Generation of iPSCs from Genetically Corrected Brca2 Hypomorphic Cells: Implications in Cell Reprogramming and Stem Cell Therapy

Abstract

Fanconi anemia (FA) is a complex genetic disease associated with a defective DNA repair pathway known as the FA pathway. In contrast to many other FA proteins, BRCA 2 participates downstream in this pathway and has a critical role in homology-directed recombination (HDR). In our current studies, we have observed an extremely low reprogramming efficiency in cells with a hypomorphic mutation in Brca2 (Brca2), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2 D27/D27 induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2 D27/D27 iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2 D27/D27 mouse embryonic fibroblasts. Gene-corrected Brca2 D27/D27 iPSCs could be differentiated in vitro toward the hematopoietic lineage, although with a more limited efficacy than WT iPSCs or mouse embryonic stem cells, and did not engraft in irradiated Brca2 D27/D27 recipients. Our results are consistent with previous studies proposing that HDR is critical for cell reprogramming and demonstrate that reprogramming defects characteristic of Brca2 mutant cells can be efficiently overcome by gene complementation. Finally, based on analysis of the phenotype, genetic stability, and hematopoietic differentiation potential of gene-corrected Brca2 D27/D27 iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed. STEM CELLS 2014;32:436-446

Fanconi anemia: a model disease for studies on human genetics and advanced therapeutics

“…Because FA fibroblasts are difficult to reprogram, this study uncovered a novel role of the FA pathway in cell reprogramming. Consequently, correcting FA genes restores the reprogramming efficiency of FA fibroblasts to IPS cells [93][94][95][96][97][98]. Reprogramming induces the DDR [99] and activates the FA pathway [94], leading to P53-mediated apoptosis and low reprogramming efficiency [96].…”

High-Risk Human Papillomavirus E6 Protein Promotes Reprogramming of Fanconi Anemia Patient Cells through Repression of p53 but Does Not Allow for Sustained Growth of Induced Pluripotent Stem Cells

“…Recent studies have established that cells from both mice and humans with defects in the FA pathway undergo somatic cell reprogramming at dramatically reduced efficiencies (25)(26)(27)(28)39). We previously immortalized skin keratinocytes from individuals with FA using the high-risk HPV E6 and E7 oncogenes (36).…”

Therapeutic gene editing in hematopoietic progenitor cells from a mouse model of Fanconi anemia

“…MEFs both from FVB/NJ WT and FVB FA-A mice were obtained from the chorion of 13.5 E pregnant females as has been described previously (Navarro et al, 2014). MEFs from WT or FA-A mice were immortalized by a transient transfection with pLXSN 16 E6E7 and pCL-ECO-gag-pol (Naviaux et al, 1996) using the CaCl 2 DNA precipitation method and cultured also in DMEM 1X with GlutaMAX TM (Gibco) with 10% Hyclone (GE Healthcare) and 1% Penicillin/Streptomycin (P/S) (Gibco).…”