Increasing Gene Editing Efficiency for CRISPR-Cas9 by Small RNAs in Pluripotent Stem Cells.

Gene manipulations of human induced pluripotent stem cells (iPSCs) by CRISPR-Cas9 genome engineering are widely used for disease modeling and regenerative medicine applications. There are two competing pathways, non-homologous end joining (NHEJ) and homology directed repair (HDR) that correct the double-strand break generated by CRISPR-Cas9. Here, we improved gene editing efficiency of gene knock-in (KI) in iPSCs with minimum components by manipulating the Cas9 expression vector.

Generation of a human iPSC line harboring a biallelic large deletion at the INK4 locus (HMGUi001-A-5).

The INK4 locus is considered as a hot-spot region for the complex genetic disorders, including cancer, type 2 diabetes (T2D) and coronary artery disease (CAD). By CRISPR/Cas9 gene editing, we generated a human induced pluripotent stem cell (hiPSC) line (HMGUi001-A-5) deleting an 8 kb genomic DNA encompassing six T2D-associated SNPs at the INK4 locus. The resulting hiPSC line revealed a normal karyotype, preserved pluripotency and was able to differentiate towards germ layers, endoderm, mesoderm and ectoderm.

Generation of a homozygous ARX nuclear CFP (ARX) reporter human iPSC line (HMGUi001-A-4).

The aristaless related homeobox (ARX) transcription factor plays a crucial role in glucagon-producing α-cell differentiation. Here, we generate an ARX reporter iPSC line by 3' fusion of an intervening viral T2A sequence followed by a nuclear-localized histone 2B-cyan fluorescent protein (nCFP). The resulting cells have a normal karyotype and preserved pluripotency.

Endogenous WNT signaling regulates hPSC-derived neural progenitor cell heterogeneity and specifies their regional identity.

Neural progenitor cells (NPCs) derived from human pluripotent stem cells (hPSCs) are a multipotent cell population that is capable of nearly indefinite expansion and subsequent differentiation into the various neuronal and supporting cell types that comprise the CNS. However, current protocols for differentiating NPCs toward neuronal lineages result in a mixture of neurons from various regions of the CNS. In this study, we determined that endogenous WNT signaling is a primary contributor to the heterogeneity observed in NPC cultures and neuronal differentiation.

Analysis of SOX2-expressing cell populations derived from human pluripotent stem cells.

SOX2 is involved in several cell and developmental processes, including maintenance of embryonic stem cells, differentiation of neural progenitor cells, and patterning of gut endoderm. To study its role in a human system, we generated a human embryonic stem cell (hESC) line harboring a reporter gene encoding GFP in the SOX2 locus. This SOX2 reporter line faithfully recapitulates expression of the SOX2 gene in undifferentiated human pluripotent stem cells (hPSCs), neural progenitor cells (NPCs), and anterior foregut endoderm (AFE).

A hormone-dependent module regulating energy balance.

Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however.