期刊
STEM CELLS
卷 32, 期 2, 页码 436-446出版社
WILEY
DOI: 10.1002/stem.1586
关键词
Gene therapy; Induced pluripotent stem cells; Hematopoietic stem cells; Bone marrow aplasia; Cellular therapy; Fanconi anemia
资金
- European Commission [222878]
- Spanish Ministry of Economy and Competitiveness [Ref PLE 2009/0100, SAF 2009-07164, SAF2012-39834]
- Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III [RETICS-RD06/0010/0015, RD12/0019/0023]
- Direccion General de Investigacion de la Comunidad de Madrid (CellCAM) [Ref S2010/BMD-2420]
- La Fundacio Privada La Marato de TV3 [121430/31/32]
- ICREA Funding Source: Custom
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(27/27)), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2(27/27) induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2(27/27) iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2(27/27) mouse embryonic fibroblasts. Gene-corrected Brca2(27/27) 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(27/27) 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(27/27) iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed. Stem Cells2014;32:436-446
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