PhD project mark 2

Some of you have offered help to make this happen, for which I am extremely grateful. Thus, for anyone who has the time and passion, I have put together a few questions. (Hopefully, they will start to make sense after you read the proposal...) I am of course working on these myself, but I would highly appreciate different opinions. Furthermore, your feedback on anything else is always most welcome!

- How would you priorize the various objectives?
- What further aging markers should be assayed?
- Do you have better, or more "classical" references at hand, for the ones I list?
- What is a good way to diagnose myeloid and lymphoid of malignancies in the mouse?
- How many mice would it take to statistically measure the incidence of spontaneous blood malignancies? I've got some confusing results form a preliminary search. Do you know a waterproof reference?
- In how far do you think the mouse is a suitable model for the cancer issues? The references I cite on carcinogenesis theory are mostly about humans. The mouse has fewer effective tumor suppressor mechanisms, which should take less time to evolve away and this might compromise my argument. How strong do you think is this objection?
- Due to certain drawbacks of the mouse (such as the above), do you think it is worth adapting the whole proposal to a different animal? What could be done in that animal and what not?
- As for potential supervisors, I have been in contact with one or two, but I would like to wait until after having a close look at everyone at SENS2 before making a definite decision. Until then, I would always appreciate other opinions on who is suitable.

[Edit: added more questions]

Edited by John Schloendorn, 01 July 2005 - 07:50 AM.

Proposal for a PhD project: Tissue rejuvenation of aged mice

By John Schloendorn, prospective PhD student.
Email: Zauberkugel@yahoo.com

This proposal should be seen as an inspiration and starting point for developing a useful project. I am well aware that many points need much further research and discussion. I will always be happy to discuss any type of feedback with you.


Abstract

Aging is the greatest single cause of physical suffering in developed countries, because it is the primary risk factor for most serious diseases and frailty. It is widely accepted that aging is caused by molecular damage that accumulates during the life-span, mostly inside our cells. Biomarkers of aging, including measures of oxidative stress, DNA damage, telomere length and others, are roughly correlated with aging. All somatic body cells age, but the germ-line does not. Cell replacement therapy has the potential to replace our aged body cells with ones that are closer to the germ line in terms of time and molecular damage. Complete blood cell replacement is possible in animals and humans.
Thus, it would be interesting to ask whether the complete replacement of blood cells in experimental animals can reverse biomarkers of aging. If so, then it should be determined whether the extent of such reversal correlates with the distance from the germ line of the replacement stem cells in terms of time and molecular damage levels. In the project proposed here, replacement cells would be obtained from aged, young adult, embryonic or embryonic-cloned sources and evaluated for their ability to reverse biomarkers of blood cell aging in old recipients. If successful, the experiment might further ask whether repeated cell replacements can sustainably suppress blood aging biomarkers, while the animals' other tissues continue to age. Furthermore, the project might be extended with various types of systemic (cell-extrinsic) therapy.


1. Opinion on aging

Age-related diseases are diseases the incidence of which increases with chronological age in the group of organisms studied. Aging reflects the increasing probability of an organism to develop age-related diseases, get increasingly frail, and die with time. In developed countries, all major causes of death, including heart disease[1], Alzheimer's disease[2] and most types of cancer[3] are age-related diseases.
Observing that medical research funding continues to increase, while medical progress, measured by healthy life-extension of the oldest is stagnating, some argue that the prevalent medical research strategy, which seeks to improve health[4] in the later parts of life by research into individual age-related diseases, has progressed into the realm of diminishing returns.[5],[6] In fact, as of June 2005, a PubMed search of "diminishing returns" gave over 120 results, a substantial fraction of which referred in one way or another to this phenomenon. Some go as far as to prophesize an impending decline in life expectancy in the US due to the rise of bad lifestyle habits that may overwhelm geriatric treatment options.[7]
Thus, it would seem defensible to say that medicine needs a new paradigm, which seeks not only to remedy individual age-related diseases, but also their increasing probability to occur and recur with age.[8] One paradigm that has the potential to rescue geriatric medicine from the realm of diminishing returns is thus: "Cure aging!".

A detailed case can be made that certain types of irreversible, accumulating cell damage almost exclusively cause aging in the above sense.[9],[10],[11] According to this view, age-related diseases could best be combated by the periodic repair of cell damage.[12],[13] Specifically, if the damage levels of young cells (i.e. ones that are close to the germ-line in terms of chronological time, number of cell divisions and stress exposure) were used as a reference[14], then their levels of age-related cell damage, and hence the incidence of age-related diseases (including frailty), could be periodically reset to the very low childhood or embryonic levels, thereby rendering disease and frailty effectively non-age-related.
Methods exist to replace all cells of certain types in the body with cells from exogenous sources. Although in some tissues such methods have been clinical routine for decades (e.g. solid organ transplantation for end-stage organ failure, bone-marrow transplantation for leukemia), it was to my knowledge rarely investigated whether such forms of complete cell replacement, when using donor cells that are younger than the recipient, can reverse the cellular effects of aging in the respective tissue. Such is the aim of this project, which will focus on blood cell replacement. After myeloablative therapy, bone-marrow repopulating cells from different developmental backgrounds would be evaluated for their ability to reverse various biomarkers of aging upon transplantation.


2. Biomarkers of aging

Given the tissue-specific nature of the intervention, the effect on aging cannot be monitored as organismal life-span extension. Tissue-specific biomarkers of aging may serve as a substitute. Unfortunately, such markers do not enjoy a reputation of being the most adequate predictors of remaining health and life-span.[15] However, positive effects on a set of commonly used markers, mostly gleaned from studies on caloric restriction (the only known intervention that consistently slows aging in mammals[16]), are likely to yield publishable results[17]-[25]. Classical biomarkers of aging that could in principle be applied to any type of nucleated blood cell include (but are not limited to) protein and lipid oxidation[18], decreased proteasome function[19], DNA damage levels[20], cytogenetic aberrations[21], rate of apoptosis[22], superoxide production[23], glutathione peroxidase, GSH and GSSG levels[24], impaired growth factor response, chemotaxis and phagocytosis of immune cells[25]. For all of these exist relatively simple commercial or homemade assay procedures.
Telomere length is a less suitable aging marker in mice than it is often said to be in humans, because mouse telomeres are much longer and telomerase is much more active in mice than in humans.[26] Specifically, in mouse nucleated blood cells, telomeres do not shorten with age like in their human counterparts.[27] Depending on the technical and financial possibilities, gene expression profiling by microarray technology would provide an additional means to assay markers of aging, again inspired by work on caloric restriction.[28]

Furthermore, the incidences of lymphoid and myeloid malignancies deserve to be monitored, not only as an aging biomarker, but also as an undesirable consequence of getting old per se. Cancer progression, including age-related cancers of the blood, involves the interaction of a few mutated dividing cells, which are thought to be most often stem cells[29], with a permissive tissue.[30] The competition of clones with particular nuclear DNA mutations for the tissue's resources is thought to give rise to a microevolutionary process that confers on cancers their aggressive systemic flexibility and allows them to respond in a seemingly intelligent manner to endogenous and exogenous therapeutic initiatives.[31] It is plausible to expect that, in order to accumulate enough mutations to become life threatening, cancers need time to undergo lots and lots of cell divisions[32]. Thus, it would seem reasonable to ask whether replacement cells that have been derived relatively fresh from the germ-line can immediately form life-threatening tumors, and when they can begin to do so. To this end, monitoring the incidence of age-related cancers after cell replacement therapy deserves urgent attention, especially when replacement cells are derived relatively fresh from the germ-line.
I would argue that this is indeed the highest virtue of cell replacement therapy: Unlike any other proposed anti-aging intervention[33],[34], it has the theoretical potential to remedy the universal age-related increase of cancer incidence[35]. Furthermore, few would argue that cancer was caused mostly by extracellular factors. On the contrary, because the roots of cancer are thought to be mostly (stem[36]) cell-intrinsic (perhaps with the major exception of tumor promotion by an impaired immune response), cancer is the most likely aspect of aging to be preventable by cell replacement alone. The time and resources allotted for monitoring spontaneous lymphoid and myeloid malignancies should reflect this twofold relevance.

In addition to the universal biomarkers of aging, the immune system would also offer a number of age-related changes of cellularity and gene-expression, some of which appear as well-defined predictors of immune function and remaining life-span[37].
Most notably, the CD4+ CD8+ memory T-cell compartment expands with age, at the expense of naive CD4+ helper T-cells and CD8+ cytotoxic T-cells.[38] This includes more fine-grained changes in the respective sub-populations, such as extreme expansion of the CD4+ CD8+ P-glycoprotein-expressing compartment of memory T-cells[39]. Interestingly, CD28 extinction, a consistent marker of human T-cell aging[40], does not seem to accompany immunosenescence in the mouse.
B-cell aging, too, is well-defined in mice and humans: The numbers of pro-B-cells expressing recombinase activating gene (RAG) decline with age and the remaining RAG+ pro-B-cells express less RAG.[41]
However, not only age-related T-cell population changes[42], but intriguingly also B-cell progenitor changes[43] seem to be in part caused the age-related involution of the thymus. Indeed, bone-marrow transplantation from young donors to aged hosts is not sufficient to rejuvenate either the thymus or the lymphocyte populations.[44]
Due to these complications and extensive competition that is already working to address them, lymphocyte population changes should not be a primary focus of the project proposed here. Specifically, the importance of systemic causes of aging (in addition to cell-intrinsic causes) make lymphocyte population changes a less than ideal target for purely cell-replacement based rejuvenation therapy. However, depending on future developments in the field, thymic rejuvenation might be considered via a combination of cell therapy with systemic approaches that are not normally considered together, such as interleukin-7 (IL-7) supplementation[45] plus bone-marrow transplantation. IL-7 can reverse age-related changes in some, but not all stages of thymopoiesis.[46] Given the extent of hematopoietic stem cell intrinsic age-related changes[47], on the other hand, it may not be unreasonable to ask whether extrinsic and intrinsic types of therapy can complement each other to achieve a substantially greater rejuvenating effect that each type of therapy by itself.
Bone-marrow transplantation addresses by definition all intrinsic changes, whereas IL-7 as a single extrinsic mediator cannot possibly address all extrinsic changes. A much more comprehensive type of systemic intervention was famously pioneered by Conboy et al.[48], who established a shared circulatory system between young and old mice to successfully reverse a number of age-related changes in satellite cells and hepatocytes. This approach, together with the transplantation of young cells might serve to test the hypothesis that comprehensive reversal of systemic plus cell-intrinsic age-related changes are sufficient to reverse all detrimental effects of cellular aging in a given tissue.


3. Constructive interventions

In order to test the hypothesis that cell replacement with young cells will fix some of the damage of aging, cells from different developmental backgrounds will be tested for their ability to reverse biomarkers of aging in the aged mouse.
There is little reason to believe that transplantations from aged donor to aged recipient would affect aging a lot. Such transplantations may serve as controls, together with no transplantation.
As discussed above, young donor to aged recipient transplantations fail to amend several extrinsic age-related changes, and have been reported to suffer from additional problems related to repopulation kinetics.[49] It should be asked in the project, whether cell-intrinsic aging biomarkers are affected by this type of therapy.
Bone marrow repopulating hematopoietic stem cell-like cells can now also be derived from embryonic stem cells.[50] Embryonic stem cells can be obtained by in-vitro by fertilization from the germ line.[51],[52] Since individual organisms are generated from the germ-line's viable and evolving genetic material throughout all organismal generations, the germ-line is often said to be "immortal", i.e. not accumulating age-related cell damage.[53] Thus, it should be worth asking whether bone-marrow repopulating cells, derived fresh from the germ-line via fertilization and embryonic stem cells, can outperform cells from young adult donors at reversing the biomarkers of aging and carcinogenesis.
A different way to obtain embryonic stem cells is by somatic cell nuclear transfer (SCNT).[54] The use of SCNT-derived cells for therapy (therapeutic cloning) was proof-of-principled in mice by the correction of a model of severe combined immunodeficiency[55], both via hematopoietic progenitor cell differentiation and reproductive cloning of young bone-marrow donor animals.
SCNT-derived cells carry the cytoplasm of the germ line, but the nucleus of a somatic cell. Thus, in this project, they can be used to estimate the relative contribution of nuclear vs. cytoplasmic effects on the ability of replacement cells to reverse different aging biomarkers, in relation to the source of the somatic cell used for cloning. The main distinction of potential interest would be dividing vs. post-mitotic cells. This might improve our understanding of the nuclear vs. cytoplasmic components of the aging process and help to optimize the choice of somatic cells for therapeutic cloning purposes.

If a sufficient degree of hematopoietic rejuvenation can be achieved, a second milestone of the project would be multiple repetition of the cell replacement procedure in the same animals to see if blood cell aging can be continually suppressed, while the animals' other tissues continue to age, ultimately limiting the extent to which the procedure can be repeated longitudinally.


4. Destructive interventions

In many tissues, stem cell niches must be emptied before efficient engraftment can occur.[56] The first, wild-type "generation" of cells is probably the most difficult to ablate. In bone marrow, the standard destructive intervention to achieve this is whole-body irradiation.[57] However, to accurately measure the effects of the transplantation on aging and cancer incidence, especially during multiple, longitudinal transplantations, a more gentle and specific treatment may become desirable.
Later generations of cells can be made to die on demand by incorporating suicide genes. Cells that express suicide genes will convert a normally non-toxic pro-drug into a toxic drug. For example, herpes virus thymidine kinase has become prominent for its use in the cure of graft-versus-host disease upon gancyclovir administration in mice[58] and men[59] by removing the alloreactive donor T-cells responsible for the disease. Thus, suicide genes seem like promising candidates to ablate later generations of hematopoietic stem cells.
In addition, in order to control for potential effects of the myeloablative therapy, transgenic animals may be derived that express suicide genes under various hematopoietic-system specific promoters. In these animals, even the first generation of hematopoietic cells might be removed using the gentle and specific suicide gene approach. This might be a better model of a futuristic rejuvenation therapy that might be developed for use in healthy humans. (This is not the place to speculate how such selective cell removal may actually be achievable in non-transgenic humans in the future.) Different suicide genes might be used in subsequent generations of hematopoietic cells, so that they can be selectively removed, without harming other generations or the aging animals' other tissues. This might provide a model for a futuristic comprehensive rejuvenation therapy in multiple tissues.
If cancers should arise from the replacement cells, then the suicide genes should be investigated as a curative option. This may include teratoma from contaminating embryonic stem cells. While teratoma can be avoided by rigorous purification of hematopoietic stem- and progenitor cells (see reference cited above), it may be a useful exercise to omit the purification step for a control and to investigate the use of suicide genes for teratoma therapy.


5. Conclusion:

The project proposed here would attempt an interdisciplinary approach to estimate the utility of cell-replacement as an anti-aging intervention. The aim shall be to establish a point of reference for the development of futuristic rejuvenation therapies for human use. This proposal is inspired by a vision of similar therapies that might one day go far beyond the hematopoietic system. The project shall be understood as a tiny, but very purposeful step towards one of the most ambitious and remarkable endeavours that humans set out on: Undoing involuntary age-related suffering and death.


6. References

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57 Shank B. Total body irradiation for marrow or stem-cell transplantation. Cancer Invest. 1998; 16(6): 397-404.

58 Cohen JL, Boyer O, Salomon B et al. Prevention of graft-versus-host disease in mice using a suicide gene expressed in T lymphocytes. Blood 1997; 89(12): 4636-4645.

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[Edit: added an abstract]

Edited by John Schloendorn, 24 August 2005 - 02:59 PM.

Sorry John, only just found this. Maybe write a PM next time.

If you are in the lucky position of going into a lab where funds can be diverted to do what you like, then it doesn't really matter. (Rare) In that case I'd simply side with Mondey's comments about mice.

However, if you intend to get funding for this project, you won't get far with the above. Way to much (!!!), way to broad.

What is not evident to me atm. is "where is the unique idea?" what are you proposing to REALISTICALLY do that nobody has done before. Age benchmarking is happening all over the place. So is cell therapy. Discuss previous work, and show how you will expand it in the meager space of 3 years.

Keep it tight. The aim is to take on extra projects during the PhD not to be struggling with a given workload.


Also, its not exactly easy reading in that you mix and conflate theory, discussion, wishes, possibilities, proposals, and methods.

If you want serious suggestions, you might wish to post a concise project outline with clear timelines and protocols. You will need to do that for funding anyway.
(And cut crap like "tiny, but very purposeful step" that is unnecessary in this forum and won't cut any ice with the funders unless they happen to be private.)

Finally, I'm sure that you are aware that affiliation to the lab is the decisive factor. In most cases, it will be down to what you can do and what your supervisor is good at. So I suggest you start working on proposals that take account and advantage of the specific expertise of an existing group.

Thanks Caliban,
I'm not sure what makes you and Mondey take this proposal as a grant application. Although I have been interested in funding options on occasion, actually applying with funders is not what phd students are normally required to do before they even start or have a supervisor, is it? This piece is meant to be handed to prospective supervisors (or third parties such as you ;-) as an "inspiration and starting point for developing a useful project", i.e. to show off, break a discussion and ultimately make them think "well then, why not let this guy have a shot". At this stage, I do not concentrate yet on any particular supervisor, and therefore should not and cannot have the degree of specificity that would be required of a grant application.
You are very right that this should and will change as I get closer to picking someone. This is also the reason the proposal still describes more work than can normally be done in three person-years. It will depend on the lab and negotiations with the supervisor which parts will be modified, omitted (or added). This is really my question about priorization. So perhaps I should make these things even clearer than I already do.

Also, its not exactly easy reading in that you mix and conflate theory, discussion, wishes, possibilities, proposals, and methods.

Good point, thanks, organization along these lines would definitely help.

"where is the unique idea?"

It is to combine cell therapy and aging biomarkers to ask if the former can have a beneficial effect on the latter. Except for that thymus work I was citing, I'm not aware of any such experiments.

I'd simply side with Mondey's comments about mice.

I'm trying to quantify these ideas, and back them with data. But if a number of experienced scientists tell me that their "gut feeling" is against using mice, that will have substantial impact on my choice, too. I'm actually close to coming around on this point. In particular, I cannot see any clear-cut advantage of using mice, except possibly the experience that my particular lab might have with them. Most labs have experience with mice, rather than other animals and that's the main reason I used mice in this generic proposal.

Thanks John, not laying on too hard I hope.

I'm not sure what makes you and Mondey take this proposal as a grant application.

My guess is that 90% of prospective supervisors would read from that angle. Because

actually applying with funders is not what PhD students are normally required to do before they even start or have a supervisor, is it?

Applying? Hell no. Having on in place would be highly unsusal, because most PhDlers start of doing as they are told, not what they WANT to do. Remember that almost every PhD post is funded for a specific project. It takes ~6 months to apply.

This is also the reason the proposal still describes more work than can normally be done in three person-years. It will depend on the lab and negotiations with the supervisor which parts will be modified, omitted (or added). This is really my question about priorization. So perhaps I should make these things even clearer than I already do.

People are not interrested in the big picture, they know how it shifts. People are interrested in single, snappy ideas that will produce some data and a paper or two. If you say "I wanna do gene therapy to cure aging prettyplease" everyone yawns. If you say "I want to use plasmid A with vector B, to effect celltype C, and measure agemarker D - here is the protocol and five references to show why this is interresting" they can engage you eye-to-eye.

It is to combine cell therapy and aging biomarkers to ask if the former can have a beneficial effect on the latter. Except for that thymus work I was citing, I'm not aware of any such experiments.

Now we are getting somewhere. Can you remind me what exactly you mean by 'cell therapy'?

Developing efficient suicide gene mechanisms could be a project in itself. Some thoughts on that... adding a suicide gene that makes cells sensitive to a drug is not efficient since a mutation can knock out the suicide gene. There are two options to avoid this: make the cells dependent for survival on an added gene (like antibiotic selection in bacteria culture), or knock out a gene and make them dependent on a supplement that allows for survival. This way mutations cannot get around your suicide mechanism.

Applying? Hell no. Having on in place would be highly unsusal, because most PhDlers start of doing as they are told, not what they WANT to do. Remember that almost every PhD post is funded for a specific project. It takes ~6 months to apply

People are not interrested in the big picture

I strongly disagree with these sentiments. Since you're not applying for the grant, take advantage of the opportunity to work on something that will be difficult to get grant money for. Also, there are some people interested in the big picture and you should make it a priority to find one of those to work with. Like you say, SENS2 will be a good opportunity to find them. If you have a good and well developed idea you can control your PhD project to a large degree.

My guess is that 90% of prospective supervisors would read from that angle

Forget the 90% and seek out the 10.

BTW, thanks for the citation

Yes, what I'd expect is that single suicide genes make a poor cancer treatment because they'd mutate away. However, when they do not get malignant, mouse nucleated blood cells show as good as no genomic instability. Therefore I would wager that before the animal is well on the way to leukemia, only very few cells can escape the suicide gene. Remember how well they clean up well with GVHD, despite the enormous potential for expansion of a single alloreactive T-cell. And the goal is only to ablate the majority of cells in one or a few sequential generations.

For cancer treatment it may be an option to simply use a huge number of suicide genes (for example by stuffing them into all the pseudo-acceptor sites in the mouse genome with a site-specific integrase?!) but that would obviously strongly depend on the specific lab.

I was looking into a positive selection marker system for solid organs based on MHC II allelic variation (i.e. a purposeful graft-versus-host disease), but that is obviously far beyond the scope of actually being done here. With your supplement based stuff, I guess you will face huge difficulties getting the pharmacological stuff right, i.e. maintain the effective concentration in all cell-types at once and pharmacokinetics. Can't begin to speculate about that though, it would have to be tested and optimized to see if comes anywhere near to work. So yes, this would indeed be a project of its own and it seems like a risky one at that.

As for the supervisor-strategic stuff, thanks Osiris, you found the right words to express my thoughts.

So how did the project go?

Wow! Way to revive and interesting old thread Caston. Thank you. Hopefully John has time to comment.

I wish more people would search for and add to old threads here at Imminst. The Imminst forums are becoming a vast and valuable historical record on the progress and evolution of aging theories, biomedical breakthrough, and supplement science. I encourage members to search some old threads first before starting a new topic as this would be beneficial to all.



"Party on, wooohoo, I just OD'd on piracetam!!" just isn't all that helpful (and has been posted hundreds of times).

Wow! Way to revive and interesting old thread Caston. Thank you. Hopefully John has time to comment.

I wish more people would search for and add to old threads here at Imminst. The Imminst forums are becoming a vast and valuable historical record on the progress and evolution of aging theories, biomedical breakthrough, and supplement science. I encourage members to search some old threads first before starting a new topic as this would be beneficial to all.



"Party on, wooohoo, I just OD'd on piracetam!!" just isn't all that helpful (and has been posted hundreds of times).

Thanks Mind. I couldn't agree more and I'd like to take this opportunity to thank you for the great work you are doing as executive director.

Oh dear! The good old days. This must have been before I ever heard of the MF.

So how did the project go?

None of it ever happened.

Well, it looks like you found good use of your time and brainpower working on LysoSENS.