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.