A Boolean Logical model for Reprogramming of Testes-derived male Germline Stem Cells into Germline pluripotent stem cells

Abstract

Background and ObjectiveMale germline stem (GS) cells are responsible for the maintenance of spermatogenesis throughout the adult life of males. Upon appropriate in vitro culture conditions, these GS cells can undergo reprogramming to become germline pluripotent stem (GPS) cells with the loss of spermatogenic potential. In recent years, voluminous data of gene transcripts in GS and GPS cells have become available. However, the mechanism of reprogramming of GS cells into GPS cells remains elusive. This study was designed to develop a Boolean logical model of gene regulatory network (GRN) that might be involved in the reprogramming of GS cells into GPS cells. MethodsThe gene expression profile of GS and GPS cells (GSE ID: GSE11274 and GSE74151) were analyzed using R Bioconductor to identify differentially expressed genes (DEGs) and were functionally annotated with DAVID server. Potential pluripotent genes among the DEGs were then predicted using a combination of machine learning [Support Vector Machine (SVM)] and BLAST search. Protein isoforms were identified by pattern matching with UniProt database with in-house scripts written in C++. Both linear and non-linear interaction maps were generated using the STRING server. CellNet is used to study the relationship of GRNs between the GS and GPS cells. Finally, the GRNs involving all the genes from integrated methods and literature was 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. ResultsThrough the use of machine learning and logical modeling, the present study identified 3585 DEGs and 221 novel pluripotent genes including Tet1, Cdh1, Tfap2c, Etv4, Etv5, Prdm14, and Prdm10 in GPS cells. Pathway analysis revealed that important signaling pathways such as core pluripotency network, PI3K-Akt, WNT, GDNF and BMP4 signalling pathways were important for the reprogramming of GS cells to GPS cells. On the other hand, CellNet analysis of GRNs of GS and GPS cells revealed that GS cells were similar to gonads whereas GPS cells were similar to ESCs in gene expression profile. A logical regulatory model was developed, which showed that TGFβ negatively regulated the reprogramming of the GS to GPS cells, as confirmed by perturbations studies. ConclusionThe study identified novel pluripotent genes involved in the reprogramming of GS cells into GPS cells. A multivalued logical model of cellular reprogramming is proposed, which suggests that reprogramming of GS cells to GPS cells involves signalling pathways namely LIF, GDNF, BMP4, and TGFβ along with some novel pluripotency genes.