THE SEARCH FOR A TREATMENT: RESEARCHING THE USE OF
MESENCHYMAL STEM CELLS THAT PRODUCE INSULIN-LIKE GROWTH
FACTOR 1 AS A TREATMENT FOR AMYOTROPHIC LATERAL SCLEROSIS
Michelle Renée Ohlson
B.S., California State University, Sacramento, 2009
PROJECT
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF ARTS
in
BIOLOGICAL SCIENCES
(Stem Cell)
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
SPRING
2011
THE SEARCH FOR A TREATMENT: RESEARCHING THE USE OF
MESENCHYMAL STEM CELLS THAT PRODUCE INSULIN-LIKE GROWTH
FACTOR 1 AS A TREATMENT FOR AMYOTROPHIC LATERAL SCLEROSIS
A Project
by
Michelle Renée Ohlson
Approved by:
__________________________________, Committee Chair
Thomas Peavy, Ph.D.
__________________________________, Second Reader
Jan Nolta, Ph.D.
__________________________________, Third Reader
Thomas Landerholm, Ph.D.
____________________________
Date
ii
Student: Michelle Renée Ohlson
I certify that this student has met the requirements for format contained in the University
format manual, and that this project is suitable for shelving in the Library and credit is to
be awarded for the Project.
__________________________, Graduate Coordinator
Susanne Lindgren, Ph.D.
Department of Biological Sciences
iii
________________
Date
Abstract
of
THE SEARCH FOR A TREATMENT: RESEARCHING THE USE OF
MESENCHYMAL STEM CELLS THAT PRODUCE INSULIN-LIKE GROWTH
FACTOR 1 AS A TREATMENT FOR AMYOTROPHIC LATERAL SCLEROSIS
by
Michelle Renée Ohlson
Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative
disease. Each year, about 5,000 people in the US are diagnosed with ALS. Treatment
options for ALS patients are very limited with only one FDA approved drug that extends
survival by a few months. Mesenchymal stem cells (MSCs) have created a lot of interest
as a possible treatment option for many difficult to treat diseases. The purpose of this
project is to characterize genetically altered MSCs that overproduce IGF-1 (MSC-IGF-1),
as well as, GFP (MSC-GFP) as a control as a possible, future treatment for ALS. During
this research, it was found that MSC-IGF-1 produced IGF-1 at 18µg/mL of supernatant,
while MSC-GFP produced no detectable IGF-1. MSC-IGF-1 proliferated at a slower rate
than MSC-GFP during a proliferation assay. MSC-IGF-1 was found to have decreased
osteogenic differentiation potential as compared to MSC-GFP. MSC-IGF-1 migrated and
proliferated slower than MSC-GFP during a motility assay. In conclusion, there is still a
large amount of future experimentation to be completed before this treatment plan can
become an option for ALS patients, but there is a lot of potential in this research bringing
iv
hope for an effective treatment for patients that fight a tragic and fatal disease with very
limited options
_______________________, Committee Chair
Thomas Peavy, Ph.D.
_______________________
Date
v
ACKNOWLEDGMENTS
First, I would like to acknowledge and thank Dr. Thomas Peavy for his guidance
and support during this program. Additionally, I would like to thank Dr. Thomas
Landerholm for his guidance and support during the writing of this project.
Second, I would like to acknowledge and thank Dr. Jan Nolta, Dr. Nanette Joyce,
Fernando Fierro, Stefanos Kalomoiros, Dr. Scott Olson, Kari Pollock, and other members
of the Nolta laboratory for their guidance and help during the research of this project.
Finally, I would like to thank my husband, family, and friends for their unfaltering
support throughout this program. Their love and support means the world to me.
vi
TABLE OF CONTENTS
Page
Acknowledgments....................................................................................................... vi
List of Tables ............................................................................................................. viii
List of Figures .............................................................................................................. ix
INTRODUCTION .........................................................................................................1
MATERIALS AND METHODS ...................................................................................7
Transduction of Bone Marrow Derived MSCs and Vectors Used ................... 7
IGF-1 Enzyme-linked Immunosorbent Assay .................................................. 9
Proliferation Assay.......................................................................................... 10
Differentiation Assay ...................................................................................... 11
Motility Assay................................................................................................. 13
RESULTS ....................................................................................................................15
Transduction of Bone Marrow Derived MSCs ............................................... 15
IGF-1 Enzyme-linked Immunosorbent Assay ................................................ 17
Proliferation Assay.......................................................................................... 19
Differentiation Assay ...................................................................................... 21
Motility Assay................................................................................................. 25
DISCUSSION ..............................................................................................................27
Literature Cited ............................................................................................................32
vii
LIST OF TABLES
Page
Table 1. Molecular components of experimental and control vectors ................................8
viii
LIST OF FIGURES
Page
Figure 1. Transduction of bone marrow derived MSCs with doxycycline-inducible
IGF-GFP (MSC-IGF-1) or GFP vector (MSC-GFP)........................................16
Figure 2. IGF-1 ELISA of supernatant collected after MSC-IGF-1 were in
doxycycline media for 24 hours........................................................................18
Figure 3. Proliferation assay to compare proliferation rates of MSC-IGF-1 and
MSC-GFP after cell culture in doxycycline media ...........................................20
Figure 4. Osteogenic differentiation assay after 21 days under doxycycline
osteogenic cell culture conditions .....................................................................22
Figure 5. Adipogenic differentiation assay after 14 days under doxycycline
adipogenic cell culture conditions ....................................................................24
Figure 6. Motility assay to determine the rate of cell motility and proliferation
between MSC-IGF-1 and MSC-GFP after culture in doxycycline media ........26
ix
1
INTRODUCTION
Mesenchymal stem cells (MSCs) have created a lot of interest in the scientific
community as a possible treatment option for many diseases that are currently untreatable
or difficult to treat including, but not limited to, Huntington’s disease, Parkinson’s
disease, and muscular dystrophy. This interest in MSCs is due to the fact that MSCs are
immune-privileged and have an intrinsic paramedic response when injected into the body
(1). In previous studies, MSCs injected into the body have been shown to travel to the
damaged parts of the body, particularly to hypoxic and inflamed tissues (1). Once the
MSCs have reached damaged tissues in the body, they are known to produce many
cytokines and growth factors like hepatocyte growth factor, fibroblast growth factor 2,
vascular endothelial growth factor, and insulin-like growth factor 1 that aid in the healing
of damaged tissues in the body (1). Additionally, MSCs have the ability to be genetically
altered to produce specific proteins or growth factors that have the potential to heal or
stop the further degeneration of the diseased tissues within the body (1). In the lab,
MSCs are easily extracted and grown from bone marrow samples, as well as, from fat
tissue samples from liposuctions. MSCs could be the tool needed by the scientific
community to create effective treatment options, and possibly cures, for many diseases
that are currently difficult to treat and/or incurable like those mentioned previously.
More specifically, MSCs hold a lot of promise in the future treatment of neurological
diseases that affect the central nervous system (CNS) like amyotrophic lateral sclerosis
(ALS).
2
ALS, also known as Lou Gehrig’s disease, is a progressive, fatal
neurodegenerative disease (2). ALS is a motor neuron disease, characterized by the
degeneration and subsequent loss of motor neurons (2). In ALS, the upper and lower
motor neurons found in the brain, brain stem, and spinal cord are affected specifically (2).
Within the body, upper motor neurons in the brain carry signals to lower motor neurons
in the spinal cord that continue the signals to the voluntary muscles (3). As the muscles
receive fewer signals from the brain and/or spinal cord, the result is continued muscle
weakness leading to muscle atrophy of voluntary muscles and the loss of voluntary
movement for ALS patients (3). The clinical presentation of ALS patients can vary
widely, but there are some common symptoms seen among patients (2). Some of these
symptoms include, but are not limited to: muscle weakness, muscle twitching called
fasciculations, muscle cramping, and muscle stiffness also known as spasticity (3).
Symptom onset is asymmetric and often begins in the hands and feet, then progress to
affect the muscles of the trunk of the body (3). Additionally, there is a bulbar form of
ALS that affects the face, mouth and tongue of ALS patients (4). Symptoms of the
bulbar form of ALS include motor speech impairments known as dysarthria with poor
verbal articulation, as well as, difficulty swallowing (4). Progression of the disease
inevitably leads to death by respiratory failure and often occurs within two to three years
in bulbar ALS patients and three to five years in limb onset ALS patients (4). Diagnosis
of ALS patients is difficult and based on clinical criteria (2). There is no definitive test or
biomarker for ALS (2). Doctors must rule-out other causes of treatable motor neuron
3
disease, and the patient’s symptoms must progress both over time and over areas of the
body before an ALS diagnosis is given (2).
Each year, about 5,000 people in the US are diagnosed with ALS (2). Of those
diagnosed with ALS, 90% of patients have developed ALS for unknown reasons and are
labeled as sporadic ALS cases (4). The remaining 10% of those diagnosed each year in
the US have a genetically transmitted familial form of ALS (fALS) (4). Of those patients
with fALS, 20% will carry a mutation in the Copper Zinc Superoxide Dismutase 1
(SOD1) gene (4). To date, there are at least 120 identified mutations in the SOD1 gene
that are known to cause ALS (5). It is hypothesized that these mutations create a toxic
gain of function in SOD1, caused by the misfolding of the SOD1 protein likely leading to
endoplasmic reticulum stress and motor neuron cell apoptosis (5). This endoplasmic
reticulum stress induced by the accumulation of SOD1 misfolded proteins activates the
unfolded protein response pathway, which is caused by an increase in the amount of
misfolded proteins in the endoplasmic reticulum (6). The unfolded protein response
pathway leads to the induction of chaperones, stress sensor kinases, and apoptotic
mediators, as well as, the activation of the endoplasmic reticulum associated degradation
system (6, 9). If there continues to be a buildup of misfolded proteins in the endoplasmic
reticulum despite the activation of the unfolded protein response and endoplasmic
reticulum associated degradation system, then apoptotic cell death occurs (9). This
phenomenon has been seen in the cell and rodent models of fALS, as well as, in the
spinal cord of human ALS patients (6). Although there is some information known on
4
the mechanisms in fALS, the ALS disease pathophysiology is still largely unknown, but
appears to be multifactorial (13).
Treatment options for ALS patients are very limited with only one FDA approved
drug, Riluzole, which extends survival by only a few months (10). Riluzole must be
taken daily with the possibility of many detrimental side effects with some examples
being lowered ability to fight infection, muscle weakness and aches, fatigue, and loss of
appetite (11). Unfortunately, patients that qualify for treatment with Riluzole must take it
without the possibility of knowing whether it is prolonging their life because there is no
way to gauge if it is working or not (13). During recent research of possible future ALS
treatments, growth factors have been at the center of many experiments (10, 15). Growth
factors are proteins that are necessary for the survival of neurons, as well as, neuron and
support cell differentiation during development (6). The growth factor, insulin-like
growth factor 1 (IGF-1) has been considered in the possible treatment of ALS by many
researchers. In the body, IGF-1 binds to IGF-binding proteins and then binds the IGF-1
receptor (7). IGF-1 receptors have been shown to have high expression during tissue
development and expression remains elevated in motor neurons after development (7).
Previously, IGF-1 and other growth factors have been tested using experimental models
of ALS (10, 16, 17, 18). The most common rodent experimental model is the SOD1G93A mouse model of ALS (8, 19). This model uses transgenic mice that have a
mutated human copy of the SOD1 gene that develops a disease similar to fALS (8, 19).
In rodent models of ALS that carry SOD1 mutations, the results have shown
improvements in motor neuron survival and neuromuscular function in the animals being
5
tested (10, 16, 17, 18). However, human trials with ALS patients using classic protein
delivery methods have shown a lack of improvement in neuromuscular function and
patient survival (10, 12). These negative results could be due to inadequate dosing,
issues with the protein crossing the blood-brain barrier, the short half-life of the protein,
and/or the side effects of the protein in non-targeted areas of the body (10).
For many neurological diseases, MSCs that are genetically altered to produce
specific proteins or growth factors like IGF-1 are a promising alternative to classic
protein delivery methods. This is because they have the potential to be injected directly
into the CNS through a spinal tap or surgical means to promote healing and/or halt
further degeneration of tissues through their innate paramedic properties and the
continuous production and secretion of specific proteins like the growth hormone, IGF-1.
There has previously been a Phase 1 clinical trial completed where ALS patients had
MSCs surgically implanted into the CNS to determine the safety of this procedure (14).
This study showed no deaths or serious adverse events related to the surgical
transplantation of MSCs into the CNS further showing the promise of MSCs in the
treatment of neurological diseases (14).
The purpose of this project is to characterize genetically altered MSCs that
overproduce IGF-1, as well as, GFP as a control. The ultimate goal of this project is to
create a relevant therapy for ALS patients using MSCs that produce prolonged and
targeted IGF-1 secretion thus changing the microenvironment of the motor neurons
thereby slowing disease progression and improving quality of life. The broader
significance of this experimental research is to increase future treatment options for ALS
6
patients while also evaluating the potential of mesenchymal stem cell drug delivery in the
treatment of neurodegenerative diseases.
7
MATERIALS AND METHODS
Transduction of Bone Marrow Derived MSCs and Vectors Used
The purpose of MSC transduction was to create MSCs that stably express either
the IGF-GFP vector (MSC-IGF-1) or GFP control vector (MSC-GFP). Both vectors are
based on the third-generation lentiviral vector pCCLc backbone with tetracycline
response elements (TRE). The IGF-GFP retroviral construct is: pCCLc-TREIGF1/p2A/EGFP-PGK-rtTA and is 8,948 bases in size. The GFP retroviral construct is
pCCLc-TRE-EGFP-PGK-rtTA and is 8,417 bases in size. Both vectors are described in
detail below in Table 1. Additionally, both vectors are under the control of a
doxycycline-inducible promoter ensuring that the transduced cell only produces the
vector product when there is doxycycline in the cell culture media. To transduce MSCs,
first, cultured MSCs were grown in normal media (HyClone MEMα with 10% Atlanta
Biologicals Premium Select FBS). After cultured MSCs reach 80% confluency, they
were plated into six well plates at 50,000 cells per well. Two days after plating, cells
were incubated with the desired vector in a viral mixture (normal media, 0.002%
protamine sulfate, and virus). After transduced cells become 80% confluent, they were
plated for future experiments or cryopreserved in liquid nitrogen.
8
Table 1. Molecular components of experimental and control vectors.
Elements in vectors used1
Definition of the element
pCCLc
Lentiviral vector backbone with cytomegalovirus
promoter driving high levels of expression
TRE
Tetracycline response element which must be bound by
doxycycline-bound rtTA allowing for gene expression
IGF-1
Insulin-like growth factor 1 gene segment
p2A
Linker segment
EGFP
Green fluorescent protein gene segment
PGK
Housekeeping phosphoglycerate kinase promoter
rtTA
Tet-on advanced transactivator which must be bound
by doxycycline before binding TRE allowing for gene
expression
1
Experimental IGF-1 vector (pCCLc-TRE-IGF1/p2A/EGFP-PGK-rtTA);
Control GFP vector (pCCLc-TRE-EGFP-PGK-rtTA).
9
In order to know the amount of the desired virus needed to transduce 50,000 cells,
a previous titer using GFP expression on the FC500 FACS machine was performed. To
complete the titer, cells are plated at a known cell number into a 12 well cell culture dish
and are transduced at various viral concentrations to determine what concentration of vius
produces a high percentage of GFP positive cells. These transduced cells are then run
through the FC500 flow cytometer to determine the amount of GFP positive cells. To
complete this, cells were released from the plate with the protease, trypsin, before being
centrifuged to create a cell pellet. The media surrounding the cell pellet was decanted
and the cell pellet was re-suspended in PBS prior to being run through the FC500 flow
cytometer to determine the amount of GFP positive cells.
IGF-1 Enzyme-linked Immunosorbent Assay
The purpose of the enzyme-linked immunosorbent assay (ELISA) for the IGF-1
protein was to determine the amount of IGF-1 expressed per cell after transduction with
either the IGF-GFP vector or GFP control vector. This was done in order to figure out
the number of cells needed for treatment dosages for future mice experimentation and,
possibly, clinical trials. Additionally, three different doxycycline medias with three
doxycycline concentrations were used to see how much IGF-1 was produced under
different doxycycline concentrations. To achieve this, previously transduced MSCs were
grown in normal media. After cells became 80% confluent, they were plated into 12 well
cell culture plates at 4,000 cells per well in normal media. Two days after plating, media
was changed to media with lowered FBS concentrations to prevent it from affecting the
10
results and doxycycline medias with three different doxycycline concentrations of 2X,
1X, and 0.5X (HyClone MEMα with 2% Atlanta Biologicals Premium Select FBS with
doxycycline at 4µL/mL, HyClone MEMα with 2% Atlanta Biologicals Premium Select
FBS with doxycycline at 2µL/mL, HyClone MEMα with 2% Atlanta Biologicals
Premium Select FBS with doxycycline at 1µL/mL). Supernatant from each condition
was collected after 24 hours incubation with the transduced cells. The IGF-1 ELISA was
completed following the instructions from the R & D Systems Human IGF-1 ELISA Kit
(DG100). According to these instructions, first, add assay diluent, then sample or
provided standard to each well of one of the 96 well plate provided and incubate. After
incubation, aspirate and wash each well with wash buffer before incubation with IGF-1
conjugate. Then, the wash step is repeated before addition and incubation with the
substrate solution. Finally, the stop solution is added and the plate is read using a
microplate reader set at the wavelength of 450 nm.
Proliferation Assay
The purpose of the proliferation assay was to determine and compare the
proliferation rate of MSCs expressing either the IGF-GFP vector or GFP control vector.
To achieve this, previously transduced MSCs were grown in normal media. After cells
became 80% confluent, they were released from the plate with the protease, trypsin, and
counted with a hemacytometer prior to being plated into 12 well cell culture plates at
4,000 cells per well in normal media. Two days after plating, media was changed to
doxycycline media (HyClone MEMα with 10% Atlanta Biologicals Premium Select FBS
11
with doxycycline at 2µL/mL). The first time point of the assay is called Day 0 and is two
days after media was changed from normal media to doxycycline media. The rest of the
time points occurred every 48 hours and were called Day 2, 4, 6, 8, and 10. During each
time point, two wells of the previously plated 12 well cell culture plates were trysinized
and counted in duplicate using trypan blue in a standard hemocytometer. Additionally, at
each time point, the sample was run through the FC500 flow cytometer to determine the
amount of GFP positive cells. To complete this, cells were released from the plate with
the protease, trypsin, before being centrifuged to create a cell pellet. The media
surrounding the cell pellet was decanted and the cell pellet was re-suspended in PBS prior
to being run through the FC500 flow cytometer to determine the amount of GFP positive
cells.
Differentiation Assay
The purpose of the differentiation assay was to determine and compare the ability
for MSCs expressing either the IGF-GFP vector or GFP control vector to differentiate
into either adipocytes or osteocytes. To achieve this, previously transduced MSCs were
grown in normal media. After cells became 80% confluent, they were released from the
plate with the protease, trypsin, and counted with a hemacytometer prior to being plated
into 6 well cell culture plates at 50,000 cells per well in normal media. Two days after
plating, media was changed to doxycycline media (HyClone MEMα with 10% Atlanta
Biologicals Premium Select FBS with doxycycline at 2µL/mL). Two days later,
doxycycline media was changed to either osteogenic (HyClone MEMα with 10% Atlanta
12
Biologicals Premium Select FBS, 0.01% betaglycerol phosphate, 0.01% ascorbic acid,
0.0002% dexamethasone) or adipogenic differentiation media (HyClone MEMα with
10% Atlanta Biologicals Premium Select FBS, 0.001% 3-Isobutyl-1-methylxanthine
(IBMX), 0.001% dexamethasone, 0.001% indomethacin). The osteogenic and
adipogenic medias were made in the laboratory using the necessary components.
For cells incubated in osteogenic media, media changes were completed every 3
days until day 21 in culture was reached. On day 21, RNA was collected and processed
following the Qiagen RNeasy Kit instructions. The stored RNA was made into cDNA
following the Quantitect Reverse Transcription Kit instructions. The stored cDNA was
then used to complete qPCR for osteogenic markers bone sialoprotein (BSP) and
osteocalcin (OCN). Additionally, on day 21 in osteogenic media, the alkaline
phosphatase (ALP) concentration was determined. To achieve this, cells are trypsinized
and centrifuged to create a cell pellet. After the cell pellet is made, the media is decanted
and the cells are incubated in cell lysis solution (1.5M Tris-HCl solution with 1.0mM
ZnCl2, 1.0mM MgCl2, 1% Triton X-100). After incubation, the cells are centrifuged to
create a cell pellet prior to incubation with p-nitrophenylphosphate (p-NPP). After
incubation, cells are read using a microplate reader set at the wavelength of 405 nm.
Additionally, the protein concentration must be calculated by comparing to bovine serine
albumin (BSA) standards. First, samples are diluted 1:5 with molecular grade water.
Next, 10µL of sample or standard is added to 200µL Coomassie and cells are read using
a microplate reader set at the wavelength 595 nm. Finally, ALP concentration is
determined as “µg p-NPP/µg of protein”.
13
For cells incubated in adipogenic media, media changes were completed every 3
days until day 14 in culture was reached. On day 14, RNA was collected and processed
following the Qiagen RNeasy Kit instructions. The stored RNA was made into cDNA
following the Quantitect Reverse Transcription Kit instructions. The stored cDNA was
then used to complete qPCR for adipogenic markers peroxisome proliferator-activated
receptor gamma (pparg) and fatty acid binding protein 4 (FABP4). Additionally, on day
14 in adipogenic media, the lipid structures were stained with the nonfluorescent lipid
stain nile red. To achieve this, cells are trypsinized and centrifuged to create a cell pellet.
After the cell pellet is made, the media is decanted and a nile red solution (0.01% nile red
in PBS) is incubated with the cells in darkness. After incubation, cells are centrifuged to
create a cell pellet, then the media is decanted and cells are resuspended in PBS. Finally,
the nile red stained cells are run in the FC500 flow cytometry and to determine the nile
red positive percentage.
Motility Assay
The purpose of the motility assay was to determine and compare the rate of cell
migration and proliferation of MSCs expressing either the IGF-GFP vector or GFP
control vector. To achieve this, previously transduced MSCs were grown in doxycycline
media. After cells became 80% confluent, they were released from the plate with the
protease, trypsin, and counted with a hemacytometer prior to being plated into 24 well
cell culture plates at 30,000 cells per well in doxycycline media. There is a defined area
within the well of the cell culture plate that is absent of MSCs. Under a microscope, this
14
area is seen as a line across the middle of the well of the cell culture plate that is absent of
MSCs. To create this define area absent of cell growth, a plastic divider is positioned in
the well of the cell culture plate before MSCs were plated. MSCs are then plated in
doxycycline media with 15,000 cells on each side of the divider for a total of 30,000 cells
per well. Twenty four hours after plating, the dividers are carefully removed to prevent
disturbing plated MSCs along the edge of this define area. The first time point of the
assay is called Hour 0 and occurs when the dividers are removed. The rest of the time
points are at hours 3, 6, 12, 24, and 30. At each time point, pictures are taken under the
microscope at the area where the defined area without cell growth is within the well of
the cell culture plate. The rate of closure of the defined area without cell growth was
determined by TScratch computer software, available for download from the
Computational Science and Laboratory of the ETH Zurich University website (www.cselab.ethz.ch).
15
RESULTS
Transduction of Bone Marrow Derived MSCs
Cultured MSCs were transduced with either the doxycycline-inducible IGF-GFP
vector (MSC-IGF-1) or GFP control vector (MSC-GFP) to produce MSCs that stably
express either vector product within the transduced cells. After transduction, MSCs were
cultured in doxycycline media. After three days in doxycycline media, MSC-IGF-1 were
shown to have 30% GFP positive cells while MSC-GFP were shown to have 80% GFP
positive cells after transduction with 8µLof either lentivirus as seen in Figure 1. When
discussing MSC-IGF-1, GFP positive cells represent IGF-1 secreting MSCs.
Additionally, a picture of MSC-GFP can be seen in Figure 1B and MSC-IGF-1 can be
seen in Figure 1C.
16
Figure 1. Transduction of bone marrow derived MSCs with doxycycline-inducible IGFGFP (MSC-IGF-1) or GFP vector (MSC-GFP). (A) Transduction of MSCs with IGFGFP or GFP control vector. (B) Representative field of GFP positive cells in MSC-GFP
(100X magnification). (C) Representative field of GFP positive cells in MSC-IGF-1
(100X magnification).
17
IGF-1 Enzyme-linked Immunosorbent Assay
To determine the amount of IGF-1 expressed per cell after transduction in MSCIGF-1 or MSC-GFP cells were grown in doxycycline media and supernatant was
collected after 24 hours in culture. Additionally, three different doxycycline
concentrations were used to compare the amount of IGF-1 produced when the
doxycycline-inducible promoter is induced with cell culture media with varying
doxycycline concentrations. The supernatant collected was used in the R and D Systems
Human IGF-1 ELISA Kit. It was found that MSC-GFP produced no detectable IGF-1
with or without doxycycline media. Additionally, it was found that MSC-IGF-1 with
normal (1X) doxycycline media produced IGF-1 at 18µg/mL of supernatant as seen in
Figure 2. Additionally, there was minimal dose dependence seen in the supernatant on
cells in cultures with 2X and 0.5X doxycycline concentrations as seen in Figure 2.
18
Figure 2. IGF-1 ELISA of supernatant collected after MSC-IGF-1 were in doxycycline
media for 24 hours. This demonstrated the amount of IGF-1 protein produced by MSCIGF-1 in two donor cell lines with three different concentrations of Doxycycline media.
19
Proliferation Assay
A proliferation assay was completed to compare the proliferation rate of MSCIGF-1 and MSC-GFP. It was found that MSC-IGF-1 proliferated at a slower rate than
MSC-GFP as seen in Figure 3A. Additionally, MSC-IGF-1 showed a progressive
decrease in GFP positive cells as proliferation increases and time passes as seen in Figure
3B. Additionally, MSC-GFP showed an equal amount of GFP positive cells as
proliferation increases and time passes as seen in Figure 3B.
20
Figure 3. Proliferation assay to compare proliferation rates of MSC-IGF-1 and MSCGFP after cell culture in doxycycline media. (A) The average proliferation rate from two
donor cell lines of MSC-IGF-1 and MSC-GFP. (B) The average GFP positive cells
during proliferation of two donor cell lines of MSC-IGF-1 and MSC-GFP.
21
Differentiation Assay
A differentiation assay was completed to compare the ability of MSC-IGF-1 and
MSC-GFP to differentiate into osteocytes and adipocytes. After 21 days in osteogenic
media, MSC-IGF-1 showed decreased ALP concentration as compared to MSC-GFP as
seen in Figure 4A.
22
Figure 4. Osteogenic differentiation assay after 21 days under doxycycline osteogenic
cell culture conditions. (A) The ALP concentration of MSC-IGF-1 and MSC-GFP after
osteogenic differentiation. (B) The BSP mRNA levels in MSC-IGF-1 and MSC-GFP in
two donor cell lines after osteogenic differentiation. (C) The OCN mRNA levels in MSCIGF-1 and MSC-GFP in two donor cell lines after osteogenic differentiation.
23
Additionally, BSP and OCN mRNA levels were decreased in MSC-IGF-1 as
compared to MSC-GFP as seen in Figure 4B and Figure 4C. After 14 days in adipogenic
media, PPARG and FABP4 mRNA levels were consistent between MSC-IGF-1 and
MSC-GFP as seen in Figure 5A and Figure 5B.
24
Figure 5. Adipogenic differentiation assay after 14 days in doxycycline adipogenic cell
culture conditions. (A) The PPARG mRNA levels in MSC-IGF-1 and MSC-GFP in two
donor cell lines after adipogenic differentiation. (B) The FABP4 mRNA levels in MSCIGF-1 and MSC-GFP in two donor cell lines after adipogenic differentiation.
25
Motility Assay
A motility assay was completed to compare the rate of cell migration and
proliferation of MSC-IGF-1 and MSC-GFP. It was found that MSC-GFP showed a faster
rate of migration and proliferation as compared to MSC-IGF-1 in both donor cell lines as
seen in Figure 6A and Figure 6B. Additionally, pictures of MSC-GFP and MSC-IGF-1 at
various time points can be seen in Figure 6C.
26
Figure 6. Motility assay to determine the rate of cell motility and proliferation between
MSC-IGF-1 and MSC-GFP after culture in doxycycline media. (A) The time it takes to
fill in a defined space in MSC-IGF-1 and MSC-GFP in 11.6 donor cell line. (B) The time
it takes to fill in a defined space in MSC-IGF-1 and MSC-GFP in 11.6 donor cell line.
(C) Images of MSC-IGF-1 and MSC-GFP at various time points during the motility assay
(100X magnification).
27
DISCUSSION
First, cultured MSCs were transduced with either the doxycycline-inducible IGFGFP vector (MSC-IGF-1) or GFP control vector (MSC-GFP) to create MSCs that stably
express either vector product when there is doxycycline in the cell culture media. MSCIGF-1 had 30% GFP positive cells while MSC-GFP had 80% GFP positive cells after
transduction with 8µLof either lentivirus. Even though MSC-IGF-1 had a much lower
percentage of GFP positive cells than MSC-GFP, this was expected since the IGF-GFP
vector is larger and more complex and, therefore, harder to transduce MSCs with than the
GFP control vector. In the future, these transduction methods will continue to be used.
Next, to determine the amount of IGF-1 expressed per cell after transduction in
MSC-IGF-1 or MSC-GFP, supernatant was collected after 24 hours in cell culture
conditions. It was found that MSC-GFP did not produce any detectable amounts of IGF1 in cell culture media with or without doxycycline in the cell culture media. It was
found that MSC-IGF-1 produced IGF-1 at 18µg/mL of supernatant when there was 1X
concentration of doxycycline in the media at 2µL/mL of media. Additionally, two other
concentrations of doxycycline were used and it was found that there was minor dose
dependence. In the 2X concentration of doxycycline in the media at 4µL/mL of media,
there was a slightly elevated amount of IGF-1 produced as compared to the 1X
concentration, but it was not statistically significant. In the 0.5X concentration of
doxycycline in the media at 1µL/mL of media, there was a slight lower amount of IGF-1
produced as compared to the 1X concentration, but it was also not statistically significant.
28
In future experimentation, we would like to test additional concentrations of doxycycline
that are lower than those tested previously in an attempt to find a difference in IGF-1
production as related to varying doxycycline concentrations that are statistically
significant. Additionally, we would like to repeat this experiment with sorted MSC-IGF1 and MSC-GFP. This would allow us to test a homogenous population of cells instead
of a mixed population of transduced and non-transduced cells. In order to sort cells, we
would need to transduce cells with either the doxycycline-inducible IGF-GFP vector
(MSC-IGF-1) or GFP control vector (MSC-GFP) to create MSCs that stably express
either vector product when there is doxycycline in the cell culture media. These
transduced cells can then be sorted for GFP positive cells in both heterogeneous cell
populations. By doing this, we can create a homogeneous mixture of GFP positive cells
only for both cell populations.
A proliferation assay was completed to compare the proliferation rate of MSCIGF-1 of and MSC-GFP. It was found that MSC-IGF-1 proliferated at a slower rate than
MSC-GFP. It was also found that MSC-IGF-1 showed a progressive decrease in GFP
positive cells with MSC-GFP showed an equal amount of GFP positive cells as
proliferation increases and time passes. We have hypothesized that the amount of GFP
positive cells decrease as proliferation increases due to faster proliferation of nontransduced MSCs in the heterogenous mixture of cells as compared to transduced, GFP
positive cells. In future experiments, we would like to repeat this experiment with sorted
MSC-IGF-1 and MSC-GFP. This would allow us to test a homogenous population of
cells instead of a mixed population of transduced and non-transduced cells. The methods
29
described above would be used to sort cells for GFP positive cells to create homogeneous
cell populations.
A differentiation assay was completed to compare the ability of MSC-IGF-1 and
MSC-GFP to differentiate into osteocytes or adipocytes. For differentiation into
osteocytes, it was found that MSC-IGF-1 showed decreased ALP concentration as
compared to MSC-GFP. The mRNA levels for osteogenic markers, BSP and OCN, were
decreased in MSC-IGF-1 as compared to MSC-GFP. For differentiation into adipocytes,
it was found that mRNA levels were consistent between MSC-IGF-1 and MSC-GFP. In
future experiments, we would like to repeat the differentiation into osteocytes and use
additional osteogenic mRNA markers to see if there continues to be differences between
MSC-IGF-1 and MSC-GFP. Since there were no significant differences in adipocyte
differentiation between MSC-IGF-1 and MSC-GFP, there are no future experiments
planned.
Finally, a motility assay was completed to compare the rate of cell migration and
proliferation of MSC-IGF-1 and MSC-GFP by looking at the time it takes to fill a define
space vacant of cell growth for both cell types. It was found that MSC-GFP showed a
faster rate of migration and proliferation as compared to MSC-IGF-1. It is thought that
this occurred because MSC-GFP proliferates faster than MSC-IGF-1 as shown in the
proliferation assay. For future experiments, we would like to repeat this experiment with
additional bone marrow donors, as well as, with a homogeneous population of sorted
MSC-IGF-1 and MSC-GFP to see if that affects the results. The methods described
30
above would be used to sort cells for GFP positive cells to create homogeneous cell
populations.
The purpose of this project was to characterize genetically altered MSCs that
overproduce IGF-1, as well as, GFP as a control. The ultimate goal of this project was to
create a relevant therapy for ALS patients using MSCs that produce prolonged and
targeted IGF-1 secretion thus changing the microenvironment of the motor neurons and
thereby slowing disease progression and improving quality of life. Previous research has
shown that using classical protein delivery methods of IGF-1 in humans, like
subcutaneous injections, did not show increased patient survival or neuromuscular
function (10, 12). This is thought to have occurred due to inadequate dosing, problems
with IGF-1 passing the blood-brain barrier, the short half-life of IGF-1, and/or side
effects of IGF-1 in non-targeted areas of the body (10). Additionally, IGF-1 has shown
some positive effects in the rodent models of ALS that carry mutations in the SOD1 gene
when injected into the muscles of these animals (10). Clearly, these positive effects seen
in the rodent models of ALS have not easily translated into humans when
experimentation has previously been done. There is a potential problem with translating
a relatively small number of muscle injections in the ALS mouse to the possibly high
number of injections needed in a patient with ALS if this experimental method became a
treatment option in the future. Additionally, there is the question of whether injections
into easily accessible muscles of the human body will positively affect the diaphragm,
which is ultimately the muscle that leads to respiratory failure and death in ALS patients
once it becomes affected by the disease and can’t function properly (13).
31
The ultimate goal of the research presented here is to create a treatment option for
ALS patients where MSCs that stably express IGF-1 are transplanted into the CNS and
allowed the positively affect the microenvironment of the disease affected motor neurons
to increase muscle function, quality of life, and patient survival. This potential treatment
plan could bypass many of the problems previously discussed that are associated with
other experimental treatment plans. In conclusion, there is still a large amount of
experimentation to be completed before this treatment plan can become an option for
ALS patients, but there is a lot of potential in this research bringing hope for an effective
treatment for patients that fight a tragic and fatal disease with very limited options.
32
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