Stem Cells and Iron Chelators

ironjustice · Guest

RS1-00154-1: Increasing the number and survival of hESC and the
derived lineages in tissue culture.Recommendation: Not recommended for
funding
Public Abstract (provided by applicant)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 (provided by applicant)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.

Review 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 human embryonic stem cells (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 Principal Investigator (PI) proposes
to examine the effects of two compounds - N-tert-butyl hydroxylamine
and methylene blue- 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 survival and senescence of hESC, and
differentiation into neurons, is a good idea.

STRENGTHS: The proposal is responsive to the spirit of the Request for
Applications (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. Also, the applicant does not
adequately justify the choice of the specific antioxidants. Why do
these compounds (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, and there is
no acknowledgement that oxidant stress has a considerable non-
mitochondrial contribution or how to dissect these two. The reactive
oxygen species (ROS) measurements at the cutting edge of this field
are moving towards more specific measures of peroxide vs. superoxide.
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 proposed assays will address 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.

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 hESC, which will be left to a post-doctoral fellow
to be named. There is mention of one experienced technician serving as
a consultant, but the extent of his/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 so there may be
difficulty completing the assays in two years. In this regard, the
length of time for different phases of expansion and differentiation
should be spelled out better. Also, the conditioned medium experiments
proposed (not surprisingly since the medium will change based on
reagent lots) have been hard to replicate in many labs, so the
neuronal differentiation plans need to be defined better, including
the plan to determine whether the generated neurons are central
nervous system types or peripheral types (literature is quoted for
both).

The description of collaborators is also an issue. The exact role of
the co-PI listed is not clear, and the applicant states that there
will be collaborations to use 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. Finally, while this proposal is not eligible for NIH funding
because of the hESC lines chosen, their rationale for using new lines
in these studies is not strong.

DISCUSSION: There was no further discussion following the reviewers'
comments.

The following Working Group members had a conflict of interest with
this application and were therefore recused from participating in
review of, discussion of, and voting on the application:
None

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Tom

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Guest · Posted: Sun Mar 02, 2008 2:53 am Post subject: Re: Stem Cells and Iron Chelators

"DISCUSSION: There was no further discussion following the reviewers'
comments."

In other words, the grant proposal was tossed as being poor in concept
and execution just as the reviewer described.

This is a strong candidate for the iron hall of shame,ie. those posts
which contridict the poster's agenda.

Jesus ate a mediterranean diet.