1. 1
Do mouse embryonic stem cells begin to differentiate toward dental
pulp stem cells in culture medium containing added L-proline?
Timothy Doolin1, Tara Formisano2, Nalini Chandar, Lon Van Winkle3*
1Timothy Doolin, Department of Biomedical Science, Midwestern University, Downers Grove, IL,
USA. 2Tara Formisano, Department of Biomedical Science, Midwestern University, Downers
Grove, IL, USA. 3Lon Van Winkle, Department of Biochemistry, Midwestern University, Downers
Grove, IL, USA.
2. 2
ABSTRACT
Introduction
Over the past few decades stem cell research has become mainstream due to its potential abilities to
revolutionize the health care field. Researchers continue to look for ways to better control stem cell
proliferation and achieve differentiation. One way to help regulate stem cell growth and
differentiation is through the use of amino acids. The following research will explain the
importance of L-Proline in the differentiation of mouse embryonic stem (mES) cells toward dental
pulp stem cells (DPSCs). Dental stem cells have been successfully used to grow teeth in mice and
rats. The genes that are upregulated in these cases are the same genes that can be observed to
impact the pluripotency of embryonic stem cells. Previous research has determined that L-Proline
has a significant effect on differentiation of mES cells toward the ectodermal development. While
the origin of dental pulp stem cells is relatively unknown in vitro, the research that has been done
shows that dental pulp stem cells come from the neural crest cells which we know to be ectoderm
cells. Proline is also known to have a significant role in the formation of teeth. Current literature
fails to make a connection between the differentiation of mES cells towards mDPSCs and the
effect of proline. For these reasons, this research will attempt to show, that proline is a major
component of differentiation for mouse embryonic stem cells being pushed toward mouse dental
pulp stem cells in vitro. This will be shown through characterization of the pathway of
differentiation of mES cells under various conditions. The conditions included growth of mES cells
in media supplemented with L-proline and compared to cells grown in media supplemented with
bone morphogenetic protein (BMP2), a substance known to have significant effect on
odontoblastic differentiation. The cells were grown, imaged, and tested side by side, and the
differences were recorded to determine if proline’s effect on differentiation is significant. After
RT-PCR analysis, it can be said that while there is no conclusive evidence to show that our mES
cells have differentiated to DPSCs, there is reason to believe that a high proline concentration of L-
Proline does have a significant effect on mES cells as they differentiate. When testing primers not
specific to mRNA of dental cells, which were expected to show correlation between proline
concentration and gene expression, the cells showed very little gene expression at high
concentrations. Furthermore when testing dental related genes the cells treated with the same high
concentration of proline showed significant up regulation. Specifically we found that mRNAs were
upregulated as the concentration of L-Proline was increased for the genes osterix, CBFA, and
col3a1 which are dental related. Interestingly, the mRNAs for col2a1 and cola5a1, which are not
dental related, followed this trend at first but when 2000μM was reached, expression was down-
regulated. It was concluded that cells treated with a high concentration of L-Proline show a
significant trend in up-regulation of genes specific to DPSCs, and we believe for this reason that in
the presence of a high concentration of L-Proline cells may be pushed to differentiate towards
DPSCs.
3. 3
INTRODUCTION
In recent years there has been increased interest in research with stem cell technology. Whether
from political controversy or the prospect of creating full organs in a lab, the conversation of the
possible outcomes that result from stem cell research have become common conversation in
today’s society. Among the first organs made from stem cells were teeth. Now as the research
progresses the possibility of growing teeth in humans as a replacement for dental implants may
become a reality. It has already been successful in mice and rats [1] however as research expands
different complications have developed along the way. Most importantly, researchers are
concerned with aspects of money, ability to obtain the stem cells needed, and the time table
required for complete tooth regeneration. All of which become increasingly important once this
procedure enters the health care field.
Unfortunately, the process of differentiation towards DPSCs is relatively unknown in vitro. There
are different hypotheses but very little can be found in the literature. The following research will
set out to complete just that. Find evidence to show that mES cells differentiate into mDPSCs
through ectodermal tissue and characterize the pathway and its use of amino acids in vitro.
Some of the current studies with ES cells are investigating the effects of amino acids on cellular
differentiation and proliferation. ES cells are ideal for studies like this because they are highly
pluripotent and have the ability to differentiate into many types of cell which could then be used as
a focus for a study [4]. With the help of amino acids and other factors ES cells potentially can
differentiate into a wide variety of other cells which can be used for the purposes previously
mentioned.
ES cells are not the same as adult stem cells, while both can be used for differentiation into
different types of cells and self-renewal, only ES cells are pluripotent and therefore have the ability
to differentiate into cells of the ectoderm, endoderm, and mesoderm [6]. On the other hand adult
stem cells, like DPSCs, can only differentiate into cells for specific tissue development because
they are multipotent. This will be important throughout this study while looking at and comparing
ES cells to DPSCs. [2]
The current research of the lab of Dr. Van Winkle involves tracking the effects of amino acids on
ES cells. In general we know that amino acids are essential for the human body. Most notably they
are the building blocks for proteins and are very important in our biological pathways. Less
commonly known is that amino acids can act as signaling molecules in regulation of cellular
processing. Research has suggested that the transporters for amino acids could work as signals for
some of our pathways in embryologic development. [8] More importantly in terms of the objective
at hand it is important to take note of situations where proline has shown to be important for cell
proliferation and differentiation of stem cells.
There are a couple different situations where stem cells have been shown to be directly impacted
by amino acids. These amino acids could have the effect of changing the morphology of the cells,
enhancing proliferation, as well as improving cell differentiation. More specifically in the case of
proline, research looks at the impact it has had on ES cells in different environments. Recently
research has shown that L-proline has been identified as a component of media required for ES cell
differentiation. [11] Research shows that this is due to the mechanism of L-proline being taken up
4. 4
via a sodium neutral amino acid transporter (SNAT), which in turn makes the transporter an
important aspect for cell differentiation. This process can also be inhibited by other amino acids
and the researchers have shown that the proline is important for cellular differentiation due to the
fact that when the transporters of proline are inhibited there is significantly less cellular
differentiation. [7, 11] There are many different chemical alterations that can be made to proline
however.
In another experiment using DPSCs, researchers tested to see the effects of many different forms of
proline and found that the best effect came from media that contained greater than 100μm of L-
Proline. In fact those that did not contain L-proline did not have any changes from cells growing in
general media. These included amino acid analogs of Proline like D-Proline, which showed to have
no effect what so ever. What the research showed was that L-Proline is the most important
derivative to be included in media if one is looking for differentiation of ES cells. [15Similar
research involving differentiation of DPSCs has shown that peptides that simply contained proline
such as Gly-Pro and Ala-Pro could be used as well to induce differentiation. This resulted in
significant positive effects, similar to L-proline. Interestingly, when reversed (Pro-Ala and Pro-
Gly) there was no significant effect on differentiation. This specificity that is seen for L-proline
could suggest that there is a specific receptor cite on the cells that require a certain conformation of
active proline in order to induce conformation change and activate differentiation. This is why
when the proline is attached to other amino acids it is imperative that it is located at the C-terminus
of the peptide so it has proper access to the receptor. [14, 15]Due to the experiments above
researchers have concluded that proline is bioactive, therefor one might suggest that proline could
be used as a key ingredient for the matrix suggested above for stem cell tooth formation. For the
current project, this shows a potential similarity for proline’s effect on differentiation in both
DPSCs and ES cells.
L-Proline has been shown to have a significant effect on morphology of stem cells it interacts with.
L-proline promotes the change of ES cells into spindle-shaped, mesenchymal-like stem cells that
are highly mobile, furthermore they have been shown to have an invasive and pluripotent tendency.
This shows that L-proline could be a microenvironment cue for cell morphological change. [14]
This becomes very important when looking to grow the cells into viable tissue. The cells must take
a shape that is usable for the tissue that it will be creating. If the cells are changed into something
else the tissue will not grow, however if we can have the ability to control how the cells change we
could use this to potentially grow specific tissues and specific types of cells. [13]
While we know that L-proline has significant impact on differentiation of mES cells, in recent
studies this has been more specifically studied. What has been found is that L-proline has a
tendency to increase markers for ectodermal cells. The other effects that have been reported with
this are that the markers for neural precursors are increased and the markers for pluripotency
decrease for the most part. This opens the door for future studies as there is now a phenotypic
identity for differentiated ES cells. Furthermore since we know that the ectoderm is responsible for
tooth formation this could provide a link between ES cells and DPSCs. [3, 15]
BMP2 is among the key proteins that allow tooth formation through the differentiation of DPSCs.
Furthermore it has been shown to have a significant impact on differentiation of stem cells used for
tissue engineering in vitro. [16] Finally BMP2 has been shown to be responsible for the initiation,
promotion, and maintenance of odontogenesis. [17] It is for these reasons that BMP2 will be used
along with proline to attempt to differentiate mES cells toward DPSCs in vitro. This will allow for
5. 5
a better characterization for the importance of proline in the differentiation process as we will be
able to analyze both cells groups beside one another and compare their gene upregulation and
morphological changes.
We know that proline has a significant effect on mES cells differentiation toward ectodermal
tissue. However, after becoming ectodermal tissue the route to DPSCs is relatively unknown in
vitro. It is known that dental tissues are derived from the ectoderm, and for that reason we hope
that with the help of proline or other amino acid combinations the cells can be driven to
differentiate to mDPSCs. Therefore, the purpose of this study is to begin to determine whether
proline can cause mES cells to differentiate towards mDPSCs.
RESEARCH METHODS AND DESIGN
mES cell Culture
For all the experiments described below, ATCC American Tissue Type Culture Collection CE1
Mouse Embryonic Stem Cells taken from the preimplantation blastocyst that is formed four days
following fertilization in mice [7]. These cells were placed in T75 Tissue Culture Flasks with mES
cell media for 2 days. Media includes DMEM F12 (Gibco), Leukemia Inhibiting Factor (LIF)
(Gibco), 2% Fetal Bovine Serum (Bio west), non-essential amino acid (Gibco), Penicillin-
streptomycin (Hydroclone) and glutamine solution containing DMEM F12 (Gibco), glutamine
(Sigma), and 2-mercaptoethanol (Bio Rad). Cells were harvested at day 4 and media changed every
2 days. When splitting was necessary for cells not differentiating the procedure was as follows;
cells were trypsinized with Tryspin-EDTA (Gibco) for 5 minutes and then collected in a 15 ml
conical centrifuge tube. The cells will be spun down at 3400 RPM for 1 minute and then re-
suspended in 5 ml of mES cell media by tituration. Cells will then be placed in approximately 20
ml of mES cell media in a 50 ml conical tube and counted using a TC20 Automated Cell Counter
(Bio Rad) and then brought to a concentration of 1X106 cells/ml mES cell media.
Groups
ES cells were cultured in the presence of exogenous amino acids and seeded at density of 1x106
cells/cm2 onto tissue v culture-treated plastic ware in ES cell medium. There were seven total
treatment groups as well as a separate control group. The treatment groups were treated with
different concentrations of L-proline, BMP-2, and BMP-2 and Proline together. Proline was treated
at increasing concentrations from 200μM which had been used in previous studies [3] to 2000μM,
in total there were five L-proline groups: 200μM, 500μM, 1000μM, 1500μM, and 2000μM. BMP-2
was treated at 10ng/ml along with a heparin supplement. The control group was grown in a media
containing LIF so that no differentiation would occur.
Primers
Primers were found via research on the pubmed database, finding the cited materials and suggested
primers listed in table 1. Primers without a listed reference were used in previous studies of our lab.
Primers were used based on their specific characteristics which are listed in table 1. All primers
that were considered for use are listed in table 1. Some primers were determined not useful in the
study after PCR was done on a gel. Others not included in results were result of poor melt curve
results and lack of up-regulation.
6. 6
Figure 1: The mouse-specific primer sequences
GENE Characteristic Primers Referance
B2M Housekeeping F: CCTGAATTGCTATGTGTCTGGG
R: TGATGCTGCTTACATGTCTCGA
Osterix Odontoblast Specific F: GAAAGGAGGCACAAAGAAG
R: CACCAAGGAGTAGGTGTGTT
CBFA Odontoblast Specific F: CGCTCTCCTTCCAGGATGGT
R: GCTTCCGTCAGCGTCAACA
mCol2a1 CT and Cartilage F: GCCACCATGCCCGAAAATTAG
Specific Collagen R: CTCTGGGTCCTTCACCTG
mCol3a1 Dentin Specific F: TGACTGTCCCACGTAAGCAC
Collagen R: GAGGGCCATAGCTGAACTGA
mCol5a1 CT specific Collagen F: TTGGATATGGCGAGGGTGTG
R: CTGGAGCTGGATTGGAGGTG
Brachyury Mesodermspecific F: GCTGTGACTGCCTACCAGCAGAATG Ginis et al.
R: GAGAGAGAGCGAGCCTCCAAAC [11]
Mesp2 Mesodermspecific F: CCCCAAATACAGTCACCCTTACAC Janebodin et al.
R: CGCTCCTGGAAGATGGTG [1]
foxa mES cell specific F: GATCCTATGATTTTGTAATGGGGC Long et al.
R: GATCGCCCCATTACAAAATCATAG [28]
blimp 1b Masterregulator of F: TCGGGTCGTTTACCCCATC Morgan et al.
differentiation R: CACAGCGCTCAGGCCATTA [29]
GATA6 Neural crest specific F: AGCAGGACCCTTCGAAACG Janebodin et al.
R: GCGCTTCTGTGGCTTGATG [1]
Oct4 Pluripotency marker F: GGAGAGGTGAAACCGTCCCTAGG Ginis et al.
and differentiation
promoter R: AGAGGAGGTTCCCTCTGAGTTGC [11]
Nanog Transcription factor F: TCTCAAGTCCTGAGGCTGACAAG Janebodin et al.
for undifferentiated
mES cells R: GTGCTGAGCCCTTCTGAATCA [1]
c-Kit Pluripotency F: AGCCTGGCGTTTCCTACGT Janebodin et al.
R: GCCCGAAATCGCAAATCTTT [1]
Sox2 Pluripotency F: CCGGACCGCGTCAAGAG Janebodin et al.
R: TCATGAGCGTCTTGGTTTTCC [1]
BSP Odontoblast specific F: AGAACAATCCGTGCCACTCACT Janebodin et al.
R: CCCTGGACTGGAAACCGTTT [1]
DMP1 Odontoblast specific F: TTGGGAGCCAGAGAGGGTAGA Janebodin et al.
R: AGTCCACCAGCCGGTCTGTA [1]
Dsp-PP Obligate component F: CCCCTCGGAGGCTTTGA Janebodin et al.
of desmosomes R: ACTCGGAGCCATTCCCATCT [1]
7. 7
Through testing in gel PCR, five primers were chosen to be further investigated quantitatively with
RT-PCR. These primers included the dental specific primers Col3a1, Osterix, and CBFA as well
as non-dental primers Col2a1 and Col5a1.
Before any investigation took place, a proper housekeeping mRNA gene expression needed to be
found that provided a consistent expression value in all samples. This was accomplished using all
the samples that would be tested with multiple potential housekeeping genes. After triplicate was
performed the gene which yielded the most consistent expression values was chosen and used for
further testing. Through this process it was decided that B2M which is associated with nearly all
proteins was found to be the most appropriate housekeeping gene.
Cellular-Imaging
Using magnifications of 100x and 400x the cells were imaged at days 0 and 4. The groups that
were imaged include 2000μM, BMP2, and BMP2+2000μM. After imaging, cells were harvested
using methods listed below.
Real Time-PCR
Mouse ES cells are extracted for total RNA by using the RNeasy Mini Kit (Qiagen) according to
the manufacturer’s protocol. Quantity and purity of RNA is determined by 260/280nm absorbance.
A 260/280 absorbance greater than 2.0 was considered appropriate with a 260/230 absorbance
greater than 1.75. First-strand cDNA is synthesized from 500 ng of RNA using High Capacity
cDNA synthesis kit from Applied Biosystems via manufactures protocols using a randomized
primer. cDNA is then diluted in a final volume of 20μl per reaction using SYBR green PCR master
mix from Applied Biosystems. PCR was performed using the following thermal cycling
conditions; 95C 7 min for initial activation followed by 95C/30s; 57C/30s; 72C/45s, for 40 cycles
with a final 5-min extension at 72C. A melt curve was run on all primers. Mouse-specific primers
are listed in Figure 1.
RESULTS
Cellular-Imaging
Imaging found that even at day 2 cells had begun to change dramatically. There was an obvious
increase in cellular volume with very little cell death. At this time point cells had started forming
protrusions for what could possibly be communication. Cells also became more globular and round
in shape. These changes continued to day 4 and day 8. Day 8 did however show increased cellular
death. Figure 2 shows these changes at day 4. At this time point very obvious protrusions are
extending outward from the cells in virtually all directions. One can also see a difference in the size
and shape of the cells as they are much larger than cells imaged at day 0 and they are more globular
and round in shape. These results allowed for the conclusion that differentiation is occurring in the
cells. Unfortunately there was no determining what type of cells they had differentiated to with just
the use of imaging.
8. 8
Figure 2: Cellular imaging
Image a: +LIF
Image b: Day 0 +Proline 400x
Image c: Day 4 +Proline 400x
Image d: Day 0 +BMP 400x
Image e: Day 4 +BMP 400x
Image f: Day 0 +BMP and Proline
400x
Image g: Day 4 +BMP and Proline
400x
b c
d
gf
a
e
9. 9
Reverse Transcriptase PCR analysis
Cells were first tested using reverse transcriptase PCR on gels to test for the upregulation of the
genes GATA6, foxa, Oct4, Sox2, and Nanog. The genes included were either specific to both mES
cells and DPSCs or only to mES cells. In Figure 3 the images taken of the gels are shown. The
mRNA primers used to test whether or not our cells were differentiating away from mES cells were
foxa and GATA6 [40]. However, while some research has suggested that there is no relationship
between DPSCs and GATA6 because GATA6 is specific to mesodermal and endodermal cells,
other research, [1] has found through experimentation that this is not the case. There is evidence to
show that GATA6 is upregulated in DPSCs. In either case, after 4 days of differentiation none of
the cells used by us show an upregulation of the mRNA for GATA6. You can see that only the
samples treated with leukemia inhibiting factor express the genes foxa and GATA6 after 4 days of
growth which is expected due to their inability to differentiate.
Real-Time PCR analysis
After running a reverse transcriptase PCR on gels, the cells were tested quantitatively through the
use of RT-PCR. Figures 4 and 6 show the relative expression of each mRNA as compared to the
control which is cells treated with leukemia inhibiting factor so that no differentiation will take
place. The general expectation for collagen genes is that there should be a consistent increase in the
upregulation of the collagen gene being examined as the concentration of proline is increased in
our media. If the cells are not differentiating toward dental cells there should be no significantly
greater upregulation of the osterix and cbfa genes in the proline treated cells. It is expected that the
BMP treated cells differentiate toward dental cells.
The results from PCR testing have shown that in almost all cases there is a consistent increase of
gene expression in relation to the concentration of proline that cells were treated with. In the cases
this is not true, what should be noted is that high concentration of proline does not express a gene
unless it is dental related. As seen in Figure 4 our dental related genes are Col3a1, Osterix and
CBFA. In Genes Col2a1 and Col5a1 you can see a switch point between 1500 and 2000μM L-
Proline. At some point between 1500 and 2000μM L-Proline a reaction must take place that causes
the cells to show very little upregulation of the mRNA encoding for these genes when compared to
the cells treated with 1500μM L-Proline.
BMP2 treated cells very consistently showed the expression that was expected for dental cells as
seen in Figure 6. You can see that for BMP2 cells there was very little expression of mRNA for
Col2a1 and Col5a1, which are not dental related, compared to that of the proline samples in Figure
4. One can also see upregulation in osterix, CBFA and Col3a1 which are all genes associated with
dental cells.
The cells treated with BMP and Proline showed decreased expression for all primers related to
DPSCs. However in Col2a1 which is not associated with dental cells significant upregulation was
shown for mRNA of this combination group.
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Figure 4: RT-PCR
Day 4 samples, the concentrations listed
represent the amount of Proline
supplemented into each cell group. One
way ANOVA with a post hoc Donnet’s
test showed statistically significant
differences indicated by #.
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12. 12
CBFA Col2a1
200 uM vs. 2000 uM 200 uM vs. 500 uM
500 uM vs. 2000 uM 200 uM vs. 1000 uM
1000 uM vs. 2000 uM 200 uM vs. 1500 uM
1500 uM vs. 2000 uM 500 uM vs. 1000 uM
2000 uM vs. BMP2 500 uM vs. 1500 uM
2000 uM vs. BMP2 + 2000 uM 500 uM vs. 2000 uM
500 uM vs. BMP2
Osterix 1000 uM vs. 1500 uM
200 uM vs. 2000 uM 1000 uM vs. 2000 uM
500 uM vs. 2000 uM 1000 uM vs. BMP2
500 uM vs. BMP2 1000 uM vs. BMP2 + 2000 uM
500 uM vs. BMP2 + 2000 uM 1500 uM vs. 2000 uM
1000 uM vs. 2000 uM 1500 uM vs. BMP2
1000 uM vs. BMP2 1500 uM vs. BMP2 + 2000 uM
1500 uM vs. 2000 uM 2000 uM vs. BMP2 + 2000 uM
1500 uM vs. BMP2 BMP2 vs. BMP2 + 2000 uM
2000 uM vs. BMP2 + 2000 uM
BMP2 vs. BMP2 + 2000 uM Col3a1
200 uM vs. 2000 uM
Col5a1 500 uM vs. BMP2 + 2000 uM
200 uM vs. 1500 uM 1000 uM vs. 2000 uM
500 uM vs. 1500 uM 1500 uM vs. 2000 uM
1500 uM vs. 2000 uM 1500 uM vs. BMP2 + 2000 uM
1500 uM vs. BMP2 2000 uM vs. BMP2
1500 uM vs. BMP2 + 2000 uM 2000 uM vs. BMP2 + 2000 uM
BMP2 vs. BMP2 + 2000 uM
Figure 5: list of RT-PCR significant
relationships. Donnet’s test was used
to find statistically significant
differences.
13. 13
Figure 6: RT-PCR analysis for
mRNA expression in mES cells
treated with 2000uM and BMP2 for
4 days. One way ANOVA with a
post hoc Donnet’s test showed
statistically significant differences
indicated by #.
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DISCUSSION
Cellular-Imaging
Due to a large volume of possibilities with imaging we limited our search to only what was viewed
as most important in terms of determining if differentiation to DPSCs was occurring. It was
deduced from previous studies that day 4 would be one of the key points for differentiation to
monitor. It was originally believed that through imaging we would get our first signs that our cells
had or had not differentiated toward mDPSC. Unfortunately what was found was that while the
cells treated with Proline and BMP2 looked similar, they did not show the expected morphology of
dental stem cells. Dental stem cells are usually described as looking similar to neurons with many
protrusions which are spindle shaped and mesenchymal-like. [32] While mES cells look similar to
the described mDPSC they do lack protrusions and show very few signs of cell to cell
communication before differentiation. Examples of this can be seen in the images at day 0 and in
the cells treated with leukemia inhibiting factor seen in figure 2.
When examining Figure 2, you can see that the actual results are much different than what was
expected. There are a couple key features of the cells that should be noted however. While the cells
have changed in morphology to be more globular and not spindle shaped they do increase the
number of protrusions immensely. In all the cases shown there seems to be this similarity between
the different treatment groups. The one major difference that is noted is what seems to be an
increase in cell size for the combination group as seen in image g in Figure 2.
While these cells seem to be differentiating in a similar manner, the images show cells that do not
look like the expected mDPSC. A potential future experiment to look at would be adding a solid
component to the media so that the cells have a three-dimensional environment rather than a two
dimensional plate. For this reason our conclusions were made mainly on the results of the PCR.
Reverse Transcriptase-PCR
The reverse transcriptase PCR run on a gel was used as a qualitative method of confirming the
presence of mRNA that we expected to be present in either all samples or in none of the
differentiated samples. This can be seen with the primers Sox2, Oct4, Nanog, GATA6, and foxa in
Figure 3. These results from Figure 3 became an important starting off point because they
confirmed that our cells had done two things. The most important note from these results is that our
cells for the most part are remaining consistent to what was expected for DPSCs which can be seen
in Figure 7. From this figure we can see how consistent cells treated with 2000μM have followed
the expected expression for DPSCs before real-time PCR was used. There was one major
difference however; as mentioned previously GATA6 was studied and found to be upregulated in
DPSCs [1], however, we have also learned that GATA6 is specific to the mesoderm and endoderm
and for this reason we may not expect upregulation in cells differentiating down an ectodermal
path. Our cells were found to lack upregulation but this could be viewed as a negative point since
previous research has shown DPSCs to actually express upregulation of GATA6. From what we
have collected with reverse transcriptase PCR we believe that the cells treated with 2000μM
proline are differentiating towards ectodermal cells.
15. 15
Figure 7: mRNAs upregulated in DPSC and mES compared to our cells.
mRNA
Upregulated
in mES cells
Upregulated
in DPSC
Shows
concentration
effect with
Proline
Upregulated
in 2000μM
Proline at
day 4
Upregulated
in BMP2 at
day 4
Referance
Osterix X X X X Hirata et al. [37]
CBFA X X X #
D’Souza et al.
[38]
Col2a1 X
Yes when
compared
to LIF
Col3a1 X X X Ferre et al. [35]
Col5a1 X
Yes when
compared
to LIF
Runx2 X N/A N/A N/A
D’Souza et al.
[38]
DSPP X N/A N/A N/A
D’Souza et al.
[38]
OCN X N/A N/A N/A
Janebodin et al.
[1]
Sox2 X # = =
Janebodin et al.
[1]
Oct4. X # = =
Janebodin et al.
[1]
Nanog X # = =
Janebodin et al.
[1]
GATA6 X #
Janebodin et al.
[1]
foxa X Long et al. [28]
N/A: Melt curve revealed that primer did not work in RT-PCR
=: expression is at relatively same level as mES cells
#: Upregulation is decreased
Real Time-PCR
For some of the the mRNAs primers we concluded it was best that a quantifiable number be found
regarding the amount of upregulation, these primers included col2a1, col3a1, col5a1, Osterix, and
CBFA. Unfortunately, DPSCs were not able to be obtained, for this reason, as mentioned
previously, cells were grown side-by-side with cells treated with BMP2 which has been shown to
have effect on DPSC growth and differentiation. [16, 17] Because the effects of BMP2 on mES
cells are not fully understood, we will not rely on this as proof of differentiation toward DPSCs. As
well as since we do not have information regarding the values of upregulation in DPSCs we cannot
make conclusions but just analyze what we record as interesting facts of differentiation as cells are
affected by our media samples. The most important relationship that we would like to see in our
dental specific primers is a concentration effect for L-Proline which would signify that there is a
relationship between L-Proline and the upregulation of the mRNA encoding for dental specific
16. 16
primers. In Figure 7 we have listed the most important mRNAs that we analyzed in our study and
their relationship to the different cells. These relationships were analyzed in both our research and
in previous studies found in the literature.
Primers to detect mRNA encoding three different collagen genes (Col2a1, Col3a1, and Cola5a1)
were used to compare the effects of the concentration of proline on genes that are related to
different collagen specific tissues. Osterix and CBFA mRNA expression are markers for
DPSCs[37, 38]. It is known that proline is a major component of collagens [33], for this reason we
deduced that with an increase in proline we would expect an increase in up-regulation of all the
collagen genes, whether dental related or not. Interestingly this was not always the case.
Col2a1 was chosen because the gene Col2a1 it is not related to dental cells. In fact it is mainly
found only in relation to the eye; most notably a mutation in this gene is shown to have correlation
with the prevalence of Stickler Syndrome. [34]
Col3a1 expression is correlated with proper dental formation, when mutations occur, problems
with dentin and the periodontal ligaments are found. [35]
Col5a1 expression is related to tendons, ligaments, and cartilages. A mutation in this gene will lead
to very easily damaged tendons and ligaments. [36]
While a consistent up-regulation was observed from 200μM to 1500μM in just about all cases, at
2000μM the regulation did not follow the expected trend for all samples. At 2000μM proline,
expression of Col2a1 and Col5a1 was much lower than at 1500 uM proline, while Col3a1, osterix
and CFBA mRNA expression continued to increase up to 2000 uM proline as expected (Figure 4).
While these results for a relatively small change in proline concentration between 1500 and 2000
uM proline might seem surprising, other signals have been induced with an increase in intracellular
concentration of only 10% of the amino acid, like in the case of L-leucine. It has been show that
just a 10% change in the concentration of L-Leucine is responsible for the difference between a
successful and not successful trigger of mRNA expression [39]. We shall discuss more regarding
this switch point in a bit, for now we must note the most interesting result in the amount of
upregulation of mRNAs for the collagen genes that are not related to dental cells (col2a1 and
col5a1) for the 2000μM proline samples. In these collagens we see a significant drop in the amount
of upregulation at this switch point. Since we do not have DPSCs to compare our results with we
can only say that something of importance is happening between these two concentrations that
causes mES cells to differentiate differently than expected.
Osterix is an early marker in DPSC differentiation [37]. It is a transcription factor for osteoblast
formation and proper function of odontoblasts. Furthermore the up regulation of osterix is required
for root formation in teeth. For these reasons we investigated its expression in our cell groups. We
hoped to find two different effects for this gene. First is that both BMP2 and 2000μM Proline
treated cells have significantly differentiated to express osterix in similar amounts. (Figure 6)
Because BMP2 is known to have effect on growth of cells toward dental cells this would promote
the notion that proline might have a similar effect. Second there is a concentration effect of proline
on osterix. (Figure 4) If a concentration effect is seen than we can conclude that there is a
relationship between a gene specific to dental cells and cells that are treated with Proline. Because
osterix is upregulated by 2000μM L-Proline, these cells may be differentiating towards DPSCs.
17. 17
The cells not only show upregulation of the expression of mRNA encoding the gene osterix
specific to dental collagen, but also upregulation in odontoblast specific genes.
Similar to osterix, CBFA is required in early stages of tooth development [3]. It is the main
transcription factor for osteoblast formation and a key regulator for odontoblasts. Some forms of
CBFA are also required for the formation of the periodontal ligament. Unlike osterix, the cells that
were treated with 2000μM Proline expressed significantly more upregulation of CBFA than the
cells treated with BMP2. This provides an intriguing twist to our results as we do not know the
expression of CBFA in DPSCs so we do not know what is better. However, again we see a positive
relationship between proline concentration and the expression of our gene of interest. This
upregulation of the expression of mRNA encoding for CBFA in cells treated with 2000μM shows
that these cells are showing characteristics of DPSCs. To what extent we are unsure how similar
our cells may be to DPSCs, but we do see significant changes in what we hope to be in the right
direction. These results leave us with interesting information regarding the effects of Proline
unfortunately we cannot confirm or deny whether the effect is in fact differentiation toward
DPSCs.
From the results of mRNA expression of the genes we have chosen, we would like to say that there
seems to be a significant correlation and trend between our cells differentiation towards DPSCs and
L-Proline concentration. Unfortunately, with the data we have collected this cannot be concluded.
Instead we have gathered very interesting information for future studies. One of the aspects that
must be further investigated is this switch point that can be best seen when observing the dental
specific genes regulation.
This switch point is one of the most interesting aspects of the proline effect on our cells and it
happens somewhere between 1500 and 2000μM L-Proline. As stated previously, similar reactions
have been found with other amino acid triggers, where just a small change in concentration can
trigger a dramatic reaction. With the results that we have gathered it is clear that at some point
between these two concentrations a trigger is switched that causes a dramatic change in the cells.
The most notable cases we have recorded are with our collagens that are not dental related (Col2a1
and Col5a1). This switch has brought us to the conclusion that our cells may, in some ways be
differentiating toward DPSCs. This theory is only further enhanced as we analyze the dental related
genes (Col3a1, osterix, and CBFA) mRNA expression; one can observe a significant upregulation
difference between 1500 and 2000μM L-Proline.
As we consider proline uptake by the cell it is important to look at the expression in all of the
samples when both proline and BMP2 are supplemented in the media. All samples except Col2a1
showed lower expression in the combination sample than both of the individual samples. This
could represent that BMP2 and Proline actually are antagonistic towards one another. It is also
important to note that the only sample that did not show this effect was Col2a1 which is also not
related to dental cells. In the future we must examine this uptake in a clinical situation, as BMP2 is
a normal supplement for dental cell growth in dental offices around the nation. If this relationship
is in fact significant, and is inhibiting the cells to grow towards dental cells, a Dr. using BMP to
promote dental cell growth must also be careful to avoid the use of proline enriched toothpastes or
solutions during the treatment of a patient, as it may have a significant effect on the outcome of a
patient’s treatment.
18. 18
The results of the above experiments leave us with many more questions than answers however
there is evidence to show a relationship between proline and dental gene expression. We are not
sure if our cells are differentiating specifically toward DPSCs but we do know that they are
differentiating. Furthermore, we have shown positive results that must be further investigated both
in the laboratory and in a clinical setting. L-proline does have a significant effect on mES cell
differentiation but we cannot give a definite conclusion on the question originally asked, instead
we pose many more questions that must be further investigated for the scientific community.
19. 19
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Correspondance:
Dr. Lon Van Winkle
Midwestern University
Department of Biochemistry
555 31st St.
Downers Grove, IL, 60515, USA
lvanwi@midwestern.edu
