chir-99021 and Teratoma

Advances in differentiation of cardiomyocytes from human induced pluripotent stem cell (hiPSC) were emerged as a tool for modeling of cardiovascular disease that recapitulates the phenotype for the purpose of drug screening, biomarker discovery, and testing of single-nucleotide polymorphism (SNP) as a modifier for disease stratification. Here, we describe the (1) retroviral reprogramming strategies in the generation of human iPSC, (2) methodology in characterization of iPSC in order to identify the stem cell clones with the best quality, and (3) protocol of cardiac differentiation by modulation of Wnt signaling and β-catenin pathway.

Topics: Amides; Animals; beta Catenin; Biomarkers; Cell Culture Techniques; Cell Differentiation; Cellular Reprogramming; Collagen; Drug Combinations; Embryo, Mammalian; Embryoid Bodies; Enzyme Inhibitors; Feeder Cells; Fibroblasts; Gene Expression; Genetic Vectors; Humans; Induced Pluripotent Stem Cells; Intercellular Signaling Peptides and Proteins; Laminin; Mice; Myocytes, Cardiac; Primary Cell Culture; Proteoglycans; Pyridines; Pyrimidines; Retroviridae; Teratoma; Wnt Signaling Pathway

The human naive pluripotent stem cell (PSC) state, corresponding to a pre-implantation stage of development, has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate, a normal karyotype, the ability to form teratomas, transcriptional similarities to human pre-implantation embryos, reduced heterochromatin levels, and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP(-/-) cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state, with implications for early human embryology.

Topics: Adaptor Proteins, Signal Transducing; Benzamides; Blastocyst; Cell Differentiation; Cells, Cultured; CRISPR-Cas Systems; Diphenylamine; Embryonic Stem Cells; Female; Gene Expression Regulation; Glycogen Synthase Kinase 3; Heterochromatin; Humans; Induced Pluripotent Stem Cells; Karyotype; Lysophospholipids; Male; Phosphoproteins; Pluripotent Stem Cells; Pyridines; Pyrimidines; RNA Interference; Teratoma; Transcription Factors; Wnt Signaling Pathway; YAP-Signaling Proteins

Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3β and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.

Topics: Animals; Animals, Genetically Modified; Benzamides; Blastocyst; Cattle; Cells, Cultured; CRISPR-Cas Systems; Diphenylamine; Fertilization in Vitro; Fibroblasts; Gene Knock-In Techniques; Genetic Engineering; Genetic Enhancement; Genetic Loci; Genetic Vectors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Homologous Recombination; Induced Pluripotent Stem Cells; Mice; Mice, SCID; Polymorphism, Restriction Fragment Length; Pyridines; Pyrimidines; Recombinant Fusion Proteins; Teratoma; Transcription Factors; Valproic Acid