Our research for methods to protect the cells during aging revealed that N-t-butyl hydroxylamine (NtBHA, active form of a drug in clinical trial for stroke) and methylene blue (MB, a well known drug) significantly extend the vigor and lifetime of cells. MB or NtBHA at very low concentrations delay senescence of the cells; increase the number of the cells by 6-7 million cells/week; enhance cellular resistance to oxidative stress and toxic agents; and improve energy production. When fed to old rats MB and NtBHA enhance cognitive function and improve muscle strength. Preliminary observations suggest that NtBHA and MB, accomplish this, in part, by improving the quality of the mitochodnria, which are the cellular source for energy. MB and NtBHA also lowered the levels of oxidative damage to the cell compnents. We will use MB and NtBHA to improve the maintenance of hESC and the derived neuronal lineages in culture. We are expecting MB and NtBHA to improve the culturing of hESC and derived cells when added to the established growth media. Thus, we hypothesize that maintaining hESC and derived neuronal lineages with MB or NtBHA considerably increases the total number of cells, efficiency of derivation, and survival in the tissue culture conditions. If the goals of this proposal are successfully accomplished, then the quality and the number of the available stem cell units for cell therapy should improve. Additionally, the storage of stem cells at –80 °C will be made safer. A future longer-term objective of our proposed research is to test whether MB and NtBHA promote the function of endogenous stem cells when administered in experimental animal models. The basis for this hypothesis is that MB and NtBHA enhanced the cognitive function and improved muscle strength in rats. A one possible explanation for this effect is that MB and NtBHA, in part, improve the function of endogenous stem cells in the rat’s tissues (e.g. increased the secretion of specific hormones). Additionally, MB and NtBHA may enhance the metabolic activity of the somatic cells adjacent to the stem cells, which, based on recent research, should preserve the function of the endogenous stem cells in the rat’s tissues. Promoting the function of endogenous stem cells has been proposed as a therapeutic strategy to prevent and cure several diseases in human. A pharmacological approach to enhancing the function of native stem cells in tissue (e.g. the use of MB and NtBHA) to prevent disease (e.g. Alzheimer disease) may complement stem-cell transplantation therapy.
Statement of Benefit to California:
In order for the objectives of Proposition 71 to be accomplished and patients in Californian to benefit from the use of human embryonic stem cells (hESC) to regenerate tissues to replace tissues damaged by disease and injury, researchers must first be able to grow and maintain sufficient numbers of healthy and pure hESC and their derived lineages. Current hESC research is impeded by the technical difficulties and safety concerns in getting hESC, especially the derived lineages, to grow in adequate numbers under laboratory conditions and being able to maintain the cells in a healthy state. We are proposing to test the effect of two compounds, which have been shown to improve the health of aging cells from old rats, without harmful side effects. These two compounds appear to accomplish this by preventing the dysfunction of key organelles known as mitochondria, which produce energy that cells use for all biological processes, including cell division, cell repair, and the production of enzymes and hormones. If our research is successful, it will help speed up basic hESC research, use of hESC in the private biotechnology sector, and cell replacement therapy in California. This research, in conjunction with the ongoing research at many of California’s institutes, should help position California at the forefront of stem cells research.
SYNOPSIS: The applicant proposes to look at the effect of two antioxidants, (i) N-tert-butyl hydroxylamine and (ii) methylene blue added to the culture medium of hESCs, on a variety of parameters including cell growth, clonal efficiency, survival, yields after cryopreservation, genomic stability, and mitochondrial function. The hypothesis underlying the proposal is that either or both of these antioxidants will mediate beneficial effects on many levels that result in increased ability to scale up the cultivation of hESC. SIGNIFICANCE AND INNOVATION: The PI proposes to examine the effects of two compounds – methylene blue and N-t-Butyl hydroxylamine – on hESC cells in culture. The PI has worked previously with these compounds and found that they have an effect on primary cells in culture. While this proposal is not innovative in its approach, it is significant in that these compounds may lead to new and improved culture conditions for hESC lines. A critical look at oxidant handling and redox regulation of hESC would be a welcome innovation to the field. Mitochondrial function and biogenesis are certainly important factors in hESC maintenance, differentiation and translation, and deserve to be studied since little good work has been done in this area. In that respect the idea of studying the role of mitochondrial function for (Aim 1) survival and senescence of hESC and (Aim 2) differentiation into neurons is a good idea. STRENGTHS: The proposal is responsive to the spirit of the RFA. The strength of the proposal is that the PI has picked an area of hESC biology that is neglected despite its importance in virtually all aspects of cell function - the role of oxidant signaling and redox regulation. Another strength of this proposal lies in the use of the two novel mitochondrial compounds in hESC culture. WEAKNESSES: There are several significant weaknesses in this proposal. To begin, the Research Design does not contain enough detail. No clear plan for quantifying mitochondrial biogenesis is presented. No mention is made of the time intervals, overall time period, or number of passages that will be used for the analysis. There seems to be a lack of proper controls and depth of analysis. The applicant does not adequately justify her choice of the specific antioxidants. Why do these (and not low O2 culturing or CO2 mimetics or NAC or any of myriad others) justify a look in hESC rather than other promising antioxidant strategies? The applicant is familiar with these reagents but does that mean they are the best reagents for improving hESC viability and differentiation? In addition, the tools necessary to make progress in this area are not being used, thus there is no clear plan for getting to the heart of mitochondrial function. All the assays are descriptive. There is also no acknowledgement that oxidant stress has a considerable non-mitochondrial contribution or how to dissect these two. The approaches are fairly superficial and not at the cutting edge of redox biology. The ROS measurements at the cutting edge of this field are moving away from DCFH to more specific measures of peroxide vs. superoxide. Even luminol measures of H2O2 are probably more reliable than DCFH where the chemistry is proprietary. In addition, though peroxides are important, there are now ways to assay for superoxide as well, if the focus is on mitochondrial function (and there are ways to assay for non-mitochondrial sources of superoxide). The HPLC measures of the GSH-GSSG redox couple will provide cellular redox potential but a more cutting edge approach would be to try and measure mitochondrial redox potential. The applicant proposes to look at ‘levels’ of antioxidant enzymes, where the activity of antioxidant enzymes would be significantly more informative. The proposal is based around culturing hESC lines and analyzing the effects of the mitochondrial compounds on the cells. The proposal is written in a way that suggests that the applicant has no experience with the technical details involved in hESC cultivation. Since this is a SEED grant, lack of experience is not a major issue, but it does not appear that anyone in the PI’s lab currently has experience handling hES cells, which will be left to a post-doc TBN. There is mention of one experienced technician serving as a consultant, but the extent of her involvement and level of commitment is not clear. The literature is clear that certain of the assays proposed will take a really long time (karyotypic stability in particular will require long, continuous passage of the cells) so that there may be difficulty completing the assays in two years. The length of time for different phases of expansion and differentiation should be spelled out better. Only enzymatic treatment for passaging is described, without any description of how the MEFs will be dealt with as contaminants in their assays. Also the HepG2 conditioned medium experiments (not surprisingly since the medium will change based on reagent lots) have been hard to replicate in many labs, so that the neuronal differentiation plans are not well thought out. There is no plan to determine whether the generated neurons are CNS types or peripheral types (literature is quoted for both). Ongoing quality control of the hESC also is not well described. Depending on the cell line used, it may be difficult (but not impossible) to get single cell suspensions for the sorting and counting assays. The description of collaborators is an issue. Dr. Ames is listed as co-PI but his exact role in the project is not well defined. The PI states that there will be collaborations with Drs. Martin and Kuypers using their as yet underived hESC lines. These collaborations appear to begin only following the completion of all the Specific Aims of this proposal, so the pertinence here is not clear. Dr. Kuypers is listed as a collaborator and will do some work “on the effect of lipid composition in the media” but the only mention of his contribution is in the cover sheet, so it is unclear how his collaboration contributes to the effort. Dr. Martin is also listed as a collaborator because he is in the process of deriving new hESC lines. Why are the Melton lines then used? While this proposal is not eligible for NIH funding because of the hESC lines chosen, there is no rationale for using new lines in these studies. The PI proposes to use 4 of the Harvard-HHMI HUES cell lines but presents no discussion of why to use these lines and not any of the NIH-eligible hESC lines. Finally, two minor problems were found in the Biosafety section – hESC lines should be considered biohazardous materials as they are human tissue – and in the Budget – travel to the annual CIRM meeting was not budgeted. DISCUSSION: There was no further discussion following the reviewers' comments.