Human embryonic stem cells, by virtue of their capacity to proliferate indefinitely and to differentiate into almost all types of somatic cells, hold the potential to help us understand and cure, some of our most devastating diseases. The nucleus of a cell controls the expression of its genes and is responsible or the eventual coding of all the proteins that make up the different kinds of functionalities attributed to such a cell. However, each of our cells also contains a large number of copies of mitochondria, which contain their own DNA and proteins responsible for generating most of the energy needed for the functioning of these cells. In addition to energy generation, the mitochondrion is also the site of much biosynthesis. This explains why, even after millions of years of evolution, human cells have learnt to coexist with the mitochondrion, which are derived from bacteria, rather than take on the task of energy generation as part of their nuclear function.
Under normal circumstances, we assume that each cell will contain a perfectly functioning set of mitochondria. A cell will then possess normal metabolism similar to the other cells found within our body. When we talk about transplanting stem cells that have grown on a dish for a while, or when we derive hIPS cells from mature skin cells that are reprogrammed, we test the cells repeatedly for their nuclear coding capacity before use in therapy. Less attention, we believe, has been paid to the metabolic status and the mitochondrial function of the cell. We find it critical that each cell line be given a “metabolic health check” before its use in therapy.
This is important from many standpoints. Mutations in mitochondrial DNA can cause disease, so we intend looking for such changes in the stem cell lines being used. Our studies suggest that the mitochondria are highly dynamic and their morphology varies from one cell line to another although each of these lines is considered virtually synonymous with the other in all other aspects. Our collaborator, [REDACTED} finds that the greatest variation between an ES and an IPS cell line is in the expression of mitochondrial genes. Finally, we find that stem cells in which we induce mitochondrial defects will cause tumors in mice under circumstances where normal stem cells will not. All these issues suggest that the metabolic status of a cell needs to be thoroughly examined before its use in therapy. The problem is in defining what the range of “normal” states is going to be. For this, we need to do controlled experiments with large numbers of lines that we query through many experimental paradigms. From this basic scientific interrogation will result the Standard Operating Procedure for assessing the metabolic component of an acceptable stem cell line. Creating such a protocol based on initial mechanistic studies is the central focus of this proposal.
Statement of Benefit to California:
California is at the forefront of Stem Cell and Regenerative Medicine Research. At [REDACTED], under the auspices of the [REDACTED] we wish to move the basic science of stem cells from the bench to the clinic as efficiently and as safely as possible. This proposal furthers these goals in two ways. As a basic science proposal, it seeks to ask mechanistic questions about the role of metabolism in stem cell function. As importantly, its more practical goal is to ensure safety in the procedures followed. We hope that with the guidelines that we will develop as a result of this proposal, all scientists, particularly those in California, will have a rapid access to obtain a “metabolic check” on the lines that they anticipate using in therapy. In conjunction with our [REDACTED}, we hope, for the future, to be able to push the limits of such detection to a finer scale. This will be a critical step in achieving California’s goal in setting operating procedures and setting guidelines for stem cell research around the world.
This proposal focuses on the role of mitochrondrial activity in the self-renewal and early differentiation of human embryonic stem cells (hESCs). The applicant presents preliminary data suggesting that blocking mitochondrial function impairs hESC proliferation without affecting pluripotency. In Aim 1 the applicant proposes to follow up on these observations by knocking down mitochondrial proteins in hESCs using RNA interference and performing a variety of assays for mitochondrial function, cell proliferation, apoptosis, senescence and teratoma formation. In Aim 2 the applicant will assess mitochondrial mutation load in existing hESC lines, newly derived hESC lines and induced pluripotent stem cell (iPSC) lines derived from different sources. Finally, in Aim 3, the applicant proposes to develop a standard operating procedure for generating a “metabolic bill of health” for pluripotent cells prior to therapeutic use.
Reviewers agreed that this proposal addresses an important and neglected issue in stem cell biology: the role of mitochondria in the proliferation, maintenance of pluripotency, and potential for differentiation of hESCs and iPSCs. However, one reviewer felt that the impact would be limited by the restriction of the study to only the two hESC and two iPSC lines listed in the application. Reviewers did not find the project to be particularly innovative, noting that no novel techniques or experimental systems are being developed. One commented that there is a significant body of work already published in this area, some of which is cited but not described in the research proposal. The proposal would have been strengthened by inclusion of findings from these publications, such as those describing mitochondrial size, number, morphology, location and transcriptional activity in hESCs and their differentiated progeny, related to the preliminary data in this proposal.
The reviewers raised a number of concerns about the experimental design that led them to doubt the project’s feasibility. One reviewer noted that the preliminary data are all based on the blockade of mitochondrial function by an uncoupling agent and cautioned that this may not be a good model of mitochondrial dysfunction caused by genetic mutations, which are the primary concern in pluripotent cells. The reviewer called this uncoupling agent a “sledgehammer” and was not surprised that it slows cell proliferation. Furthermore, while the formation of a branched mitochondrial network may characterize differentiation, it was unclear what cellular or metabolic properties and consequences were related to this change in mitochondrial morphology.
Additional concerns were raised about the feasibility of Specific Aim 2. For example, a reviewer cautioned that heteroplasmy below 30% is almost impossible to detect by conventional or hybridization sequencing, so the applicant is unlikely to detect any new mutations. A large number of homoplasmic polymorphic sequence variants between existing lines will be detected, but assessing whether these have any phenotypic consequence will be extremely difficult. This reviewer also raised issue with the assumption that iPSC lines derived from fibroblasts are likely to contain mitochondrial DNA (mtDNA) mutations. Based on the applicant’s hypothesis, cells harboring such mutations should have a growth disadvantage and therefore be selected against during the generation of iPSCs. Furthermore, even in patients carrying large-scale mtDNA deletions, it has been quite difficult to identify fibroblasts harboring a significant proportion of these deletions. Regarding Aim 3, while reviewers praised its goal of producing a “metabolic report card” for stem cell lines, it was unclear how the applicant would integrate the various functional measurements to come up with a “score”. Finally, a reviewer noted that there was little discussion of alternative strategies should the proposed experiments not yield results as expected.
The reviewers praised the principal investigator as a highly-respected scientist who has made important, high-impact contributions to the field of Drosophila developmental biology. They noted that the research team clearly has the expertise required for the mitochondrial aspects of the project but were less sure about the aspects involving hESCs and iPSCs, and noted that the applicant has not published previously on work with hESCs. The applicant’s resources were judged to be more than adequate for the proposed studies.
Overall, while the reviewers acknowledged the importance of the scientific questions addressed in this proposal, they raised a number of serious concerns about the research plan that led them to question its impact and feasibility.