Detailed Study on Molecular Mechanism of Pluripotency and Reprogramming in Testicular Stem Cells – In Silico Approach

Abstract

Testis-derived male germ-line stem (GS) cells are the in vitro counterpart of spermatogonial stem cell (SSC). Under appropriate culture conditions, GS cells can acquire pluripotency to become multipotent GS or Germ-line pluripotent stem (GPS) cells with the loss of spermatogenic properties. The molecular mechanism of GS niche and, the origin and reprogramming into GPS is elusive. This study hypothesize that, analysis and annotation of high-throughput omics data of GS and GPS cells, by computational methods, may provide insight in drawing the landscape of regulatory network involved in maintenance of stemness in GS cells and their reprogramming into GPS cells. In the first part of the study, the RNA-seq data of both GS and GPS cells were retrieved and subjected to Tuxedo protocol and Network analysis. A novel approach was adopted for the prediction of novel pluripotent genes. Five clusters were identified and ranked according to their score. Novel pluripotent genes like Cdh5, Cdh10 were predicted. The co-expression, clustering of the transcriptome and variation of the transcriptome of GS and GPS cells was studied using fuzzy clustering by AutoSOME. Transcriptome analysis using the proposed approach intuitively and consistently characterized the variation in cell-cell significantly. The study also analyzed the Alternative Polyadenylation (APA) pattern and 3' Untranslated Regions (3'-UTR) length in differentially expressed mRNAs during the reprogramming of GS cells to GPS cells using APADetect. Obtained results suggest that 3'-UTR is longer in GS cells compared to GPS cells The APA-regulated genes were found to be involved in the regulation of mRNA processing and RNA splicing with shortened 3'-UTR. In the next part of the study, CellNet analysis and Boolean models were used to study the relationship of Gene Regulatory Networks (GRNs) between the GS and GPS cells. The GRNs involving all the genes from integrated methods and literature were constructed and qualitative modelling for reprogramming of GS to GPS cells were done by considering the discrete, asynchronous, multivalued logical formalism using the GINsim modeling and simulation tool. Result suggests that reprogramming of GS cells to GPS cells involves signaling pathways namely LIF, GDNF, BMP4, and TGF-β along with some novel pluripotency genes. The study also predicted miRNAs among GS and GPS cells to construct miRNA synergistic networks (MSN) and identify regulatory miRNA modules. Synergistic network involving mmu miR-200b-3p, mmu-miR-429-3p and mmu-miR-141-3p, mmu miR-200a-3p and mmu-miR-200c-3p was found to conjecture and control the pluripotency and reprogramming by promoting Mesenchymal to Epithelial Transition (MET). These data may be useful in analyzing the regulatory network in acquiring pluripotency by male GPS cell, and predicting novel cocktails (mRNA, miRNA, transcription factors (TFs)) that may collectively target the members of regulatory network to induce pluripotency.