Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A Phase contrast microscope images showing the phenotype and morphology of the cells in the course of conversion of fibroblasts to iHeps at different times points after transduction with a cocktail of three TFs HNF1A, HNF4A and FOXA3 . B Generation of highly proliferative iHep cells by transducing iHeps with two pools of liver cancer-specific oncogenic drivers, a list of xenograft experiments in nude mice that were used to test the tumorigenicity of different conditions, and mutation rates of the oncogenic drivers as reported in the COSMIC database for HCC and MYC amplification as reported in . CMT pool contains three oncogenes CTNNB1 T41A , MYC, and TERT, and CMT + sg TP53 pool contains the same oncogenes along with constructs for TP53 inactivation by CRISPR-Cas9. Phase contrast microscope images showing the phenotype and morphology of the cells. Oncogenes are co-transduced with fluorescent reporter mCherry for the detection of transduced cells. Oncogene transduction to fibroblasts fails to transform the cells, passaging of oncogene-expressing fibroblasts results in cellular senescence as demonstrated by β-galactosidase staining and loss of mCherry-positive oncogene-expressing cells from the fibroblast population. iHeps maintained in defined culture medium become senescent around week four of transdifferentiation although they can survive in culture for several weeks after that if not passaged. Passaging of iHeps without oncogenes results in apoptosis after few passages. Scale bar 1000 μm unless otherwise specified.

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Microscopy, Transduction, Mutagenesis, Amplification, Construct, CRISPR, Passaging, Expressing, Staining

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A Subcutaneous injection of transformed iHeps results in xenograft tumors in nude mice (tumor size of 1.5 cm ~23 weeks after xenotransplantation). Proliferative iHeps transduced with defined CMT oncogenes with TP53 inactivation (CMT + sg TP53 ) or control iHeps without oncogenes were used in the injections. B In vivo imaging of xenograft tumors ~12 weeks after implantation. Two biological replicate experiments are shown for CMT + sg TP53 cells with iHep conversion and oncogene transduction with TP53 inactivation performed in two separate human fibroblast cell lines (foreskin fibroblast [left panel] and fetal lung fibroblast [middle]) as well as proliferative CMT iHeps without TP53 inactivation (right). Fluorescence signal emitted by mCherry co-transduced with the oncogenes is detected in vivo using the Lago system (scale bar = radiance units). Control mice are injected with either fibroblasts or iHeps. C Histological analysis of CMT + sg TP53 tumor tissue harvested at 20 weeks. Hematoxylin-eosin (H&E) staining for general histology and immunohistochemical staining for Ki-67 for cell proliferation (100x magnification). Note that the appearance of the tumor is consistent with both poorly differentiated hepatic tumor or sarcoma. Differential diagnosis from sarcoma is accomplished by analysis of marker gene expression (see Figs. and ). D Analysis of chromosomal aberrations in the transformed iHeps by spectral karyotyping. CMT + sg TP53 cells were analyzed at passage 18 (early) and passage 50 (late) and CMT cells at passage 18. Fibroblasts have normal diploid karyotype (46, XY, representative spectral image on left) and transformed iHeps show aneuploidies as indicated in the figure. Early passage CMT + sg TP53 cells show two different populations with two distinct modal chromosome numbers (45, XY and 67-92, XY, representative spectral image for 45, XY on middle-left). Late passage CMT + sg TP53 cells have modal chromosome number 67-92, XY (middle-right) and CMT cells 75, XY (right). In the text box below the images, recurrent chromosomal aberrations seen in majority (>90%) of the cells are reported. E Frequencies of chromosomal alterations reported for human HCC samples [see ].

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Injection, Transformation Assay, Transduction, Control, In Vivo Imaging, Fluorescence, In Vivo, Staining, Immunohistochemical staining, Biomarker Discovery, Marker, Gene Expression

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A Gene set enrichment analysis (GSEA) results for CMT-iHeps and CMT + sg TP53 -iHeps compared to control fibroblasts against liver cancer signature [HCC Subclass 2 ] from molecular signatures database (MSigDB). Positive normalized enrichment score (NES) reflects overrepresentation of liver cancer signature genes among the top-ranked differentially expressed genes in CMT-iHep and CMT + sg TP53 -iHep conditions compared to control fibroblasts. B Differential expression levels [log 2 (fold change)] of marker genes for fibroblasts, hepatocytes, and liver cancer in bulk RNA-seq measurements from CMT + sg TP53 -iHeps and CMT-iHeps at p20 (~22 weeks after oncogene transduction) as well as xenograft tumor from CMT + sg TP53 cells against control fibroblasts (mean ± standard error, n = 3). C IGV snapshots for promoter regions of representative genes from fibroblast markers ( MMP3 ), liver markers ( SERPINA1/α-1-antitrypsin ), and liver cancer markers ( SAA1 ) showing ATAC-seq enrichment from fibroblast and CMT + sg TP53 -iHeps. D Chromatin accessibility and CpG methylation of DNA measured using NaNoMe-seq. Cytosine methylation detected using Nanopore sequencing from CMT + sg TP53 -iHeps and control fibroblasts is shown for promoter regions of representative genes from fibroblast markers ( MMP3 ), liver markers ( SERPINA1/α-1-antitrypsin ), and liver cancer markers ( SAA1 ) using a window of TSS ± 1500 bp. GpCpH methylation (all GC sequences where the C is not part of a CG sequence also, top) reports on chromatin accessibility, whereas HpCpG methylation reports on endogenous methylation of cytosines in the CpG context. E CpG methylation detected using bisulfite-sequencing from primary human foreskin fibroblasts and from the normal adult liver [data from the Roadmap Epigenomics Consortium ]. IGV snapshots from the genomic loci corresponding to the MMP3 , SERPINA1 , and SAA1 promoters (same regions as indicated in Fig. 5D) showing methylation proportions [methylated calls/(methylated calls + unmethylated calls)] for all CpGs covered by at least 4 reads.

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Control, Quantitative Proteomics, Marker, RNA Sequencing, Transduction, CpG Methylation Assay, Methylation, Nanopore Sequencing, Sequencing, Methylation Sequencing

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A Immunohistochemical analysis of xenograft tumor tissue from CMT + sg TP53 harvested at 20 weeks and xenograft tumor from the HuH7 HCC cell line. Staining for glypican-3 and arginase-1 are shown along with negative control without primary antibody (100x magnification). B (Top) Beta-galactosidase staining as a marker of cellular senescence in primary human hepatocytes (control), after transduction of CMT oncogenes, or after transduction with iHep-TFs (HNF1A, HNF4A, FOXA3) followed by CMT oncogene transduction one week later (stained three weeks after first transduction). (Middle) Beta-galactosidase staining as a marker of cellular senescence in control fibroblasts and fibroblasts transduced with CMT oncogenes and stained at p4. (Bottom) Fluorescent microscope images of induced neurons with and without transduction of neuroblastoma-specific oncogenes (at three weeks of neuronal differentiation) visualized using EGFP at ten weeks after neuronal conversion. C Schematic presentation of the molecular approach for identifying minimal determinants of tumorigenesis in specific tissues. Lineage-specific transcription factors are used to reprogram human fibroblasts to precise cellular identity (left), whose transformation by specific combinations of oncogenes (right) can then be tested. This approach combined with single-cell RNA-seq and RNA velocity analyses allows also analysis of which cell type along the stem cell to terminally differentiated cell axis (top to bottom) is susceptible for transformation.

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Immunohistochemical staining, Staining, Negative Control, Marker, Control, Transduction, Microscopy, Transformation Assay, RNA Sequencing

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A , B t-SNE plots of 3,500 single cells from fibroblasts, iHeps at one to three weeks after iHep induction, iHeps transduced with CMT oncogenes at one week and harvested for scRNA-seq two weeks later, and fibroblasts transduced with CMT oncogenes and harvested at one and three weeks. Cells are colored by sample ( A ), and distinct clusters ( B ) based on their expression profiles with sample collection time points indicated. C Principal component analysis (PCA) projection of single cells from control fibroblasts, iHeps at one to three weeks after iHep induction, and CMT-iHeps two weeks after oncogenes shown with velocity field with the observed states of the cells shown as circles and the extrapolated future states shown with arrows for the first two principal components. Cells are colored by cluster identities corresponding to Fig. 4B. D Relative expression of the genes from the Notch signaling pathway (panel on the right) across pseudotime in the single-cell RNA-seq data from control fibroblasts, iHeps at one to three weeks after iHep induction, and CMT-iHeps two weeks after oncogenes (the expression of a gene in a particular cell relative to the average expression of that gene across all cells). Relative expression of the senescence marker genes (panel on the left) from control fibroblasts and fibroblasts transduced with CMT oncogenes and harvested at one and three weeks after transduction. Color codes illustrating sample and cluster identities correspond to the colors in Fig. 4A, B, respectively. E Expression levels [log 2 (transcripts per million, tpm)] for LGR5 as well as lentiviral and endogenous HNF4A, TERT , and CTNNB1 in bulk RNA-seq measurements from control fibroblasts, iHeps at four weeks of differentiation, CMT + sg TP53 -iHeps at two and 22 weeks after oncogene transduction, xenograft tumor from CMT + sg TP53 cells, and from liver cancer cell lines HepG2 and HuH7 (mean ± standard error, n = 3). Nanopore sequencing was performed from the CMT + sg TP53 cells at 22 weeks after oncogene transduction as indicated in the figure and used for identifying the genomic insertions of the lentiviral constructs (Supplementary Table ).

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Transduction, Expressing, Control, RNA Sequencing, Marker, Nanopore Sequencing, Construct