Efficient gene editing in induced pluripotent stem cells enabled by an inducible adenine base editor with tunable expression.

The preferred method for disease modeling using induced pluripotent stem cells (iPSCs) is to generate isogenic cell lines by correcting or introducing pathogenic mutations. Base editing enables the precise installation of point mutations at specific genomic locations without the need for deleterious double-strand breaks used in the CRISPR-Cas9 gene editing methods. We created a bulk population of iPSCs that homogeneously express ABE8e adenine base editor enzyme under a doxycycline-inducible expression system at the AAVS1 safe harbor locus. These cells enabled fast, efficient and inducible gene editing at targeted genomic regions, eliminating the need for single-cell cloning and screening to identify those with homozygous mutations. We could achieve multiplex genomic editing by creating homozygous mutations in very high efficiencies at four independent genomic loci simultaneously in AAVS1-iABE8e iPSCs, which is highly challenging with previously described methods. The inducible ABE8e expression system allows editing of the genes of interest within a specific time window, enabling temporal control of gene editing to study the cell or lineage-specific functions of genes and their molecular pathways. In summary, the inducible ABE8e system provides a fast, efficient and versatile gene-editing tool for disease modeling and functional genomic studies.

Generation and characterization of induced pluripotent stem cell line IITGi001-A derived from adult human primary dermal fibroblasts.

Adult human primary dermal fibroblasts [ATCC (PCS-201-012)] were reprogrammed by transfection of oriP/EBNA-1 based episomal plasmids expressing OCT3/4, SOX2, KLF4, L-MYC, LIN28 and a p53 shRNA (Okita et al., 2011) to give rise to induced pluripotent stem cells (iPSCs). These iPSCs expressed core pluripotency markers, maintained normal karyotype, and showed tri-lineage differentiation potential. Further, the absence of episomal plasmid integration in this iPSC line was confirmed by genomic PCR. In addition, DNA fingerprinting of fibroblast and iPSC DNA by microsatellite analysis confirmed the genetic identity of this cell line. This iPSC line was shown to be free from mycoplasma contamination.

Derivation of Clinical-Grade Induced Pluripotent Stem Cell Lines from Erythroid Progenitor Cells in Xenofree Conditions.

One of the major hurdles in realizing the therapeutic potential of human-induced pluripotent stem cells (iPSC) is the generation of clinical-grade iPSC lines and their differentiated progenies for preclinical and clinical applications. Therefore, there is a need to have standardized protocols for efficient generation of clinical-grade iPSC lines from easily accessible somatic cells in feeder-free, xenofree GMP grade culture conditions without genomic integration of the reprogramming factors. Here, we provide a detailed protocol for expansion of erythroid progenitor cells from peripheral blood mononuclear cells (PBMNC) and generation of iPSC lines in feeder-free and xenofree culture conditions from these cells by using GMP grade reagents. With this optimized protocol, clinical-grade iPSC lines can be derived from erythroid progenitor cells expanded from peripheral blood, which is easy-to-access, minimally invasive, and can be obtained from any donors. It will have implications in developing a large number of iPSC lines from individual healthy donors, diseased patients, or donors with homozygous human leukocyte antigen (HLA) for "haplobanking."