Author Correction: Maternal vitamin C regulates reprogramming of DNA methylation and germline development.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Maternal vitamin C regulates reprogramming of DNA methylation and germline development.

Development is often assumed to be hardwired in the genome, but several lines of evidence indicate that it is susceptible to environmental modulation with potential long-term consequences, including in mammals1,2. The embryonic germline is of particular interest because of the potential for intergenerational epigenetic effects. The mammalian germline undergoes extensive DNA demethylation3-7 that occurs in large part by passive dilution of methylation over successive cell divisions, accompanied by active DNA demethylation by TET enzymes3,8-10. TET activity has been shown to be modulated by nutrients and metabolites, such as vitamin C11-15. Here we show that maternal vitamin C is required for proper DNA demethylation and the development of female fetal germ cells in a mouse model. Maternal vitamin C deficiency does not affect overall embryonic development but leads to reduced numbers of germ cells, delayed meiosis and reduced fecundity in adult offspring. The transcriptome of germ cells from vitamin-C-deficient embryos is remarkably similar to that of embryos carrying a null mutation in Tet1. Vitamin C deficiency leads to an aberrant DNA methylation profile that includes incomplete demethylation of key regulators of meiosis and transposable elements. These findings reveal that deficiency in vitamin C during gestation partially recapitulates loss of TET1, and provide a potential intergenerational mechanism for adjusting fecundity to environmental conditions.

Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells.

DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germ line, and may be mediated by Tet (ten eleven translocation) enzymes, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet enzymes have been studied extensively in mouse embryonic stem (ES) cells, which are generally cultured in the absence of vitamin C, a potential cofactor for Fe(II) 2-oxoglutarate dioxygenase enzymes such as Tet enzymes. Here we report that addition of vitamin C to mouse ES cells promotes Tet activity, leading to a rapid and global increase in 5hmC. This is followed by DNA demethylation of many gene promoters and upregulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site of genes affected by vitamin C treatment. Importantly, vitamin C, but not other antioxidants, enhances the activity of recombinant Tet1 in a biochemical assay, and the vitamin-C-induced changes in 5hmC and 5mC are entirely suppressed in Tet1 and Tet2 double knockout ES cells. Vitamin C has a stronger effect on regions that gain methylation in cultured ES cells compared to blastocysts, and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A particle retroelements, which are resistant to demethylation in the early embryo, are resistant to vitamin-C-induced DNA demethylation. Collectively, the results of this study establish vitamin C as a direct regulator of Tet activity and DNA methylation fidelity in ES cells.

Larotrectinib for paediatric solid tumours harbouring NTRK gene fusions: phase 1 results from a multicentre, open-label, phase 1/2 study.

BACKGROUND: Gene fusions involving NTRK1, NTRK2, or NTRK3 (TRK fusions) are found in a broad range of paediatric and adult malignancies. Larotrectinib, a highly selective small-molecule inhibitor of the TRK kinases, had shown activity in preclinical models and in adults with tumours harbouring TRK fusions. This study aimed to assess the safety of larotrectinib in paediatric patients. METHODS: This multicentre, open-label, phase 1/2 study was done at eight sites in the USA and enrolled infants, children, and adolescents aged 1 month to 21 years with locally advanced or metastatic solid tumours or CNS tumours that had relapsed, progressed, or were non-responsive to available therapies regardless of TRK fusion status; had a Karnofsky (≥16 years of age) or Lansky (<16 years of age) performance status score of 50 or more, adequate organ function, and full recovery from the acute toxic effects of all previous anticancer therapy. Following a protocol amendment on Sept 12, 2016, patients with locally advanced infantile fibrosarcoma who would require disfiguring surgery to achieve a complete surgical resection were also eligible. Patients were enrolled to three dose cohorts according to a rolling six design. Larotrectinib was administered orally (capsule or liquid formulation), twice daily, on a continuous 28-day schedule, in increasing doses adjusted for age and bodyweight. The primary endpoint of the phase 1 dose escalation component was the safety of larotrectinib, including dose-limiting toxicity. All patients who received at least one dose of larotrectinib were included in the safety analyses. Reported here are results of the phase 1 dose escalation cohort. Phase 1 follow-up and phase 2 are ongoing. This trial is registered with ClinicalTrials.gov, number NCT02637687. FINDINGS: Between Dec 21, 2015, and April 13, 2017, 24 patients (n=17 with tumours harbouring TRK fusions, n=7 without a documented TRK fusion) with a median age of 4·5 years (IQR 1·3-13·3) were enrolled to three dose cohorts: cohorts 1 and 2 were assigned doses on the basis of both age and bodyweight predicted by use of SimCyp modelling to achieve an area under the curve equivalent to the adult doses of 100 mg twice daily (cohort 1) and 150 mg twice daily (cohort 2); and cohort 3 was assigned to receive a dose of 100 mg/m2 twice daily (maximum 100 mg per dose), regardless of age, equating to a maximum of 173% of the recommended adult phase 2 dose. Among enrolled patients harbouring TRK fusion-positive cancers, eight (47%) had infantile fibrosarcoma, seven (41%) had other soft tissue sarcomas, and two (12%) had papillary thyroid cancer. Adverse events were predominantly grade 1 or 2 (occurring in 21 [88%] of 24 patients); the most common larotrectinib-related adverse events of all grades were increased alanine and aspartate aminotransferase (ten [42%] of 24 each), leucopenia (five [21%] of 24), decreased neutrophil count (five [21%] of 24), and vomiting (five [21%] of 24). Grade 3 alanine aminotransferase elevation was the only dose-limiting toxicity and occurred in one patient without a TRK fusion and with progressive disease. No grade 4 or 5 treatment-related adverse events were observed. Two larotrectinib-related serious adverse events were observed: grade 3 nausea and grade 3 ejection fraction decrease during the 28-day follow-up after discontinuing larotrectinib and while on anthracyclines. The maximum tolerated dose was not reached, and 100 mg/m2 (maximum of 100 mg per dose) was established as the recommended phase 2 dose. 14 (93%) of 15 patients with TRK fusion-positive cancers achieved an objective response as per Response Evaluation Criteria In Solid Tumors version 1.1; the remaining patient had tumour regression that did not meet the criteria for objective response. None of the seven patients with TRK fusion-negative cancers had an objective response. INTERPRETATION: The TRK inhibitor larotrectinib was well tolerated in paediatric patients and showed encouraging antitumour activity in all patients with TRK fusion-positive tumours. The recommended phase 2 dose was defined as 100mg/m2 (maximum 100 mg per dose) for infants, children, and adolescents, regardless of age. FUNDING: Loxo Oncology Inc.

Soluble growth factors stimulate spermatogonial stem cell divisions that maintain a stem cell pool and produce progenitors in vitro.

Spermatogonial stem cells (SSCs) support life-long spermatogenesis by self-renewing and producing spermatogonia committed to differentiation. In vitro, SSCs form three-dimensional spermatogonial aggregates (clusters) when cultured with glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2); serial passaging of clusters results in long-term SSC maintenance and expansion. However, the role of these growth factors in controlling patterns of SSC division and fate decision has not been understood thoroughly. We report here that in a short-term culture, GDNF and FGF2 increase the number of dividing SSCs, but not the total SSC number, compared to a no-growth-factor condition. Since the total germ cell number increases with growth factors, these results suggest that GDNF and FGF2 promote a SSC division pattern that sustains the size of the stem cell pool while generating committed progenitors. Our data also show that SSC numbers increase when the cluster structure is disintegrated and cell-cell interaction in clusters is disrupted. Collectively, these results suggest that in this culture system, GDNF and FGF2 stimulate SSC divisions that promote self-renewal and differentiation in the SSC population, and imply that the destruction of the cluster structure, a potential in vitro niche, may contribute to SSC expansion.

The application of biomarkers of spermatogonial stem cells for restoring male fertility.

Spermatogonial stem cells (SSCs) are defined by their ability to both self-renew and produce differentiated germ cells that will develop into functional spermatozoa. Because of this ability, SSCs can reestablish spermatogenesis after testicular damage caused by cytotoxic agents or after transplantation into an infertile recipient. Therefore, SSCs are an important target cell for restoring male fertility, particularly for cancer patients who have to undergo sterilizing cancer therapies. In the mouse, the identification of SSC markers allows for the isolation of a highly enriched population of stem cells. This enriched stem cell population can be expanded in culture for an indefinite period of time, cryopreserved, and transplanted into infertile recipients to restore fertility. Thus, the identification of markers and the establishment of a long-term culture system for human SSCs will be crucial for realizing the potential of these cells in a clinical setting. In this article, we focus on the markers that have been identified for mouse SSCs and discuss how human SSC markers may be used in the restoration of fertility.

Vitamin C induces specific demethylation of H3K9me2 in mouse embryonic stem cells via Kdm3a/b.

BACKGROUND: Histone methylation patterns regulate gene expression and are highly dynamic during development. The erasure of histone methylation is carried out by histone demethylase enzymes. We had previously shown that vitamin C enhances the activity of Tet enzymes in embryonic stem (ES) cells, leading to DNA demethylation and activation of germline genes. RESULTS: We report here that vitamin C induces a remarkably specific demethylation of histone H3 lysine 9 dimethylation (H3K9me2) in naïve ES cells. Vitamin C treatment reduces global levels of H3K9me2, but not other histone methylation marks analyzed, as measured by western blot, immunofluorescence and mass spectrometry. Vitamin C leads to widespread loss of H3K9me2 at large chromosomal domains as well as gene promoters and repeat elements. Vitamin C-induced loss of H3K9me2 occurs rapidly within 24 h and is reversible. Importantly, we found that the histone demethylases Kdm3a and Kdm3b are required for vitamin C-induced demethylation of H3K9me2. Moreover, we show that vitamin C-induced Kdm3a/b-mediated H3K9me2 demethylation and Tet-mediated DNA demethylation are independent processes at specific loci. Lastly, we document Kdm3a/b are partially required for the upregulation of germline genes by vitamin C. CONCLUSIONS: These results reveal a specific role for vitamin C in histone demethylation in ES cells and document that DNA methylation and H3K9me2 cooperate to silence germline genes in pluripotent cells.

Bivalent chromatin marks developmental regulatory genes in the mouse embryonic germline in vivo.

Developmental regulatory genes have both activating (H3K4me3) and repressive (H3K27me3) histone modifications in embryonic stem cells (ESCs). This bivalent configuration is thought to maintain lineage commitment programs in a poised state. However, establishing physiological relevance has been complicated by the high number of cells required for chromatin immunoprecipitation (ChIP). We developed a low-cell-number chromatin immunoprecipitation (low-cell ChIP) protocol to investigate the chromatin of mouse primordial germ cells (PGCs). Genome-wide analysis of embryonic day 11.5 (E11.5) PGCs revealed H3K4me3/H3K27me3 bivalent domains highly enriched at developmental regulatory genes in a manner remarkably similar to ESCs. Developmental regulators remain bivalent and transcriptionally silent through the initiation of sexual differentiation at E13.5. We also identified >2,500 "orphan" bivalent domains that are distal to known genes and expressed in a tissue-specific manner but silent in PGCs. Our results demonstrate the existence of bivalent domains in the germline and raise the possibility that the somatic program is continuously maintained as bivalent, potentially imparting transgenerational epigenetic inheritance.

Male germ line stem cells have an altered potential to proliferate and differentiate during postnatal development in mice.

Spermatogonial stem cells (SSCs) continuously support spermatogenesis after puberty. However, accumulating evidence suggests that SSCs differ functionally during postnatal development. For example, mutant mice exist in which SSCs support spermatogenesis in the first wave after birth but cease to do so thereafter, resulting in infertility in adults. Studies using a retroviral vector have shown that the vector transduces pup SSCs more efficiently than adult SSCs, which suggests that pup SSCs divide more frequently. Thus, it is hypothesized that the SSCs in pup and adult testes have different characteristics. As an approach to testing this hypothesis in the present study, we investigated the proliferation kinetics of pup SSCs (6-9 days old) and their self-renewal/differentiation patterns for the first 2 mo after transplantation, and compared them to those of adult SSCs. Using serial transplantation, we found that the number of pup SSCs declined over the first week after transplantation. Thereafter, it increased ~4-fold by 1 mo and ~9-fold by 2 mo after transplantation, which indicates that pup SSCs continuously proliferate from 1 wk to 2 mo after transplantation. Compared to the proliferation of SSCs derived from adult intact testes, that of pup SSCs was lower at 1 mo but similar at 2 mo, indicating the delayed proliferation of pup SSCs. However, the pup SSCs regenerated spermatogenic colonies at 1 mo that were similar in length to those of SSCs from adult intact testes. Therefore, these results suggest that some functional differences exist in SSCs during postnatal development, and that these differences may affect the abilities of SSCs to self-renew and differentiate.

Aging of male germ line stem cells in mice.

In the present study, we investigated the effect of aging on spermatogonial stem cells (SSCs) and on the testicular somatic environment in ROSA26 mice. First, we examined testis weights at 2 mo, 6 mo, 1 yr, and 2 yr of age. At 1 and 2 yr, bilateral atrophied testes were observed in 50% and 75% of the mice, respectively; the rest of the mice had testis weights similar to those of young mice. Next, we evaluated the number and the activity of aged SSCs using spermatogonial transplantation. Numbers of SSCs in atrophied testes decreased in an age-dependent manner to as low as 1/60 of those in testes of young mice. Numbers of SSCs in nonregressed testes were similar regardless of age. The colony length, which is indicative of the potential of SSCs to regenerate spermatogenesis, was similar with donor cells from atrophied testes of 1-yr-old mice and those from testes of young mice, suggesting that SSCs remaining in 1-yr atrophied testes were functionally intact. Colonies arising from SSCs derived from 2-yr atrophied testes were significantly shorter, however, indicating that both SSC numbers and activity declined with age. Finally, we transplanted donor cells from young animals into 1- and 2-yr atrophied testes. Although the weight of 2-yr testes did not change after transplantation, that of 1-yr testes increased significantly, indicating that 1-yr, but not 2-yr, atrophied testes are permissive for regeneration of spermatogenesis by SSCs from young mouse testes. These results demonstrate that both SSCs and somatic environment in the testis are involved in the aging process.

Expression patterns of cell-surface molecules on male germ line stem cells during postnatal mouse development.

Spermatogonial stem cells (SSCs) are stem cells of the male germ line. In mice, SSCs are quiescent at birth but actively proliferate during the first postnatal week, while they rarely divide in adult, suggesting an age-dependent difference in SSC characteristics. As an approach to evaluate this possibility, we studied the expression pattern of cell-surface molecules on neonatal, pup, and adult mouse SSCs. Using immunomagnetic cell sorting, testis cells were selected for the expression of alpha(6) integrin, alpha(v) integrin, c-kit receptor tyrosine kinase (Kit), or a binding subunit of glial-cell-line-derived neurotrophic factor (GDNF) receptor, GFRalpha1. Selected cells were assayed for their stem cell activity using spermatogonial transplantation. The results showed that SSCs expressed alpha(6) integrin, but not alpha(v) integrin and Kit, regardless of age. The SSC activity in pup GFRalpha1(+) cells was higher than that in adult and neonatal cells, indicating that the expression pattern of GFRalpha1 varied age-dependently. To evaluate if SSCs show an age-dependent difference in their response to GDNF, we cultured highly enriched pup and adult SSCs with GDNF: we could not observe such an age-dependent difference in vitro. In addition, we failed to immunologically detect the expression of two types of GDNF receptor signaling subunits on SSCs. These results indicate that SSCs may change the expression patterns of cell-surface molecules during postnatal development, and suggest that GDNF receptor molecules may not be abundantly or specifically expressed in the in vivo population of mouse SSCs.

Genetic analysis of the clonal origin of regenerating mouse spermatogenesis following transplantation.

Spermatogonial transplantation provides a straightforward approach to quantify spermatogonial stem cells (SSCs). Because donor-derived spermatogenesis is regenerated in the form of distinct colonies, the number of functional SSCs can be obtained by simply counting the number of colonies established in recipient testes. However, this approach is legitimate only when one colony arises from one stem cell (one colony-one stem cell hypothesis). In this study, we evaluated the validity of this hypothesis. Two populations of donor cells were obtained from the testes of two transgenic mouse lines and mixed at a 1:1 ratio. Following transplantation of the cell mixture, donor-derived colonies were visualized and individually excised, and genomic DNA was extracted from each colony. Based on unique marker genes of the two transgenic lines, the genotype of the cells contained in a colony was examined by polymerase chain reaction. A colony was determined to be clonal when only one transgene was detected. The results showed that 100% and 90% of colonies were clonal when <5 and 19 colonies were formed per recipient testis, respectively. However, the clonality of colonies decreased as the colony number per recipient testis or the length of each colony increased. These results support the one colony-one stem cell hypothesis and demonstrate that spermatogonial transplantation provides a highly quantitative assay for SSCs; however, these conclusions are applicable under a defined transplantation condition.