Transcriptional profiling of pluripotent and multipotent stem cells to decipher pluripotency and lineage specification

Abstract

Stem cells hold great promise for the fields of regenerative medicine, gene therapy and disease modeling. Understanding the transcriptional machinery involved in their maintenance is critical to their successful isolation and experimentation. Careful statistical analysis of high throughput transcriptome data can provide novel insights into the gene networks and patterns active in these cells. Public repositories are a source of gene expression data from various studies involving stem cells. This expression data can be overlaid on functional interactions maps of the genome to predict functional association. Further on, comparison of stem cells of different potencies can help identify key genes involved in the maintenance of pluripotency. The hypotheses derived from such transcriptional profiling can be tested experimentally to confirm expression and interactions. Transcriptome data from studies involving human and mouse pluripotent stem cells was collected from repositories such as GEO and Arrayexpress (EBI). Non-negative matrix factorization was used as a dimensionality reduction tool to detect biological patterns and clusters in the data. Following the classification of data into biologically meaningful classes, a ‘metagene’ profile characteristics of pluripotent stem cells was determined in both species. Reverse engineering was performed for predicting gene networks and signaling ‘hubs’. An algorithm was also developed to overlay this predicted gene signature onto functional networks that combine a large amount of genetic and genomic iv data from various sources, for detecting small subnetworks that are conserved in expression in the pluripotent stem cells of both species. Embryonic and induced pluripotent stem cells are considered as in vitro counterparts of pluripotent cells seen in the early embryo, namely the inner cell mass (ICM) and the epiblast. Multipotent adult progenitor cells (MAPCs), although isolated from the bone marrow of an adult rat, bear a striking similarity with another cell types in early embryonic development-the primitive endoderm or nascent hypoblast cells. Due to the developmental proximity of the pluripotent cells and the primitive endoderm cells in the early embryo, MAPCs have been used as a model system for probing gene interactions in pluripotent cells. On the basis of comparative transcriptome analysis, as well as, experimental studies, a model of gene regulation in MAPCs has been developed. The study of dynamics of this gene network provides novel insights into the transcriptional regulation of key pluripotency-associated genes. Also, using the concepts of cellular reprogramming, these multipotent stem cells (MAPCs) have been reprogrammed to a pluripotent state. This represents a unique reprogramming system where an Oct4 expressing extraembryonic cell has been transformed to an embryonic stem cells (ESC) like state. While this was performed using the traditional reprogramming cocktail consisting of Oct4, Sox2, Klf4 and c-Myc, future studies are likely to narrow down this number to one or two genes.