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1.
PLoS One ; 13(7): e0200657, 2018.
Article in English | MEDLINE | ID: mdl-30024920

ABSTRACT

Mutations in the gene Adenomatous Polyposis Coli or APC appear in most sporadic cases of colorectal cancer and it is the most frequent mutation causing hereditary Familial Adenomatous Polyposis. The detailed molecular mechanism by which APC mutations predispose to the development of colorectal cancer is not completely understood. This is in part due to the lack of accessibility to appropriate models that recapitulate the early events associated with APC mediated intestinal transformation. We have established a novel platform utilizing human induced Pluripotent Stem cells or iPSC from normal or FAP-specific APC mutant individuals and evaluated the effect of the mutation in the cells before and after differentiation into intestinal organoids. In order to minimize genetic background effects, we also established an isogenic platform using TALEN-mediated gene editing. Comparison of normal and APC mutant iPSC revealed a significant defect in cell identity and polarity due to the presence of APC in heterozygosity as well as chromosomal aberrations including abnormal anaphases and centrosome numbers. Importantly, upon specification into intestinal progeny, APC heterozygosity was responsible for a major change in the transcriptional identity of the cells with dysregulation of key signaling pathways, including metabolic reprogramming, abnormal lipid metabolism and intestinal-specific cadherin expression. In conclusion, we have developed a novel iPSC/intestinal model of APC mutagenesis and provide strong evidence that APC in heterozygosity imparts a clear phenotypic and molecular defect, affecting basic cellular functions and integrity, providing novel insights in the earlier events of APC-mediated tumorigenesis.


Subject(s)
Adenomatous Polyposis Coli Protein/genetics , Adenomatous Polyposis Coli/genetics , Induced Pluripotent Stem Cells/metabolism , Mutagenesis , Mutation , Adenomatous Polyposis Coli/pathology , Cell Differentiation/genetics , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Gene Expression Regulation, Developmental , HEK293 Cells , Humans , Induced Pluripotent Stem Cells/cytology , Intestines/pathology , Organoids/metabolism , Signal Transduction/genetics
2.
Stem Cell Reports ; 8(4): 1076-1085, 2017 04 11.
Article in English | MEDLINE | ID: mdl-28111279

ABSTRACT

Sickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH-funded NextGen Consortium, we generated a diverse, comprehensive, and fully characterized library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs) from patients of different ethnicities, ß-globin gene (HBB) haplotypes, and fetal hemoglobin (HbF) levels. iPSCs stand to revolutionize the way we study human development, model disease, and perhaps eventually, treat patients. Here, we describe this unique resource for the study of sickle cell disease, including novel haplotype-specific polymorphisms that affect disease severity, as well as for the development of patient-specific therapeutics for this phenotypically diverse disorder. As a complement to this library, and as proof of principle for future cell- and gene-based therapies, we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS) mutation.


Subject(s)
Anemia, Sickle Cell/genetics , Anemia, Sickle Cell/therapy , CRISPR-Cas Systems , Genetic Therapy , Hemoglobin, Sickle/genetics , Induced Pluripotent Stem Cells/metabolism , beta-Globins/genetics , Adolescent , Adult , Anemia, Sickle Cell/blood , Anemia, Sickle Cell/ethnology , Base Sequence , Cell Line , Cells, Cultured , Child , Child, Preschool , Erythroid Cells/cytology , Erythroid Cells/metabolism , Female , Fetal Hemoglobin/analysis , Genetic Therapy/methods , Haplotypes , Humans , Induced Pluripotent Stem Cells/cytology , Male , Middle Aged , Point Mutation , Polymorphism, Genetic , Transcriptome , Young Adult
3.
PLoS One ; 7(12): e51711, 2012.
Article in English | MEDLINE | ID: mdl-23272148

ABSTRACT

Delivery of the transcription factors Oct4, Klf4, Sox2 and c-Myc via integrating viral vectors has been widely employed to generate induced pluripotent stem cell (iPSC) lines from both normal and disease-specific somatic tissues, providing an invaluable resource for medical research and drug development. Residual reprogramming transgene expression from integrated viruses nevertheless alters the biological properties of iPSCs and has been associated with a reduced developmental competence both in vivo and in vitro. We performed transcriptional profiling of mouse iPSC lines before and after excision of a polycistronic lentiviral reprogramming vector to systematically define the overall impact of persistent transgene expression on the molecular features of iPSCs. We demonstrate that residual expression of the Yamanaka factors prevents iPSCs from acquiring the transcriptional program exhibited by embryonic stem cells (ESCs) and that the expression profiles of iPSCs generated with and without c-Myc are indistinguishable. After vector excision, we find 36% of iPSC clones show normal methylation of the Gtl2 region, an imprinted locus that marks ESC-equivalent iPSC lines. Furthermore, we show that the reprogramming factor Klf4 binds to the promoter region of Gtl2. Regardless of Gtl2 methylation status, we find similar endodermal and hepatocyte differentiation potential comparing syngeneic Gtl2(ON) vs Gtl2(OFF) iPSC clones. Our findings provide new insights into the reprogramming process and emphasize the importance of generating iPSCs free of any residual transgene expression.


Subject(s)
Epigenesis, Genetic , Gene Expression Profiling , Induced Pluripotent Stem Cells/metabolism , Transcription Factors/genetics , Transcription, Genetic , Animals , Cell Differentiation/genetics , Cell Line , Cellular Reprogramming , Cluster Analysis , DNA Methylation , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression , Gene Expression Regulation, Developmental , Gene Silencing , Hepatocytes/cytology , Hepatocytes/metabolism , Induced Pluripotent Stem Cells/cytology , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/metabolism , Male , Mice , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Transcription Factors/metabolism , Transgenes
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