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The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5'-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis using methods dependent on bisulfite conversion needs to be established for each experiment, taking effects of incomplete bisulfite conversion and template impurity or topology into consideration.

Ancestral environmental exposures to non-mutagenic agents can exert effects in unexposed descendants. This transgenerational inheritance has significant implications for understanding disease etiology. Here we show that exposure of F0 mice to the obesogen tributyltin (TBT) throughout pregnancy and lactation predisposes unexposed F4 male descendants to obesity when dietary fat is increased. Analyses of body fat, plasma hormone levels, and visceral white adipose tissue DNA methylome and transcriptome collectively indicate that the F4 obesity is consistent with a leptin resistant, thrifty phenotype. Ancestral TBT exposure induces global changes in DNA methylation and altered expression of metabolism-relevant genes. Analysis of chromatin accessibility in F3 and F4 sperm reveals significant differences between control and TBT groups and significant similarities between F3 and F4 TBT groups that overlap with areas of differential methylation in F4 adipose tissue. Our data suggest that ancestral TBT exposure induces changes in chromatin organization transmissible through meiosis and mitosis. Early life exposure to endocrine disrupting chemicals has been linked to increased adiposity during adulthood. Here Chamorro-García et al. show that ancestral exposure to the obesogen tributyltin causes obesity in untreated F4 generation male descendants by inducing heritable changes in genome architecture that promote a thrifty phenotype.

Induced pluripotent stem cells (iPSCs) have been generated by enforced expression of defined sets of transcription factors in somatic cells. It remains controversial whether iPSCs are molecularly and functionally equivalent to blastocyst-derived embryonic stem (ES) cells. By comparing genetically identical mouse ES cells and iPSCs, we show here that their overall messenger RNA and microRNA expression patterns are indistinguishable with the exception of a few transcripts encoded within the imprinted Dlk1 – Dio3 gene cluster on chromosome 12qF1, which were aberrantly silenced in most of the iPSC clones. Consistent with a developmental role of the Dlk1 – Dio3 gene cluster, these iPSC clones contributed poorly to chimaeras and failed to support the development of entirely iPSC-derived animals (‘all-iPSC mice’). In contrast, iPSC clones with normal expression of the Dlk1 – Dio3 cluster contributed to high-grade chimaeras and generated viable all-iPSC mice. Notably, treatment of an iPSC clone that had silenced Dlk1 – Dio3 with a histone deacetylase inhibitor reactivated the locus and rescued its ability to support full-term development of all-iPSC mice. Thus, the expression state of a single imprinted gene cluster seems to distinguish most murine iPSCs from ES cells and allows for the prospective identification of iPSC clones that have the full development potential of ES cells. Key gene cluster in iPS cells The extent to which iPS (induced pluripotent stem) cells are equivalent to embryonic stem (ES) cells is an open question. Some reports claim that hundreds of genes are aberrantly expressed in iPS compared with ES cells, yet iPS cells can satisfy one of the most stringent tests for developmental potential by producing all-iPS cell mice via tetraploid embryo complementation. To address this question with a minimum of complicating factors, Stadtfeld et al . compared gene expression in genetically identical mouse ES and iPS cells. They find overall mRNA and microRNA expression patterns that are indistinguishable, apart from a few transcripts and fewer than 20 microRNAs encoded by an imprinted gene cluster on chromosome 12qF1. The developmental potential of iPS cells depends on whether genes are silenced or active on this site. Induced pluripotent stem cells (iPSCs) are generated by the enforced expression of particular transcription factors in somatic cells. The extent to which such cells are equivalent to embryonic stem (ES) cells is an open question. Here, genetically identical mouse ES cells and iPSCs have been compared; the overall expression patterns of messenger RNAs and microRNAs are the same, with the exception of a few transcripts encoded within an imprinted gene cluster on chromosome 12qF1.

Prostate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging; hence, sampling circulating tumor cells (CTCs) may reveal drug-resistance mechanisms. We established single-cell RNA-sequencing (RNA-Seq) profiles of 77 intact CTCs isolated from 13 patients (mean six CTCs per patient), by using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of AR gene mutations and splicing variants. Retrospective analysis of CTCs from patients progressing under treatment with an AR inhibitor, compared with untreated cases, indicates activation of noncanonical Wnt signaling (P = 0.0064). Ectopic expression of Wnt5a in prostate cancer cells attenuates the antiproliferative effect of AR inhibition, whereas its suppression in drug-resistant cells restores partial sensitivity, a correlation also evident in an established mouse model. Thus, single-cell analysis of prostate CTCs reveals heterogeneity in signaling pathways that could contribute to treatment failure.

Proper coordination of cholesterol biosynthesis and trafficking is essential to human health. The sterol regulatory element-binding proteins (SREBPs) are key transcription regulators of genes involved in cholesterol biosynthesis and uptake. We show here that microRNAs (miR-33a/b) embedded within introns of the SREBP genes target the adenosine triphosphate-binding cassette transporter A1 (ABCA1), an important regulator of high-density lipoprotein (HDL) synthesis and reverse cholesterol transport, for posttranscriptional repression. Antisense inhibition of miR-33 in mouse and human cell lines causes up-regulation of ABCA1 expression and increased cholesterol efflux, and injection of mice on a western-type diet with locked nucleic acid-antisense oligonucleotides results in elevated plasma HDL. Our findings indicate that miR-33 acts in concert with the SREBP host genes to control cholesterol homeostasis and suggest that miR-33 may represent a therapeutic target for ameliorating cardiometabolic diseases.

Polo et al . show that early-passage induced pluripotent stem cells retain an epigenetic memory of their cell type of origin. These epigenetic differences affect the cells’ differentiation potential and might be exploited to generate particular cell types of interest. Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages.

Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.

Exposure to environmental stressors is known to increase disease susceptibility in unexposed descendants in the absence of detectable genetic mutations. The mechanisms mediating environmentally-induced transgenerational disease susceptibility are poorly understood. We showed that great-great-grandsons of female mice exposed to tributyltin (TBT) throughout pregnancy and lactation were predisposed to obesity due to altered chromatin organization that subsequently biased DNA methylation and gene expression. Here we analyzed DNA methylomes and transcriptomes from tissues of animals ancestrally exposed to TBT spanning generations, sexes, ontogeny, and cell differentiation state. We found that TBT elicited concerted alterations in the expression of “ chromatin organization ” genes and inferred that TBT-disrupted chromatin organization might be able to self-reconstruct transgenerationally. We also found that the location of “ chromatin organization ” and “ metabolic ” genes is biased similarly in mouse and human genomes, suggesting that exposure to environmental stressors in different species could elicit similar phenotypic effects via self-reconstruction of disrupted chromatin organization.

An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and several types of somatic cells. Among these, granulosa cells play a key role in follicle formation and support for oogenesis. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs), a method of generating granulosa cells has been elusive. Here, we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of hiPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology and may enable the development of therapies for female reproductive health. Ovaries are responsible for forming the eggs humans and other mammals need to reproduce. Once mature, the egg cell is released into the fallopian tube where it can be potentially fertilized by a sperm. Despite their crucial role, how eggs are made in the ovary is poorly understood. This is because ovaries are hard to access, making it difficult to conduct experiments on them. To overcome this, researchers have built artificial ovaries in the laboratory using stem cells from the embryos of mice which can develop into all cell types in the adult body. By culturing these embryonic stem cells under special conditions, researchers can convert them in to the two main cell types of the developing ovary: germ cells which go on to form eggs, and granulosa cells which help eggs grow and mature. The resulting lab-grown ovary can make eggs that produce live mice when fertilized. This approach has also been applied to human induced pluripotent stem cells (iPSCs), adult human cells which have been reprogrammed to a stem-like state. While this has produced human germ cells, generating human granulosa cells has been more challenging. Here, Pierson Smela, Kramme et al. show that activating a specific set of transcription factors (proteins that switch genes on or off) in iPSCs can make them transition to granulosa cells. First, the team tested random combinations of 35 transcription factors which, based on previous literature and genetic data, were likely to play a role in the formation of granulosa cells. This led to the identification of a small number of factors that caused the human iPSCs to develop features and carry out roles seen in mature granulosa cells; this includes producing an important reproductive hormone and supporting the maturation of germ cells. Pierson Smela, Kramme et al. found that growing these granulosa-like cells together with germ cells (also generated via iPSCs) resulted in structures similar to ovarian follicles which help eggs develop. These findings could help researchers build stable systems for studying how granulosa cells behave in human ovaries. This could lead to new insights about reproductive health.

Pluripotent stem cell-derived human primordial germ cell-like cells (hPGCLCs) provide important opportunities to study primordial germ cells (PGCs). We robustly produced CD38⁺ hPGCLCs [∼43% of FACS-sorted embryoid body (EB) cells] from primed-state induced pluripotent stem cells (iPSCs) after a 72-hour transient incubation in the four chemical inhibitors (4i)-naïve reprogramming medium and showed transcriptional consistency of our hPGCLCs with hPGCLCs generated in previous studies using various and distinct protocols. Both CD38⁺ hPGCLCs and CD38⁻ EB cells significantly expressed PRDM1 and TFAP2C, although PRDM1 mRNA in CD38⁻ cells lacked the 3′-UTR harboring miRNA binding sites regulating mRNA stability. Genes up-regulated in hPGCLCs were enriched for cell migration genes, and their promoters were enriched for the binding motifs of TFAP2 (which was identified in promoters of T, NANOS3, and SOX17) and the RREB-1 cell adhesion regulator. In EBs, hPGCLCs were identified exclusively in the outermost surface monolayer as dispersed cells or cell aggregates with strong and specific expression of POU5F1/OCT4 protein. Time-lapse live cell imaging revealed active migration of hPGCLCs onMatrigel. Whereas all hPGCLCs strongly expressed the CXCR4 chemotaxis receptor, its ligand CXCL12/SDF1 was not significantly expressed in the whole EBs. Exposure of hPGCLCs to CXCL12/SDF1 induced cell migration genes and antiapoptosis genes. Thus, our study shows that transcriptionally consistent hPGCLCs can be readily produced from hiPSCs after transition of their pluripotency from the primed state using various methods and that hPGCLCs resemble the early-stage PGCs randomly migrating in the midline region of human embryos before initiation of the CXCL12/SDF1-guided chemotaxis.