Current
Pharmaceutical Biotechnology
ISSN: 1389-2010
Current Pharmaceutical Biotechnology
Volume 8, Number 3, June 2007
Contents
Analysis of Progenitor Cells in the Brain before and
after Treatment
Guest Editors: M.A. Curtis and L. Paulson
Editorial Pp. 115
Defining Primary and Secondary Progenitor Disorders
in the Brain: Proteomic Approaches for Analysis of Neural
Progenitor Cells Pp. 117-125
L. Paulson, P.S. Eriksson and M.A. Curtis
[Abstract]
Bromodeoxyuridine and the Detection of Neurogenesis
Pp. 127-131
H.G. Kuhn and C.M. Cooper-Kuhn
[Abstract]
Fluorescent Activated Cell Sorting: A Window on the
Stem Cell Pp. 133-139
K.W. Johnson, M. Dooner and P.J. Quesenberry
[Abstract]
Using the Neurosphere Assay to Quantify Neural Stem
Cells In Vivo Pp. 141-145
G.P. Marshall II, B.A. Reynolds and E.D. Laywell
[Abstract]
Adult Neurogenesis: Can Analysis of Cell Cycle Proteins
Move Us “Beyond BrdU” Pp. 147-165
A.J. Eisch and C.D. Mandyam
[Abstract]
Microarray RNA/DNA in Different Stem Cell Lines
Pp. 167-175
A.C. Piscaglia, T. Shupe, A. Gasbarrini and B.E. Petersen
[Abstract]
Techniques and Strategies to Analyze Neural Progenitor
Cell Migration Pp. 177-185
I. Comte, P.B. Tran and F.G. Szele
[Abstract]
Adult Neurogenesis in Mesial Temporal Lobe Epilepsy:
A Review of Recent Animal and Human Studies Pp. 187-194
Y.W.J. Liu, E.W. Mee, P. Bergin, H.H. Teoh, B. Connor,
M. Dragunow and R.L.M. Faull
[Abstract]
Abstracts
[Back to top]
Editorial
In 1913, Santiago Ramon Y Cajal, one of the fathers
of neuroscience and a Nobel prize laureate, wrote “...
the functional specialization of the brain imposes on
the neurons two great lacunae; proliferation inability and
irreversibility of intraprotoplasmic differentiation. It is
for this reason that, once the development was ended, the
founts of growth and regeneration of axons and dendrites dried
up irrevocably. In adult centers, the nerve paths are something
fixed, ended and immutable. Everything may die, nothing may
be regenerated. It is for the science of the future to change,
if possible, this harsh decree”. However, today,
in what Cajal may have termed the future, neuroscientists
know that this ‘harsh decree’ is not as harsh
as first thought. Rather, the brain’s ability to produce
new neurons via neurogenesis in the two developmentally active
germinal zones is highly regulated and appears vital for cognition,
memory and for the repair and replacement of damaged neurons
after injury. Although the first studies showing that progenitor
cell proliferation and neurogenesis occur in the mammalian
brain were first reported in the 60’s, these studies
did not receive the attention they deserved and until the
90’s, we were on the heel of the learning curve that
was about to become exponential. In particular, it was the
demonstration by Eriksson et al. in 1998 that neurogenesis
occurs in the human hippocampus that first made the field
realise that these germinal zone cells might be useful for
the treatment of disease in humans.
Although neuroscientists tend to focus on the advances in
biology, it is very evident that many biological advances
occur subsequent to the development of technology. In this
edition of JCPB, we will focus on the studies performed and
the methods used to analyse progenitor cell populations in
vivo and in vitro. Our review series begins
by defining the progenitor cell populations and clarifying
the potentially confusing nomenclature used in stem and progenitor
cell biology. We also review the techniques used to examine
the unique cohort of proteins that progenitor cells express,
thus making them distinct as a cell population. Then our series
examines the pitfalls that have entrapped many, when using
double and triple labelling techniques, due to confounding
artefacts. The next review describes neurosphere formation
as a measure of self-renewal and differentiation capacity
of progenitor/stem cells in vitro with comparison
to the in vivo situation. Then fluorescence activated
cell sorting (FACS) is reviewed as a method for the detection
of specific cell populations based on cell surface markers;
these techniques appear to be coming of age in the field of
neural progenitor cells also. From there, our series focuses
on the temporal expression of endogenous cell cycle proteins
through the progenitor cell cycle and reveals how the analysis
of these proteins may take us ‘beyond BrdU’. We
have also included a review of microarrays for high throughput
detection of differentially expressed RNA and DNA in different
progenitor cell lines. Then, the techniques used and the results
reported for analysing progenitor cell migration in vivo
and in vitro are reviewed- this detailed review is
not to be missed.
The final review is focused on progenitor cells from hippocampi
donated by patients that undergo temporal lobectomy for intractable
epilepsy. This final review hits the core of practical progenitor
cell biology; the results reviewed do not present a model
or theory but rather an insight into a human neurological
disease itself and the effect the disease has on progenitor
cells, or vice versa.
We hope that you find these reviews both timely and interesting.
We also hope that from these reviews you will be inspired
with new approaches and ideas for answering the many questions
that remain concerning progenitor cells in the brain.
M.A. Curtis and L. Paulson
Center for Brain Repair and Rehabilitation
Institute of Neuroscience and Physiology
The Sahlgrenska Academy at Göteborg University
Medicinaregatan 11, 413 19 Göteborg
Sweden
[Back to top]
Defining Primary and Secondary Progenitor
Disorders in the Brain: Proteomic Approaches for Analysis
of Neural Progenitor Cells
L. Paulson, P.S. Eriksson and M.A. Curtis
Since the discovery of endogenous progenitor cells in two
brain regions in the adult, the notion that progenitor cells
might be useful for repairing damaged neurons or replacing
dead neurons has gone from fiction to a reality, at least
in the laboratory setting. Progenitor cells have the unique
ability to be able to produce new neurons in response to endogenous
and exogenous cues from their microenvironment in the brain
and from the environment of the organism. However, in models
of several disorders and insults the regenerative potential
of the central nervous system need external enhancing. In
this review we begin by focussing on the developments in the
field of neurobiology that have led to the specific study
of neural progenitor cell biology. In particular we discuss
the two germinal niches, the subventricular zone and the subgranular
zone, as well as how various neurological diseases affect
these niches. We furthermore try to define primary progenitor
cell disorders and secondary progenitor cell responses. The
second part of this review focuses on proteomic approaches
for studying progenitor cells. These techniques allow the
array of proteins that are expressed by progenitor cells to
be determined and further more allow comparisons between diseased
and normal cells or treated and untreated cell populations.
If we can induce neural progenitor cells to generate functional
neurons in the central nervous system (CNS) then the burden
of neurological disorders may be eased in the future. The
advances in proteomic technology have and will enable further
understanding of the regulatory processes in these cells so
that progenitor cell integration and differentiation can be
enhanced. Hopefully an increase in knowledge of progenitor
cell biology will have a major impact on clinical practice.
[Back to top]
Bromodeoxyuridine and the Detection of Neurogenesis
H.G. Kuhn and C.M. Cooper-Kuhn
Bromodeoxyuridine (BrdU) is widely used for labeling dividing
cells to determine their fate. In particular, the analysis
of neurogenesis in the adult mammalian brain has made significant
progress through the use of this technique. However; when
using BrdU for labeling, there are several issues to consider
in order to minimalize possible cytotoxicity or false-positive
labeling. This current review summarizes methodological and
technical aspects of BrdU administration and detection, compares
alternative methods and gives recommendations on how to avoid
labeling artifacts.
[Back to top]
Fluorescent Activated Cell Sorting: A Window on the
Stem Cell
K.W. Johnson, M. Dooner and P.J. Quesenberry
Fluorescence activated cell sorting (FACS) in the field of
stem cell biology has become an indispensable tool for defining
and separating rare cell populations with a high degree of
purity. Steady progress has been made in this regard, but
the intrinsic lability of the stem cell phenotype presents
a different challenge and there are many technical caveats.
FACS remains, however, the technology of choice for reporting
and characterizing rare cell populations such as stem cells.
[Back to top]
Using the Neurosphere Assay to Quantify Neural Stem
Cells In Vivo
G.P. Marshall II, B.A. Reynolds and E.D. Laywell
Since their initial description in 1992, neurospheres have
appeared in some aspect of more than a thousand published
studies. Despite their ubiquitous presence in the scientific
literature, there is little consensus regarding the fundamental
defining characteristics of neurospheres; thus, there is little
agreement about what, if anything, the neurosphere assay can
tell us about the relative abundance or behavior of neural
stem cells in vivo. In this review we will examine
some of the common features of neurospheres, and ask if these
features should be interpreted as a proxy for neural stem
cells. In addition, we will discuss ways in which the neurosphere
assay has been used to evaluate in vivo treatment/manipulation,
and will suggest appropriate ways in which neurosphere data
should be interpreted, vis-à-vis the neural stem cell.
Finally, we will discuss a relatively new in vitro
approach, the Neural-Colony Forming Cell Assay, which provides
a more meaningful method of quantifying bona fide
neural stem cells without conflating them with more growth-restricted
progenitor cells.
[Back to top]
Adult Neurogenesis: Can Analysis of Cell Cycle Proteins
Move Us “Beyond BrdU”
A.J. Eisch and C.D. Mandyam
One of the greatest scientific discoveries of the 20th
century is that the mammalian brain can give rise to new neurons
throughout the lifespan. The phenomenon of adult neurogenesis
raises hopes of harnessing neural stem cell for brain repair,
and has sparked interest in novel roles for these new neurons,
such as olfaction, spatial memory, and even regulation of
mood. Traditionally, studies on adult neurogenesis have relied
on exogenous markers of DNA synthesis, such as bromodeoxyuridine
(BrdU), to label and track the birth of new cells. However,
the exponential increase in our knowledge of endogenous markers
of cycling cells has ushered in a new era of stem cell biology.
Here we review the strides made in using endogenous cell cycle
proteins to study adult neurogenesis in vivo. We
(1) discuss the distribution of endogenous cell cycle proteins
in proliferative regions of the adult mammalian brain; (2)
review cell cycle phase-specific information gained from analyzing
a combination of endogenous cell cycle proteins; and (3) provide
data on the regulation of cell cycle proteins by a robust
inhibitor of proliferation, morphine. The ability of BrdU
to birthdate cells ensures it will always serve a role in
studies of adult neurogenesis, thus preventing us from moving
entirely ‘beyond BrdU’. However, it is hoped that
this review will provide interested researchers with the tools
needed to apply the powerful and relatively novel approach
of analyzing endogenous cell cycle proteins to the study of
stem cells in general and adult neurogenesis in particular.
[Back to top]
Microarray RNA/DNA in Different Stem Cell Lines
A.C. Piscaglia, T. Shupe, A. Gasbarrini and B.E. Petersen
Stem cells represent the key to tissue genesis, regeneration,
and turnover. This notion has spawned the concept of regenerative
medicine, or stem cell based therapies to supplement degenerating
or damaged tissues. However, stem cells may also represent
a preferential target of carcinogens. The unique ability of
stem cells to self-renew and to differentiate into multiple
phenotypes implies that all stem cells share a common transcriptional
signature. A better knowledge of the stem cell transcriptome
appears to be fundamental to fully achieve the potential of
regenerative medicine, and may lead to new strategies for
cancer prevention and treatment. Elucidation of the transcriptional
programming and molecular mechanisms which direct stem cell
self-renewal, differentiation, and tumorigenesis should provide
key insights into deciphering exactly how “stemness”
is maintained, as well as the molecular basis of cell plasticity
and cancer development. cDNA and oligonucleotide microarrays
are the most accessible transcriptome profiling methods to
date, providing the unique opportunity to compare global gene
expression patterns among different cell populations. Microarray
technologies have been applied to three major areas of stem
cell research: maintenance of pluripotency, development of
uniform and regulated differentiation, and microenvironment
analyses. The aim of the present review is to summarize state-of-the-art
transcriptional profiling of different stem cell lines, cancer
stem cells, and the niches these cells occupy in vivo.
[Back to top]
Techniques and Strategies to Analyze Neural Progenitor
Cell Migration
I. Comte, P.B. Tran and F.G. Szele
One of the most surprising aspects of neural development is
that cells do not remain in their birthplace but actively
migrate along a variety of routes to their final destinations.
This review traces past, present, and future techniques used
to analyze progenitor cell migration in the brain, and also
discusses their relevant strengths and weaknesses. The large
majority of information regarding cell migration is from studies
where migratory cells have been labeled, but in which the
actual movements are not observed, ie., from static experiments.
More recently, dynamic imaging of cell migration in living
slices and, even in vivo, has provided a glimpse
of how complex these phenomena truly are. A variety of new
techniques, such as 2-photon videomicroscopy, are emerging
that will continue to add to our body of knowledge concerning
the migration of cells in the central nervous system.
[Back to top]
Adult Neurogenesis in Mesial Temporal Lobe Epilepsy:
A Review of Recent Animal and Human Studies
Y.W.J. Liu, E.W. Mee, P. Bergin, H.H. Teoh, B. Connor,
M. Dragunow and R.L.M. Faull
Mesial temporal lobe epilepsy (mTLE) is a neurological condition
characterized by the occurrence of spontaneous recurrent seizures
originating from mesial structures involving the hippocampus
within the temporal lobe. This condition is often associated
with pathological features in the hippocampus such as neuronal
cell loss, widening of the granule cell layer, astrogliosis
and mossy fibre spouting. At present, the mechanisms underlying
these pathological features are unclear. However, recent advances
in adult neurogenesis studies in mTLE animals and patients
suggest that newly generated neurons may contribute to the
pathogenesis of ongoing epileptogenesis. This article will
review the recent animal and human studies on adult neurogenesis
in mTLE and discuss how these results suggests that adult
endogenous neurogenesis may not always be reparative in the
mTLE and may be targeted in new therapeutic strategies for
mTLE.
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