Basic Biology V
$1 132 720
There is little consensus on whether current analysis is sufficient to predict stability or true phenotypic pluripotency in human Embryonic Stem Cells (hESC) and induced Pluripotent Stem Cells (iPSC). hESC and iPSC lines are unstable over time, and inefficient in functional assays. Thus, additional screens may be valuable in moving forward clinical applications for stem cell technology. Work over several years has found that proper control (silencing) of a human endogenous retroelements (ERVs) is crucial for establishment of a robust and stable ESC phenotype. These elements represent so called "junk DNA". During normal development, the vast majority of junk DNA elements are silenced, and never re-expressed after birth. Silencing takes place rather quickly during normal development. But the process is random, and therefore, can contribute to diversity in individual clones if the process is done under low stringency conditions, as is the case with all iPSC generation. In this proposal, we will compare the expression patterns of ERVs across a panel of hESC and iPSC to identify "signatures of control" that can be predictive of which cell lines will be most productive to maintain and propagate for human clinical trials. Most existing expression analysis of hESCs and iPSCs is useless for analysis of ERVs because of their repetitive nature. Thus the current proposal will use direct sequencing methods (RNAseq and ChIPseq methods) that can capture this previously ignored complexity.
Statement of Benefit to California:
CIRM is tasked with moving forward human embryonic stem cell technology toward clinical utility. While major strides have been accomplished, there are elements of complexity that limit and delay application. For instance, hESC and iPSCs are rather difficult to generate as the process isolates rare events, requiring vast amounts of characterization of existing clones. Additionally, functional diversity of ES/iPSC cell lines has been revealed, such that some lines of cells appear better able to perform certain types of functions. Is this diversity an innate feature of different individual genomes, or is is it due to randomness of processes involved in establishing hESC and iPSC lines in the 1st place? While some aspects maybe due to individuality of genomes, a much larger amount of variation is likely due to the process of ES in iPS generation. We have found that one aspect of this stochastic process can be measured reproducibly through analysis of the expression of repetitive genetic elements called human endogenous retroelements (ERVs). We propose that inexpensive assays can be developed to monitor the silencing of specific ERV elements which are critical to pluripotency. Once we have determined which elements are most predictive of a stable ES-like state. This work can not be carried out in model genetic systems, because ERVs vary greatly between humans and other animals. Thus the work is specifically tailored to improving understanding of existing hES and iPSC lines.
The goal of this Fundamental Mechanisms Award proposal is to understand the extent and role of endogenous retroelements (ERV) activity in human pluripotent stem cells. ERVs consist of repeated sequences derived from ancient viral-like elements. During early embryogenesis, ERVs are mostly re-activated before getting silenced at the blastocyst stage. This dynamic is dependent by epigenetic mechanisms among which a prominent role is played by demethylation of DNA and chromatin. Thus, functional control of this large array of elements is tightly regulated and their residual activity might influence the expression of nearby genetic units. This proposal seeks to test the hypothesis that proper control of ERVs is required for the establishment of a stable embryonic stem cell (ESC) phenotype. Three specific aims are proposed to test this hypothesis; first to identify and evaluate patterns of ERV expression in a panel of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) lines; second, to determine the stability of ERV expression after extensive passaging in hESCs and hiPSCs lines; and third to examine the clonality of ERV expression in second generation hiPSCs. Significance and Innovation - The overall significance of this proposal is modest. - The likelihood of the metrics developed having an immediate utility in either basic or translational research is small. Feasibility and Experimental Design - The proposal is well structured; however the scope of the proposal is very limited in the experimental plan and lacks ambition. For example, there was a lack of effort to correlate ERV expression changes with the different traits of pluripotency and its associated differentiation potential. - Similar studies have been performed in murine ESCs, which could have been utilized for a functional understanding of ERV misregulation. - The project will require strong bioinformatics that were not developed in the proposal. It was not clear if such bioinformatics are present or available to the applicant. Principal Investigator (PI) and Research Team - PI is well-established investigator primarily working in cancer biology but has no previous publications on this particular line of research. - The team has not yet been appointed. Responsiveness to the RFA - The proposal is responsive to the RFA.