1 The John P. Roberts Research Institute, 100 Perth Drive, London, ON, N6A 5K8,
Canada
2 Center for Regenerative Medicine, Maine Medical Center Research Institute, 81
Research Drive, Scarborough, ME 04074, USA
3 Center for Molecular Medicine, Maine Medical Center Research Institute, 81
Research Drive, Scarborough, ME 04074, USA
Author for correspondence (e-mail:
verdij{at}mmc.org)
Accepted 8 June 2004
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SUMMARY |
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Key words: Plasticity, Neural stem cells, Myogenesis
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Introduction |
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NSCs in the central nervous system, and their in vitro model, neurospheres
are restricted to a differentiation profile of neurons, astrocytes and
oligodendrocytes (Davis and Temple,
1994; Reynolds et al.,
1992
; Reynolds and Weiss,
1992
; Reynolds and Weiss,
1996
; Tsai and McKay,
2000
). Whether NSCs are tripotent in vivo is still unclear
(Gabay et al., 2003
;
Pevny and Rao, 2003
). However,
NSCs expanded from either cell-adherent or aggregate cultures differentiate
into immunoreactive myocytes when co-cultured with either primary myoblasts,
ES cells or when introduced into preimplantation embryos
(Clarke et al., 2000
;
Galli et al., 2000
;
Rietze et al., 2001
;
Tsai and McKay, 2000
). The
differentiation of NSC into myogenic derivatives has been reported to be
density dependent in some microenvironments
(Tsai and McKay, 2000
) and
dependent upon pre-existing myocytes in other environments
(Galli et al., 2000
;
Rietze et al., 2001
). It has
also been reported that bone morphogenetic protein (BMP) induces smooth muscle
differentiation of NSCs, and it has been suggested that BMP induces a
transformation of central nervous system NSC into peripheral nervous system
neural crest stem cells (Tsai and McKay,
2000
).
Although studies using the neurosphere assay have begun to discern the
molecular mechanism of specification of NSCs to specific neural sublineages
(Hitoshi et al., 2002a;
Hitoshi et al., 2002b
;
Tropepe et al., 2001
), the
molecular mechanisms of non-neural specification and differentiation of NSCs
remain unknown. Thus far, studies demonstrating the myogenic differentiation
potential of NSCs leave unanswered the question of whether myogenic and
neurogenic differentiated progeny arise from a single multilineage neural stem
cell (MLNSC), as well as the issue of which factors propagate these MLNSC
populations and instruct myogenic differentiation. It is also not known
whether NSCs can generate biologically functional myogenic derivatives.
We have attempted to address these questions by demonstrating that differentiated neural and myogenic progeny can originate from a single isolated cell and can be propagated in serum-free insulin-containing media. MLNSCs arise as a consequence of insulin-mediated survival and not as a result of cell fusion events. Moreover, myogenic differentiation is insulin dose sensitive; low concentrations of insulin promote cardiomyocyte differentiation, whereas elevated concentrations induce skeletal muscle formation. At any dose, myogenic differentiation occurred at the expense of neural differentiation. MLNSC-derived cardiomyocytes were metabolically coupled, contracted spontaneously, responded to sympathetic and parasympathetic stimulation, and engrafted and differentiated appropriately when introduced into damaged cardiac muscle. These studies provide support for the hypothesis that NSCs have a broad differentiation potential, including phenotypes outside the neuroectodermal lineage. As such, MLNSCs may provide a population of cells suitable for regenerative medicine strategies inside and outside of the nervous system.
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Materials and methods |
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Flow cytometry
Embryonic cortical precursors were immunolabeled using monoclonal insulin
receptor (IR) (Pharmingen) and FITC-GAM antibodies. In addition, cells were
labeled with anti-CD35 and anti-CD31 conjugated to rhodamine and PE,
respectively. CD31- CD35- cells were sorted into IR
high, low and non-expressing subpopulations using a BD FACS Vantage SE cell
sorter.
Transfection and retroviral transduction
293GPG packaging cells were transfected with the AP2-IRES-EGFP retroviral
construct (Galipeau et al.,
1999). Enhanced green fluorescent protein (EGFP) retroviral
particles were collected every 24 hours. Flow cytometric analysis of EGFP
fluorescence was used to determine the viral titer
(Galipeau et al., 1999
). NSCs
were infected with 1.2x1010 cfu/ml EGFP-retroviral
particles.
Southern blotting analysis
Clones were digested overnight in 400 µg/ml proteinase K at 50°C.
The resulting DNA (25 µg) was digested with BglII and run on a
0.7% agarose TAE gel and transferred to a Nytran Plus membrane.
Prehybridization, hybridization and washing of the resulting membrane were
performed using standard methods.
RT-PCR analysis
RT-PCR analysis was performed according to published methods
(Verdi and Anderson, 1994)
with the following parameters: 35 cycles of denaturing at 94°C (30
seconds), annealing at 53-58°C for 1 minute and elongation at 72°C for
1 minute. Primer sequences are available upon request.
Antibodies
The antibodies used included monoclonal antibodies to ßIII-tubulin
(Chemicon), microtubule associated protein 2a+2b, ß-actin (Sigma), 47A
skeletal myosin heavy chain (MHC), BA-G5 cardiac MHC (New England Biolabs);
CD31, CD35, tropomyosin and troponin1 (Chemicon); and polyclonal antibodies to
caspase 3 (Pharmingen), phospho-Akt (Ser473 and Thr308; New England Biolabs),
glial fibrillary acidic protein, (Sigma), nestin. Secondary antibodies used
were purchased from Jackson ImmunoResearch Laboratories.
Immunocytochemistry
Cells were fixed with 70% ethanol and 0.15 M NaCl. Non-specific binding
sites were blocked with 10% goat serum and 0.1% Nonidet P-40 for 30 minutes.
Cells were incubated 1 hour at room temperature with primary antibody prior to
washing and application of the secondary antibody for 1 hour at room
temperature. The immunoreactivity was examined with an Olympus IX70
fluorescent microscope.
For immunohistochemical analysis of tissues, specimens were fixed with 4% paraformaldehyde and embedded in paraffin wax. Serial 15 µm sections were dewaxed in xylene, hydrated in a graded ethanol series (100%, 90%, 70%, 50%), for 5 minutes per hydration and washed with PBS.
Western blot analysis
Cells were lysed in ice-cold buffer composed of 10 mM NaCl, 20 mM Tris (pH
8.0), 0.5 mM EDTA, 10% glycerol, 1% Nonidet P-40, 1 mM phenyl-methylsulfonyl
fluoride, 1 mM sodium orthovanadate, 2 µg/ml leupeptin and 10 µg/ml
aprotinin. Clarified protein lysate (30 µg) was separated on a reducing 15%
SDS-PAGE and blotted onto Immobilon-P (Millipore). Membranes were blocked with
5% blotto/10 mM tris-saline pH 7.4 overnight. Membranes were incubated with
either anti-phospho-Akt (1:4000), anti-caspase 3 (1:1000) or anti-ß-actin
(1:5000) antibody prior to incubation with horseradish peroxidase-conjugated
goat anti-rabbit or anti-mouse antibodies. Immunoreactivity was detected using
an enhanced chemiluminescent methodology (Amersham Biosciences).
DNA synthesis analysis
Cells were incubated with 10 µM BrdU in triplicates, at 37°C, 5%
CO2 for 6 or 12 hours after 24, 72 and 120 hours of expansion in MB
media. Cells were fixed in 4% paraformaldehyde (RT-20 minutes) and labeled
with a monoclonal anti-BrdU, according to manufacturer's specifications
(Becton-Dickinson Biosciences). BrdU+ cells were counted using an
epifluorescent microscope.
Dye preloading
NSC-derived cardiomyocytes were incubated for 20 minutes in a solution
containing: isotonic glucose, 0.1% calcein AM, 0.1%
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyaine
perchlorate (DiI) (Molecular Probes) at 37°C
(Goldberg et al., 1995).
Single preloaded (donor) cells were seeded over contracting NSC-derived
cardiomyocyte clusters (one donor cell per cluster) and incubated for 2 hours
at 37°C.
Dye microinjection
NSC-derived cardiomyocytes were incubated in cold medium 5 minutes before
microinjection to slow contraction. A single cell within each cardiomyocyte
cluster was pressure-microinjected with 10 mM 5(6)-carboxyfluorescein
(Mr=376, Molecular Probes) using a Leitz Labovert FS
Microinjector. Dye transfer was examined after 10-30 minutes.
Live imaging of the NSC-derived cardiomyocytes
Time-lapse images of individual clusters were used to examine the
contraction rate of cardiomyocyte clusters. Fluorescent images were taken
using a 488 nm argon/krypton laser line on a Zeiss LSM 410 inverted confocal
microscope. Optical sections were scanned at a speed of 32 seconds/image and
collected continuously for up to 10 minutes. Focus, contrast and brightness
settings remained constant during acquisition.
Organotypic cultures
Adult CD1 hearts were isolated and placed in Hanks balanced salt solution,
freed of blood and placed in trans-well inserts for six-well Nunc plates.
Organs were covered with MB-media such that a meniscus formed over the top of
the explant. To induce injury, one ventricle was injured with an etched
tungsten micro-needle. Immediately after injury, medium was refreshed and
EGFP-tagged NSC (5000 cells grown in MB-media) were injected into the injured
area. Organs were maintained in the culture for 10 days and then fixed for
analysis. Manual counting of z-sections taken through the entire
heart (n=5) was used to calculate the percentage of cardiac
MHC+/EGFP+ cells engrafted in the host.
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Results |
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|
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To confirm that the sphere was generated from a single retrovirally infected cell, aliquots from five random wells prior to adherent culturing were subjected to Southern analysis. Every clone had a single unique integration event (Fig. 1I). Moreover, as shown in Fig. 1D, both neurogenic and myogenic EGFP expressing differentiated progeny were observed within a single clone, strongly suggesting that both lineages arose from one founding cell and not as an aggregate of EGFP+ and EGFP- founders.
MLNSC progeny express markers of mature skeletal and cardiac muscle
We further characterized the course of maturation of the myogenic progeny
produced from the differentiation of MLNSCs. CD31- CD35-
NSC did not express skeletal (sk)-MHC or cardiac-MHC immunoreactivity prior to
differentiation in MB-media (data not shown). However, during differentiation,
neurospheres grown in MB media began to express markers identifying some
progeny as potential skeletal (Fig.
2A-D) or cardiac myocytes (Fig.
2A,E). In a limited time course, it was demonstrated that MyoD
expression preceded that of myogenin (data not shown) and subsequent tissue
type-specific expression (Fig.
2A). Differentiating clones expressed sk-MHC immunoreactivity as
early as day 9 and formed clusters of skeletal myoblasts and slender myofibers
that joined those of other clusters (Fig.
2B-D). In addition to formation of skeletal muscle, NSC cultures
also differentiated into cardiac myocytes with the appearance of terminal
differentiation markers beginning (day 13) after the onset of skeletal
muscle terminal differentiation (
day 9). NSC-derived cardiac myocytes
grew clusters of cells expressing cardiac
-actin (data not shown),
cardiac-MHC and connexin43 (Cx43) (Fig.
2E), and these clusters over time (
3 weeks) began to contract
synchronously (see Movies 1-3 at
http://dev.biologists.org/supplemental).
This contraction was used as an additional phenotypic marker to distinguish
skeletal and cardiac myocytes, as well as Cx43, a standard marker to
distinguish between the two cell types.
|
A criterion that defines stem cell populations is the existence of the
population throughout the lifetime of the animal
(Weissman et al., 2001). We
examined the prevalence of MLNSCs within cortical precursors during gestation
and within the adult counterpart, the frontal ventricular subependymal layer
(6 weeks post partum). The absolute number and percentage of MLNSCs declined
through development (Table 2).
However, MLNSCs could be isolated from the adult animal, suggesting that the
cell population exists for the lifetime of the animal and thus meets this
criterion defining stem cells.
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As insulin signaling appeared to be crucial for the formation of MLNSC neurospheres, we used insulin receptor (IR) to further demarcate the MLNSC population. We prospectively isolated CD31- CD35- IR+ embryonic cortical precursors at embryonic day 14 and found that 65% of the population was IR expressive (Fig. 4A). Neurospheres generated from the IRhigh (the brightest 20% of the IR+ cells) population generated more multilineage differentiated progeny in comparison to neurospheres generated from the IRneg and IRlow (the lowest 20% of IR+ cells) populations (Fig. 4B). Although a minimum number of insulin receptors were required for cells to differentiate into myocytes, cells with more receptors were more competent to generate myocytes, suggesting a role for insulin beyond that of a survival factor.
|
We examined the differentiation profiles of NSC subsets after 6 days of expansion based on IR immunoreactivity. IRneg cells gave rise exclusively to clones with neural differentiated progeny (61±8% neuron + astrocytes; 22±11% neurons only; astrocytes only 16±4%). IRlow cells gave rise to multilineage clones. Of the 227 clones examined, 83 contained myogenic progeny, of which 36 (47±14%) contained neurons, astrocytes, and both skeletal and cardiomyocytes. The IRhigh cells gave rise to the broadest differentiation profile, composed of all combinations of differentiation outcomes including oligodendrocytes that were not present in the IRneg or IRlow differentiated progeny. Eighty-one out of 102 multilineage clones (80±15%) were composed from neuronal, astrocytes and myogenic progeny; of the 81 clones, 17 also contained oligodendrocytes (21±7%). Importantly, all three IR subpopulations contain multipotential cells, but myogenic potential was contained only within IR expressive cells, and higher percentage of myogenic differentiation correlated with higher levels of IR expression.
Insulin is a dose-dependent instructive myogenic differentiation signal for NSC
Because sorting NSCs by IR levels suggested the possibility that insulin
was acting as more than a survival agent for MLNSCs, we examined the
propensity of MLNSCs to produce clones with myogenic progeny as the insulin
dose varied (Fig. 5A). The
percentage of clones expressing myogenic derivatives increased in a
dose-dependent manner. Surprisingly, the type of muscle cells generated was
also dose sensitive. At lower insulin concentrations, both cardiomyocyte and
skeletal muscle differentiation were equally favored within a particular
clone. Increasing the concentration of insulin strongly favored skeletal
muscle differentiation at the expense of cardiomyocyte differentiation
(Fig. 5B). In all cases,
myogenic differentiation occurred at the expense of neuronal and glial
differentiation as the percentage of neurons and glial progeny within myogenic
clones was reduced.
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We challenged the myogenic signal by co-exposing NSCs to insulin plus
either EPO, a neuronal enhancer (Shingo et
al., 2001), or CNTF (Johe et
al., 1996
), a promoter of glial differentiation, after the fourth
day of expansion. In all cases, the myogenic differentiation signals of
insulin were subordinate to EPO, LIF and CNTF
(Table 4). This may be one
explanation of why myogenic differentiation does not occur in vivo despite the
observations that subsets of NSCs maintain the potential.
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MLNSCs do not pass through a neural crest stem cell-like intermediate
As it has been suggested that NSCs treated with BMP2 transform into neural
crest stem cells (NCSCs) (Tsai and McKay,
2000), we wondered if insulin was transforming NSCs into NCSCs. By
two independent measures, one phenotypic and one functional, no such
transition occurred. Dissociated neurospheres in MB-media did not express two
salient markers of NCSCs, p75 and
4 integrin. Moreover, neither BMP2
(Shah et al., 1996
), a
neuronal instructive factor, nor TGFß
(Shah et al., 1996
;
Shah and Anderson, 1997
), a
myogenic instructive factor, augmented the differentiation profiles of
dissociated neurospheres generated in MB-media when subjected to clonal NCSC
differentiation assays in a factor-rich media known to support NCSCs expansion
and differentiation. Importantly, TGFß, despite being an instructive
smooth muscle signal of NCSCs, did not induce the generation of a single
smooth muscle actin-expressive progeny from cortical-derived NSCs in these
experiments (data not shown).
NSC-derived cardiomyocytes are functionally active
Looking beyond immunological and molecular markers, we asked whether the
MLNSC-derived cardiomyocytes might be biologically functional. A hallmark of
cardiac myocytes is that they are metabolically coupled through
Cx43-expressing gap junctions (van Veen et
al., 2001). We tested for the presence of metabolic coupling
within each myogenic cluster by injecting fluorescent dyes into a single
putative cardiomyocyte and following the dye transfer into the adjacent cells,
as well as by the preloading technique
(Fig. 6A,B). Dye readily passed
from the dye-labeled cell to the adjacent cardiomyocytes and neurons within
the clone. Conversely, when a preloaded cell was placed upon a cardiomyocyte
cluster, no dye transfer was observed. Using either assay, no dye transfer was
observed between putative skeletal myocyte progeny (data not shown).
|
Until recently, it was believed that the mammalian myocardium did not
contain reserve cells and that terminally differentiated cardiomyocytes are
incapable of regeneration after injury
(Carbone et al., 1995;
Nadal-Ginard, 1978
). Embryonic
stem cells (Maltsev et al.,
1993
; Wu et al.,
2004
), bone marrow cells
(Badorff et al., 2003
;
Deb et al., 2003
) and the
recently identified cardiac stem cells
(Beltrami et al., 2003
) have
been shown to generate cells with the appropriate differentiation profile, and
the heart has been shown to be a receptive area that can support myocardial
repair. We asked whether NSC-derived cardiomyocytes have the capacity to
integrate within injured heart tissue (Fig.
7A). Differentiating EGFP+ CD31-
CD35- NSCs were injected into mechanically injured hearts as soon
as progeny began to cluster (
day 10) but prior to the onset of cardiac
myogenic differentiation (Fig.
7B-D). Sixteen percent (±5; n=5) of the
EGFP+ cells integrated with the host organ within 48 hours. The
majority of these cells completed their differentiation process following
injection as determined by the induction of expression of cardiac-MHC
(Fig. 7D). Confocal analysis of
the damaged heart tissue revealed that the EGFP+
cardiac-myosin+ cells integrated deep within the tissue
(Fig. 7D). By contrast,
EGFP+ cells grown in serum-containing medium neither integrated nor
differentiated into cardiac myocytes (data not shown). This experiment
highlights the potential use of this population in cell replacement and
regenerative medicine strategies inside and outside the nervous system.
|
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Discussion |
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We have attempted to show in a rigorous manner that a subpopulation of insulin-responsive NSCs indeed has multilineage differentiation potential. In these studies, great care was taken to examine the forming clones after plating to identify wells containing a single EGFP+ cell after seeding, thus diminishing any possibility of fusion events prior to expansion. Although we cannot completely rule out the possibility that an EGFP- cell was present, more than one EGFP+ cell was ruled out as clones and subclones displayed a single and unique viral integration site. We cannot formally exclude the possibility that cell fusion between differentiating and undifferentiated cells within a clone occurs and that such an event leads to the reprogramming of the cell and its progeny allowing for multilineage differentiation. However, as MLNSCs gave rise to secondary MLNSC clones and lineage-restricted NSCs, and lineage-restricted founders never gave rise to secondary MLNSC clones (Table 3), it seems unrealistic to suggest that this event occurs with regularity in one population compared with another.
The expansion regime initially used both bFGF and 5-azacytidine. In our
hands, bFGF is acting to support the expansion of the neural differentiation
restricted stem cell pool. One could argue that 5-azacytidine, a histone
deacylase, is the artificial equivalent of reprogramming the nucleus. Whereas
this may potentially enhance myogenic potential
(Wakitani et al., 1995), it is
not solely responsible, as we were able to perform the majority of the
experiments in the absence of 5-azacytidine
(Table 1 and subsequent
experiments).
Linearity and maturation of NSCs
The myogenic differentiation of NSCs in MB-media does not involve a
transition into NCSCs. This does not rule out the possibility that BMP induces
such a transition. Our population, and the population used by Tsai and McKay
(Tsai and McKay, 2000), were
not prospectively isolated to homogeneity. It is plausible that there are
multiple subsets of NSCs and those capable of such a transition by BMP are not
enriched by insulin treatment in vitro.
Our serial subcloning experiments suggest an intriguing model of how the differentiation potential of MLNSC may be restricted in the central nervous system, as secondary and tertiary stem cells undergo a restriction in their differentiation potential during expansion (Table 3). We cannot yet determine which clones will be multilineage, but we can infer that there are developmental and restrictive forces taking place through cell-cell contacts or by secreted factors in the medium that slowly diminish myogenic differentiation potential. Multilineage founder clones gave rise to secondary subclones with both multilineage and neural lineage-restricted characteristics. Likewise, secondary multilineage clones give rise to both tertiary multilineage and neural lineage-restricted clones suggests that a neural restricted stem cell may arise from a more plastic MLNSC population during development. This hypothesis is supported by the observation that insulin receptor immunoreactivity decreases in NSCs during expansion. This drop in receptor immunoreactivity correlates with a decrease in the percentage of myogenic clones observed. It is also possible that MLNSCs arise only by the removal of `myogenic inhibitory forces' or removal of other dominant instructive differentiation signals encountered in vivo. The actions of insulin to instruct myogenic differentiation were subordinate to a variety of factors at physiological relevant concentrations (Table 4). Analyzing MLNSCs in vitro may be the only way to determine the widest potential of these stem cells and the mechanisms regulating multilineage differentiation in contrast to their competence in vivo.
Insulin mechanism of action
We demonstrate that insulin serves both as a potential cell survival signal
and an instructive myogenic differentiation signal for MLNSCs. It is possible
that during expansion and differentiation in this environment, cells may
encounter other instructive cues within the developing clone leading them to a
myogenic fate, and that insulin acts as a permissive rather than an
instructive agent. Nevertheless, whether permissive or instructive, insulin is
required for myogenic differentiation not only in the expansion phase to keep
MLNSCs viable but also during the differentiation phase.
Insulin uses an activated form of Akt to mediate cell survival, a mechanism
used in other cell systems (Datta et al.,
1996; Datta et al.,
1999
). It is interesting to speculate as to the role that Akt may
play in cardiovascular development and repair. Bone marrow-derived stem cells
have been used with success in treating ischemic damage in the heart via an
Akt survival mechanism that allows engraftment and differentiation to occur
(Koc and Gerson, 2003
). In
this model, MLNSC selective expansion via insulin is correlated with activated
Akt. Our demonstration that MLNSCs can engraft in damaged heart tissue is
suggestive that perhaps both bone marrow-derived stem cells and MLNSCs use a
similar survival/differentiation mechanism to produce functional and
engraftable cardiomyocytes.
Interestingly, although insulin mainly affects the type of muscle cells
formed in culture, higher levels of insulin receptors on the surface of NSCs
enhance their total myogenic capacity. However, only a small concentration of
insulin passes the blood-brain barrier into the cortex, and insulin uptake
into the brain does not correlate with the localization of its receptor or the
site of action (Banks and Kastin,
1998; Schulingkamp et al.,
2000
). The concentrations of insulin that support the survival and
differentiation of MLNSCs in this study exceed any in vivo concentrations in
the brain. As a result, the acquisition of myogenic fate in the brain by MLNSC
existing in vivo becomes even less probable. If the brain is a privileged
environment restricting myogenic differentiation potential, do MLNSCs have any
role and can they be said to exist in vivo? Our results support previous
studies suggesting that NSCs possess intrinsic potentials evident only upon
exposure to different microenvironments. Some NSCs clearly have the potential
to form myogenic derivatives, and we demonstrate that resulting myocytes may
well be functional. These results challenge our concepts of neural lineage
commitment and NSC differentiation, highlighting the role of microenvironments
in limiting the competence of stem cells relative to their potential. Finally,
if the reports from Gabay and colleagues
(Gabay et al., 2003
) prove to
be true and tripotent NSCs do not exist in vivo, are all data from neurosphere
assays insignificant? The neurosphere model has been a cornerstone in
understanding the instructive molecular and cellular mechanism of neural
development. Likewise, we contend that MLNSCs may be an important model system
with which to study the cellular and molecular basis of germ line and lineage
restriction and the mechanisms by which stem cell progression and maturation
may occur.
Transplantation advantages
In transplantation biology, a reliable source of cardiomyocyte precursors
is of paramount concern. Cardiomyocytes have been produced from ES cells and
bone marrow-derived stem cells. This is the first description of
cardiomyocytes coming from NSC populations. We used contraction ability,
metabolic coupling and neurotransmitter responsiveness as criteria to
determine whether NSC-derived cardiac myocytes are functional. These
characteristics of the NSC-derived cardiac myocyte suggest that they may have
therapeutic potential. Upon injection into damaged heart tissue, 16% of the
NSCs grown in MB-media were able to engraft and complete their differentiation
program based on the induction of cardiac-MHC expression. Future studies will
require demonstrating electrophysiological integration into the host tissue of
these NSC-derived cardiomyocytes to definitively prove engraftment and
functionality. In any event, our studies support the growing consensus that
tissue-derived stem cells are more plastic than originally believed, and these
studies further demonstrate that the non-neural progeny of NSCs can generate
biologically functional myogenic derivatives.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Neurogenesis and Brain Repair, Institute for Biological
Sciences, National Research Council of Canada, 1500 Montreal Road, Building
M-54, Ottawa, ON, K1A 0R6, Canada
Present address: USB Corporation, 2611 Miles Road, Cleveland, OH 44128,
USA
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