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The increasing understanding of stem cell biology provides
us with significant new opportunities to understand development,
mechanisms of injury and approaches to regeneration. Development
of a sophisticated understanding of the general principles
underlying precursor cell function will increase our ability
to use stem cells, or the more restricted precursor cells
derived from them, to successfully repair damaged tissue either
by cell transplantation or by recruitment of endogenous stem
cells/precursor cells.
Our studies on the biology of stem cells are interconnected
with our studies on cancer in multiple ways. Many cancers
are derived from stem cells, and understanding how normal
development goes awry may help us to develop ways of controlling
cancer cell growth. In addition our studies on the sensitivity
of normal cells of the brain to chemotherapeutic agents help
us to understand why some cancers are chemosensitive while
others are chemoresistant. Critically, our studies are also
revealing that a number of the chemical agents used to treat
cancer exhibit a disturbing level of toxicity on multiple
normal brain cell populations. The ability of standard chemotherapeutic
agents to kill normal brain cells may help to explain the
cognitive impairment that increasingly is being recognized
as a potential side effect of cancer therapy. The comparative
analysis of stem cells, lineage-restricted precursor cells
and differentiated cells of the normal brain with brain tumor
cells may enable us to develop means of selectively protecting
normal cells. It also may be possible to use brain cell transplantation
to restore normal function, much as bone marrow transplantation
is used to restore normal function of the hematopoietic system
following cancer treatment.
Examples of specific research areas: One of our major goals
is to identify general physiological principles that apply
to the understanding of many different kinds of stem cells.
For example, for all stem cells and lineage-restricted precursor
cells, regulation of the balance between self-renewing divisions
and differentiation is at the heart of understanding normal
development and tissue repair. We were the first team to discover
that appropriate combinations of growth factors can dramatically
subvert this balance, and convert precursor cells with an
apparently limited mitotic lifespan to cells able to undergo
continuous division for long periods in the absence of differentiation.
The finding that it was possible to expand precursor cells
in essentially unlimited numbers by exposing them to appropriate
combinations of mitogens was a key step in allowing the generation
of sufficient numbers of such cells to carry out tissue repair
by cell transplantation. In subsequent experiments, we and
others have discovered multiple growth factors that modulate
the balance between division and differentiation of oligodendrocyte
precursor cells (a cell type so well understood as to allow
studies that cannot be readily carried out in other lineages).
These studies have revealed a complex and sophisticated interplay
between cell-intrinsic regulatory mechanisms and the response
of a precursor cell to environmental signals. Many of our
studies are now directed towards both increasing our understanding
of this interplay and exploiting such understanding to enhance
tissue repair.
The controlled application of growth factors to modulate
precursor cell behavior in lesion repair is a matter of central
concern in current experiments focused on the repair of, for
example, spinal cord injury. Through collaborations with colleagues
here at the University of Rochester (in particular, Margot
Mayer-Pröschel and Howard Federoff), and at other campuses,
we are defining the comparative utility of different precursor
cell populations in lesion repair, and are using regulable
genetic modification of precursor cells, stem cells and host
tissue to enhance precursor cell function and outcome following
transplantation.
Another critical aspect of our attempts to deepen our understanding
of precursor cell function is to decipher the biochemical
processes involved in modulating the balance between division
and differentiation. This is a critical challenge in cellular
biology, as it involves discovering how cells integrate the
multiple different kinds of signaling information provided
in their environments with their own intrinsic cellular programs
to provide the correct physiological outcome.
We have recently made the rather surprising discovery that
intracellular redox state is a crucial component of the apparatus
regulating the balance between self-renewal and differentiation.
We discovered that precursor cells that exhibit a more reduced
state in vivo have a greater capacity to undergo self-renewal
while those that are more oxidized in vivo tend to differentiate.
Moreover, growth factors that enhance self-renewal cause precursor
cells to become more reduced, while exogenous signaling molecules
that cause differentiation cause cells to become more oxidized.
These redox changes seem to be an essential component of the
mechanism by which these diverse growth factors modify behavior,
as antagonizing the effects of these signaling molecules on
redox state blocks their effects on division and differentiation.
Our results indicate that redox modulation may be both sufficient
in its own right to modify the balance between division and
differentiation in dividing precursor cells, and necessary
for the action of classic signaling molecules that control
these processes. Still other studies going on in the laboratory
support the role of redox modulation in modulating normal
development. In the next stages of this work, we are exploring
the sensor mechanisms by which cells sense redox changes,
the intracellular signaling pathways modified by these changes
and the regulatory networks by which growth factors cause
redox state modulations in cells.
Our studies on normal developmental processes have also provided
important clues for understanding developmental abnormalities.
In particular, we have been puzzled by the observations -
first made over eighty years ago - that developmental insults
frequently only have severe effects if they occur during very
specific developmental periods. For example, an absence of
thyroid hormone, or iron, during early development is associated
with profound reductions in myelin production and with subsequent
neurological impairment. In contrast, if an adult becomes
hypothyroid, or iron deficient, their myelin does not go away.
Moreover, it is well established that if nutritional or hormonal
deficiency during development is normalized quickly enough,
significant recovery occurs - yet, if normalization of nutritional
or hormonal status is delayed, the damage done is irreversible.
What is the biological basis for the existence of these windows
of vulnerability? Our research indicates the answer is likely
to be that these windows correspond to very specific stages
in precursor cell development and that the various agents
that cause - whether by their absence or their inappropriate
presence - these abnormalities are responsible for regulating
progression through these stages. In other words, it is becoming
quite clear that many of these developmental maladies are
precursor cell diseases, an insight that has considerable
implications for understanding such syndromes and developing
new means of treating them. One of the cornerstones of further
work on this problem is to identify the common biochemical
and molecular endpoints on which these different kinds of
insults converge so as to similarly alter normal development.
Cancer represents another form of abnormal development of
considerable interest to us. The major driving force behind
studies on cancer is the hope that a better understanding
of this complex constellation of diseases will be associated
with the development of improved treatments. In this context,
we have three related interests in this large field of activity.
First, in many ways, a cancer cell can be thought of as the
evil sibling of a stem cell, sharing the capacity of stem
cells for migration and division but lacking in a normal responsiveness
to environmental signals and the ability to undergo terminal
differentiation. Understanding the molecular and biochemical
features that distinguish cancer cells from normal cells may
enable cancer-specific treatments to be developed. This is
an extremely serious issue, as existing front-line therapies
for cancer- radiation and chemotherapy - can cause severe
damage to the normal cells of the body. It thus follows that
it is necessary to increase our understanding of how existing
cancer treatments cause damage to normal tissue, our second
area of cancer-related research.
One of the side-effects of cancer treatment increasingly
recognized as being of importance is cognitive impairment.
Available information is alarming. It has been known for some
years that cognitive impairment may be associated with the
treatment of brain tumors, or with application of prophylactic
therapy to the brain in treatment of children with certain
hematopoietic tumors. More recent studies are demonstrating,
however, that cognitive impairment is even seen in a significant
proportion of patients treated with chemotherapeutic agents
for non-CNS cancers. Such findings should perhaps come as
no surprise, as those involved in long-term follow-up of cancer
survivors are well aware of their patients' complaints that
they cannot function mentally as well after treatment as before
treatment, a phenomenon that often is referred to as "chemobrain."
The only way to prevent or treat cognitive impairment associated
with cancer therapy is to understand why it occurs, our second
area of cancer-related studies. Our studies are indicating
that the most likely cause of this impairment is through killing
of both oligodendrocytes and of the lineage-restricted precursor
cells that we think play a role in repair of CNS damage. Even
for drugs that are used for treating cancers outside of the
brain, penetration of some of these drugs to the brain is
likely to be at high enough levels to kill these vulnerable
cells, and cognitive impairment is indeed a real problem in
these patients. Our third area of cancer-related research
thus returns us full circle to some of the central questions
motivating our research on normal development: Can we prevent
damage to the cells of the brain and spinal cord? If we can't
prevent the damage, can we repair it by stem cell or precursor
cell transplantation?
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1. Raff, M.C., Miller, R.H. and Noble,
M. (1983) A glial progenitor cell that develops in vitro
into an astrocyte or an oligodendrocyte depending on the culture
medium. Nature 303, 390-396.
2. Noble, M., Fok-Seang, J. and
Cohen, J. (1984) Glia are a unique substrate for the in vitro
growth of CNS neurons. J. Neurosci. 4, 1892-1903.
3. Noble, M. and Murray, K. (1984)
Purified astrocytes promote the division of a bipotential
glial progenitor cell. EMBO J. 3, 2243-2247.
4. Small, R., Riddle, P. and Noble, M.
(1987) Evidence for migration of oligodendrocyte-type-2 astrocyte
progenitor cells into the developing rat optic nerve. Nature
328, 155-157.
5. Noble, M., Murray, K., Stroobant,
P., Waterfield, M. and Riddle, P. (1988) Platelet-derived
growth factor promotes division and motility, and inhibits
premature differentiation, of the oligodendrocyte-type-2 astrocyte
progenitor cell. Nature 333, 560-562.
6. Raff, M.C., Lillien, L.E., Richardson, W.D., Burne,
J.F. and Noble, M.D. (1988) Platelet-derived
growth factor from astrocytes drives the clock that times
oligodendrocyte development in culture. Nature 333,
562-565.
7. Wolswijk, G. and Noble, M. (1989)
Identification of an adult-specific glial progenitor cell.
Development 105, 387-400.
8. Bögler, O., Wren, D., Barnett, S.C., Land,
H. and Noble, M. (1990) Cooperation between
two growth factors promotes extended self-renewal, and inhibits
differentiation, of O-2A progenitor cells. Proc.
Natl. Acad. Sci. U.S.A. 87, 6368-6372.
9. Wren, D., Wolswijk, G. and Noble,
M. (1992) In vitro analysis of origin and maintenance
of O-2Aadult progenitor cells J. Cell Biol. 116,
167-176.
10. Wolswijk, G. and Noble, M. (1992)
Cooperation between PDGF and FGF converts slowly dividing
O-2Aadult progenitor cells to rapidly dividing cells with
characteristics of their perinatal counterparts. J. Cell
Biol. 118, 889-900.
11. Groves, A.K., Barnett, S.C., Franklin, R.J.M.,
Crang, A.J., Mayer, M., Blakemore, W.F. and Noble,
M. (1993) Repair of demyelinated lesions by transplantation
of purified O-2A progenitor cells Nature 362,
453-455.
12. Urenjak, J., Williams, S., Gadian, D. and Noble,
M. (1993) Proton nuclear magnetic resonance spectroscopy
unambiguously identifies different neural cell types. J. Neurosci. 13, 981-989.
13. Groves, A.K., Entwistle, A., Jat, P.S. and Noble,
M. (1993) The characterisation of astrocyte cell lines
that display properties of glial scar tissue. Dev.
Biol.
159, 87-104.
14. Bögler, O. and Noble, M.
(1994) Measurement of time in oligodendrocyte-type-2 astrocyte
(O-2A) progenitors is a cellular process distinct from differentiation
or division. Dev. Biol. 162, 525-538.
15. Mayer, M. and Noble, M. (1994)
N-Acetyl-L-cysteine is a pluripotent protector against cell
death and enhancer of trophic factor-mediated cell survival
in vitro. Proc. Natl. Acad. Sci. U.S.A. 91,
7496-7500.
16. Florian, C.L., Preece, N.E., Bhakoo, K.K. Williams,
S.R. and Noble, M. (1995) Cell-type specific
fingerprinting of meningioma and meningeal cells by proton
nuclear magnetic resonance spectroscopy. Cancer
Res. 55,
420-427.
17. Bhakoo, K. K., Williams, S. R., Florian, C. L.,
Land, H. and Noble, M. (1996) Immortalisation
and transformation are associated with specific alterations
in choline metabolism. Cancer Res. 56, 4630-4635.
18. Ibarrola, N., Mayer-Pröschel, M., Rodriguez-Pena
and Noble, M. (1996) Evidence for the existence
of at least two timing mechanisms that contribute to oligodendrocyte
generation in vitro. Dev. Biol. 180, 1-21.
19. Rao, M., Noble, M. and Mayer-Pröschel,
M. (1998) A novel tripotential glial precursor cell is present
in the developing spinal cord. Proc. Natl. Acad. Sci. U.S.A.
95, 3996-4001.
20. Yakovlev, A. Y., Boucher, K., Mayer-Pröschel,
M. and Noble, M. (1998) Quantitative insight
into proliferation and differentiation of O-2A progenitor
cells in vitro: The clock model revisited. Proc.
Natl. Acad. Sci. U.S.A. 95, 14164-14167.
21. Smith, J., Ladi, E., Mayer-Pröschel, M. and
Noble, M. (2000) Redox state is a central
modulator of the balance between self-renewal and differentiation
in a dividing glial precursor cell. Proc. Natl. Acad. Sci.
U.S.A. 97, 10032-10037.
22. Nutt, C.L., Noble, M., Chambers,
A. F., Cairncross, J.G.(2000) Differential expression of drug
resistance genes and chemosensitivity in cells of different
glial lineages correlates with differential sensitivity of
oligodendrogliomas and astrocytomas to chemotherapy. Cancer
Res. 60, 4812-4818.
Synopses of Papers:
1. This paper reports the original isolation
of the oligodendrocyte-type-2 astrocyte progenitor cell of
the perinatal optic nerve. This cells is also referred to
as an oligodendrocyte precursor cell.
2. One of our aspects of the analysis of
glia, early on, was to analyze interactions of neurons with
the glial cell surface. These studies described the finding
that CNS neurons grow on glial cells in a manner that is quite
distinct from that seen when these neurons are plated on non-glia.
3. These studies led to the identification
of a wholly new kind of cell-cell interaction in the CNS,
wherein a differentiated glial cell population (the astrocyte)
promotes division of a CNS precursor cell from a separate
lineage.
4. These were the first studies indicating
that the cells that give rise to oligodendrocytes appear to
populate myelinated tracts by migration from germinal zones
in located in other regions of the CNS.
5. One of the key questions raised by our
earlier studies demonstrating that astrocytes promote division
of O-2A progenitor cells was to identify the mitogenic factor
involved. This paper demonstrated that this factor was likely
to be platelet-derived growth factor.
6. Previous studies from Martin Raff and
colleagues had amplified on our discovery that growth of O-2A
progenitor cells in the presence of purified astrocytes restored
a normal pattern of division and differentiation to show,
quite surprisingly, that growth of embryo-derived progenitor
cells in the presence of purified astrocytes was associated
with the first appearance of oligodendrocytes at a time precisely
equivalent to the time that these cells appeared in vivo.
Such studies were critical in promoting research on cell-intrinsic
clocks and the contribution of such clocks to the timing of
normal development. These studies on PDGF showed that all
one had to do to create this normal timing was stimulate embryonic
O-2A progenitor cells to divide by exposure to an appropriate
mitogen.
7. There is no room in the white matter tracts
of the adult brain for rapidly dividing progenitor cells that
make lots of new cells, yet one wants to have some precursor
cells present to participate in tissue repair. In these studies
we discovered that the O-2A progenitor cells of the adult
nervous system exhibit a fundamentally different biology from
those derived from younger animals. The adult progenitors
divided slowly and expressed other properties more suitable
for the physiological requirements of the adult central nervous
system. These studies represented one of the first descriptions
of adult-specific progenitor cells.
8. Previous studies had indicated that O-2A
progenitor cells had a defined mitotic lifespan, and underwent
differentiation into oligodendrocytes within a limited number
of cell divisions. In these studies we discovered that simply
exposing these cells to a combination of platelet-derived
growth factor and fibroblast growth factor caused them to
undergo continuous division in the absence of differentiation.
Along with demonstrating that cell-intrinsic biological clocks
could be overridden by appropriate environmental signals,
this research provided a means of expanding precursor cells
in sufficient numbers to enable their use in tissue repair.
Subsequent work in other laboratories demonstrated that this
same principle could be applied also to precursor cells in
other developmental lineages.
9. Having identified biologically distinct
populations of O-2A progenitor cells in developing and adult
animals, the next question that had to be solved was what
the relationship between these two populations might be. Remarkably,
it turned out that adult-specific progenitors were derived
directly from the progenitors present in the younger animal.
These studies may still represent the only case in which the
developmental origin of an adult-specific precursor cell has
been identified.
10. The properties of adult-specific O-2A
progenitor cells are not favorable for repair, particularly
in respect to their long cell cycle times. When we exposed
these cells to combinations of growth factors known to be
expressed in CNS lesions, however, the adult-specific cells
transiently re-expressed the properties (including rapid division)
of progenitor cells isolated from the CNS on developing animals.
Thus, the capacity to express these earlier properties is
not irreversibly lost, but is placed under a different control
mechanism.
11. The natural extension of our capacity
to expand O-2A progenitor cells by growth factor cooperation
was to examine the ability of such cells to repair demyelinated
lesions. In these studies we demonstrated that we could remyelinate
>90% of demyelinated axons by precursor cell transplantation.
12. One of the extremely useful tools for
analyzing cellular biochemistry and tissue function in vivo
is proton-NMR spectroscopy. When we initiated these studies,
the problem we sought to examine was the extent to which different
cell types differed in expression of the small metabolites
detected by this analytical method (which, with the NMR magnet
used in these studies, principally represented free amino
acids). The findings obtained were quite surprising, and revealed
that the different cell kinds and quantities of free amino
acids expressed by each cell type was highly specific, and
the profiles obtained through this analysis could be used
to distinguish cells as unambiguously as did analysis of antigenic
phenotype.
13. Although our own studies are focused
on identifying conditions that allow the extended growth of
normal cells of interest to our research program, such growth
conditions have only been identified for a small number of
cell types. Thus, there is still great importance in being
able to generate cell lines of interest, a goal that is best
achieved through the expression of conditionally active immortalizing
genes. To this end, we worked in collaboration with Parmjit
Jat and Dimitris Kioiussiss to generate transgenic mice that
harbor a temperature sensitive variant of SV40 large T antigen,
a potent immortalizing gene, under the control of the Class
I antigen promoter. Growth of cells from this mouse at 33°C
in the presence of interferon-gamma turns on expression of
a functionally active T antigen. Such mice have been used
in the generation of a wide variety of cell lines. In these
particular studies, lines were generated with the properties
of glial scar tissue. Of particular interest in these studies
was the finding that these scar like cells inhibited the migration
of O-2A progenitor cells, a finding of potential relevance
in understanding the failure of remyelination in scarred regions
of the brain and spinal cord.
14. Multiple studies have demonstrated that
measurement of elapsed time in dividing cells can be associated
with the induction of differentiation. But is this a necessary
link? These studies demonstrated that these are distinct processes,
and that precursor cells kept from differentiating by growth
in the presence of growth factor combinations appear to have
a clear memory of how "old" they are. Strikingly,
the manner in which this aging occurs cannot be explained
by any existing hypotheses of aging (including such diverse
hypotheses as shortening of telomere length and accumulation
of DNA damage).
15. These studies were initiated to examine
the role of redox regulation in cell survival, at an early
point in the recognition of the role of oxidative stress in
induction of cell death. Along with some fundamental discoveries
related to antioxidants and cell survival, we made the surprising
discovery that one can enhance the efficacy of survival factors
quite strikingly by applying them in combination with antioxidants.
These studies began our analysis of novel roles for redox
modulation in development, for which see Smith et al., 2000.
16. Following up on our previous studies
that different cell types have highly distinctive patterns
of expression of free amino acids and other small metabolites,
we asked whether these patterns were conserved from rat to
human, and whether the patterns seen in tumor cells were like
the patterns seen in normal cells. These first studies were
supportive of such an outcome, and further work (not yet published)
has confirmed that such conservation occurs. This speaks to
an intriguing biological question: What is so important about
the kinds and quantities of free amino acids that a particular
cell type makes that these patterns of expression are conserved
across species boundaries and even during transformation?
17. Our comparative studies on cancer cells
and normal cells by 1H-NMR spectroscopy revealed not only
striking similarities (see above), but also began to indicated
the existence of some specific metabolic differences. In this
study we describe one of those differences, which thus far
seems to be a specific metabolic correlate of transformation.
18. These studies were initiated to follow
up on studies by many others demonstrating that humans, and
experimental animals, that have a thyroid hormone deficiency
during development have severe delays in myelination. Thinking
that this might mean that thyroid hormone contributed to the
timing of oligodendrocyte generation, we initiated these studies.
The findings revealed a previously unsuspected complexity
of cell-intrinsic timing mechanisms, showed that the balance
between self-renewal and differentiation in a dividing precursor
cell was subject to exquisitely sensitive environmental regulation,
and overthrew a decade-old dogma regarding the importance
of symmetric differentiation in timed differentiation processes.
19. Understanding the relationship between
O-2A progenitor cells and early development, and understanding
the complexity of precursor cell populations that contribute
to CNS development is a major concern. This study describes
the in vivo isolation and biological properties of a new glial-restricted
precursor cell that may be the ancestor of all glia. For more
information on this cell, and this line of research, please
refer to the web site (in the Center for Cancer Biology) of
Dr. Margot Mayer-Pröschel.
20. One of the big problems in biology is
that so much of it is a narrative science. One of the arenas
in which this creates enormous problems is in the evaluation
of precursor cell behavior. How does one know whether subtle
differences in behavior are significant? In addition, can
one use the power of mathematics to derive insights that can't
be obtained by just narratively interpreting the data? This
paper is one in a series of studies attempting to develop
quantitative approaches to the analysis of differentiation.
21. These studies return us to the problem
of control of the balance between self-renewal and differentiation
in dividing precursor cells. Controlling this balance is at
the heart of understanding normal development and tissue repair,
and also may provide insights into the biology of cancer.
As we, and others, have discovered multiple growth factors
and other signaling molecules that regulate this balance,
we were very interested in determining whether there is a
physiological point where these pathways converge. Surprisingly,
one of the points of convergence is on redox regulation. These
studies demonstrate that redox modulation is sufficient to
modulate the balance between self-renewal and differentiation,
that signaling molecules that modulate this balance regulate
redox state, that this regulation may be a necessary component
of the action of these signaling molecules, and that redox
state in vivo is predictive of self-renewal capacity.
22. This study was initiated to try and
begin dissecting a puzzling paradox in brain tumor therapy.
Patients with oligodendrogliomas frequently respond quite
well to chemotherapy, yet patients with astrocytomas - even
of the same malignancy grade- relatively rarely benefit from
such treatment. We wished to know whether these properties
might have something to do with the properties of the putative
cells of origin of these different tumors. We found that oligodendrocytes
are indeed very sensitive to BCNU, an alkylating agent used
to treat brain tumors, while astrocytes are quite resistant.
This work is the starting point for our current studies on
chemosensitivity of neural stem cells, which are referred
to in the description of our research program.
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