EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele.

The maintenance of genome stability is critical for the suppression of diverse human pathologies that include developmental disorders, premature aging, infertility and predisposition to cancer. The DNA damage response (DDR) orchestrates the appropriate cellular responses following the detection of lesions to prevent genomic instability. The MRE11 complex is a sensor of DNA double strand breaks (DSBs) and plays key roles in multiple aspects of the DDR, including DNA end resection that is critical for signaling and DNA repair. The MRE11 complex has been shown to function both upstream and in concert with the 5'-3' exonuclease EXO1 in DNA resection, but it remains unclear to what extent EXO1 influences DSB responses independently of the MRE11 complex. Here we examine the genetic relationship of the MRE11 complex and EXO1 during mammalian development and in response to DNA damage. Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 leads to severe developmental impairment, embryonic death and chromosomal instability. While EXO1 plays a minimal role in normal cells, its loss strongly influences DNA replication, DNA repair, checkpoint signaling and damage sensitivity in NBS1 hypomorphic cells. Collectively, our results establish a key role for EXO1 in modulating the severity of hypomorphic MRE11 complex mutations.

Positional Enrichment by Proton Analysis (PEPA): A One-Dimensional 1 H-NMR Approach for 13 C Stable Isotope Tracer Studies in Metabolomics.

A novel metabolomics approach for NMR-based stable isotope tracer studies called PEPA is presented, and its performance validated using human cancer cells. PEPA detects the position of carbon label in isotopically enriched metabolites and quantifies fractional enrichment by indirect determination of 13 C-satellite peaks using 1D-1 H-NMR spectra. In comparison with 13 C-NMR, TOCSY and HSQC, PEPA improves sensitivity, accelerates the elucidation of 13 C positions in labeled metabolites and the quantification of the percentage of stable isotope enrichment. Altogether, PEPA provides a novel framework for extending the high-throughput of 1 H-NMR metabolic profiling to stable isotope tracing in metabolomics, facilitating and complementing the information derived from 2D-NMR experiments and expanding the range of isotopically enriched metabolites detected in cellular extracts.

Autologous Induced Pluripotent Stem Cell-Based Cell Therapies: Promise, Progress, and Challenges.

The promise of human induced pluripotent stem cells (iPSCs) lies in their ability to serve as a starting material for autologous, or patient-specific, stem cell-based therapies. Since the first publications describing the generation of iPSCs from human tissue in 2007, a Phase I/IIa clinical trial testing an autologous iPSC-derived cell therapy has been initiated in the U.S., and several other autologous iPSC-based therapies have advanced through various stages of development. Three single-patient in-human transplants of autologous iPSC-derived cells have taken place worldwide. None of the patients suffered serious adverse events, despite not undergoing immunosuppression. These promising outcomes support the proposed advantage of an autologous approach: a cell therapy product that can engraft without the risk of immune rejection, eliminating the need for immunosuppression and the associated side effects. Despite this advantage, there are currently more allogeneic than autologous iPSC-based cell therapy products in development due to the cost and complexity of scaling out manufacturing for each patient. In this review, we highlight recent progress toward clinical translation of autologous iPSC-based cell therapies. We also highlight technological advancements that would reduce the cost and complexity of autologous iPSC-based cell therapy production, enabling autologous iPSC-based therapies to become a more commonplace treatment modality for patients. © 2021 The Authors.

EXD2 governs germ stem cell homeostasis and lifespan by promoting mitoribosome integrity and translation.

Mitochondria are subcellular organelles that are critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, and on their coordinated translation, import and respiratory complex assembly. Here, we characterize EXD2 (exonuclease 3'-5' domain-containing 2), a nuclear-encoded gene, and show that it is targeted to the mitochondria and prevents the aberrant association of messenger RNAs with the mitochondrial ribosome. Loss of EXD2 results in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of the Drosophila melanogaster EXD2 orthologue (CG6744) causes developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that is reversed with an antioxidant diet. Our results define a conserved role for EXD2 in mitochondrial translation that influences development and ageing.

Null diffusion-based enrichment for metabolomics data.

Metabolomics experiments identify metabolites whose abundance varies as the conditions under study change. Pathway enrichment tools help in the identification of key metabolic processes and in building a plausible biological explanation for these variations. Although several methods are available for pathway enrichment using experimental evidence, metabolomics does not yet have a comprehensive overview in a network layout at multiple molecular levels. We propose a novel pathway enrichment procedure for analysing summary metabolomics data based on sub-network analysis in a graph representation of a reference database. Relevant entries are extracted from the database according to statistical measures over a null diffusive process that accounts for network topology and pathway crosstalk. Entries are reported as a sub-pathway network, including not only pathways, but also modules, enzymes, reactions and possibly other compound candidates for further analyses. This provides a richer biological context, suitable for generating new study hypotheses and potential enzymatic targets. Using this method, we report results from cells depleted for an uncharacterised mitochondrial gene using GC and LC-MS data and employing KEGG as a knowledge base. Partial validation is provided with NMR-based tracking of 13C glucose labelling of these cells.

Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro.

Progress toward finding a cure for muscle diseases has been slow because of the absence of relevant cellular models and the lack of a reliable source of muscle progenitors for biomedical investigation. Here we report an optimized serum-free differentiation protocol to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (hPSCs) in vitro. By mimicking key signaling events leading to muscle formation in the embryo, in particular the dual modulation of Wnt and bone morphogenetic protein (BMP) pathway signaling, this directed differentiation protocol avoids the requirement for genetic modifications or cell sorting. Robust myogenesis can be achieved in vitro within 1 month by personnel experienced in hPSC culture. The differentiating culture can be subcultured to produce large amounts of myogenic progenitors amenable to numerous downstream applications. Beyond the study of myogenesis, this differentiation method offers an attractive platform for the development of relevant in vitro models of muscle dystrophies and drug screening strategies, as well as providing a source of cells for tissue engineering and cell therapy approaches.

CEP63 deficiency promotes p53-dependent microcephaly and reveals a role for the centrosome in meiotic recombination.

CEP63 is a centrosomal protein that facilitates centriole duplication and is regulated by the DNA damage response. Mutations in CEP63 cause Seckel syndrome, a human disease characterized by microcephaly and dwarfism. Here we demonstrate that Cep63-deficient mice recapitulate Seckel syndrome pathology. The attrition of neural progenitor cells involves p53-dependent cell death, and brain size is rescued by the deletion of p53. Cell death is not the result of an aberrant DNA damage response but is triggered by centrosome-based mitotic errors. In addition, Cep63 loss severely impairs meiotic recombination, leading to profound male infertility. Cep63-deficient spermatocytes display numerical and structural centrosome aberrations, chromosome entanglements and defective telomere clustering, suggesting that a reduction in centrosome-mediated chromosome movements underlies recombination failure. Our results provide novel insight into the molecular pathology of microcephaly and establish a role for the centrosome in meiotic recombination.

Testing of nine different xeno-free culture media for human embryonic stem cell cultures.

BACKGROUND: Human embryonic stem cells (hESC) are excellent candidates for cell replacement therapies. However, currently used culture conditions contain animal-derived components that bear a risk of transmitting animal pathogens and incorporation of non-human immunogenic molecules to hESC. METHODS: Nine xeno-free culture media were compared with the conventional serum replacement (ko-SR) containing media in the culture of hESC on human feeder cells. Cultured hESC were characterized immunocytochemically and by fluorescence-activated cell sorter analysis. The differentiation potential of hESC cultured with xeno-free media was determined with the RT-PCR analysis. RESULTS: The hESC cultured in xeno-free media differentiated or the proliferation decreased substantially. Under some test conditions, the morphology of the feeder cells was altered considerably. The hESC cultured with human serum underwent excessive differentiation in the beginning of culture, but a fraction of hESC was able to adapt to culture conditions containing 20% of human serum. CONCLUSIONS: None of the studied xeno-free media was able to maintain the undifferentiated growth of hESC. The medium containing 20% human serum was found to sustain undifferentiated hESC proliferation to some extent, yet was inferior to the conventional ko-SR-containing medium.