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Schwann Cell Differentiation from Stem Cells of
Neurofibromatosis 1 Patients and Normal Controls
Amrita Mohanty
Table of Contents
Abstract …………………………………………………………………………….……
2
Introduction …………………………………………………………………..…….…….. 3
Materials and Methods ……………………………………………..……...……...….…. 7
Results and Discussion ….……………………………………………………….……
11
Future Work ……………………………………………………….………..…….…..
16
Conclusions …………………………………………………………………………….
17
Bibliography ……………………………………………………………………………… 18
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Abstract
One in every 3,500 newborns is diagnosed with Neurofibromatosis 1 (NF1), a common
genetic disorder. Many NF1 patients develop painful tumors along their peripheral nervous
system. The NF1 disease usually consists of cancerous Schwann cells called Schwannomas. This
research is focused on the development of Schwann cells in NF1 patients. The goal of this
project was to determine if NF1 Schwann cells undergo a different process of differentiation
compared to non-NF1 Schwann cells. Induced-pluripotent stem (iPS) cells of non-NF1 and NF1
patients were successfully differentiated into Schwann cells and samples were collected on day 0
(iPS), day 19 (neural precursor), and day 30 (Schwann). These samples were analyzed using
quantitative polymerase chain reaction to detect gene expression and to determine the rate of
differentiation.
The display of correct gene expression levels in this experiment demonstrated that
Schwann cells can be successfully differentiated from iPS cells. The findings of this study
disproved the experimental hypothesis and found that NF1 iPS cells differentiate into Schwann
cells at an accelerated rate compared to non-NF1 iPS cells. In addition, these findings indicate
that the genetic mechanism that brings forth the accelerated differentiation of Schwann cells
occurs during the transition from the iPS cell stage to the neural precursor stage. By studying
differences in development and gene expression between NF1 and non-NF1 Schwann cells, a
model that depicts how Schwann cells form was created. This research offers insight into the
NF1 disease by modeling Schwann cell development and gene activity of NF1 and non-NF1
patients.
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Introduction
This past year, nine-year-old Zac Ann of Minnesota lost his fight with Neurofibromatosis
1 (NF1). Zac was diagnosed with NF1 at the age of one when doctors discovered benign tumors
in his brain. Zach was a vocal advocate for NF1 research and the Zachary NF1 Fund lives on in
his honor. It is patients like Zach who inspire research in NF1. There are thousands of children
just like Zach who struggle with NF1. In fact, NF1, a common genetic disorder, affects one in
every 3,500 newborns and is the most commonly inherited tumor predisposition syndrome in the
world1. About 13% of patients with NF1 have their disease progress into malignant peripheral
nerve sheath tumors (MPNST), which is a fatal form of NF1.
A person is diagnosed with NF1 if they have a mutated form of the neurofibromin gene
which is a known cancer suppressor gene. When mutated, neurofibromin is either completely
silenced or faulty, and causes unregulated cell proliferation. Patients can inherit NF1 from a
parent or develop it through spontaneous mutation2. Patients with NF1 usually develop café au
lait spots and both malignant and nonmalignant tumors called neurofibromas. According to
Johns Hopkins Medical School3 there are various types of neurofibromas. For example, dermal
neurofibromas consist of small nodules that grow on or just below the skin’s surface. One
specified form of dermal neurofibromas is Schwannomas which occur when neural cells called
Schwann cells form benign tumors. Schwann cells are a part of the myelin sheath in neurons
which are present throughout the entire body, including the peripheral and central nervous
1
Sedani, Cooper, and Upadhyaya 2012
Zhang, Wernig, Duncan, Brustle, Thomson 2001
3
Johns Hopkins Comprehensive Neurofibromatosis Center, 2012
2
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system. When Schwann cells become Schwannomas in NF1, large floret-shaped benign tumors
form beneath the epidermis throughout the entire body. There is no cure for NF1 and the only
forms of treatment are radiation, chemotherapy, or surgery to remove tumors.
Many researchers suspect that human pluripotent stem cell derived neural crest cells
present a valuable tool to model human Schwann cell development, cell fate specification,
pluripotency, and cell migration. By monitoring Schwann cell development, a model of the NF1
disease can be created4. First, however, a strong understanding of how a normal Schwann cell
differentiates must be established. To begin, the term differentiation refers to the process of an
unspecialized stem cell becoming a specialized cell with an assigned job, like a liver or blood
cell. A neural crest is derived from stem cells in the ectoderm layer of an embryo. The fate of
cell differentiation then depends on where it migrates. For instance, a cranial neural crest cell can
become cartilage while a trunk neural crest cell can become a melanocyte. The surrounding
extracellular matrices of the neural tube control the path taken by migrating neural crest cells5.
Jessen and Mirsky differentiated neural crest precursors into Schwann cells of a mouse. They
noted two main developmental transitions: neural precursors to immature Schwann cells and
immature Schwann cells to fully formed mature Schwann cells. The researchers concluded their
findings by stating that the survival of Schwann cell precursors depends on axonal survival
signals mediated by neuregulin6.
Current research in the NF1 disease involves microRNA profiling. miRNAs are noncoding RNAs that regulate gene expression. The level of complementarity that exists between
miRNA and the target miRNA decides how the miRNA will regulate gene expression. For
4
Lee, Chamber, Studer and Tomishima 2010
Gilbert 2000
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Jessen and Mirsky 1997
5
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instance, complete complementarity will silence or degrade the miRNA7. Scientists study
miRNA expression to see when Schwann cells become Schwannomas and what causes this loss
of controls of cell dynamics8. Based on patterns in gene expression, they are able to establish a
correlation between protein activity and Schwannoma development to determine what genetic
mechanisms cause Schwann cells to become cancerous. Using quantitative polymerase chain
reaction (qPCR), Presneau and Eskandarpour found that the neurofibromin 1 gene (a tumor
repressing gene) was less active in Schwannomas than in normal Schwann cells.
Like Presneau and Eskandarpor, many researchers have found qPCR to be a very
valuable tool when studying NF1. In qPCR, a control gene is used that has the same copy
number in all sample cells and does not change significantly in expression. After a number of
cycles, more abundant transcripts from highly transcribed genes will yield more product
(indicated by fluorescent dye) than weakly transcribed genes. Thus, researchers can determine
when mRNA coding for a protein is most highly transcribed and further elucidate the protein’s
function. By assessing the expression level of a gene at various time points, researchers get a
better idea of whether a protein is important to a specific process9. Presneau and Eskandarpour
found that 16 miRNAs were more significantly expressed in Schwannomas compared to the nonNF1, normal Schwann cells. 14 of these 16 miRNAs were down regulated. Many of these
miRNAs affected cell motility, proliferation, and migration.
Researchers Zelkowitz and Stambouly performed NF1 research when they cultivated five
patient cells lines of NF1 and normal dermal fibroblasts (cells that live within the dermis layer of
7
Sedani, Cooper, and Upadhyaya 2012
Presneau and Eskandarpour 2012
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Hunt 2010
8
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the skin).10 Their work was done prior to the existence of stem cell technology and was solely
focused on the proliferation of a particular mature cell type. They hypothesized that NF1 dermal
cell growth would correlate with the malignant qualities associated with the NF1 disease and
display an accelerated rate of proliferation. The researchers found, however, that the NF1 cell
lines displayed an equal rate of growth compared to the non-NF1 controls. Zelkowitz and
Stambouly’s findings influenced the hypothesis of my experiment.
The following research was performed to track and analyze the developmental behavior
of NF1 Schwann cells. In this experiment, two developmental transitions were studied: inducedpluripotent stem cells to Rosettes (neural precursors) and Rosettes to Schwann cells. Four cell
lines were used to see if NF1 Schwann cell development was similar to normal Schwann cell
development.
Cell Line Name
Condition
Normal
Non-NF1
Patient #1
NF1
Patient #2
NF1
Patient #3
NF1
Table 1: Cell lines used in this experiment
In addition, qPCR was used to measure gene expression during these time points. The
purpose of this experiment was to demonstrate normal Schwann cell differentiation and to
compare Schwann cell differentiation between normal and NF1 patients. It was hypothesized that
Schwann cell development can be modeled through iPS differentiation and that Schwann cell
differentiation will display correct gene expression at the distinguished time points (days 0,19,
and 30). In addition, it was also hypothesized that NF1 iPS cells differentiated into Schwann
10
Zelkowitz and Stambouly 1981
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cells at the same rate as non-NF1 iPS cells. These hypotheses were tested based on gene
expression. Gene expression was detected by qPCR assays performed on consecutive days of
development.
Materials and Methods
The expression level of specific genes at each stage of cell development was detected by
the genetic markers Oct4, Ap2α, HNK-1, P75, and S100. These markers were used in qPCR in
order to target specific genes and relay information of their levels of expression.
The following table indicates the uses of these genetic markers.
Marker
What Marker Indicates
OCT4
Induced pluripotent stem (iPS) cells
Ap2α
Rosette cells (neural precursor stage)
HNK-1
Neural crest cells (involved in cell adhesion)
P75
Immature Schwann cells (tumor suppressor)
S100
Mature Schwann cells (involved in cell motility)
Table 2: Markers used in this experiment
Table 3 below indicates the stages at which the markers are expected in the control cell line
OCT4
AP2a
HNK-1
P75
S100
Stem cell (day 0)
Neural precursor
Mature neural precursor/
rosette (day 19)
Immature Schwann cell
Schwann cell (day 30)
Table 3: Markers expected during stages of normal Schwann cell differentiation
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Four iPS patient cell lines developed from patient skin cells were taken and differentiated
into Rosettes. Lastly, these Rosette cells were differentiated into Schwann cells.
Materials used that were unique to this differentiation:
Day 0 to Day 4 Medium
400 ml: DMEM/F12
100 mL Knockout Serum Replacer (KSR)
5 mL MEM Nonessential Amino Acids Solution (NEAA)
5 mL L-glutamine Solution (L-glut)
3.5 µL Beta-mercaptoethanol
Neural Induction Medium
Fill to 500 mL with DMEM/F12
5 mL Nonessital Amino Acids Solution (NEAA)
5 mL N2 Supplement
1 mL of 1 mg/mL Heparin
200 ml: DMEM/F12
2 mL N2 Supplement
2 mL MEM Nonessital Amino Acids Solution (NEAA)
400 µL of 1 mg/mL Heparin
RLT Buffer Plus BME
1 mL RLT Buffer
1 mL Beta-mercaptoethanol
Schwann Cell Differentiation Medium
194.5 mL DMEM/F12
2 mL N2 Supplement
200 µL of 10 µg/mL CNTF (Ciliary Neurotrophic factor)
40 µL of 100 ng/mL NRG1 (Neuregulin 1)
200 µL of 10 µg/mL FGF2 ( Fibroblast Growth Factor 2)
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1 mL of 100 mM cAMP ( Cyclic adenosine monophosphate )
0.310 g Glucose
0.400 g NaHCO3
2. 5 mL (0.0050 g) Insulin
Table 4 below summarizes the 12 samples that were taken.
Normal
NF1 Patient #1
NF1 Patient #2
NF1 Patient #3
iPS cell (Day 0)
iPS cell (Day 0)
iPS cell (Day 0)
iPS cell (Day 0)
Rosette (Day 19)
Rosette (Day 19)
Rosette (Day 19)
Rosette (Day 19)
Schwann (Day 30)
Schwann (Day 30)
Schwann (Day 30)
Schwann (Day 30)
Table 4: Cell samples taken for qPCR
After the cell lines were developed, messenger RNA (mRNA) was isolated from each
sample. Following this procedure, complementary DNA (cDNA) was synthesized from the
mRNA because mRNA is single stranded and will not function in a qPCR machine. In addition,
cDNA was created from mRNA because mRNA has undergone modification and does not
contain introns (non-coding genetic material). By utilizing reverse transcriptase and DNA
polymerase, mRNA was turned into a double stranded cDNA molecule. The genetic markers
(also called primers) Oct4, Ap2α, HNK-1, p75, and s100 were used to monitor their respected
genes through PCR.
The first of eight qPCRs was executed on Day 0 (normal), Rosette (normal), Schwann
(normal), and melanoma cDNA samples using GAPDH, OCT4, HNK-1, and Ap2a as markers.
The second qPCR was run on the same cDNA samples but used GAPDH, P75, and S100 as
markers instead. The six other qPCRs were run with the same primers as the first two but used
the NF1 patient cDNA samples instead. Melanoma cDNA was used as a positive control for the
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markers to make sure that the primers were not faulty. RNase free water was used as a negative
control because H2O should have no gene expression after 40 cycles of qPCR and can indicate
possible contamination. GAPDH is a control marker (it displays a constant rate of gene
expression) and was used on all qPCR plates that were performed.
Figure 1: Project overview
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Results and Discussion
Expression Relative to Day 0
Gene Expression of normal sample
Figure 2: Relative gene expression of all tested genes in normal sample
All of the genes studied in this experiment were compared in the normal, non-NF1
sample. Figure 2 shows that the stem cell marker OCT4 had its highest expression on day 0 and
the neural precursor markers HNK-1 and Ap2a had their highest expression on day 19 (Rosette).
Expression Relative to Day 0
Lastly, the Schwann cell markers P75 and S100 had their highest expression on day 30.
Figure 3: Relative gene expression of OCT4
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In figure 3 all expression levels matched anticipated levels. OCT4 was expressed most
highly on day 0 in all of the patient samples. This is because at the beginning of each
Expression Relative to Day 0
differentiation, all samples began primarily as undifferentiated iPS cells.
Figure 4: Relative gene expression of HNK-1
The HNK-1 gene (figure 4), which is characteristic of neural precursors, had the
predicted, highest expression on day 19 in the normal sample and NF1 samples. All samples had
a high presence of neural precursors on day 19.
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Expression Relative to Day 0
Figure 5: Relative gene expression of Ap2a
In all samples, the AP2a gene (figure 5) presented predicted expression levels. As an
Expression Relative to Day 0
indicator of neural precursors, the gene was expected to have its highest expression on day 19.
Figure 6: Relative gene expression of P75
In figure 6, the normal sample displayed its highest level of P75 expression on day 30. In
the NF1 samples, however, linear growth appeared normal only up until day 19. After day 19 the
gene’s expression level dropped drastically and showed minimal presence on day 30. The high
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appearance of fluoresce of P75 in the patient lines on day 19 demonstrates that there was a high
Expression Relative to Day 0
presence of Schwann cells in those samples on day 19.
Figure 7: Relative gene expression of S100
In figure 7, the normal sample followed the expected route of differentiation by
presenting its highest level of S100 expression on day 30. On the other hand, the NF1 samples
presented their highest S100 expression level on day 19. This demonstrates that the patient
samples had a high presence of Schwann cells on day 19.
The appearance of predicted relative levels of gene expression in the normal sample
(figure 2) demonstrates that correct Schwann cell differentiation occurred. This is important
because it models normal Schwann cell development and indicates that Schwann cells can be
differentiated from iPS cells. Now that correct Schwann cell differentiation was executed, a
comparison between the normal and NF1 patient lines can occur.
The contrasting patterns of gene expression in the Schwann cell markers P75 and S100
between the normal and NF1 samples disprove the previous hypotheses and demonstrate that the
normal sample differentiated differently compared to the NF1 patient samples. Gene expression
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shows that the NF1 samples differentiated more quickly than the normal sample and also
indicates that a trigger occurs during the iPS cell to Rosette cell differentiation transition that
causes cells to differentiate at an accelerated rate.
To begin, figure 3 (OCT4) indicates that the vast majority of cells presented on day zero
were undifferentiated stem cells. This strengthens the assumption that the NF1 samples and
normal sample began guided differentiation in the same starting condition. In addition, the high
expression of the S100 gene (figure 7) in the NF1 samples on day 19 indicates that mature
Schwann cells were present and fully formed by day 19, a time when only neural precursors
should have been present. Also, low expression of the P75 gene (figure 6) in the NF1 samples on
day 30 suggests that immature Schwann cells were no longer present in those samples. From
these data I can conclude that the NF1 patient cell lines differentiated at an accelerated rate
compared to the normal, control cell line. In addition, these findings indicate that accelerated
differentiation began during the iPS cell to Rosette developmental transition in all NF1 patient
samples.
It is beneficial to acknowledge that in this experiment possible errors could have occurred
due to imprecise pipetting technique, the quality of cDNA used, and the quality of the primers.
For example, imprecise pipetting causes a varied ratio of primer to cDNA per well which could
slightly impact results. In addition, a low amount of cDNA in a sample could affect the results
because gene expression would not be measured consistently among all of the samples. An error
could also be traced back to the differentiation of the cells. Lastly, these cell lines could have
varied in their capacity to expand and viability, thus presenting inconsistencies between samples.
This would have also affected the quality of the cell samples that underwent qPCR analysis
because each sample is unique to the patient.
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Future Work
In the future, it would be beneficial to observe the differentiation of NF1 and normal cells
past day 30 of Schwann cell differentiation. Perhaps more information will be gained by looking
at cell developmental and proliferative behavior beyond day 30.
In addition, work will be continued on this project by taking samples more frequently
during the differentiation experiment. For example, in this experiment samples were taken on
days 0, 19, and 30. In the future, samples would be additionally collected on days 3, 6, 9, and 12.
More day samples would provide greater insight on the course of differentiation the cells take as
well as offer an in-depth look at gene expression. For example, when directing the differentiation
of the NF1 patient lines, extra samples were taken. In addition to the samples taken on day 0, day
19, and day 30; extra samples were taken on day 11 and day 15. These samples provided even
more information regarding the differentiation of stem cells to Schwann cells. For instance, the
neural precursor markers HNK-1 and Ap2a had relatively high expression on day 11. These
findings provide even more evidence that the NF1 samples began differentiating more quickly
and give a better idea of when this accelerated differentiation began.
Additional future work includes differentiating more lines of patient iPS cells into
Schwann cells. Increased batches of patient cell samples will contribute greater knowledge of
Schwann cell development by adding more trials and increasing overall sample size. microRNA
analysis and gel staining could be executed on these samples and provide an even greater
understanding of how Schwann cells develop in NF1 patients by directly studying protein
behavior.
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Conclusion
This experiment utilized Schwann cells that were successfully differentiated from iPS
cells. In addition, it modeled correct Schwann cell gene expression and development in a normal
patient. This feat itself tapped a new realm of Schwann cell development. Also, because the NF1
samples consistently had high expression of the P75 gene and S100 gene, genes that indicate
immature and mature Schwann cells, on day 19 (Rosette), there is evidence that the NF1 cell
samples had accelerated differentiation and this accelerated differentiation occurred during the
iPS cell to Rosette cell stage of development. The accelerated differentiation found in the NF1
cell lines could be correlated to the proliferative tumor behavior that is characteristic of
Schwannomas and can serve as a marker for the disease. By studying the developmental
transition where the acceleration occurs, we could possibly find the genetic mechanism that is
responsible for accelerated differentiation. This mechanism could also be linked to loss of
control of cell dynamics, causing the formation of Schwannomas.
Knowledge of the transition from iPS to Rosette at which this acceleration occurs could
help narrow the search to identify the genetic mechanism that causes both accelerated
differentiation and cancerous Schwannomas to form in NF1 patients. By using accelerated
differentiation as a marker of the disease and further studying the iPS to Rosette transition, new
knowledge can be offered as to how gene activity causes neurofibromas form. This study
contributed new findings to our understanding of the NF1 disease and how Schwann cells
develop in NF1 patients. Hopefully this work, joined with others, will help thousands of children
just like Zac in their struggle against NF1.
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