Cardenas, Fabian Leonardo
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PublicationModeling Monogenic Diabetes Mody3 Using Human Pluripotent Stem Cells(2018-01-01) Cardenas, Fabian LeonardoUnderstanding monogenic diabetes has been challenging due to the lack of human model but also because mouse models do not recapitulate the disease. Here, we use human pluripotent stem cells (ESCs) and CRISPR-CAS9 (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas9) nucleases to understand the most common form of monogenic diabetes, Maturity Onset Diabetes of the Young 3 (MODY3) caused by heterozygous mutations in the transcription factor HNF1A. In this thesis research, we found that the lack of HNF1A does not impair the generation of beta-like cells; Wild-type (WT) and mutant cell lines presented similar differentiation efficiency between genotypes. HNF1A is necessary to repressed alpha cell fate; loss of HNF1A leads skew beta-like cell differentiation towards alpha cell type. We also explore beta cell function related to insulin secretion and cellular bioenergetics; we found that HNF1A needs it for proper insulin secretion and cellular respiration leading to lower glycolysis metabolism and mitochondrial respiration. We explore the HNF1A relationship between phenotype and genotype by generating a genome profiling analysis of HNF1A. We MODY3 models confirmed that many of the genes that are dis-regulated in the Hnf1α mouse model such us HNF4A, PCSK1, GC, DDP4, KIF12 6GPC2 and others. Besides, we also found a large number of genes to be uniquely dis-regulated in the human model, which could explain the species-specific phenotype. These genes include the transcription factors RFX6, GLIS3 and PAX4, the metallothionein gene family a novel long non-coding (lnc)RNA LINC01139. This lncRNA is only present in primates, and also it is expressed in human endocrine cells. We interrogate the role of this lncRNA, and we found that it recapitulates a subset of phenotypes seen in HNF1A, related with defects in glycolysis, mitochondrial respiration, and downregulation of group of genes downstream of HNF1A. Our work offers a model to define the unique biology of HNF1A in humans that may be distinct from rodent models leading to a better understanding of human beta cell physiology with clinical implications for MODY3 patients but also with broader applications to diabetes. PublicationEssential Role for Endogenous siRNAs during Meiosis in Mouse Oocytes(2015-02-19) Cárdenas, Fabián L; Davydenko, Olga; Stein, Paula; Vandivier, Lee; Rozhkov, Nikolay V; Gregory, Brian D; Li, Fan; Schultz, Richard M; Hannon, Gregory JIn animals, the three main classes of small RNAs are microRNAs, short interfering RNAs, and PIWI-interacting RNAs. All three RNA species silence gene expression post-transcriptionally through interaction with the ARGONAUTE family of proteins. In mammals in particular, microRNAs are ubiquitously expressed, are essential for development, and perform numerous functions in a variety of cells and tissues. piRNAs are expressed almost exclusively in the germline, and are essential for male fertility and defense against transposons. Endogenous siRNAs are only expressed in germ cells and embryonic stem cells and have not been ascribed a functional role. By engineering a mouse that expresses a modified ARGONAUTE protein, we disrupt the function of endo-siRNAs exclusively in oocytes and find that females are infertile. Oocytes with an impaired siRNA pathway fail to complete meiosis I, and display severe spindle formation and chromosome alignment defects. Their transcriptome is widely perturbed and expression of the most abundant transposon is increased. These findings indicate that endo-siRNAs are essential for female fertility in mouse, are required for spindle formation, chromosome congression, and defense against transposons. This study unequivocally demonstrates an essential function for siRNAs in mammals, mediated through endonucleolytic cleavage of targets, and provides an explanation for the selective pressure that one AGO protein retains catalytic activity.