BRCA1 and 53BP1 regulate reprogramming efficiency by mediating DNA repair pathway choice at replication-associated double-strand breaks.

Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency. In contrast, cells deficient in HDR, but proficient in SFP and RGS, reprogram with reduced efficiency. Conversely, the restoration of HDR function through inactivation of 53bp1 rescues reprogramming in Brca1-deficient cells, and 53bp1 loss leads to elevated HDR and enhanced reprogramming in mouse and human cells. These results demonstrate that somatic cell reprogramming is especially dependent on repair of replication-associated double-strand breaks (DSBs) by the HDR activity of BRCA1 and BRCA2 and can be improved in the absence of 53BP1.

Reduced replication fork speed promotes pancreatic endocrine differentiation and controls graft size.

Limitations in cell proliferation are important for normal function of differentiated tissues and essential for the safety of cell replacement products made from pluripotent stem cells, which have unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest or by impairing S phase entry or S phase completion and determined growth potential, differentiation, and function of insulin-producing endocrine cells. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells toward insulin-producing cells and could substitute for endocrine differentiation factors. Reduced replication fork speed during differentiation improved the stability of insulin expression, and the resulting cells protected mice from diabetes without the formation of cystic growths. The proliferative potential of grafts was proportional to the reduction of replication fork speed during pancreatic differentiation. Therefore, a compromised ability to enter and complete S phase is a functionally important property of pancreatic endocrine differentiation, can be achieved by reducing replication fork speed, and is an important determinant of cell-intrinsic limitations of growth.

Homologous recombination DNA repair genes play a critical role in reprogramming to a pluripotent state.

Induced pluripotent stem cells (iPSCs) hold great promise for personalized regenerative medicine. However, recent studies show that iPSC lines carry genetic abnormalities, suggesting that reprogramming may be mutagenic. Here, we show that the ectopic expression of reprogramming factors increases the level of phosphorylated histone H2AX, one of the earliest cellular responses to DNA double-strand breaks (DSBs). Additional mechanistic studies uncover a direct role of the homologous recombination (HR) pathway, a pathway essential for error-free repair of DNA DSBs, in reprogramming. This role is independent of the use of integrative or nonintegrative methods in introducing reprogramming factors, despite the latter being considered a safer approach that circumvents genetic modifications. Finally, deletion of the tumor suppressor p53 rescues the reprogramming phenotype in HR-deficient cells primarily through the restoration of reprogramming-dependent defects in cell proliferation and apoptosis. These mechanistic insights have important implications for the design of safer approaches to creating iPSCs.

Comparative analysis of diagnostic methods in meniscal lesions.

UNLABELLED: The aim of this study is to determine the accuracy of clinical and MRI diagnosis in comparison with arthroscopy for detection of meniscal lesions. Also, to answer if MRI diagnosis impacts on the decision of the surgeon for the choice of treatment (operative or conservative). MATERIAL AND METHODS: We examined 70 patients with knee injuries. Clinical diagnosis was established using the case-history of the patient and positive clinical tests for meniscal injuries (McMurray and Aplay). All patients underwent MRI on a 1.5 T magnet for MRI diagnosis. This was followed by arthroscopy for final diagnosis. Clinical and MRI diagnoses were correlated with the arthroscopic diagnosis which was used as a gold standard. RESULTS: Of 70 patients with knee injuries, 55 had a clinical diagnosis of meniscal lesions out of whom 44 patients had a medial meniscal lesion and 11 had a lateral meniscal lesion. Arthroscopy confirmed the clinical diagnosis in 32 patients (72.72%) (44 vs 32) in medial meniscal lesion, and 8 patients (72.7%) (11 vs 8) with a lateral meniscal lesion. In MRI diagnosis of 56 patients with medial meniscal lesion arthroscopy confirmed the diagnosis in 34 patients (60.7%) (56 vs 34) and pf 10 patients with lateral meniscal lesion arthroscopy confirmed the diagnosis in 6 patients (60%) (10 vs 6). The sensitivity, specificity, PPV and NPV of clinical diagnosis versus MRI for medial meniscus were (79.9% vs 79.5%); (58.1% vs 38.1%); (69.8% vs 69.6%); (69.2% vs 69.2%). The sensitivity, specificity, PPV and NPV of clinical diagnosis versus MRI for lateral meniscus were (50% vs 40%); (92.7% vs 92.7%); (63.6% vs 60%); (87.9% vs 85.5%). CONCLUSIONS: Carefully performed clinical examination can give an equal or better diagnosis of meniscal lesions in comparison with MRI diagnosis. Any experienced orthopaedic surgeon can trust his clinical diagnosis as an indication of arthroscopy. When the clinical diagnosis is established, with no doubts due to positivity of the clinical tests, the MRI is not essential. In suspected cases where there is a dilemma, MRI is very helpful in making a decision for arthroscopy. The diagnostic accuracy of clinical and MRI diagnosis of meniscal lesions is high. Their reliability in diagnosing meniscal lesions is evident. lesion, clinical diagnosis, MRI, arthroscopy.