratomas reproducibly contain approximatelyat least 20 cell types across all three germ layers, and the cell type heterogeneity between teratomas was comparable to that found in organoid systems. We found that a significant fraction of injected PSCs engrafted, suggestingused lentiviral barcoding to determine there is minimalno bottlenecking during teratoma formation. Additionally, we mapped our teratoma data to published human fetal data and showed that the teratoma gut and brain cell types correspond well to existing fetal gut and brain scRNA-seq datasetscells. We then performed a CRISPR-Cas9 knockout screen in teratomas targeting key genes known to be embryonic lethal, and found that TWIST1, RUNX1, ASCL1, CDX2, and KLF6 knockouts resulted in assessed shifts in lineage abundance. using scRNA-seq. We found that TWIST1, RUNX1, ASCL1, CDX2, and KLF6 knockouts resulted in reproducible shifts in lineage abundance consistent with known biology. All of these These knockouts had effects on cell types from multiple germ layers, showing that the teratoma can serve as a platform to studymodel all major human lineages of human development simultaneously. Additionally, we ran a CRISPR-Cas9 screen targetingWe also knocked out the genes underlying Rett, Pitt-Hopkins, and L1 Syndromes and identified cell type specific shifts in gene expression. Finally, we engineered a novel miRNA-regulated suicide gene circuit that enriches for tissues in the teratoma that express a specific miRNA that selectively depletes cells that do not express a tissue-specific endogenous miRNA, allowing us to enrich for desired lineages in the teratoma, which we demonstrated using miR-124 to enrich for neuro-ectoderm. Specifically, we used miR-124 to successfully enrich for neuro-ectoderm. Taken together, we believe the teratoma is a promising platform for modeling multi-lineage human development, functional genetic screening, and cellular engineering

Project description:Human pluripotent stem cells (hPSCs) tend to acquire genomic aberrations in culture, the most common of which is the trisomy of chromosome 12. Interestingly, trisomy 12 is also prevalent in germ cell tumors (GCTs). Here, we aimed to dissect the cellular and molecular implications of trisomy 12 in hPSCs. A genome-wide gene expression analysis revealed that trisomy 12 profoundly affects the global gene expression profile of hPSCs, inducing a transcriptional program very similar to that of CGTs. Direct comparison of the proliferation, replication, differentiation and apoptosis between diploid and aneuploid hPSCs revealed that trisomy 12 significantly increases the proliferation rate of hPSCs. Increased replication largely accounts for the increased proliferation observed, and may explain the selection advantage that trisomy 12 confers to hPSCs. A comparison of the tumors induced by diploid and aneuploid hPSCs further demonstrated that trisomy 12 increases the tumorigenicity of hPSCs, inducing transcriptionally-distinct teratomas, from which pluripotent cells can be recovered. Lastly, a chemical screen of 89 anticancer drugs against diploid and aneuploid hPSCs discovered that trisomy 12 raises the sensitivity of hPSCs to several replication inhibitors, suggesting that the increased proliferation and tumorigenicity of these aberrant cells also makes them more vulnerable, and might potentially be used for their selective elimination from culture. Together, our findings demonstrate the extensive effect of trisomy 12 on the gene expression signature and on the cellular behavior of hPSCs, and highlight the danger posed by this trisomy for the successful use of hPSCs in basic research and in regenerative medicine. Expression data from diploid and aneuoploid human pluripotent stem cells, teratomas derived from them, and pluripotent-like cells recovered from these teratomas total RNA was isolated from undifferentiated human pluripotent stem cells grown under standard human ES conditions, or from teratomas derived from them, or from ES-like cells recovered from these teratomas.

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Expression data from diploid and aneuoploid human pluripotent stem cells-derived teratomas

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