Optimal culture conditions for neurosphere formation and neuronal differentiation from human dental pulp stem cells

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

Germline and somatic mtDNA mutations in mouse aging.

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

Early embryonic and NCR mtDNA mutations in <i>Polg</i> mice.

<p>(A) Bar graphs representing changes in mean heteroplasmy of early embryonic mutations with <i>Polg</i> mice aging. Error Bars, mean ± SEM. (B) Distribution of non-synonymous early embryonic mtDNA mutations among different genes. Error bars, mean ± SEM. (C) Quantification of mtDNA mutations in the non-coding region (NCR) in <i>Polg</i> mice (n = 12 for 2 months; n = 24 for 9 months). Error bars, mean ± SEM. (D) Bar graphs representing mean heteroplasmy levels of NCR mtDNA mutations in <i>Polg</i> mice with aging. Error bars, mean ± SEM. (E) Summary of mtDNA mutations found in the NCR region (mtDNA15423-16299) in <i>Polg</i> mice. Dots represent mtDNA mutations and numbers under dots represent the heteroplasmy levels. ETAS indicates the extended termination associated sequence and CSB indicates the conserved sequence block.</p

Dynamics of mtDNA mutations during mouse aging.

<p>Dynamics of mtDNA mutations during mouse aging.</p

Summary of mtDNA mutations detected in wild type and <i>Polg</i> mice.

<p>Summary of mtDNA mutations detected in wild type and <i>Polg</i> mice.</p

Characterization of mtDNA mutations in homozygous <i>Polg</i> mice with aging.

<p>(A). Comparison of mean number of germline mutations in wt and <i>Polg</i> mice at young and old age (n = 31 for young wt, n = 8 for young <i>Polg</i>, n = 44 for old wt and n = 14 for old <i>Polg</i>). Error bars, mean ± SEM. Asterisk indicates a significant increase in the number of mutations per tissue in old <i>Polg</i> compared to the old wt (P < 0.05, Student’s t-test). (B). Mean heteroplasmy levels of non-synonymous germline mutations with ≥2% heteroplasmy in <i>Polg</i> mice (mean ± SEM; asterisk, P < 0.05, Student’s t-test). (C) Pie charts showing gene distributions of non-synonymous germline mutations in young <i>Polg</i> mice (2 months, left) and old <i>Polg</i> mice (9 months, right). (D) Bar graphs representing the mean heteroplasmy levels of non-synonymous germline mutations in protein-coding and RNA-coding genes in <i>Polg</i> mice (asterisks, P < 0.05, Student’s t-test). (E) Pie charts showing the distribution of shared and unique mtDNA mutations detected in single skin fibroblast (SF) clones in young and old <i>Polg</i> mice. (F) Mean heteroplasmy changes for non-synonymous somatic mutations with ≥15% heteroplasmy in <i>Polg</i> mice. Error bars, mean ± SEM. Asterisk, P < 0.05, Student’s t-test. (G) Changes in number of non-synonymous somatic mutations with heteroplasmy levels ≥ 15% among different gene types with <i>Polg</i> mice aging. Error bars, mean ± SEM. Asterisks, P < 0.05, Student’s t-test.</p

Characterization of mtDNA mutations in wild type mice with aging.

<p>(A) Quantification of mtDNA mutations (mean ± SEM; asterisk, P < 0.05, Student’s t-test) for different mutation types in young wt mice at age 2–13 months (green bars; n = 31) and old mice at age 18–34 months (orange bars; n = 44). Somatic mutations were undetectable in wt mice. Asterisk represents a significant increase in number of germline mutations with age. (B) Mean heteroplasmy levels of non-synonymous germline and early embryonic mutations as a function of age. Error bars, mean ± SEM. Asterisk represents a significant increase in the mean heteroplasmy levels of non-synonymous germline mutations in old wt mice compared to the young group (P < 0.05, Student’s t-test). (C) Pie chart showing gene distribution of non-synonymous germline mutations in protein-coding and RNA coding genes in old wt mice. (D) Bar graphs showing mean heteroplasmy levels for non-synonymous germline mutations in protein-coding and RNA-coding genes in old wt mice. (E) Heteroplasmy of non-synonymous mutations in protein-coding and RNA-coding genes of early embryonic origin. (F) Pie chart showing relative proportion of mutation types in young and old wt mice. (G) Mitochondrial OXPHOS complex I activity in iPS cells carrying non-synonymous mutations in protein-coding genes and in age-matched control ESCs or iPS cells. The complex I activity was measured in cell homogenates (n = 12 per cell line, technical replicates) and was expressed as “% rotenone inhibition”. 10m-iPS7, 34m-iPS9 and 34m-iPS10 cells displayed reduced activities compared to controls. (H) Mitochondrial OXPHOS complex IV activity in iPS cells carrying non-synonymous mutations in protein-coding genes and in age-matched control ES or iPS cells. The complex IV activity was measured in cell homogenates (n = 12 per cell line, technical replicates) and was expressed as “nmol / min / mg protein”. 18m-iPS2 cells showed decreased activity compared to controls. In (G and H), ns denotes p ≥ 0.05.</p