Dataset Information

Generation of Human Fatty Livers Using Custom-Engineered Induced Pluripotent Stem Cells with Modifiable SIRT1 Metabolism.

ABSTRACT: The mechanisms by which steatosis of the liver progresses to non-alcoholic steatohepatitis and end-stage liver disease remain elusive. Metabolic derangements in hepatocytes controlled by SIRT1 play a role in the development of fatty liver in inbred animals. The ability to perform similar studies using human tissue has been limited by the genetic variability in man. We generated human induced pluripotent stem cells (iPSCs) with controllable expression of SIRT1. By differentiating edited iPSCs into hepatocytes and knocking down SIRT1, we found increased fatty acid biosynthesis that exacerbates fat accumulation. To model human fatty livers, we repopulated decellularized rat livers with human mesenchymal cells, fibroblasts, macrophages, and human SIRT1 knockdown iPSC-derived hepatocytes and found that the human iPSC-derived liver tissue developed macrosteatosis, acquired proinflammatory phenotype, and shared a similar lipid and metabolic profiling to human fatty livers. Biofabrication of genetically edited human liver tissue may become an important tool for investigating human liver biology and disease.

SUBMITTER: Collin de l'Hortet A

PROVIDER: S-EPMC6691905 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Publications

Generation of Human Fatty Livers Using Custom-Engineered Induced Pluripotent Stem Cells with Modifiable SIRT1 Metabolism.

Collin de l'Hortet Alexandra A Takeishi Kazuki K Guzman-Lepe Jorge J Morita Kazutoyo K Achreja Abhinav A Popovic Branimir B Wang Yang Y Handa Kan K Mittal Anjali A Meurs Noah N Zhu Ziwen Z Weinberg Frank F Salomon Michael M Fox Ira J IJ Deng Chu-Xia CX Nagrath Deepak D Soto-Gutierrez Alejandro A

Cell metabolism 20190801 2

The mechanisms by which steatosis of the liver progresses to non-alcoholic steatohepatitis and end-stage liver disease remain elusive. Metabolic derangements in hepatocytes controlled by SIRT1 play a role in the development of fatty liver in inbred animals. The ability to perform similar studies using human tissue has been limited by the genetic variability in man. We generated human induced pluripotent stem cells (iPSCs) with controllable expression of SIRT1. By differentiating edited iPSCs int ...[more]

PMID: 31390551

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Project description:BackgroundPigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown.MethodsiPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis.ResultsIn this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable "dome-shaped" morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%.ConclusionsOur findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.

Dataset Information

Generation of Human Fatty Livers Using Custom-Engineered Induced Pluripotent Stem Cells with Modifiable SIRT1 Metabolism.

Publications

Generation of Human Fatty Livers Using Custom-Engineered Induced Pluripotent Stem Cells with Modifiable SIRT1 Metabolism.

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