FOXL2 and NR5A1 induce human fibroblasts into steroidogenic ovarian granulosa-like cells.

Human granulosa cells in different stages are essential for maintaining normal ovarian function, and granulosa cell defect is the main cause of ovarian dysfunction. To address this problem, it is necessary to induce functional granulosa cells at different stages in vitro. In this study, we established a reprogramming method to induce early- and late-stage granulosa cells with different steroidogenic abilities. We used an AMH-fluorescence-reporter system to screen candidate factors for cellular reprogramming and generated human induced granulosa-like cells (hiGC) by overexpressing FOXL2 and NR5A1. AMH-EGFP+ hiGC resembled human cumulus cells in transcriptome profiling and secreted high levels of oestrogen and progesterone, similar to late-stage granulosa cells at antral or preovulatory stage. Moreover, we identified CD55 as a cell surface marker that can be used to isolate early-stage granulosa cells. CD55+ AMH-EGFP- hiGC secreted high levels of oestrogen but low levels of progesterone, and their transcriptome profiles were more similar to early-stage granulosa cells. More importantly, CD55+ hiGC transplantation alleviated polycystic ovary syndrome (PCOS) in a mouse model. Therefore, hiGC provides a cellular model to study the developmental program of human granulosa cells and has potential to treat PCOS.

Multi-Omics Analysis Reveals Translational Landscapes and Regulations in Mouse and Human Oocyte Aging.

Abnormal resumption of meiosis and decreased oocyte quality are hallmarks of maternal aging. Transcriptional silencing makes translational control an urgent task during meiosis resumption in maternal aging. However, insights into aging-related translational characteristics and underlying mechanisms are limited. Here, using multi-omics analysis of oocytes, it is found that translatomics during aging is related to changes in the proteome and reveals decreased translational efficiency with aging phenotypes in mouse oocytes. Translational efficiency decrease is associated with the N6-methyladenosine (m6A) modification of transcripts. It is further clarified that m6A reader YTHDF3 is significantly decreased in aged oocytes, inhibiting oocyte meiotic maturation. YTHDF3 intervention perturbs the translatome of oocytes and suppress the translational efficiency of aging-associated maternal factors, such as Hells, to affect the oocyte maturation. Moreover, the translational landscape is profiled in human oocyte aging, and the similar translational changes of epigenetic modifications regulators between human and mice oocyte aging are observed. In particular, due to the translational silence of YTHDF3 in human oocytes, translation activity is not associated with m6A modification, but alternative splicing factor SRSF6. Together, the findings profile the specific translational landscapes during oocyte aging in mice and humans, and uncover non-conservative regulators on translation control in meiosis resumption and maternal aging.

DAZL regulates proliferation of human primordial germ cells by direct binding to precursor miRNAs and enhances DICER processing activity.

Understanding the molecular and cellular mechanisms of human primordial germ cells (hPGCs) is essential in studying infertility and germ cell tumorigenesis. Many RNA-binding proteins (RBPs) and non-coding RNAs are specifically expressed and functional during hPGC developments. However, the roles and regulatory mechanisms of these RBPs and non-coding RNAs, such as microRNAs (miRNAs), in hPGCs remain elusive. In this study, we reported a new regulatory function of DAZL, a germ cell-specific RBP, in miRNA biogenesis and cell proliferation. First, DAZL co-localized with miRNA let-7a in human PGCs and up-regulated the levels of >100 mature miRNAs, including eight out of nine let-7 family, miR21, miR22, miR125, miR10 and miR199. Purified DAZL directly bound to the loops of precursor miRNAs with sequence specificity of GUU. The binding of DAZL to the precursor miRNA increased the maturation of miRNA by enhancing the cleavage activity of DICER. Furthermore, cell proliferation assay and cell cycle analysis confirmed that DAZL inhibited the proliferation of in vitro PGCs by promoting the maturation of these miRNAs. Evidently, the mature miRNAs up-regulated by DAZL silenced cell proliferation regulators including TRIM71. Moreover, DAZL inhibited germline tumor cell proliferation and teratoma formation. These results demonstrate that DAZL regulates hPGC proliferation by enhancing miRNA processing.

Single-Cell Dissection of the Multiomic Landscape of High-Grade Serous Ovarian Cancer.

High-grade serous cancer (HGSC) is the most common subtype of ovarian cancer. HGSC is highly aggressive with poor patient outcomes, and a deeper understanding of HGSC tumorigenesis could help guide future treatment development. To systematically characterize the underlying pathologic mechanisms and intratumoral heterogeneity in human HGSC, we used an optimized single-cell multiomics sequencing technology to simultaneously analyze somatic copy-number alterations (SCNA), DNA methylation, chromatin accessibility, and transcriptome in individual cancer cells. Genes associated with interferon signaling, metallothioneins, and metabolism were commonly upregulated in ovarian cancer cells. Integrated multiomics analyses revealed that upregulation of interferon signaling and metallothioneins was influenced by both demethylation of their promoters and hypomethylation of satellites and LINE1, and potential key transcription factors regulating glycolysis using chromatin accessibility data were uncovered. In addition, gene expression and DNA methylation displayed similar patterns in matched primary and abdominal metastatic tumor cells of the same genetic lineage, suggesting that metastatic cells potentially preexist in the subclones of primary tumors. Finally, the lineages of cancer cells with higher residual DNA methylation levels and upregulated expression of CCN1 and HSP90AA1 presented greater metastatic potential. This study characterizes the critical genetic, epigenetic, and transcriptomic features and their mutual regulatory relationships in ovarian cancer, providing valuable resources for identifying new molecular mechanisms and potential therapeutic targets for HGSC. SIGNIFICANCE: Integrated analysis of multiomic changes and epigenetic regulation in high-grade serous ovarian cancer provides insights into the molecular characteristics of this disease, which could help improve diagnosis and treatment.

In vitro differentiation of human embryonic stem cells into ovarian follicle-like cells.

Understanding the unique mechanisms of human oogenesis necessitates the development of an in vitro system of stem cell differentiation into oocytes. Specialized cell types and organoids have been derived from human pluripotent stem cells in vitro, but generating a human ovarian follicle remains a challenge. Here we report that human embryonic stem cells can be induced to differentiate into ovarian follicle-like cells (FLCs) in vitro. First, we find that two RNA-binding proteins specifically expressed in germ cells, DAZL and BOULE, regulate the exit from pluripotency and entry into meiosis. By expressing DAZL and BOULE with recombinant human GDF9 and BMP15, these meiotic germ cells are further induced to form ovarian FLCs, including oocytes and granulosa cells. This robust in vitro differentiation system will allow the study of the unique molecular mechanisms underlying human pluripotent stem cell differentiation into late primordial germ cells, meiotic germ cells and ovarian follicles.

Generating a Genome Editing Nuclease for Targeted Mutagenesis in Human Cells.

Gene targeting and editing is an essential tool for both basic research and clinical application such as gene therapy. Several endonucleases have been invented to fulfill these purposes, including zinc finger nucleases, TALEN, and CRISPR/Cas9. Although all of these systems can target DNA sequence with high efficiency, they also exert off-target effects and genotoxicity. The off-target effects might not hinder their usage in animal models because the correctly targeted cells can be selected for further studies. However, the off-target effects could cause mutations which may be damaging or cancerous to the patients. In this chapter, we describe a genome-editing nuclease method which relies on modifying specific amino acids on a monomeric endonuclease, I-SceI, to recognize a targeted sequence in the human genome. This nuclease is small in size and shows a much lower genotoxicity compared to other nucleases including CRISPR/Cas9.

A dominant negative mutation at the ATP binding domain of AMHR2 is associated with a defective anti-Müllerian hormone signaling pathway.

STUDY QUESTION: Does a heterozygous mutation in AMHR2, identified in whole-exome sequencings (WES) of patients with primary ovarian insufficiency (POI), cause a defect in anti-Müllerian hormone (AMH) signaling? SUMMARY ANSWER: The I209N mutation at the adenosine triphosphate binding domain of AMHR2 exerts dominant negative defects in the AMH signaling pathway. WHAT IS KNOWN ALREADY: Previous studies have demonstrated the associations of several sequence variants in AMH or AMHR2 with POI, but no functional assay has been performed to verify whether there was any defect on AMH signaling. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Ninety-six unrelated female Chinese Han patients were diagnosed with idiopathic POI and subjected to WES. In silico analysis was done for the sequence variants followed by molecular assays to examine the functional effects of the sequence variants in human granulosa cells. In silico analysis, immunostaining, Western analysis, genome-wide expression analysis, quantitatively polymerase chain reaction were applied to the characterization of the sequence variants. MAIN RESULTS AND THE ROLE OF CHANCE: We identified one novel heterozygous missense variant, p.Ala17Glu (A17E), in AMHR2. Subsequently, A17E and two independently reported missense variants, p.Ile209Asn (I209N) and p.Leu354Phe (L354F), were evaluated for effects on the AMH signaling pathway. In silico analysis predicted that all three variants may be deleterious. However, only one variant, I209N, showed severe defects in transducing the AMH signal as well as impaired SMAD1/5/8 phosphorylation. Furthermore, using genome-wide gene expression analysis, we identified genes whose expression was affected by the mutation, these included genes previously reported to participate in AMH signaling as well as newly identified genes. They are EMILIN2, FAM155A, GATA2, HES5, ID1, ID2, RLTPR, SMAD7, CBL, MALAT1 and SMARCA2. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Although the in vitro assays demonstrated the causative effect of I209N on AMH signaling, further studies need to validate its long-term effects on folliculogenesis and POI. WIDER IMPLICATIONS OF THE FINDINGS: These results will aid both researchers and clinicians in understanding the molecular pathology of AMH signaling and POI to develop diagnostic assays or therapeutics approaches. STUDY FUNDING AND COMPETING INTERESTS: Research funding is provided by the Ministry of Science and Technology of China [2012CB944704; 2012CB966702], and the National Natural Science Foundation of China [Grant number: 31171429]. The authors declare no conflict of interest.

HPV16 early gene E5 specifically reduces miRNA-196a in cervical cancer cells.

High-risk human papillomavirus (HPV) type 16, which is responsible for greater than 50% of cervical cancer cases, is the most prevalent and lethal HPV type. However, the molecular mechanisms of cervical carcinogenesis remain elusive, particularly the early steps of HPV infection that may transform normal cervical epithelium into a pre-neoplastic state. Here, we report that a group of microRNAs (microRNAs) were aberrantly decreased in HPV16-positive normal cervical tissues, and these groups of microRNAs are further reduced in cervical carcinoma. Among these miRNAs, miR196a expression is the most reduced in HPV16-infected tissues. Interestingly, miR196a expression is low in HPV16-positive cervical cancer cell lines but high in HPV16-negative cervical cancer cell lines. Furthermore, we found that only HPV16 early gene E5 specifically down-regulated miRNA196a in the cervical cancer cell lines. In addition, HoxB8, a known miR196a target gene, is up-regulated in the HPV16 cervical carcinoma cell line but not in HPV18 cervical cancer cell lines. Various doses of miR196a affected cervical cancer cell proliferation and apoptosis. Altogether, these results suggested that HPV16 E5 specifically down-regulates miR196a upon infection of the human cervix and initiates the transformation of normal cervix cells to cervical carcinoma.

Creating a monomeric endonuclease TALE-I-SceI with high specificity and low genotoxicity in human cells.

To correct a DNA mutation in the human genome for gene therapy, homology-directed repair (HDR) needs to be specific and have the lowest off-target effects to protect the human genome from deleterious mutations. Zinc finger nucleases, transcription activator-like effector nuclease (TALEN) and CRISPR-CAS9 systems have been engineered and used extensively to recognize and modify specific DNA sequences. Although TALEN and CRISPR/CAS9 could induce high levels of HDR in human cells, their genotoxicity was significantly higher. Here, we report the creation of a monomeric endonuclease that can recognize at least 33 bp by fusing the DNA-recognizing domain of TALEN (TALE) to a re-engineered homing endonuclease I-SceI. After sequentially re-engineering I-SceI to recognize 18 bp of the human ß-globin sequence, the re-engineered I-SceI induced HDR in human cells. When the re-engineered I-SceI was fused to TALE (TALE-ISVB2), the chimeric endonuclease induced the same HDR rate at the human ß-globin gene locus as that induced by TALEN, but significantly reduced genotoxicity. We further demonstrated that TALE-ISVB2 specifically targeted at the ß-globin sequence in human hematopoietic stem cells. Therefore, this monomeric endonuclease has the potential to be used in therapeutic gene targeting in human cells.

Theranostic effect of serial manganese-enhanced magnetic resonance imaging of human embryonic stem cell derived teratoma.

Although human embryonic stem cell (hESC) hold therapeutic potential, teratoma formation has deterred clinical translation. Manganese (Mn(2+)) enters metabolically active cells through voltage-gated calcium channels and subsequently, induces T(1) shortening. We hypothesized that serial manganese-enhanced MRI would have theranostic effect to assess hESC survival, teratoma formation, and hESC-derived teratoma reduction through intracellular accumulation of Mn(2+). Firefly luciferase transduced hESCs (hESC-Lucs) were transplanted into severe combined immunodeficient mouse hindlimbs to form teratoma. The chemotherapy group was injected with MnCl(2) intraperitoneally three times a week. The control group was given MnCl(2) only prior to manganese-enhanced MRI. Longitudinal evaluation by manganese-enhanced MRI and bioluminescence imaging was performed. The chemotherapy group showed significant reduction in the teratoma volume and luciferase activity at weeks 6 and 8. Histology revealed increased proportion of dead cells and caspase 3 positive cells in the chemotherapy group. Systemic administration of MnCl(2) enabled simultaneous monitoring and elimination of hESC-derived teratoma cells by higher intracellular accumulation of Mn(2+).

In vivo molecular MRI of cell survival and teratoma formation following embryonic stem cell transplantation into the injured murine myocardium.

Embryonic stem cells (ESCs) have shown the potential to restore cardiac function after myocardial injury. Superparamagnetic iron oxide nanoparticles (SPIO) have been widely employed to label ESCs for cellular MRI. However, nonspecific intracellular accumulation of SPIO limits long-term in vivo assessment of the transplanted cells. To overcome this limitation, a novel reporter gene (RG) has been developed to express antigens on the ESC surface. By employing SPIO-conjugated monoclonal antibody against these antigens (SPIO-MAb), the viability of transplanted ESCs can be detected in vivo. This study aims to develop a new molecular MRI method to assess in vivo ESC viability, proliferation, and teratoma formation. The RG is designed to express 2 antigens (hemagglutinin A and myc) and luciferase on the ESC surface. The two antigens serve as the molecular targets for SPIO-MAb. The human and mouse ESCs were transduced with the RG (ESC-RGs) and transplanted into the peri-infarct area using the murine myocardial injury model. In vivo MRI was performed following serial intravenous administration of SPIO-MAb. Significant hypointense signal was generated from the viable and proliferating ESCs and subsequent teratoma. This novel molecular MRI technique enabled in vivo detection of early ESC-derived teratoma formation in the injured murine myocardium.

Human primordial germ cell formation is diminished by exposure to environmental toxicants acting through the AHR signaling pathway.

Historically, effects of environmental toxicants on human development have been deduced via epidemiological studies because direct experimental analysis has not been possible. However, in recent years, the derivation of human pluripotent stem cells has provided a potential experimental system to directly probe human development. Here, we used human embryonic stem cells (hESCs) to study the effect of environmental toxicants on human germ cell development, with a focus on differentiation of the founding population of primordial germ cells (PGCs), which will go on to form the oocytes of the adult. We demonstrate that human PGC numbers are specifically reduced by exposure to polycyclic aromatic hydrocarbons (PAHs), a group of toxicants common in air pollutants released from gasoline combustion or tobacco smoke. Further, we demonstrate that the adverse effects of PAH exposure are mediated through the aromatic hydrocarbon receptor (AHR) and BAX pathway. This study demonstrates the utility of hESCs as a model system for direct examination of the molecular and genetic pathways of environmental toxicants on human germ cell development.

Human germ cell lineage differentiation from embryonic stem cells.

INTRODUCTIONBiological and ethical constraints hinder studies of human germ cell development despite its importance to reproductive health, including fertility and tumorigenesis. Thus, most of what we know of human germ cell development has been extrapolated from studies in model organisms. Human embryonic stem cells (hESCs) may provide an ideal system for probing the developmental genetics of germ cell formation and differentiation in vitro. The growth factors BMP (bone morphogenetic protein) 4, BMP7, and BMP8b are required for development of primordial germ cells (PGCs) in mice. It has been shown that these BMPs significantly increase germ cell differentiation from hESCs in vitro. This protocol describes a method to induce germ cell differentiation from hESCs by the addition of BMPs to hESC differentiation medium. The protocol can be used to study the basic mechanism of germ cell development in human cells.

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.