Non-coding RNA Expression for Evaluating Reprogramming Quality of Human iPSC_abstract

Project Title: Non-coding RNA Expression for Evaluating Reprogramming Quality of Human iPSC

Pilot Study Principal Investigator: Elias Zambidis, M.D., Ph.D.

Hub Site Principal Investigator: Alan Friedman, M.D.

Awarded Organizations: Johns Hopkins University School of Medicine

Abstract:

The ideal human induced pluripotent stem cell (hiPSC) line should have the capacity to differentiate into any desired clinical lineage with equivalent efficiency to human embryonic stem cells (hESCs). However, many existing hiPSC lines possess limited efficiency of differentiation into mesodermal, ectodermal, and endodermal lineages compared to bona fide hESC. This limitation may be due to incomplete reprogramming of the somatic donor cell epigenome to a bona fide embryonic stem cell (ESC)-like state, residual expression of virally-integrated transgenes, or the retention of donor lineage-specific epigenetic memory. Although protein-coding mRNAs and epigenetic marks have been described extensively in the regulation of iPSC function, it is increasingly understood that non-coding RNAs such as short microRNAs (miRNAs) and long noncoding RNA (lncRNAs) also play key roles. For example, non-coding RNAs are increasingly understood to have critical roles in regulating the protein-coding genes and networks in ESC and iPSC that regulate pluripotency. However, to date, no systematic analyses of global patterns of ncRNA expression (especially lncRNA expression) in hiPSC has been conducted that might define their quality of pluripotency, their degree of differentiation potency, and their role in contributing to somatic memory from incomplete reprogramming. This pilot project seeks to synergize the extensive transcriptomics data that is currently being amassed by the PCBC Cell Characterization Core in Cincinnati (C4)’s efforts, the exciting preliminary data by investigators from the Hopkins U01 Hub site, and the bioinformatics expertise of the Sage Bionetworks group affiliated with the FHCRC Hub Site. Our approach is to leverage the newly-supported PCBC Bioinformatics Core to identify noncoding short (microRNAs) and lncRNA signatures that define two key aspects of iPSC quality: fidelity of the reprogrammed pluripotent state, and the role of somatic memory in skewing the differentiation potency of hiPSC. In addition to the biological hypotheses developed in this pilot proposal, we believe the novel bioinformatics approaches described in this pilot will serve as a general tool and paradigm that may be used by other PCBC investigators conducting similar studies with the goal of inferring molecular signature and quantitative metrics used to assess the quality of hiPSC lines that are generated with various methodologies. We intend to make our approach available to PCBC investigators, and the wider research community, through the bioinformatics data and analysis portal developed by the Bioinformatics Core. The wealth of transcriptomics data compiled by the PCBC, and the bioinformatics tools being assembled by the PCBC Bioinformatics Core (to which Sage Bionetworks is a major contributor) are both resources with enormous possible potential. These studies will harness the latent potential of both resources to systematically expand the important findings regarding drivers of pluripotency and differentiation potential by protein-coding RNA transcripts, to the novel universe of non-coding RNA transcripts.