Advances in Bioscience and Biotechnology, 2013, 4, 1-8 ABB
http://dx.doi.org/10.4236/abb.2013.410A1001 Published Online October 2013 (http://www.scirp.org/journal/abb/)
Metabolic state alteration of neural stem cells controls
FAS-mediated apoptosis and neurogenesis
Zamawang F. Almemar1,2*, Nicole H. Urban1, Leal K. Lauderbaugh1
1Department of Mechanical Engineering, University of Colorado at Colorado Springs, Colorado Springs, USA
2Department of the Bioenergetics Institute, University of Colorado at Colorado Springs, Colorado Springs, USA
Received 27 June 2013; revised 27 July 2013; accepted 15 August 2013
Copyright © 2013 Zamawang F. Almemar et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Metabolic stress causes an increased Fas-FasL (recep-
tor-ligand pair) expression in Neural Stem Cells (NSCs)
leading to Fas-induced apoptosis. In this study, we
discuss the exposure of NSCs to different metabolic
treatments that provoke cellular stress responses. We
demonstrate that challenging cultured NSCs to etha-
nol (ETOH) increased cellular death via Fas-medi-
ated apoptosis. Moreover, we establish that NSCs cul-
tured under low lipid conditions, in which they were
deprived of essential fatty acids, demonstrated in-
creased cellular survival rates suggesting an increas-
ed ability for these lipid-starved cells to endure a
stressed environment. This was further confirmed by
exposing NSCs to low glucose levels and observing a
decrease in percent death in low lipid NSCs. When
stressed, NSCs have increased reactive oxygen levels
and are susceptible to apoptosis. These findings indi-
cate that under starved and stressed conditions, and
in the presence of Fas Ligand (FasL), NSCs pursue
fatty acid oxidation by burning fat as fuel. This may
be the key to better understand the metabolic states
of brain tumors and the characteristics of cancer.
Keywords: Neural Stem Cells; Apoptosis;
Neurogenesis; M e tabolic St r ess
1. INTRODUCTION
The growing research interest in Neural Stem Cells
(NSCs) is driven by the possible application of these
multipotent cells as a therapeu tic tool in neurodegene-ra-
tive disorders. NSCs exist in the developing central ner-
vous system (CNS) and exhibit various metabolic states
[1-3]. To create a fat deficiency in NSCs, a characteristic
of viable tumor cells, C17s (derived from developing
mouse cerebellum) were deprived of all essential lipids,
producing a low lipid (LL) cell line [4]. In this study, we
evaluate the response of Normal and LL phenotype C17
cells to observe changes in their metabolic states and
their reactions to stress. During neurogenesis, exposure
to ethanol and low leve ls of glucose lead to limited NSC
production [5-8]. They readily undergo apoptosis when
environmental conditions are not ideal [9], such as al-
tered metabolic states, hypoglycemic conditions, and etha-
nol-induced lipid-deprived environments [10-14]. Though
a considerable amount of research has focused on the ge-
neration and development of neurons, the study of NSC
health, growth and apoptosis in metabolic states which
mimic disease states, is limited [15-18].
The literature ind icates that metabolic in terference leads
to cellular stress conditions in NSCs resulting in various
disease states, including cancer [19]. We studied the Fas-
FasL expression in Normal and LL cells and observed that
the stimulation of the Fas receptor displays both apop-
totic and proliferative characteristics, affected by meta-
bolic state or cellular stress, as in lipid/glucose deprived
NSCs [20,21]. Furthermore, the Fas-FasL signaling has
been shown to be crucial for the control of tumor sup-
pression in NSCs [22]. NSCs express the Fas receptor
(CD95) on their cell surface and together with its ligand
(CD95L), Fas-FasL typically induces apoptosis [23,24],
but here we show that it can also induce growth1. In this
study, we establish that NSCs express varying responses
and tend toward either cell death or cell proliferation
when treated with different glucose levels, ETOH, or their
combinations.
2. MATERIALS AND METHODS
2.1. Cell Culture
The C17.2 cell line adopted from the cereb ellum of new-
1In this work, we assume that the predominant death mechanism is
apoptosis, and this was confirmed in our Forward Scatter and Side
Scatter data.
*Corresponding a uthor.

Z. F. Almema r et al. / Advances in Bioscience and Biotechnology 4 (2013) 1-8 2
born mice is capable of differentiating into neurons and
is very sensitive to factors in their surrounding environ-
ments. The adherent C17.2 cell line was supplied by Dr.
Evan Y. Snyder from Harvard Medical School, Boston,
MA. C17.2 NSCs were cultured in two ways. Cells term-
ed “Normal” were grown in Dulbecco’s modified Eagle’s
medium (DMEM, GIBCO 11995) and supplemented
with HEPES (GIBCO 1563-080), Pen/Strep, L-Gluta-
mi ne, 10% Fetal Bovine Serum (FBS, GIBCOBRL 10100-
139), and 5% Normal Horse Serum (NHS, GIBCO 26050-
088). Cells termed “LL” (for low lipid) were cultured in
low lipid media, also using DMEM, and supplemented
with HEPES buffer, Pen/Strep, L-Glutamine, and 15%
delipidized Bovine Calf Serum (BCS, Pel-Freeze Biolo-
gicals 37117-5), based on the established procedure [25].
2.2. C17.2—Normal and Low Lipid Cell
Preparation
Cells were cultured in normal/unaltered FBS media for
five-weeks. Once 2 × 106 to 5 × 106 cells per mL of me-
dium were grown, the Normal cells were lysed and di-
vided, with one half being cultured in normal media and
the other half cultured in low lipid media. While the Nor-
mal cells were diluted to a factor of 1:200, the low lipid
cells were diluted to 1:50, in their respective media. Due
to differing growth rates, the dilution factors were selec-
ted to achieve a similar cell density (confluency) after the
prescribed five weeks. Passages 13 - 18 were used for
these studies.
2.3. Cell Plating
Normal cells were isolated and resuspended in their ap-
propriate media (Normal or LL). 1 × 106 cells/mL of each
suspension was used for manual cell counting. The re-
maining cell suspension of each type was resuspended to
36 × 106 cells per mL of respective media. In the mean-
time, four 6-well plates were prepared and labeled ac-
cordingly, 2 for Normal and 2 for LL. Next, 6 × 106 cells
per mL of the cell suspensions are added to the proper
6-well plates. In addition, 5.0 µg/mL of FasFc (R&D
Systems Inc. 435-FA) is added to each well. Finally, all
four plates were incubated for 24 hours.
2.4. Cell Line Treatments
Normal and LL cells were treated with FasFc and in-
cubated for 24 hours. The remaining wells were treated
with 300 mM of 95% ETOH and 30 mM Glc. Once
again, the 6-well plates were incubated for 24 hours. For
viability tests, two of the treated plates, one Normal and
one LL, were used. Of the cell suspension, 100 µL was
injected into the flow cytometer for live/dead analysis;
for manual cell counts, 10 µL of the remaining cell sus-
pensions wa s used for manual cel l counting.
2.5. Fas/Fas Ligand Staining
Before treatment and staining with antibodies, cells were
fixed using 1% paraformaldehyde (Sigma P-6148). Nor-
mal and LL NSCs were suspended with Accutase (Sigma
A6964), washed three times with PBS and resuspended
in PBS. Fixative was added to each Normal and LL tube.
Both sets of tubes were incubated on ice for 20 minutes.
For antibody treatment, Fas (PE conjugated hamster anti-
Mouse CD95), IsoFas (PE anti-Armenian and Syrian ham-
ster IgG cocktail), Fas Ligand (PE labeled anti-Mouse
CD178), IsoFas Ligand (PE Rat IgG2bk isotype control)
and Fc block (Purified Rat anti-Mouse CD16/ CD32)
from BD BioSciences Pharmingen were prepared. The
Normal and LL cells treated with fixative were washed
three times and resuspended in PBS. To these cell sus-
pensions, 0.5 mg/mL of Fc block was added to Normal
and LL, and incubated on ice for 10 minutes. For anti-
body addition, 49.7 × 106 cells per mL of Normal and
11.22 × 106 cells per mL of LL were added to appropriate
wells. This was followed by the addition of 0.02 µg/mL
of the appropriate antibodies. For flow cytometry analy-
ses, plates were incubated on ice for 20 minutes followed
by resuspension and data collection. Five thousand events
of each cell population were used in the flow cytometry
analysis.
3. RESULTS
To test low-lipid and Normal phenotype expressions, the
acidity of the lysosomal contents were examined, since
lysosomes play a significant role in fatty acid storage,
hence lipid-level indicator [25]. Normal cells have an
adequate number of lysosomes, which are acidic orga-
nelles [26]. However, LL cells usually have a reduced
number of lysosomes and are less acidic due to the re-
duced availability of fatty acid. Lysosensor (Molecular
Probes-LysoSenso Green DND 189) indicates th e pH le-
vels of lysosomes. Likewise, Fas (Fas anti-Mouse PE) is
a cell surface antibody that fluoresces when bound to
surface Fas is present. The LL phenotype upregulates Fas
expression compared to the Normal phenotype.
To determine the presence of the low lipid phenotype,
Normal NSCs cultured in complete media as well as LL
NSCs cultured in lipid-deficient media were first deve-
loped in culture flasks and tested for viability. After log-
phase cell growth, microscopic photographs of each sub-
cell line were taken (Figures 1(A) and (B)) to provide a
visual aid of the phenotypic variation between Normal
and LL NSCs. As apparent from the photographs, Nor-
mal NSCs showed increased cell growth and their cell
configuration elongated.
Fas anti-Mouse PE was used for determining the pre-
sence of the Fas receptor, and Lysosensor Fluorescent
probe was used to measure lysosome pH. NSCs were

Z. F. Almema r et al. / Advances in Bioscience and Biotechnology 4 (2013) 1-8 3
Figure 1. Normal and LL Neural Stem Cells (NSCs) display
different phenotypes. (A) Microscopic photograph of Normal
C17 NSCs show elongated shape and higher cell growth con-
fluency. (B) Microscopic photograph of LL C17 NSCs illus-
trates a more spherical cell configuration, as well as reduced
number of cells.
transferred to microplates and stained with correspond-
ing antibodies. The Flow Cytometry data collection in-
volved the analysis of 5000 events from each Normal
and LL NSC suspension. The Flow Cytometry data (Fig-
ures 2(a) and (b)) displays an amplified level of Lyso-
sensor (low pH) in Normal NSCs, indicating healthy ly-
sosomes. While the Flow Cytometry data indicates a hi-
gher expression of Fas on the low lipid NSC sur face. In-
creased cell death was also observed in LL NSCs due to
their lipid-deprived metabolic state.
The geometric mean value indicates the central ten-
dency of the data [as well as identifies the intensity of
fluorescence] and is used to analyze the logarithmic-
scaled data in Flow Cytometry [27]. LL NSCs exhibit
greater Fas expression, indicated by the higher geometric
mean value (Figure 2(a)). Although Normal NSCs dis-
play a higher peak, their fluorescence intensity is less (x-
axis log scale) than the LL NSCs. As expected, the fluo-
rescence data for the Lysosensor shows a higher geo-
metric mean value and hence Lysosensor expression (low
pH) for Normal NSCs (Figure 2(b)). Since our fluore-
scence data displays higher fluorescence for Fas in LL
than in Normal NSCs and higher Lysosensor fluorescence
in Normal than in LL NSCs, we conclude that we have
achieved Normal and LL phenotypes.
3.1. Treatment of Normal and Low Lipid C17s
Result in Apoptosis as Well as Neurogenesis
Metabolic stresses, such as Ethanol, Glucose, and com-
bination of these stressors contribute to altered response
to Fas-mediated signaling. To determine the effects of
glucose and ethanol induced stress as well as the role of
metabolic state, Normal and LL NSCs were treated with
300 mM ETOH, 30 mM Glc, and combinations of these
stressors. Additionally, FasFc was used as an experimen-
tal tool to block the Fas signaling pathway. NSCs were
treated with FasFc to obstruct the Fas-receptor signaling
an experimental treatment in our experimental design in
combination with glucose and ETOH treatments. Dis-
and inhibit apoptosis [28]. FasFc was primarily used as
rupting the Fas-FasL pathway by way of FasFc protects
NSCs from growth arrest or death.
The results are shown by the Flow data in Fig ure 3(a).
The average data, obtained from four experimental re-
peats, indicates percent death to be 9.5% for Normal
NSCs. Baseline Fas-mediated death observed in “No
Add” treatments is 8.67% in Normal NSCs (solid bars)
and the response to treatments are evaluated by compar-
ing to the “No Add.” Compared to “No Add” treatment,
percent death increases upon the addition of ETOH to
Normal NSCs, and with Glc added as an additional stres-
sor to ETOH-treated NSCs, increased apoptosis is obser-
ved. At the same time, adding FasFc to ETOH-treated
Normal NSCs gives an increase in cell death suggesting
that blocking the Fas pathway does not protect NSCs
from ETOH’s effects. Moreover, the addition of Glc does
not facilitate recovery for ETOH-affected NSCs, while
similar percent deaths are observed when adding Glc to
FasFc-treated Normal NSCs. Finally, adding Glc alone
also acts as a stressor, displaying percent death results si-
milar to the FasFc + Glc treatment.
To evaluate Fas-mediated death in LL NSCs, the same
treatments were applied to cells of the LL phenotype (da-
shed bars). The percent death averages at 23.3% for LL
NSCs for all treatments (compared to 9.5% for Normal
NSCs). The data for LL NSCs indicates an overall in-
crease in susceptibility to apoptosis by displaying higher
percentages of death. These results show that baseline
Fas-mediated death observed in LL NSCs for the “No
Add” treatments is 21.17%. Detailed examination of LL
NSC data demonstrates that adding ETOH increases the
percent death from the baseline value. An increase in cell
death was also revealed following treatment with ETOH
and Glc combined. ETOH and Glc-treated LL NSCs ex-
perienced increased losses in number. Thus higher death
rates suggest that once LL NSCs apoptose by ETOH,
adding Glc acts as a death factor causing further apop-
tosis. However, the addition of FasFc alone results in a
decrease in percent death compared to the baseline value.
For further investigation of Fas-induced apoptosis in LL
NSCs, FasFc was added to block the apoptotic pathway.
FasFc addition to ETOH-treated LL NSCs increased per-
cent death, once again supporting the fatal effects of NSC
exposure to ETOH. In addition, adding FasFc to Glc-
treated LL NSCs also increased percent death, and the
percent death result was similar to the Glc-only treat-
ment.
By and large, the percent death increases with treat-
ment compared with the data from the baseline “No dd”
treatments and the absence of lipid increases the overall
percent deaths. Furthermore, percent deaths in Normal
treated NSCs show lower percentage of death, while LL
NSCs exhibit higher death rates compared to their
Normal and more healthy predecessors. In conclusion,

Z. F. Almema r et al. / Advances in Bioscience and Biotechnology 4 (2013) 1-8 4
Figure 2. Flow Cytometry Data Illustrates Fas and Lysosensor Expressions by Normal
and LL NSCs. (a) Flow cytometry and geometric mean analysis of Fas-treated Normal
and LL NSCs indicates higher Fas expression in LL NSCs supporting the conclusion that
LL NSCs show increased Fas-mediated death. (b) Flow cytometry and geometric mean
analysis of Lysosensor-treated Normal and LL NSCs illustrates the conclusion that lack of
essential lipids in LL NSCs and hence reduced fatty acid metabolism in the lysosome
leads to decreased Lysosensor expression.
treatment in Normal NSCs (Figures 3(a) and (b)). The
Cell Count data confirms that the addition of ETOH
causes the highest amount of cell death in Normal NSCs
and that ETOH induced death is observed regardless of
whether ETOH was added independently or in combi-
nation with FasFc or Glc (Figures 3(b)). The following
results again confirm our additional conclusion that Fas-
Fc, used to block the Fas pathway, protects NSCs from
death when added to untreated Normal NSCs.
LL NSCs deprived of all lipids and essential fatty acids
die Fas-mediated death due to the inadequate A culturing
environment as well as ETOH and Glc treatments.
3.2. Cell Count Data of Fas-Induced Death of
NSCs Support Flow Cytometry Data
To valid ate the Flow Cytometry data, manual cell counts
of each treated and untreated Normal and LL NSCs were
obtained [29]. The Cell Count data conf irms the same ge-
neral trends achieved by the Flow Cytometer (collected
via Coulter Elite Epics or Excel Flow Cytometer and
analyzed via FlowJo version 7.0). Evaluating forward
scatter and side scatter of 5000 events of each sample po-
pulation provides a baseline interpretation of the data.
Fas-mediated cell death is observed from the ETOH
Further inspection of the preceding data demonstrates
that the same trends are observed in LL from the Cell
Count data as compared to the Flow data. As seen from
both the Cell Count and Flow data, FasFc addition to
ETOH-treated LL increases the percent death, again sup-
porting the conclusion that ETOH-induced death is not
Fas-induced.

Z. F. Almema r et al. / Advances in Bioscience and Biotechnology 4 (2013) 1-8 5
Figure 3. Cell Count Data Follows Similar Trends from Flow
Cytometry Data for All Treatments. (a) Average percent deaths
from Flow Cytometry data illustrate highest death rates in the
presence of ETOH. (b) Average percent death from manual cell
count for same treatments as shown in (a). Error bars represent
standard error of the mean (SEM) based on four experimental
trials.
We further examined cellular proliferation using cell
count results and next we present the data supporting th e
survival rates of Normal and LL NSCs. It is noteworthy
that average percent deaths resulting from analysis of
Flow Cytometry and cellularity data provide comparable
trends.
3.3. Cellularity Data Validate Death Trends
To further analyze Fas-mediated apoptosis and Fas-me-
diated proliferation, cell survival rates (cellularity) of
Normal and LL NSCs were examined (Figures 4). The
data show that compared to the “No Add” baseline data,
decreased survival rates were observed in treatments
with ETOH and ETOH and Glc combination in Normal
NSCs. However, adding Glc to ETOH-treated LL NSCs
reduces ETOH-induced death. Adding ETOH decreases
cellularity in both cell lines. Blocking the Fas pathway
with the addition of FasFc also decreases cellularity in
both cell lines. An interesting observation is made when
the apoptotic pathway is blocked and NSCs are induced
by ETOH. In Normal NSCs, ETOH reduces cell survival
rates despite the addition of FasFc, however in LL NSCs
upon the addition of ETOH in combination with FasFc,
higher survival rates are observed, compared to ETOH-
only and FasFc-on ly treatments. The same trend is true in
Normal and LL NSCs when Glc is added and the Fas
pathway is blocked. Cellularity decreases in Normal NSCs
whereas somewhat increases in LL NSCs, compared to
the FasFc-only treatment. Furthermore, with the apopto-
tic pathway blocked, Glc-treated Normal NSCs display
higher survival rates than ETOH-treated NSCs, while LL
NSCs demonstrate decreased cellularity than the ir ETOH-
treated counterparts. Cellularity is further decreased in LL
NSCs in the Glc-only treatment; however, expected re-
sults are obtained in Normal NSCs with increased cellu-
larity upon the addition of Glc.
3.4. Low Lipid NSCs Survive Low Glucose
Levels
NSCs were treated with low Glc levels. Glucose depri-
vation decreases the number of Normal NSCs, while in-
creasing the percent death (Figure 5). As shown by the
cellularity data, LL NSCs also decrease in number, but
interestingly their percent death decreases as well. Whe-
ther changes of death rates or cellularity are analyzed,
they both follow the same statistical trends. This suggests
the utilization of a un ique survival mechanism by the LL
phenotype when sources of lipid and glucose are scarce,
which could be speculated as fatty acid oxidation.
4. DISCUSSION
4.1. Metabolic Stress States and Survival
Mechanisms of LL NSCs
To investigate the effect of different treatments applied to
both Normal and LL NSCs, the death rate variations be-
tween the two sub-cell lines are studied. Average percent
death increases by about 14% when examining LL com-
pared to Normal NSCs for all treatments. We conclude
that LL NSCs are more sensitive to apoptotic effects of
the Fas-FasL death receptor-ligand pair, therefore more
Figure 4. Cellularity Analysis of Normal and LL NSCs
Indicate Dissimilar Outcomes upon Glc Addition. Average total
cellularity data of Normal and LL NSCs generally follow the
same trends upon treatments, except in FasFc + ETOH and Glc
treatments.

Z. F. Almema r et al. / Advances in Bioscience and Biotechnology 4 (2013) 1-8 6
Figure 5. Cellularity and Percent Death Analysis of Normal
and LL NSCs Suggest Survival Mechanism used by LL NSCs.
Normal NSCs demonstrate decreased cellularity and increased
percent death under conditions of low glucose levels. While,
LL NSCs show decreased cellularity and decreased percent death,
illustrating this phenotype’s capability of survival under severe
conditions.
death is observed in LL NSCs as a result of Fas induction.
Interestingly, one of the indicators of the LL phenotype is
an upregulation of the Fas receptor. This is exclusively
caused by depriving the cell line from lipids. LL NSCs
recover only by a small amount when treated with Glc
suggesting that Glc causes Fas activation by binding to
the Fas receptors and altering the NSC’s fate from Fas-
induced apoptosis to Fas-mediated proliferation. In LL
NSCs, Glc upregulates the Fas-FasL binding pathway and
allows more receptors to bind.
We have demonstrated that treatment of Normal NSCs
with ETOH results in increased death rates. However, LL
NSCs survive better in these stressed environments. In
fact, neurogenesis is observed in these LL NSCs upon
the addition of the ETOH + Glc and FasFc + ETOH
treatments. It is reasonable to conclude that under meta-
bolic stress conditions, su ch as under conditions of limit-
ing glucose supplies, these NSCs undergo fatty acid oxi-
dation as a source of carbon [4]. Furthermore, FasL is
provided only in presence of lipid during fatty acid oxi-
dation by burning fat as fuel, and fatty acid oxidation by
LL NSCs leads to FasL dependency [19]. This finding
establishes a link between the cellu lar metabolic strategy
used by LL NSCs and demonstrates the potential to fur-
ther study these LL NSCs to understand other diseases
that involve Glc.
4.2. Metabolic Stresses, Apoptosis and
Neurogenesis in NSCs
The surface receptor Fas (CD95) has long been identified
as the primary pathway for apoptosis (programmed cell
death). However, several researchers have documented
cell proliferation when Fas is triggered. Fas-activated
NSCs can undergo either apoptosis or cell proliferation
[23]. Our data indicate that this bifurcation is affected by
their phenotypic state and type of treatment adminis-
tered. Normal and LL C17s undergo Fas-mediated apop-
tosis, indicated by the cellularity data. These results in-
troduce a novel insight into Fas-FasL-induced apoptosis
in C17 NSCs. In this research, the effect of metabolic
stresses, which mimic known disease states was investi-
gated in NSCs. The highest death rates were detected
when ETOH was administered as one of the treatments
to the developing Normal and LL NSCs. FASD results in
neuronal loss due to ethanol-induced damage [7]. Our re-
sults coincide with these findings and show increased
apoptotic activity in Normal and LL NSCs exposed to
ETOH.
We have identified that FasFc protects NSCs from
growth arrest (death) and that administering FasFc to
NSCs exposed to ETOH protects the NSCs by blocking
the Fas-FasL receptors, decreasing Fas-dependent death.
At the same time, increased percent deaths are observed
with ETOH-treated NSCs. We conclude that ETOH-in-
duced death is Fas-independent since death is observed
in both Normal and LL NSCs regardless of whether or
not the apoptotic pathway is blocked. Likewise, we con-
clude that Glc-dependent growth is also Fas-independent
since increased numbers of cells are observed in LL
NSCs while it inhibits growth in Normal NSCs when the
apoptotic pathway is blocked. This variation between
Glc-treated Normal and LL NSCs indicates that LL NSCs
use this Glc as fuel to drive Fas-mediated cell prolifera-
tion under lipid-deprived conditions by means of fatty
acid oxidation. However, adding Glc to LL NSCs when
the apoptotic pathway is not blocked acts as an added
stressor, decreasing cellularity. Collectively, these obser-
vations demonstrate that the Fas-mediated pathway can
lead to apoptosis and proliferation, which is affected by
the metabolic state of NSCs (i.e. normal conditions or
stressed and lipid-deprived environments).
4.3. Fas and Metabolic Stressors
We have demonstrated that NSCs treated with ETOH
lead to death and that death is not Fas-mediated. We have
further identified that Glc-dependent growth in NSCs is
also not Fas-mediated. However, under metabolic stress
conditions and in NSCs that are lipid-dep rived, treatment
with Glc leads to Fas activation, altering the apoptotic
pathway to lead to cellu lar proliferation instead of death.
Our studies of ETOH, Glc, and FasFc treatments of NSCs
advocate our conclusions that ETOH-induced death is
Fas-FasL independent and that Glc-dependent growth is
also Fas-FasL independent. Unraveling the factors affect-
ing Fas activation in LL NSCs could enable the use of
Fas to further our understanding of cancer.

Z. F. Almema r et al. / Advances in Bioscience and Biotechnology 4 (2013) 1-8 7
5. ACKNOWLEDGEMENTS
The authors would like to acknowledge the technical support and gui-
dance of Dr. M. K. Newell-Rogers, and for services provided by the
University of Colorado at Colorado Springs Bioenergetics Laboratory
facility. The authors have no conflicts of interest to declare.
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