Human pluripotent stem cell-derived DDX4 and KRT-8 positive cells participate in ovarian follicle-like structure formation.

Understanding the mechanisms of human pluripotent stem cells (hPSCs) specification, development and differentiation to gametes are useful for elucidating the causes of infertility and potential treatment. This study aims to examine whether hPSCs can be induced to DDX4 extracellularly expressing primordial germ cell-like cells (DDX4ec PGCLCs) and further into ovarian follicle stage in a combined in vitro and in vivo model. The transcriptional signatures show that these DDX4ec PGCLCs are characteristic of PGCs and express ovarian folliculogenesis markers. We also verify that keratin (KRT)-8 is highly expressed in the DDX4ec PGCLCs and plays a crucial role in germ cell migration. By co-culturing DDX4ec PGCLCs with human granulosa cells (GCs), these cells are further induced into ovarian follicle-like structures in a xenograft mice model. This approach can in the future design practical strategies for treating germ cell-associated issues of infertility.

Effects of diosgenin and its derivatives on atherosclerosis.

Atherosclerosis is one of the leading causes of death in patients with cardiovascular diseases worldwide. Although some progress has been made in the treatment of cardiovascular diseases, the morbidity and mortality of cardiovascular diseases still continue to rise. At present, it is an important topic for researchers to develop safe and effective drugs from natural products to prevent and treat cardiovascular diseases. Diosgenin (DSG) is a plant sterol saponin mainly found in natural medicinal plants such as fenugreek seeds and wild yam tubers. More and more studies have reported that DSG has significant pharmacological activities such as anticancer, cardiovascular protection, hypolipidemic, anti-inflammatory, and neuroprotection. Furthermore, diosgenin is also an important basic raw material for the preparation of steroids and contraceptives in the pharmaceutical industry. Numerous preclinical studies have shown that DSG has great potential in the treatment of various cardiovascular diseases in vivo and in vitro, especially in atherosclerosis. This review mainly discusses the effects of DSG on endothelial dysfunction, lipid profile, and macrophage foam cell formation, VSMC viability, thrombosis and inflammation during the formation of atherosclerosis. Also, the mechanism of DSG on atherosclerosis was elaborated in detail. It is noteworthy that newly synthesized DSG derivatives and DSG delivery systems have good antithrombotic activity and pharmacokinetic characteristics.

Masticatory hypofunction effects induced by BTXA injection of hippocampal neurons in developing rats.

BACKGROUND AND OBJECTIVE: In clinical practice, malocclusion is often encountered during the period of growth and development of individuals. In addition to nutritional imbalance, some studies have found that mastication affects learning and memory ability. Tooth loss and masticatory hypofunction have been suggested as risk factors of Alzheimer disease. However, relatively little research has been done in developing animals. The present study evaluated the relationship between masticatory hypofunction and neuropathological changes of the hippocampus in developing rats. DESIGN: Four-week-old Wistar rats were randomly divided into saline-injected and botulinum toxin type A (BTXA)-injected groups. After an experiment period of 4 weeks, the rats were sacrificed for evaluation of neuropathological changes in the hippocampus through Nissl staining and phosphorylated cyclic AMP (cAMP) response element binding protein (CREB) immunohistochemistry. RESULTS: Nissl staining revealed a significant reduction in the density of neurons in the BTXA-injected rats. The BTXA-injected rats exhibited a decreased level of CREB phosphorylation. The degree of p-CREB immunoreactivity differed significantly between the two groups. CONCLUSION: The BTXA-injected rats exhibited a reduction in neuron density and phosphorylated CREB, indicating that mastication might influence the learning and memory ability during the growth period. Therefore, it is strongly suggested that malocclusion be corrected as soon as possible during growth and development.

Comparative global immune-related gene profiling of somatic cells, human pluripotent stem cells and their derivatives: implication for human lymphocyte proliferation.

Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), represent potentially unlimited cell sources for clinical applications. Previous studies have suggested that hPSCs may benefit from immune privilege and limited immunogenicity, as reflected by the reduced expression of major histocompatibility complex class-related molecules. Here we investigated the global immune-related gene expression profiles of human ESCs, hiPSCs and somatic cells and identified candidate immune-related genes that may alter their immunogenicity. The expression levels of global immune-related genes were determined by comparing undifferentiated and differentiated stem cells and three types of human somatic cells: dermal papilla cells, ovarian granulosa cells and foreskin fibroblast cells. We identified the differentially expressed genes CD24, GATA3, PROM1, THBS2, LY96, IFIT3, CXCR4, IL1R1, FGFR3, IDO1 and KDR, which overlapped with selected immune-related gene lists. In further analyses, mammalian target of rapamycin complex (mTOR) signaling was investigated in the differentiated stem cells following treatment with rapamycin and lentiviral transduction with specific short-hairpin RNAs. We found that the inhibition of mTOR signal pathways significantly downregulated the immunogenicity of differentiated stem cells. We also tested the immune responses induced in differentiated stem cells by mixed lymphocyte reactions. We found that CD24- and GATA3-deficient differentiated stem cells including neural lineage cells had limited abilities to activate human lymphocytes. By analyzing the transcriptome signature of immune-related genes, we observed a tendency of the hPSCs to differentiate toward an immune cell phenotype. Taken together, these data identify candidate immune-related genes that might constitute valuable targets for clinical applications.

Loss of non-coding RNA expression from the DLK1-DIO3 imprinted locus correlates with reduced neural differentiation potential in human embryonic stem cell lines.

INTRODUCTION: Pluripotent stem cells are increasingly used to build therapeutic models, including the transplantation of neural progenitors derived from human embryonic stem cells (hESCs). Recently, long non-coding RNAs (lncRNAs), including delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (DLK1-DIO3) imprinted locus-derived maternally expressed gene 3 (MEG3), were found to be expressed during neural development. The deregulation of these lncRNAs is associated with various neurological diseases. The imprinted locus DLK1-DIO3 encodes abundant non-coding RNAs (ncRNAs) that are regulated by differential methylation of the locus. We aim to study the correlation between the DLK1-DIO3-derived ncRNAs and the capacity of hESCs to differentiate into neural lineages. METHODS: We classified hESC sublines into MEG3-ON and MEG3-OFF based on the expression levels of MEG3 and its downstream microRNAs as detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A cDNA microarray was used to analyze the gene expression profiles of hESCs. To investigate the capacity of neural differentiation in MEG3-ON and MEG3-OFF hESCs, we performed neural lineage differentiation followed by neural lineage marker expression and neurite formation analyses via qRT-PCR and immunocytochemistry, respectively. MEG3-knockdown via small interfering RNA (siRNA) and small hairpin RNA (shRNA) was used to investigate the potential causative effect of MEG3 in regulating neural lineage-related gene expression. RESULTS: DLK1-DIO3-derived ncRNAs were repressed in MEG3-OFF hESCs compared with those in the MEG3-ON hESCs. The transcriptome profile indicated that many genes related to nervous system development and neural-type tumors were differentially expressed in MEG3-OFF hESCs. Three independent MEG3-knockdown assays using different siRNA and shRNA constructs consistently resulted in downregulation of some neural lineage genes. Lower expression levels of stage-specific neural lineage markers and reduced neurite formation were observed in neural lineage-like cells derived from MEG3-OFF-associated hESCs compared with those in the MEG3-ON groups at the same time points after differentiation. CONCLUSIONS: Repression of ncRNAs derived from the DLK1-DIO3 imprinted locus is associated with reduced neural lineage differentiation potential in hESCs.

Granulosa cells and retinoic acid co-treatment enrich potential germ cells from manually selected Oct4-EGFP expressing human embryonic stem cells.

Differentiation of human embryonic stem (HES) cells to germ cells may become clinically useful in overcoming diseases related to germ-cell development. Niches were used to differentiate HES cell lines, NTU1 and H9 Oct4-enhanced green fluorescence protein (EGFP), including laminin, granulosa cell co-culture or conditioned medium, ovarian stromal cell co-culture or conditioned medium, retinoic acid, stem cell factor (SCF) and BMP4-BMP7-BMP8b treatment. Flow cytometry showed that granulosa cell co-culture (P < 0.001) or conditioned medium (P = 0.007) treatment for 14 days significantly increased the percentages of differentiated H9 Oct4-EGFP cells expressing early germ cell marker stage-specific embryonic antigen 1(SSEA1); sorted SSEA1[+] cells did not express higher levels of germ cell gene VASA and GDF9. Manually collected H9 Oct4-EGFP[+] cells expressed significantly higher levels of VASA (P = 0.005) and GDF9 (P = 0.001). H9 Oct4-EGFP[+] cells developed to ovarian follicle-like structures after culture for 28 days but with low efficiency. Unlike SCF and BMP4, retinoic acid co-treatment enhanced VASA, GDF9 and SCP3 expression. A protocol is recommended to enrich differentiated HES cells with germ-cell potential by culture with granulosa cells, conditioned medium or retinoic acid, manual selection of Oct4-EGFP[+] cells, and analysis of VASA, GDF9 expression, or both.

Characterization of a plant-transformation-ready large-insert BIBAC library of Arabidopsis and bombardment transformation of a large-insert BIBAC of the library into tobacco.

Plant-transformation-ready, large-insert binary bacterial artificial chromosome (BIBAC) libraries are of significance for functional and network analysis of large genomic regions, gene clusters, large-spanning genes, and complex loci in the post-genome era. Here, we report the characterization of a plant-transformation-ready BIBAC library of the sequenced Arabidopsis genome for which such a library is not available to the public, the transformation of a large-insert BIBAC of the library into tobacco by biolistic bombardment, and the expression analysis of its containing genes in transgenic plants. The BIBAC library was constructed from nuclear DNA partially digested with BamHI in the BIBAC vector pCLD04541. It contains 6144 clones and has a mean insert size of 108 kb, representing 5.2× equivalents of the Arabidopsis genome or a probability of greater than 99% of obtaining at least one positive clone from the library using a single-copy sequence as a probe. The transformation of the large-insert BIBAC and analyses of the transgenic plants showed that not only did transgenic plants have intact BIBAC DNA, but also could the BIBAC be transmitted stably into progenies and its containing genes be expressed actively. These results suggest that the large-insert BIBAC library, combined with the biolistic bombardment transformation method, could provide a useful tool for large-scale functional analysis of the Arabidopsis genome sequence and applications in plant-molecular breeding.

A reduced oxygen tension (5%) is not beneficial for maintaining human embryonic stem cells in the undifferentiated state with short splitting intervals.

BACKGROUND: Human embryos grow naturally in vivo in lower oxygen (O(2)) tension environments than atmospheric O(2) tension. Therefore, human embryonic stem cells (hESC), a derivative of embryos, will likely grow more favorably in a reduced O(2) tension. This study aimed to compare the behavior of hESC under reduced O(2) tension (5%) versus normoxia (21%). METHODS: hESC lines were cultured in different O(2) tensions and then examined for morphology, apoptosis and gene expression profiles. RESULTS: hESC grown in 5% O(2) tension were not morphologically different from hESC grown in normoxia on day 7 of the first and fourth passages. However, after prolonged culture without splitting (10-14 days), hESC colonies were thinner and looked better morphologically in 5% O(2), but the cells proliferated more slowly and their sizes were larger. At most time points, the gene expression profiles in both O(2) tensions showed no major difference in representative stemness genes (Oct-3/4, Nanog and Cripto), differentiation genes (Desmin, Nestin, alpha-fetoprotein and GDF-9) and hypoxia-related genes (HIF-1alpha and VEGF). A lower level of cyclin-D1 mRNA (suggestive of less Wnt pathway signaling on day 7 of the fourth passage) and a higher level of Desmin (suggestive of more differentiation to mesoderm, at day 7 of the first passage) were detected in 5% O(2). CONCLUSIONS: This study suggests that for routine culture of hESC with a short splitting interval (7 days), a low O(2) tension (5% O(2)) probably does not provide significant advantages over the standard 21% O(2) tension for the maintenance of an undifferentiated state by the criteria used in this study.