The azanucleosides decitabine and azacytidine are used widely in the treatment of myeloid neoplasia and increasingly in the context of combination therapies. Although they were long regarded as being largely interchangeable in their function as hypomethylating agents, the azanucleosides actually have different mechanisms of action; decitabine interferes primarily with the methylation of DNA and azacytidine with that of RNA. Here, we examine the role of DNA methylation in the lineage commitment of stem cells during normal hematopoiesis and consider how mutations in epigenetic regulators such as DNMT3A and TET2 can lead to clonal expansion and subsequent neoplastic progression. We also consider why the efficacy of azanucleoside treatment is not limited to neoplasias carrying mutations in epigenetic regulators. Finally, we summarise recent data describing a role for azacytidine-sensitive RNA methylation in lineage commitment and in the cellular response to stress. By summarising and interpreting evidence for azanucleoside involvement in a range of cellular processes, our review is intended to illustrate the need to consider multiple modes of action in the design and stratification of future combination therapies.

The mechanism to maintain stem cell properties and to exit into differentiation pathways is a pivotal question in stem cell research. Spermatogonia are the adult stem cells of the male germ line, which are used in biomedical research as a source of undifferentiated cells. The communication between germ line stem cells and specialized somatic cells (Sertoli cells and Leydig cells) plays important roles in stem cell maintenance, germ cell proliferation, and differentiation. With regard to the biology of stem cells and spermatogenesis, the medaka (Oryzias latipes) is used as a teleost model organism, and it is also used to assess the effects of endocrine disruptors on reproductive phenomena. However, the lack of suitable molecular markers hampers the detection, isolation and analysis of different testis cells including gonial stem cells and Leydig cells. Therefore, oct4, sox2 and cyp11b were chosen to create transgenic reporter lines for the labeling of stem cells and Leydig cells, respectively. The present study had the aim to examine the temporal and spatial expression of the respective genes during embryonic development and in adult gonads of the medaka, and to describe the application of these transgenic lines in stem cell biology and reproductive biology. The mCherry expression in transgenic fish of the line FSI-Tg(sox2-mCherry)17 marks embryonic stem cells, Leydig cells and interstitial cells in adult testis. Faithful EGFP and DsRed expression in transgenic reporters strains for oct4 and cyp11b mimics the endogenous expression of oct4/pou2 and cyp11b-protein, respectively. The reporter gene expression in the strains FSI-Tg(oct4-EGFP)9 and FSI-Tg(oct4-EGFP)A allows the visualization of oct4 positive cells during embryonic development, PGCs, early germ cells and adult gonial cells. The Leydig cells express brightly green or red fluorescence in the medaka strains FSI-Tg(cyp11b-EGFP)20 and FSI-Tg(cyp11b-DsRed)1434, respectively, allowing the easy identification of Leydig cells in adult testis. The oct4-EGFP reporter labels medaka embryonic and spermatogonial stem cells, in which the spermatogonial stem cells at the ends of the testicular lobules show brightly green fluorescence. The transgenic expression in stem cells is also shown in the flow plot of primary testis cells. The spermatogonia are the largest cells and have the strongest fluorescence, which decreased upon differentiation. Therefore, the oct4-EGFP reporter strains will provide an opportunity to detect and to isolate the EGFP expressing cells for transplantation. These strains will also facilitate further experiments on the effects of drugs or hypoxia on these cells, because the strongest EGFP expressing cells can be easily detected in transgenic lines. Labeling of Leydig cells in cyp11b reporter lines opens a new area to study the seasonal variation of spermatogenesis. The medaka is a seasonal breeder in its natural habitat and the simulation of seasonal changes allows the simultaneous quantitative analysis of oct4-EGFP and cyp11b-DsRed expressing cells under such conditions.

B cells and in particular IL-10-secreting B cells emerge as important players in immune balance during pregnancy. We have recently revealed that CD19-deficient (CD19−/−), B cell-specific IL-10-deficient (BIL-10−/−) and B cell-deficient µMT pregnant mice are highly susceptible to LPSinduced preterm birth (PTB). We aimed to analyze the ability of IL-10-secreting cells to protect from PTB and the underlying mechanisms. Wild type (WT), CD19−/−, BIL-10−/− and µMT mice were treated with LPS at gd16 and the cellular immune response was investigated 24 h later. LPS-treated BIL-10−/− dams showed a more pronounced PTB phenotype compared to WT, CD19−/− and µMT females, and increased inflammatory and reduced anti-inflammatory mediator concentrations in the peritoneal cavity and serum. CD19−/−, BIL-10−/− and µMT mice displayed altered immune cell population frequencies in the blood and uterus with lower numbers of IL-10-secreting B cells and T cells. BIL-10−/− mothers presented decreased frequencies of uterine CD4+CD25+Foxp3+ Treg cells. Co-stimulatory molecules are critical for feto-maternal tolerance and IL-10 secretion. We found dysregulated PD-1 expression in peripheral blood and ICOS expression in the uterus of CD19−/−, BIL-10−/− and µMT dams. Our data show that B cell-specific IL-10-signaling is essential for a balanced maternal immune response to an inflammatory stimulant that cannot be hampered without IL-10-secreting B cells.

The mechanism to maintain stem cell properties and to exit into differentiation pathways is a pivotal question in stem cell research. Spermatogonia are the adult stem cells of the male germ line, which are used in biomedical research as a source of undifferentiated cells. The communication between germ line stem cells and specialized somatic cells (Sertoli cells and Leydig cells) plays important roles in stem cell maintenance, germ cell proliferation, and differentiation. With regard to the biology of stem cells and spermatogenesis, the medaka (Oryzias latipes) is used as a teleost model organism, and it is also used to assess the effects of endocrine disruptors on reproductive phenomena. However, the lack of suitable molecular markers hampers the detection, isolation and analysis of different testis cells including gonial stem cells and Leydig cells. Therefore, oct4, sox2 and cyp11b were chosen to create transgenic reporter lines for the labeling of stem cells and Leydig cells, respectively. The present study had the aim to examine the temporal and spatial expression of the respective genes during embryonic development and in adult gonads of the medaka, and to describe the application of these transgenic lines in stem cell biology and reproductive biology. The mCherry expression in transgenic fish of the line FSI-Tg(sox2-mCherry)17 marks embryonic stem cells, Leydig cells and interstitial cells in adult testis. Faithful EGFP and DsRed expression in transgenic reporters strains for oct4 and cyp11b mimics the endogenous expression of oct4/pou2 and cyp11b-protein, respectively. The reporter gene expression in the strains FSI-Tg(oct4-EGFP)9 and FSI-Tg(oct4-EGFP)A allows the visualization of oct4 positive cells during embryonic development, PGCs, early germ cells and adult gonial cells. The Leydig cells express brightly green or red fluorescence in the medaka strains FSI-Tg(cyp11b-EGFP)20 and FSI-Tg(cyp11b-DsRed)1434, respectively, allowing the easy identification of Leydig cells in adult testis. The oct4-EGFP reporter labels medaka embryonic and spermatogonial stem cells, in which the spermatogonial stem cells at the ends of the testicular lobules show brightly green fluorescence. The transgenic expression in stem cells is also shown in the flow plot of primary testis cells. The spermatogonia are the largest cells and have the strongest fluorescence, which decreased upon differentiation. Therefore, the oct4-EGFP reporter strains will provide an opportunity to detect and to isolate the EGFP expressing cells for transplantation. These strains will also facilitate further experiments on the effects of drugs or hypoxia on these cells, because the strongest EGFP expressing cells can be easily detected in transgenic lines. Labeling of Leydig cells in cyp11b reporter lines opens a new area to study the seasonal variation of spermatogenesis. The medaka is a seasonal breeder in its natural habitat and the simulation of seasonal changes allows the simultaneous quantitative analysis of oct4-EGFP and cyp11b-DsRed expressing cells under such conditions.