Transcriptional regulatory networks underlying the reprogramming of spermatogonial stem cells to multipotent stem cells

Spermatogonial stem cells (SSCs) are germline stem cells located along the basement membrane of seminiferous tubules in testes. Recently, SSCs were shown to be reprogrammed into multipotent SSCs (mSSCs). However, both the key factors and biological networks underlying this reprogramming remain elusive. Here, we present transcriptional regulatory networks (TRNs) that control cellular processes related to the SSC-to-mSSC reprogramming. Previously, we established intermediate SSCs (iSSCs) undergoing the transition to mSSCs and generated gene expression profiles of SSCs, iSSCs and mSSCs. By comparing these profiles, we identified 2643 genes that were up-regulated during the reprogramming process and 15 key transcription factors (TFs) that regulate these genes. Using the TF-target relationships, we developed TRNs describing how these TFs regulate three pluripotency-related processes (cell proliferation, stem cell maintenance and epigenetic regulation) during the reprogramming. The TRNs showed that 4 of the 15 TFs (Oct4/Pou5f1, Cux1, Zfp143 and E2f4) regulated cell proliferation during the early stages of reprogramming, whereas 11 TFs (Oct4/Pou5f1, Foxm1, Cux1, Zfp143, Trp53, E2f4, Esrrb, Nfyb, Nanog, Sox2 and Klf4) regulated the three pluripotency-related processes during the late stages of reprogramming. Our TRNs provide a model for the temporally coordinated transcriptional regulation of pluripotency-related processes during the SSC-to-mSSC reprogramming, which can be further tested in detailed functional studies.

Temporal and Spatial Expression Patterns of miR-302 and miR-367 During Early Embryonic Chick Development

The microRNAs (miRNAs) are small, non-coding RNAs that modulate protein expression by interfering with target mRNA translation or stability. miRNAs play crucial roles in various functions such as cellular, developmental, and physiological processes. The spatial expression patterns of miRNAs are very essential for identifying their functions. The expressions of miR-302 and miR-367 are critical in maintaining stemness of pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) but their functions in early development are not fully elucidated. So, we used Locked Nucleic Acid (LNA) probes to perform in situ hybridization and confirmed the temporal and spatial distribution patterns during early chick development. As a result, we found that miR-302 and miR-367 were expressed in various tissues such as primitive steak, neural ectoderm, neural plate, neural fold, neural tube, notochord, and oral cavity. Specially, we confirmed that miR-302 and miR-367 were strongly expressed in neural folds in HH8 to HH10. miR-302 was expressed on dorsal part of the neural tube but miR-367 was expressed on lateral and ventral parts of the neural tube. And also we performed quantitative stem-loop real-time PCR to analyze global expression level of miR-302 and miR-367. miR-302 and miR-367 expression was sustained before Hamburger and Hamilton stage (HH) 14. Thus, the temporal and spatial expression patterns of miR-302 and miR-367 may provide us information of the role of these miRNAs on tissue formation during early chick development.

Proteomic Analysis of Differently Expressed Proteins in a Mouse Model for Allergic Asthma

Allergic asthma is associated with persistent functional and structural changes in the airways and involves many different cell types. Many proteins involved in allergic asthma have been identified individually, but complete protein profiles (proteome) have not yet been reported. Here we have used a differential proteome mapping strategy to identify tissue proteins that are differentially expressed in mice with allergic asthma and in normal mice. Mouse lung tissue proteins were separated using two-dimensional gel electrophoresis over a pH range between 4 and 7, digested, and then analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MS). The proteins were identified using automated MS data acquisition. The resulting data were searched against a protein database using an internal Mascot search routine. This approach identified 15 proteins that were differentially expressed in the lungs of mice with allergic asthma and normal mice. All 15 proteins were identified by MS, and 9 could be linked to asthma-related symptoms, oxidation, or tissue remodeling. Our data suggest that these proteins may prove useful as surrogate biomarkers for quantitatively monitoring disease state progression or response to therapy.

Expression and Regulation of Latent TGF-beta Binding Protein-1 Transcripts and Their Splice Variants in Human Glomerular Endothelial Cells

Latent transforming growth factor (TGF)-beta-binding protein (LTBP) is required for the assembly, secretion, matrix association, and activation of latent TGF-beta complex. To elucidate the cell specific expression of the genes of LTBP-1 and their splice variants and the factors that regulate the gene expression, we cultured primary human glomerular endothelial cells (HGEC) under different conditions. Basal expression of LTBP-1 mRNA was suppressed in HGEC compared to WI-38 human embryonic lung fibroblasts. High glucose, H2O2, and TGF-beta1 upregulated and vascular endothelial growth factor (VEGF) further downregulated LTBP-1 mRNA in HGEC. RT-PCR with a primer set for LTBP-1S produced many clones but no clone was gained with a primer set for LTBP-1L. Of 12 clones selected randomly, Sca I mapping and DNA sequencing revealed that only one was LTBP-1S and all the others were LTBP-1S delta 53. TGF-beta1, but not high glucose, H2O2 or VEGF, tended to increase LTBP-1S delta 53 mRNA. In conclusion, HGEC express LTBP-1 mRNA which is suppressed at basal state but upregulated by high glucose, H2O2, and TGF-beta1 and downregulated by VEGF. Major splice variant of LTBP-1 in HGEC was LTBP-1S delta 53. Modification of LTBP-1S delta 53 gene in HGEC may abrogate fibrotic action of TGF-beta1 but this requires confirmation.