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Applications of genome editing ultimately depend on DNA repair triggered by targeted doublestrand breaks (DSBs). However, repair mechanisms in human cells remain poorly understood and vary across different cell types. Here we report that DSBs selectively induced on a mutant allele in heterozygous human embryos are repaired by gene conversion using an intact wildtype homolog as a template in up to 40% of targeted embryos. We also show that targeting of homozygous loci facilitates an interplay of non-homologous end joining (NHEJ) and gene conversion and results in embryos which carry identical indel mutations on both loci. Additionally, conversion tracks may expand bidirectionally well beyond the target region leading to an extensive loss of heterozygosity (LOH). Our study demonstrates that gene conversion and NHEJ are two major DNA DSB repair mechanisms in preimplantation human embryos. While gene conversion could be applicable for gene correction, extensive LOH presents a serious safety concern.

The accumulation of acquired mitochondrial genome (mtDNA) mutations with aging in somatic cells has been implicated in mitochondrial dysfunction and linked to age-onset diseases in humans. Here, we asked if somatic mtDNA mutations are also associated with aging in the mouse. MtDNA integrity in multiple organs and tissues in young and old (2–34 months) wild type (wt) mice was investigated by whole genome sequencing. Remarkably, no acquired somatic mutations were detected in tested tissues. However, we identified several non-synonymous germline mtDNA variants whose heteroplasmy levels (ratio of normal to mutant mtDNA) increased significantly with aging suggesting clonal expansion of inherited mtDNA mutations. Polg mutator mice, a model for premature aging, exhibited both germline and somatic mtDNA mutations whose numbers and heteroplasmy levels increased significantly with age implicating involvement in premature aging. Our results suggest that, in contrast to humans, acquired somatic mtDNA mutations do not accompany the aging process in wt mice.

Sensorineural hearing loss is a common disability found worldwide which is associated with a degeneration of spiral ganglion neurons (SGN). It is a challenge to restore SGN due to the permanent degeneration and viability of SGN is requisite for patients to receive an advantage from hearing aid devices. Human dental pulp stem cells (DPSC) and stem cells from human exfoliated deciduous teeth (SHED) are self-renewing stem cells that originate from the neural crest during development. These stem cells have a high potential for neuronal differentiation. This is primarily due to their multilineage differentiation potential and their relative ease of access. Previously, we have shown the ability of these stem cell types to differentiate into spiral ganglion neuron-like cells. In this study, we induced the cells into neural precursor cells (NPC) and cocultured with auditory brainstem slice (ABS) encompassing cochlear nucleus by the Stoppini method. We also investigated their ability to differentiate after 2 weeks and 4 weeks in coculture. Neuronal differentiation of DPSC-NPC and SHED-NPC was higher expression of specific markers to SGN, TrkB, and Gata3, compared to monoculture. The cells also highly expressed synaptic vesicle protein (SV2A) and exhibited intracellular calcium oscillations. Our findings demonstrated the possibility of using DPSCs and SHEDs as an autologous stem cell-based therapy for sensorineural hearing loss patients. K E Y W O R D S auditory, brainstem slice (ABS), deciduous teeth (SHED), human dental pulp stem cell (DPSC), spiral ganglion neuron (SGN), stem cell from human exfoliated

Introduction Stem cell transplantation of exogenous neural progenitor cells (NPCs) derived from mesenchymal stem cells (MSCs) has emerged as a promising approach for neurodegenerative disease. Human stem cells from apical papilla (hSCAPs) are derived from migratory neural crest stem cells and exhibit a potential of neuronal differentiation. However, their neuronal differentiation is low and unpredictable. Resveratrol has been described as a sirtuin 1 (SIRT1) activator which plays an important role in enhancing neuronal differentiation. In this study, we investigate the potential of resveratrol as an enhancer on neuronal differentiation through NPCs induction of hSCAPs. Methods Stem cells were isolated from human apical papilla and characterized as MSCs. The cellular toxicity of resveratrol treatment to the characterized hSCAPs was investigated by MTT assay. The non-cellular toxicity concentrations of resveratrol were assessed with various pre-treatment times to select the optimal condition that highly expressed the neural progenitor gene, NES. Consequently, the optimal condition of resveratrol pre-treatment was synergistically performed with a neuronal induction medium to trigger neuronal differentiation. The differentiated cells were visualized, the genes profiling was quantified, and the percentage of neuronal differentiation was calculated. Moreover, the intracellular calcium oscillation was demonstrated. Results The cellular toxicity of resveratrol was not observed for up to 50 μM for 12 h. Interestingly, hSCAPs pre-treated with 10 μM resveratrol for 12 h (RSV-hSCAPs) significantly expressed NES, which is determined as the optimal condition. Under neuronal induction, both of hSCAPs and RSV-hSCAPs were differentiated (d-hSCAPs and RSV-d-hSCAPs) as they exhibited neuronal-like appearances with Nissl substance staining. The highest expression of NES and SOX1 was observed in RSV-d-hSCAPs. Additionally, the percentage of neuronal differentiation of RSV-d-hSCAPs was significantly higher than d-hSCAPs for 4 times. Importantly, the neuronal-like cells exhibited slightly increasing pattern of calcium intensity. Conclusion This study demonstrated that pre-treatment of resveratrol strongly induces neural progenitor marker gene expression which synergistically enhances neural progenitor-like cells’ induction with neuronal induction medium.

Heritable mitochondrial DNA (mtDNA) mutations are common, yet only a few recurring pathogenic mtDNA variants account for the majority of known familial cases in humans. Purifying selection in the female germline is thought to be responsible for the elimination of most harmful mtDNA mutations during oogenesis. Here we show that deleterious mtDNA mutations are abundant in ovulated mature mouse oocytes and preimplantation embryos recovered from PolG mutator females but not in their live offspring. This implies that purifying selection acts not in the maternal germline per se, but during post-implantation development. We further show that oocyte mtDNA mutations can be captured and stably maintained in embryonic stem cells and then reintroduced into chimeras, thereby allowing examination of the effects of specific mutations on fetal and postnatal development.

Objectives: Human dental pulp stem cells (DPSCs) have been used to regenerate damaged nervous tissues. However, the methods of committing DPSCs into neural stem/progenitor cells (NSPCs) or neurospheres are highly diverse, resulting in many neuronal differentiation outcomes. This study aims to validate an optimal protocol for inducing DPSCs into neurospheres and neurons. Methodology: After isolation and characterization of mesenchymal stem cell identity, DPSCs were cultured in a NSPC induction medium and culture vessels. The durations of the culture, dissociation methods, and passage numbers of DPSCs were varied. Results: Neurosphere formation requires a special surface that inhibits cell attachment. Five-days was the most appropriate duration for generating proliferative neurospheres and they strongly expressed Nestin, an NSPC marker. Neurosphere reformation after being dissociated by the Accutase enzyme was significantly higher than other methods. Passage number of DPSCs did not affect neurosphere formation, but did influence neuronal differentiation. We found that the cells expressing a neuronal marker, β-tubulin III, and exhibiting neuronal morphology were significantly higher in the early passage of the DPSCs. Conclusion: These results suggest a guideline to obtain a high efficiency of neurospheres and neuronal differentiation from DPSCs for further study and neurodegeneration therapeutics.

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