PTN from Leydig cells activates SDC2 and modulates human spermatogonial stem cell proliferation and survival via GFRA1.

BACKGROUND: Spermatogonial stem cells (SSCs) are essential for the maintenance and initiation of male spermatogenesis. Despite the advances in understanding SSC biology in mouse models, the mechanisms underlying human SSC development remain elusive. RESULTS: Here, we analyzed the signaling pathways involved in SSC regulation by testicular somatic cells using single-cell sequencing data (GEO datasets: GSE149512 and GSE112013) and identified that Leydig cells communicate with SSCs through pleiotrophin (PTN) and its receptor syndecan-2 (SDC2). Immunofluorescence, STRING prediction, and protein immunoprecipitation assays confirmed the interaction between PTN and SDC2 in spermatogonia, but their co-localization was observed only in approximately 50% of the cells. The knockdown of SDC2 in human SSC lines impaired cell proliferation, DNA synthesis, and the expression of PLZF, a key marker for SSC self-renewal. Transcriptome analysis revealed that SDC2 knockdown downregulated the expression of GFRA1, a crucial factor for SSC proliferation and self-renewal, and inhibited the HIF-1 signaling pathway. Exogenous PTN rescued the proliferation and GFRA1 expression in SDC2 knockdown SSC lines. In addition, we found downregulation of PTN and SDC2 as well as altered localization in non-obstructive azoospermia (NOA) patients, suggesting that downregulation of PTN and SDC2 may be associated with impaired spermatogenesis. CONCLUSIONS: Our results uncover a novel mechanism of human SSC regulation by the testicular microenvironment and suggest a potential therapeutic target for male infertility.

Role of telomeric repeat-containing RNA in telomeric chromatin remodeling during the early expansion of human embryonic stem cells.

This study aimed to explore the role of telomeric repeat-containing RNA (TERRA) in telomeric chromatin remodeling during the early expansion of human embryonic stem cells (hESCs). During the derivation of hESCs, histone demethylation in the telomeric region facilitates telomerase-mediated telomere elongation. An adequate telomere repeat is essential for hESCs to acquire and/or maintain the unlimited symmetric division, which suggests that there is a link between pluripotency and telomere maintenance. The present study found that the gradual decrease in TERRA levels and related TERRA foci were correlated with telomeric length elongation in the early expansion of hESCs. In addition, TERRA participated in telomeric chromatin remodeling by cooperating with SUV39H1 (suppressor of variegation 3-9 homolog 1/2) to propagate telomeric heterochromatin marker, histone H3 trimethylation of lysine 9. Moreover, the fibroblast growth factor signaling pathway, which is activated in hESCs, could suppress TERRA levels via telomeric repeat factor 1, which results in reduced SUV39H1 recruitment by TERRA at the telomere. Taken together, these results highlight the role of TERRA in hESC telomere elongation and homeostasis in the acquisition and/or maintenance of stem cell pluripotency.-Zeng, S., Liu, L., Sun, Y., Lu, G., Lin, G. Role of telomeric repeat-containing RNA in telomeric chromatin remodeling during the early expansion of human embryonic stem cells.

Transcriptional activation of human CDCA8 gene regulated by transcription factor NF-Y in embryonic stem cells and cancer cells.

The cell division cycle associated 8 (CDCA8) gene plays an important role in mitosis. Overexpression of CDCA8 was reported in some human cancers and is required for cancer growth and progression. We found CDCA8 expression was also high in human ES cells (hESCs) but dropped significantly upon hESC differentiation. However, the regulation of CDCA8 expression has not yet been studied. Here, we characterized the CDCA8 promoter and identified its cis-elements and transcription factors. Three transcription start sites were identified. Reporter gene assays revealed that the CDCA8 promoter was activated in hESCs and cancer cell lines. The promoter drove the reporter expression specifically to pluripotent cells during early mouse embryo development and to tumor tissues in tumor-bearing mice. These results indicate that CDCA8 is transcriptionally activated in hESCs and cancer cells. Mechanistically, two key activation elements, bound by transcription factor NF-Y and CREB1, respectively, were identified in the CDCA8 basic promoter by mutation analyses and electrophoretic motility shift assays. NF-Y binding is positively correlated with promoter activities in different cell types. Interestingly, the NF-YA subunit, binding to the promoter, is primarily a short isoform in hESCs and a long isoform in cancer cells, indicating a different activation mechanism of the CDCA8 transcription between hESCs and cancer cells. Finally, enhanced CDCA8 promoter activities by NF-Y overexpression and reduced CDCA8 transcription by NF-Y knockdown further verified that NF-Y is a positive regulator of CDCA8 transcription. Our study unearths the molecular mechanisms underlying the activation of CDCA8 expression in hESCs and cancer cells, which provides a better understanding of its biological functions.

Telomerase-mediated telomere elongation from human blastocysts to embryonic stem cells.

High telomerase activity is a characteristic of human embryonic stem cells (hESCs), however, the regulation and maintenance of correct telomere length in hESCs is unclear. In this study we investigated telomere elongation in hESCs in vitro and found that telomeres lengthened from their derivation in blastocysts through early expansion, but stabilized at later passages. We report that the core unit of telomerase, hTERT, was highly expressed in hESCs in blastocysts and throughout long-term culture; furthermore, this was regulated in a Wnt-ß-catenin-signaling-dependent manner. Our observations that the alternative lengthening of telomeres (ALT) pathway was suppressed in hESCs and that hTERT knockdown partially inhibited telomere elongation, demonstrated that high telomerase activity was required for telomere elongation. We observed that chromatin modification through trimethylation of H3K9 and H4K20 at telomeric regions decreased during early culture. This was concurrent with telomere elongation, suggesting that epigenetic regulation of telomeric chromatin may influence telomerase function. By measuring telomere length in 96 hESC lines, we were able to establish that telomere length remained relatively stable at 12.02 ± 1.01 kb during later passages (15-95). In contrast, telomere length varied in hESCs with genomic instability and hESC-derived teratomas. In summary, we propose that correct, stable telomere length may serve as a potential biomarker for genetically stable hESCs.

Inferring Causal Relationships Between Metabolites and Polycystic Ovary Syndrome Using Summary Statistics from Genome­Wide Association Studies.

Polycystic ovary syndrome (PCOS) is a disorder characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology. Previous studies have suggested that metabolites may play a pivotal mediating role in the progression of phenotypic variations. Although several metabolites had been identified as potential markers for PCOS, the relationship between blood metabolites and PCOS was not comprehensively explored. Previously, Pickrell et al. designed a robust approach to infer evidence of a causal relationship between different phenotypes using independently putative causal SNPs. Our previous paper extended this approach to make it more suitable for cases where only a few independently putative causal SNPs were identified to be significantly associated with the phenotypes (i.e., metabolites). When the most significant SNPs in each independent locus (the independent lead SNPs) with p-values of < 1 × 10-5 were used, 3 metabolites (2-tetradecenoyl carnitine, threitol, 1-docosahexaenoylglycerophosphocholine) causally influencing PCOS and 2 metabolites (asparagine and phenyllactate) influenced by PCOS were identified, (relative likelihood r < 0.01). Under a less stringent threshold of r < 0.05, 7 metabolites (trans-4-hydroxyproline, glutaroyl carnitine, stachydrine, undecanoate, 7-Hoca, N-acetylalanine and 2-hydroxyisobutyrate) were identified. Taken together, this study can provide novel insights into the pathophysiological mechanisms underlying PCOS; whether these metabolites can serve as biomarkers to predict PCOS in clinical practice warrants further investigations.

Long non-coding RNA TTTY14 promotes cell proliferation and functions as a prognostic biomarker in testicular germ cell tumor.

Testicular germ cell tumors (TGCTs) commonly occur in males between the ages of 15 and 34, accounting for 98% of testicular malignancies. Long non-coding RNAs (LncRNAs) have been reported to play important roles in TGCT proliferation, invasion, and functioned as prognostic biomarkers. Testis-specific transcript, Y-linked 14 (TTTY14), a long non-coding RNA localized on Chr Y q11.222, has been found to be a potential prognostic biomarker for laryngeal squamous cell carcinoma, gastric cancer, and osteosarcoma. The biological role of TTTY14 in TGCT is not well understood. In this study, we aim to clarify the biological role of TTTY14 in TGCT, as well as its role in TGCT survival prognosis and immunotherapy efficacy prediction through the deep mining of public data combined with the verification of cell biological experiments. We found that high TTTY14 expression was a poor survival prognostic factor in TGCT patients and the expression of TTTY14 might be regulated by copy number variation and DNA methylation. TTTY14 knockdown significantly inhibited the proliferation of TGCT in vitro. TTTY14 expression was positively correlated with immune cell dysfunction, and significantly negatively correlated with B cells, CD8+ T cells, and macrophages, suggesting that TTTY14 may also affect the drug sensitivity by regulating the tumor immune microenvironment. In conclusion, we revealed that lncRNA TTTY14 was a novel oncogene and a biomarker in TGCT. TTTY14 may influence the drugs sensitivity through regulating the tumor immune microenvironment.

Generation of a human embryonic stem cell line, NERCe003-A-1, with lentivirus vector-mediated inducible CTNNB1 overexpression.

The human embryonic stem cell (hESC) line NERCe003-A-1 was generated by introducing lentiviral-vector-mediated tetracycline-inducible ß-catenin expression into a normal hESC line, NERCe003-A. The resulting cell line can overexpress the ß-catenin protein, encoded by the CTNNB1 gene, after exposure to doxycycline (Dox). CTNNB1 gene expression was confirmed by quantitative PCR (qPCR) and immunofluorescence assays. Further characterization confirmed that the NERCe003-A-1 cell line expresses typical pluripotency markers and has the ability to form the three germ layers both in vitro and in vivo.

Decreased phosphorylation of PDGFR-ß impairs the angiogenic potential of expanded endothelial progenitor cells via the inhibition of PI3K/Akt signaling.

Human umbilical cord blood-derived endothelial progenitor cells (EPCs) have been proven to contribute to post-natal angiogenesis, and have been applied in various models of ischemia. However, to date, to the best of our knowledge, there is no available data on the angiogenic properties of EPCs during the process of in vitro expansion. In this study, we expanded EPCs to obtain cells at different passages, and analyzed their cellular properties and angiogenic ability. In the process of expansion, no changes were observed in cell cobblestone-like morphology, apoptotic rate and telomere length. However, the cell proliferative ability was significantly decreased. Additionally, the expression of CD144, CD90 and KDR was significantly downregulated in the later-passage cells. Vascular formation assay in vitro revealed that EPCs at passage 4 and 6 formed more integrated and organized capillary-like networks. In a murine model of hind limb ischemia, the transplantation of EPCs at passage 4 and 6 more effectively promoted perfusion recovery in the limbs on days 7 and 14, and promoted limb salvage and histological recovery. Furthermore, the phosphorylation levels of platelet­derived growth factor receptor-ß (PDGFR-ß) were found to be significantly decreased with the in vitro expansion process, accompanied by the decreased activation of the PI3K/Akt signaling pathway. When PDGFR inhibitor was used to treat the EPCs, the differences in the angiogenic potential and migratory ability among the EPCs at different passages were no longer observed; no significant differences were also observed in the levels of phosphorylated PI3K/Akt between the EPCs at different passages following treatment with the inhibitor. On the whole, our findings indicate that the levels of phosphorylated PDGFR-ß are decreased in EPCs with the in vitro expansion process, which impairs their angiogenic potential by inhibiting PI3K/Akt signaling. Our findings may aid in the more effective selection of EPCs of different passages for the clinical therapy of ischemic disease.

Generation of induced pluripotent stem cells (iPSCs) from a retinoblastoma patient carrying a c.2663G>A mutation in RB1 gene.

Skin fibroblasts were obtained from a male patient diagnosed with retinoblastoma (RB) carrying a c.2663G>A mutation in the 25 exon of RB1 gene. RB-iPS cells was generated via delivered four reprogramming factors (OCT4, SOX2, NANOG and LIN28) into these skin fibroblasts. The RB-iPS cells retained the RB1 heterozygous mutation resulted in a truncated RB1 mRNA. Characteristic tests proved that the iPSC line presented typical markers of pluripotency and had the capability to form the three germ layers in vitro.

Generation of human embryonic stem cell line chHES-472 from abnormal embryos diagnosed with Spinocerebellar ataxia type 3.

Spinocerebellar ataxia type3 (SCA3) is an autosomal dominant neurodegenerative disorder. Human embryonic stem cell line chHES-472 was derived from abnormal embryo donated by SCA3 patient after preimplantation genetic diagnosis (PGD) treatment. This cell line had a normal karyotype and retained the disease-causing mutant in ATXN3 gene. Characteristic tests proved that the embryonic stem cell line presented typical markers of pluripotency and had the capability to form the three germlayers in vivo.