It has been known for some time that the chromosomes of stem cells in culture tend to become unstable over time. This is worrisome as cells that have lost parts of their chromosomes can turn into cancer cells. This would of course be particularly bad if it happened in a stem cell line that is then implanted in a patient as a therapy. The methods currently used to determine if stem cell chromosomes are becoming unstable have a relatively low resolution.
Currently new DNA sequencing instruments have become available that allow for the first time for an acceptable price to detect all possible chromosomal instabilities through genome-wide DNA sequencing.
We propose to apply those new DNA sequencing instruments to the question of chromosomal instability in stem cell cultures. We think that based on the new instruments and our expertise in analyzing the data they generate we will be able to develop a procedure that will allow researchers to detect chromosome instability in stem cell cultures much earlier and with much higher accuracy. This should be of great benefit to stem cell research, especially if it becomes the standard procedure so that all stem cell laboratories would be enabled to directly compare their stem cell lines and cultures on a very fundamental level of quality control before using them any further in research or even to develop therapies based on them.
Statement of Benefit to California:
Our project would be beneficial to California by
-supporting jobs at [REDACTED]
-supporting jobs at the manufacturer of the DNA sequencing instruments, [REDACTED], which is located in California
-the stem cell QC procedure would be easily available to [REDACTED] researchers and other researchers in California as they could come to our laboratory to learn it there
-this project could easily form the basis for follow up research for which we could attract federal funding to California
-it would further strengthen California as a place from where people world wide expect cutting edge innovations in stem cell research - and in this case in combination with cutting edge technology - to come
Stem cells’ genomes become increasingly unstable in culture over time, which may limit their utility and safety in clinical settings. Comprehensive detection of changes in genomes represents a current bottleneck. In this application, the Principal Investigator (PI) proposes to use state of the art high-throughput DNA sequencing technology to determine if and when human pluripotent stem cell (hPSC) cultures acquire a detrimental genomic variation load. The goal is to detect sequence variations up to the single nucleotide level, and to use that information to derive a genome stability score. The PI will first establish cell cultures for 4 hPSC lines, grow each line for about 200 passages, differentiate each line into two different cell types, and collect DNA and RNA samples at multiple passage numbers and at the differentiation endpoints. Next, these samples will be used to deep sequence the genomes, followed by assembly of complete catalogs of all sequence variation detected in each sample. RNA sequencing will be used to correlate changes in gene expression networks with genomic sequence variation load. The PI will then carry out an integrative analysis of all data sets to arrive at the stability score, which is hoped to reveal whether a stem cell line is no longer suitable for certain applications. Finally, the PI intends to develop a quality control standard operating procedure for genome sequencing, which will be disseminated to the community together with the algorithms necessary to determine the stability score.
The reviewers acknowledged that this application addresses the need in the regenerative medicine field to assess the genomic integrity of human cells used for therapies. They, however, did not find the proposed approach particularly innovative, since others are already applying high throughput sequencing technology to stem cell biology.
Based on the preliminary data, reviewers were confident that the applicants would be able to carry out the proposed experiments, which mostly involve next-generation sequencing. While reviewers supported the rationale for determining whether hPSC have acquired a detrimental genomic sequence variation load, they were strongly concerned that this proposal lacked a clear vision as to how the collected information will be utilized to come to a clear conclusion. The applicants will arrive at a stability score, but did not explain how they will determine what amount or type of sequence variation will be considered detrimental. Experiments that address how a certain stability score correlates with the cells’ phenotype or safety profile are critical to validate the meaning of the score, but were not proposed. Reviewers expressed the opinion that measuring genetic drift over a large number of population doublings is not of great significance by itself, and felt that the proposal would have benefited from an elaboration on the final paragraph in which the applicants briefly mention additional benefits that might arise from this research. With regard to the details of the proposed experimental approach, reviewers cautioned that the applicants did not consider how long the hPSC lines to be analyzed have been in culture at the start of the experiments, which could influence the practical significance of the stability score.
The PI and research team were judged to be well qualified to execute this proposal. The applicant is a young PI and has strong expertise in the sequence analysis aspect of the proposal. A Partner-PI, with whom the PI has successfully worked together in the past, is included in this proposal, but reviewers felt the proposed collaboration required more justification, since both labs will perform similar experiments. Although two stem cell experts have been recruited to the team, reviewers were not convinced their level of commitment was sufficient to support that aspect of this application.
In summary, the applicants propose to use high-throughput DNA sequencing to assess the genomic integrity of stem cell cultures and develop quality control standard operating procedures to enable reliable wide spread applicability of their approach. However, reviewers were unconvinced the proposed work will be able to arrive at a meaningful metric that defines a cell population’s suitability for transplantation into humans, as it is focused on sequencing technology without a compelling plan to relate the sequence information to the biology of stem cells.