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Topical adipose-derived stem cell therapy ameliorates radiation-induced delayed wound healing

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Topical adipose-derived stem cell therapy ameliorates radiation-induced delayed wound healing

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Topical Adipose-Derived Stem Cell Therapy Ameliorates
Radiation-Induced Delayed Wound Healing

By: Gabrielle B. Davis
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
BIOCHEMISTRY AND MOLECULAR BIOLOGY

August 2013

2

DEDICATION
Dedicated to my wonderful children,
Kierra Davis and Donovan Johnson,
for their continued love and support through the years
as I pursued all of my academic endeavors.
You both have been the motivating force of my success
and I love you dearly.

3
ACKNOWLEDGEMENTS
I would like to convey my deepest gratitude to Dr. Neli Ragina, who was
instrumental in the design and organization of this work. Her extensive stem cell
background advanced my knowledge of this rapid evolving field. Not only have
you been a dedicated mentor but a supportive friend, and I enjoyed working with
you during my years of research. I would also like to acknowledge Dr. Zoltan
Tokes, my graduate advisor, for your support and guidance through this process.
Even though I was out of the basic science arena for some time you ensured that
the transition was smooth, which facilitated completion of my work.
I would like to recognize my advisor in surgery, Dr. Namir Katkhouda. You
have devoted your career to the education and training of medical students,
residents and fellows which is truly a noble aspiration. Through your integrity and
grace, you serve as a valued role model in medical education and set the
standard professionalism in surgery and academics as a whole. You have always
advocated for my best interest and recognized my potential, and this was truly
appreciated. You are passionate, driven, inspirational, hardworking, loyal, and an
innovator and leader in your field. You legacy will be passed down for
generations and I am honored to have you as my advisor.
I would also like to thank Dr. Robert Maxson and the Department of
Biochemistry and Molecular Biology for supporting my studies in the master’s
program at the University of Southern California. I would also like to thank Dr.
Vaughn Starnes, Dr. Anthony Senagore and the Department of Surgery for
allowing me to take time away from my surgical training to pursue my research

4
interests. I would like to thank Dr. Mark Urata, Dr. Warren Garner, Dr. Alex Wong
and the Division of Plastic and Reconstructive Surgery for supporting my
research in the division. In addition, I would like to acknowledge Dr. Elizabeth
Fini who fostered financial support allowing me to complete my master’s degree
at the University of Southern California. I would also like to recognize The
California Institute of Regenerative Medicine for awarded me the clinical
fellowship for support of this project.
In conclusion, I would like to note that not only has my years in research
been intellectually stimulating but it has also allowed an opportunity for profound
self-discovery and spiritual growth. In light of this I would like to close my
acknowledgement with an original quote inspired during this time.

“What is Love?”
“Love is in the eyes of new life, filled with hope, joy and peace, awaiting to fulfill
their destiny in this world with limitless possibilities,
Love is in the beauty of nature, the serenity of the sea, the stillness of the
mountains, and the coolness of the breeze,
Love innovates, inspires and drives passion for intellectual discoveries and the
cures for disease,
Love is sustainable, unconditional, and permeates beyond all physical restraints
and barriers to overcome all things,
Love is timeless, endless, transcends generations, lifetimes, the seen and
unseen,

5
Love is in the past, the here and now and what there will ever be,
Love is the most powerful energy force in creation as it can change the
consciousness of humanity and unite nations and mankind in peace,
The message is so simple, LOVE IS THE ESSENCE OF ALL THINGS,

Yet while love is such an ubiquitous energy, why does universal love on earth
seem so hard to reach?”

6
TABLE OF CONTENTS
Dedication………………………………………………………………………..2
Acknowledgements……………………………………………………………..3
List of Figures……………………………………………………………………7
Abstract…………………………………………………………………………..8
Chapter 1: Introduction………………………………………………………...10
Chapter 2
2.1 Scientific Aims………………………………………………………13
2.2 Materials and Methods…………………………………………….13
2.3 Statistical Analysis………………………………………………….20
2.4 Results ………………………………………………………………21
2.5 Discussion…………………………………………………………...26
Chapter 3: Future Directions…………………………………………………...31
Figures……………………………………………………………………………34
References………………………………………………………………………41

7
LIST OF FIGURES
Figure 1: Radiation Wound Model
Figure 2: Pathologic Changes Induced by Radiation
Figure 3: Characterization of ADSCs
Figure 4: Cellular Treatment of Wounds
Figure 5: ADSCs treatment of Irradiation Wounds
Figure 6: q-PCR Analysis of Treated Irradiated Wounds
Figure 7: In vitro Analysis of Irradiated Fibroblast

8
ABSTRACT
Recent literature supports the notion that Adipose Derived Stem Cells
(ADSCs) in lipoaspirated fat could significantly improve radiation-induced wounds.
However the mechanism of action is not completely understood which has limited
clinical applicability of this technique. Therefore, we sought to develop a murine
model of chronic radiation skin injury and to have a better understanding of how
ADSCs exerts these beneficial effects.
We generated a murine model of chronic radiation-induced skin injury in
Balb/c mice. Animals were treated topically with ADSCs in a transglutaminase
(Tg) gel matrix. Fibroblast (FB) cells in the Tg gel or Tg gel alone without cells
were used as controls. Alterations in pathologic signaling molecules and immune
responses were evaluated. In addition, in vitro analysis was performed to
investigate paracrine influences.
Wound closure was significantly impaired after radiation exposure and
demonstrated pathological up-regulation of TGF-β, which is the hallmark finding
of chronic radiation injury. Topical delivery of ADSCs to radiated wounds resulted
in significantly accelerated wound closure compared to fibroblasts and no cells,
respectively. Interestingly no difference in wound closure was observed after
ADSCs treatment under physiologic or normal wound healing conditions. ADSCs
application reduced pathologic expression of TGF-β in healing radiated wounds.
In addition, ADSCs treatment lead to less recruitment of M2-macrophages in the
wound bed of ADSCs treated irradiated wounds. In vitro analysis revealed that
soluble factors also mediate beneficial effects in wound healing.

9
Thus, purified ADSCs delivered topically in a biologic matrix significantly
accelerated wound closure specifically in irradiated animals. Based on our data
we propose that ADSCs, in part, exert their beneficial effects by suppressing the
pathological TGF-β expression, decreasing M2-macrophage recruitment and
releasing soluble mediators that aid in ameliorating the deleterious effects of
radiation.

10
CHAPTER 1
Background
With the increasing use of external beam radiation for diagnostic purposes
or oncological therapy, greater emphasis has been placed on understanding the
pathogenesis of radiation-induced tissue injury. The National Cancer Institute
estimates that more than 50 percent of cancer patients will receive radiotherapy
as either primary cancer treatment or post-surgical tumor control.
1
Despite
improvements to delivery techniques bystander injury is inevitable and can lead
to skin breakdown, non-healing wounds and delayed wound healing after
surgical procedures. Radiation-induced soft tissue injury also poses a significant
burden to the healthcare system as there are limited non-invasive treatment
modalities for these patients. In some cases, definitive therapy requires
aggressive surgical debridement and major reconstructive surgeries, which can
accrual significant cost as well as considerable morbidity to patients. Minimally
invasive, cell-based, and/or small molecule based therapies will be a useful
modality to treating these complex and challenging wounds.
Radiation skin injury is described as dose- dependent and time-dependent
loss of cells from the epidermis and dermis and is further characterized as acute
and chronic injury.
2,3
The acute injury phase manifests within the first 6 weeks
and has phenotypical characteristics of erythema, edema, and wet and dry
desquamation.
4-6
The mechanism of acute injury is the generation of free radicals,
which leads to dsDNA breaks, mitotic failure, and apoptosis of the cell. Acute
injury is typically reversible as repair mechanisms are activated to repair DNA

11
damage.
4,7,8
On the other hand, chronic effects can manifests months to years
after the radiation exposure
9
and are typically progressive and permanent.
5,7

Chronic injury can affect all cell types including resident stem cells that normally
regulate tissue homeostasis and cellular milieu.
10
Phenotypic characteristics of
chronic injury include pigmentation changes, atrophy, fibrosis, and loss of
adnexal structures in the skin and can progress to ulceration, frank necrosis, and
tumorogenesis.
3,4,6,11

Recent clinical reports have highlighted the utility of using lipo-aspirated
fat for the treatment of radiation-induced non-healing wounds.
12-14
Rigotti et al
reported that transplantation of a autologous lipo-aspirated fat from a distant
uninjured site could significantly improve non-healing, radiation-damaged
wounds.
12
Sultan et al showed that successful autologous fat grafting from
humans alleviated radiation skin injury in a murine model.
15
Fat grafting was
found to attenuate inflammation in acute radio-dermatitis and slow the
progression of fibrosis. While the precise mechanism of this treatment modality is
not well defined, many hypothesize that it is the Adipose-Derived Stem Cells
within the adipose tissue that serve as the biological stimulus for improved
wound healing. ADSCs are an ideal source for derivation of adult stem cells
because they are easily accessible via liposuction techniques and can be
harvested with limited donor site morbidity.
16
In addition, ADSCs are abundant,
have multipotent differential potential, can be safely delivered to autologous as
well as allogenic recipients
16-19
and have immunomodulatory properties.
20-23
If
radiation damaged tissue can indeed be repaired by ADSCs, this could make

12
high-risk surgical procedures unnecessary thereby conserving healthcare
resources as well as limiting patient morbidity.

13
CHAPTER 2
2.1 Scientific Aims
The aims of this study were to develop a reproducible pre-clinical small
animal model of radiation-induced wound delay and to assess whether ADSCs
delivered topically in a biological matrix could rescue subsequent chronic
radiation-induced skin injury and impaired wound healing. Furthermore, we
wished to achieve a better understanding of the mechanism by which ADSCs
might mediate these beneficial effects.

2.2 Materials and Methods
The experimental protocol was approved by the Institutional Care and Use
Committee and Radiation Safety Office at the University of Southern California
(IACUC protocol number 11464) and was in agreement with the National Institute
of Health Guide for Care and Use of Laboratory Animals. Prior to irradiation, mice
were anesthetized by intraperitoneal injection of ketamine (100mg/kg) and
xylazine (10mg/kg). A Varian Trilogy Irradiator (Palo Alto, California) was set a
6MEV at a source to surface distance of 100cm. A 1.0cm bolus tissue equivalent
material was used to ensure a maximum penetration of 0.5cm, limiting radiation
to the skin. To generate a murine model of chronic radiation-induced wound
delay, an 8 x 4 cm field on the dorsal, lateral surface of Balb/c mice (6-8 wks old)
was irradiated with a single dose of 10Gy (Figure 1A, B). The animals were then
allowed to recover for 6 weeks and were housed at the University of Southern
California animal facility where they were fed a standard laboratory diet, and

14
monitored daily.

Cell Harvest
Adipose Derived Stem Cells (ADSCs) expressing Green Fluorescent
Protein (GFP) were isolated from the inguinal fat pads of FVB.Cg-Tg(CAG-
EGFP)B5Nagy/J mice (Jackson Laboratories, Sacramento, CA) using previously
described protocols.
24
Briefly, the fats pads were minced into fine pieces and
washed in serial dilutions of betadine. The fat was then digested with .075% type
II collagenase (Sigma-Aldrich, St Louis, MO) in Phosphate Buffered Saline (PBS)
for 30 minutes in a shaking water bath at 37C. The collagenase was then
neutralized with equal part of cell culture media (DMEM, 10% Fetal Bovine
Serum (FBS), 1% penicillin/streptomycin) and centrifuged at 1200g for 5 minutes.
The supernatant was discarded and the cell pellets were resuspended in media,
and filtered through a 50-um cell strainer and plated at 3 x 10
6
cells per 100cm
2

culture plate in standard media. The primary cells were cultured from 5-7 days.
Only early passage (P1-3) ADSCs were used for cell application in all
experiments.
As a control, primary mice fibroblasts were isolated from the dorsal skin of
6-8 week old Balb/c mice (Harlan Laboratories, Hayward, CA) by mincing full
thickness skin into small pieces, and washing in serial dilutions of betadine. The
skin was then incubated for 30min in 0.25% trypsin (Invitrogen, Grand Island,
NY), in incubator at 37C in an atmosphere of 5%CO2 of humid air. After trypsin
digestion the dermis was separated from the epidermis, and the dermis was the

15
digested with enzyme solution (HEPES containing RPMI supplemented with I
mM sodium pyruvate, 2.75mg/mL bacterial collagenase, 1.25mg/mL
hyaluronidase, and 0.1 mg/mL Dnase I) for 3 hours. After digestion, the solution
was neutralized with equal parts of standard culture media (DMEM, 10%FBS and
1% P/S), filtered through nylon mesh to remove cellular debris and centrifuged at
1200g for 10 minutes. The supernatant was discarded and the pellet was
resuspended in culture media (DMEM, 10% FBS, 1% P/S). The cells were then
plated and plated at 3 x 10
6
cells per 100cm
2
culture plate in standard media
(DMEM, 10% FBS, 1% P/S, L-Glutamine). Only early passage (P1-3) fibroblasts
were used for cell application.

Cell Culture
ADSCs and fibroblasts were cultured in Dulbecco’s Modified Eagle’s
Medium (Invitrogen, Grand Island, NY) supplemented with 2mM L- glutamine,
10% FBS, 1% of penicillin- streptomycin. All cells were maintained at 5%
CO2/95% air at 37 C in a humidified. For in vivo application the cells were
resuspended in transglutaminase (Tg) gel for topically delivery as previously
described.
25

Flow Cytometric Analysis of ADSCs
The ADSCs were processed for standard Flow Cytometric Analysis
(FACS) for mouse ADSC cell surface markers CD105 (ab93567, Abcam,
Cambridge, MA) and Sca-1 (ab51317, Abcam, Cambridge, MA) and analyzed

16
using a SORP FACS Aria II cell sorter (BD Bioscience, San Jose, CA). The
ADSCs were stained for the cell surface markers following the manufacturers’
recommendation (www.abcam.com).

Wounding and topical cell delivery protocol
Six weeks post-irradiation mice weighing 28-30g were anesthetized with
inhaled isoflurane. The irradiated side of the animal was shaved and the
remaining hair removed by depilatory cream. An 8mm circular wound was made
through the skin and panniculus carnosus muscle on the dorsal lateral irradiated
side of the 6-8 week old Balb/c female mice (Harlan Labs, Hayward, CA), utilizing
the stented wound model.
5,26
After the wound was created, mice received topical
delivery of GFP+ ADSCs in 0.025% Tg gel (1 x 10
5
or 1 x 10
6
ADSC cells/ 50ul of
gel), fibroblasts (1 x10
5
FBs cells/50ul) in Tg gel or 50ul of Tg gel alone without
cells. The rate of wound closure was measured at different time points post-
application, using the Image J program. At varying time points the mice were
euthanized by CO
2
inhalation and the wounds were excised and either formalin
fixed, or stored at -80C for further molecular analysis.

Immunohistochemical Analysis
5um sections of paraffin embedded skin samples were subjected to
immune histochemical staining for CD31 (ab28364, Abcam, Cambridge, MA),
Cytokeratin 14 (SC 17104, Santa Cruz Biotechnology, Santa Cruz, CA ), RELM

17
alpha (ab39626, Abcam, Cambridge, MA) or GFP (NB600-303, Novus
Biologicals, Littleton, CO) following the manufacturer’s recommendations.
Briefly the sections were deparaffinized and subjected to antigen retrieval in
10mmol/L citrate buffer (pH 6.0) followed by overnight incubation with primary
antibodies for CD 31 (1:200), Keratin 14 (1:50 ) or GFP (1:1000) at 4C in
blocking buffer. After the incubation sections were washed and incubated with
appropriated biotinylatated secondary antibody at 1/200, followed by incubation
with alkaline phosphatase-conjugated streptavidin and DAB (BD Bioscience, San
Jose, CA) following the manufacturer’s recommendations. The immune-stained
slides were analyzed qualitatively at magnification of 10x and 20x to determine
the location and presence of the delivered cells within the wound boundaries.
Quantitative analysis of stained cells was performed at 40x magnification
determining an average of cells per 5 high-power-fields (HPFs). Secondary
antibody without primary antibody application was used as negative controls. In
addition tissues not treated with GFP-labeled ADSCs also made negative control
slides for GFP+ ADSCs localization, to ensure the specificity of the GFP antibody
on the mouse skin.

RNA isolation and cDNA synthesis from mouse skin
At various time points, the entire wound area was excised from control
and treated animals. Total mRNA from mouse skin was isolated using the
RNeasy Isolation Kit (Qiagen, Valencia, CA) following the manufacturer’s
instructions with the following modifications: the skin was completely

18
homogenized before extraction procedure to proceed using Prolytron
homogenizor (VWR International, San Dimas, CA). Total mRNA amount was
measured using Nanodrop. Two hundred nanograms (200ng) of total RNA with
OD 260/ 280 > 2.1 was used for first-strand cDNA synthesis. cDNA was
synthesized with Superscript II (Invitrogen) using anchored Oligo (dT12-18)
primers (Invitrogen) and following the manufacturer’s instructions.

Quantitative Real Time -PCR
The quantification of all gene transcripts was performed by reverse
transcription of total RNA followed by absolute real-time quantitative RT-PCR
using SYBR Green PCR Master Mix (Applied Biosystems, Foster City,CA).
Absolute quantification using this method is described elsewhere.
27,28
Primers for
absolute real-time PCR were designed using Primer Express program (Applied
Biosystems, Foster City, CA) and derived from mouse sequences found in
GeneBank. A primer matrix was performed for each gene tested to determine
optimal concentrations. Each reaction mixture consisted of 1uL of cDNA,
optimum concentration of each forward and reverse primer, nuclease-free water,
and SYBR Green PCR Master Mix in a total reaction volume of 7 ul (96-well
plates). Reactions were performed in triplicate for each sample using an ABI
Prism X Sequence Detection System (Applied Biosystems, Foster City, CA). The
thermal cycle consisted of 40 cycles of 95C for 15 s and 60C for 1 min. Standard
curves for each gene and controls were constructed using tenfold serial dilution
and run on sample plates as standards. For gene expression normalization the

19
expression levels of b-actin mRNA was used. Copies of β-actin in each pool were
determined using standard curves constructed from the serial dilution of the
cDNA samples. Representative R2 for β-actin and the rest of the genes tested
were estimated and only the one ≥ 0.98 used. For each measurement, threshold
lines were adjusted to intersect amplification lines in the exponential portion of
amplification curve.
29

In Vitro Analysis
For in vitro migration assay mouse fibroblasts (P 3-4) were irradiated with
10Gy of irradiation in culture using the XRAD 320 irradiator (Precision XRAY,
North Bradford, CT). The Irradiated fibroblasts were subsequently seeded at
equal densities of (0.3 x 10
6
cells) into 6 well plates in standard culture media
(DMEM, 10% FBS, 1% P/S) and maintained at 37C and 5% CO2 until the plates
reached 90% confluency. A scratch defect was induced with a 1000um pipette tip
in each well of the 6 well plate, creating a defect of approximately 900um in
width.
30
The cells were then washed with PBS and were exposed to: (i) ADSCs-
conditioned (conditioned for 72 hours in DMEM, 2% FBS, 1% P/S), (ii) FB-
conditioned (conditioned for 72 hours in DMEM, 2% FBS, 1% P/S), or (iii)
standard non-conditioned media (DMEM with 2 % FBS and 1%P/S). Photos were
taken every 12 hours using the Zeiss Microscope (Thornwood, NY) with 4x
inverted phase lens. The closure of the defect was measured with Zeiss
Axiovision software (Thornwood, NY).

20
The effect of the conditioned media on the proliferation of irradiated
fibroblasts was assayed using the Click-it EdU Flow Cytometry Assay kit with
Alexa Fluor 647 azide (C10424, Invitrogen, Grand Island, NY) following the
manufacturer’s recommendations. Briefly, irradiated fibroblasts (P 3-4) were
seeded at equal densities (0.3 x 10
6
) in a 6 well plate in standard culture media
(DMEM,10 % FBS,1 % P/S) and maintained at 37C at 5% CO2. When the cells
reached 60% confluency, the media was removed, cells were washed with PBS
and media was replaced with (i) ADSCs-conditioned media, (ii) FB-conditioned
media, or (iii) standard medium. All treatment groups were incubated with 10uM
EdU for 24 hours. After 24 hours the cells were harvested following the
manufacturer’s protocol and flow cytometry was performed using 633/635nm
excitation wavelength with a red emission filter to assess for dye incorporation
into the proliferating cells.

2.3 Statistical Analysis
To determine differences between experimental groups we utilized the
Student’s t-test for comparison of two variables or one-way analysis of variance
test for the comparison of multiple variables. All values were calculated as a
mean and standard error a P-value < 0.05 was considered statistically significant.

21
2.4 Results
Characterization of radiation-induced wound healing delay
After creating full-thickness skin wounds at 6 weeks post-exposure to 10
Gray (Gy) of radiation, wound healing was found to be delayed in the irradiated
group. Statistically significant differences were observed after post-operative day
(POD) 18 (Figure 1C, D). Histological analysis revealed that re-epithelization of
the irradiated wounds was significantly impaired and there was an increased
inflammatory infiltrate in the wound bed (Figure 2A). Previous studies have
demonstrated that expression of TGF- βand Smad-3 are markedly increased in
chronic radiation skin injury.
5,31,32
To validate our radiation model, expression of
these genes was assessed using quantitative Real Time PCR (qRT-PCR) on
skin from non-irradiated and irradiated animals (Figure 2B). Gene expression in
skin 6 weeks post-radiation exposure revealed an almost 4-fold up-regulation of
TGF- βexpression in the irradiated group in comparison to the controls. In
addition Smad3 expression was also up-regulated 4-fold in the radiated group in
comparison to the control (p value= 0.003) (Figure 2B). This demonstrates that at
histological and molecular levels, 10 Gy of radiation caused significant
impairment of wound healing in mice, which was analogous to bystander injury in
human radiated skin as a consequence of therapeutic radiation exposure.

Characterization of ADSCs via FACS
The collection of the stromal vascular fraction of centrifuged adipose
tissue contains a mixed progenitor cell population that has multi-lineage

22
differentiation potential. The mixed population of cells that adheres to tissue
culture plastic are commonly referred to as ADSCs.
16,33-35
To better characterize
these cells, we assayed them for expression of two well-established mouse
mesenchymal stem cell surface markers CD 105 and Sca-1. Florescent Activated
Cell Sorting analysis showed that 50% of cells were CD105+ and Sca-1+ (Figure
3A). Thus this data indicated that our stem cell population indeed consisted of
ADSCs.

Topical Delivery of ADSCs results in ADSCs incorporation into the Epidermis and
Dermis.
The ADSCs were delivered topically in a novel transglutaminase gel (Tg)
matrix used previously by our group. Tg gel is an attractive delivery vehicle
because its adhesive properties allows localization of the cells and prevents
scattering outside of the wound after delivery.
25
Moreover, the cells’ migration
from the Tg gel is not stochastic but occurs at a rate inversely proportional to the
concentration of the gel. Previously, the gel was shown to support the growth and
proliferation of mesenchymal stem cells in vitro and in vitro.
25
In the current study,
immunohistochemical analysis of the healing wounds revealed the presence of
GFP+ ADSCs incorporated into the wound after topical application. GFP+
ADSCs were seen in both the epidermis and dermis and this persisted for more
than 3 weeks post-administration. (Figure 3 B). Thus these data support the
notion that Tg gel is an effective delivery vehicle for topical application of ADSCs.

23
Topical Delivery of ADSCs Improves Wound Healing in Irradiated Skin.
Statistically significant differences in wound closure were observed at post
operative day 18. In-depth analysis of wounds was performed on wound biopsies
from this time point. At POD 18, irradiated wound beds had 2.5x greater wound
closure with a single application of 1x10
5
ADSCs (p= 0.008). A ten-fold increase
in dosage of 1 x 10
6
ADSCs led to a similar effect on wound closure (p=0.007)
(Figure 4 A). By POD 26 the radiated wounds with ADSCs application resembled
that of control animals that did not receive any radiation (Figure 4 B).
Interestingly, there was no significant difference in wound closure in non-
irradiated animals, with or without the application of ADSCs (Figure 4 A, B).
We sought to determine if the delivery of primary fibroblast cells would yield
similar effect on wound closure to that of ADSCs application. Delivery of 1x10
5

fibroblast cells in Tg gel did result in 2x greater wound closure in irradiated
animals in comparison to irradiated animals without cellular therapy (p= 0.003)
(Figure 4 C,D). However the rate of wound closure upon ADSCs application was
significantly greater (p=0.011) than the fibroblast cells alone (Figure 4 C, D).

Topical Application of ADSCs improves epithelization and modulates the immune
response
Histological evaluation of the irradiated wounds with 1 x 10
5
ADSC
application, demonstrated improved epithelization (Figure 5A) and better
organization of the epithelium as shown by Keratin 14 staining (Figure 5B).
Although our radiation delayed wound healing model displayed an improved re-

24
epithelization, ADSC therapy did not reveal any differences in vascularization
based on staining for CD31, an endothelial cell specific marker (Figure 5C).
A recent study published in 2010 by Zhang et al demonstrated that MSC aided in
wound healing by inducing polarization of M1-macrophages into M2-
macrophages thus leading to a release of anti-inflammatory cytokines.
36
However
conflicting results were demonstrated in a more recent study, in which M2-
macrophages applied topically to wounds were detrimental to wound healing.
37
In
our models we found that M2- macrophages were 3 times less predominate (p=
0.03) in the dermis of irradiated wounds treated with ADSCs, and thus ADSCs
may facilitate less recruitment of the cells to the area (Figure 5C,D).

ADSCs application down regulates TGF-β Expression in Irradiated Wounds
Transforming growth factor -β (TGF-β) has been shown to play a key role
in radiation-induced inflammation and fibrosis in vivo. It regulates both the
accelerated synthesis and reorganization of the extracellular matrix,
and the increased destruction of epithelial and hematopoetic cells present in the
targeted radiation field.
38
Smad3 is a TGF-β -dependent nuclear transcription
factor and is also reported as a key mediator in the fibrotic signaling pathway.
Moreover Smad3-/- mice exhibited reduced radiation skin damage.
31,32

Thus we analyzed the expression of TGF-β and Smad3 in irradiated wounds at
day 18 between ADSCs treated and no cells treated wounds. Our data revealed
a down regulation of TGF- β at day 18 in irradiated wounds with ADSCs
application in reference to no cell treatment (p=0.04) (Figure 6A). There was no

25
significant difference in TGF- βexpression in the fibroblast control group. This
data demonstrates that specifically ADSCs effects on wound healing are in part
mediated by down regulation of TGF- βin irradiated skin. Interestingly, there was
no statistically significant difference in Smad-3 expression at day 18 (Figure 6B).

ADSCs Conditioned media enhances Irradiated FB Proliferation and Migration in
Culture
To evaluate the contribution of ADSCs secretion of trophic factors in
modulating wound healing effects an in vitro scratch and proliferation assay was
performed using serum reduced ADSCs and primary fibroblasts (FB) conditioned
media in comparison to standard serum reduced DMEM media (Figure 7). Serum
reduced ADSCs-conditioned media as well as FB-conditioned media lead to
faster closure of the scratch defect of irradiated fibroblasts in culture after
48hours in comparison to standard serum reduced DMEM media (Figure7A).
However there was no significant difference in the closure between ADSCs and
FB-conditioned media (Figure 7B). Next we evaluated the effect of ADSCs and
FB-conditioned media and standard DMEM media on the FB proliferation. Our
results demonstrated that an addition of ADSCs and FB-conditioned media also
leads to increased proliferation of irradiated fibroblast in comparison to standard
DMEM, however the increase was not statistically significant (Figure7C). This
data indicates that both ADSCs and Fibroblasts release trophic factors that may
play a beneficial role in wound healing (Figure 7).

26
2.5 Discussion
The exact mechanism of chronic radiation injury has not been well defined
in the literature however many studies have revealed persistent up-regulation of
many inflammatory cytokines post-radiation exposure.
8,39,40
The most well
studied pathway leading to tissue fibrosis is TGF-β and its downstream mediator
Smad3.
5,8
TGF-β is one of the most important signaling molecules in wound
healing.
41
It is released by local inflammatory cells such as lymphocytes,
monocytes /macrophages, and platelets in the wound bed
42
and promotes
fibroblasts differentiation into myofibroblasts leading to extracellular matrix
deposition and granulation tissue formation.
8,41-43
Released TGF-β can also
stimulate cells to enhance its secretion in an auto-regulatory fashion
43
thus
leading to tight regulation of expression. Under physiological conditions the
expression of TGF-β is initially up-regulated in the inflammatory phase and down-
regulated during the remodeling phase of wound healing.
41
However in a setting
of chronic radiation injury, TGF-β is persistently up-regulated, extending well
beyond the remodeling phase.
2,32
Our study demonstrates that ADSCs rescues
chronic radiation effects on wound healing in part by down regulating TGF-β.
This is consistent with previous studies in which TGF-β receptor II knock-out
animals lead to faster re-epithelization and wound closure as well as deceased
inflammation during wound healing.
43

It appears that ADSCs through unknown mechanisms, can sense
dysregulation in TGF-β pathway and release factors that restore the normal
milieu and homeostasis. In theory the use of cellular therapy may be more

27
beneficial to protein therapy because it is responsive to the body’s normal
feedback mechanisms, thus utilizing natural processes to normalize secretion of
the “dysregulated” protein. Animal studies, in which TGF-β application was used
for the treatment of wounds, demonstrated that at low amounts TGF-β may be
beneficial in wound healing in both normal and irradiated conditions.
44,45
On the
other hand, too much TGF-β can be deleterious
5,41,42,45
and thus genetic
variations between individuals would make it difficult to establish a universal,
optimal concentration of protein in all individuals. Therefore cellular therapy may
be a more ideal and feasible therapeutic option.
It is clear that TGF-β regulates pathologic fibrosis as it is up-regulated in
Angiotensin II-induced cardiac fibrosis, bleomycin-induced lung fibrosis,
interstitial renal fibrosis, scleroderma and other debilitating conditions.
46-50
In
particular, the cutaneous wound healing effects of TGF-β has shown to be
mediated in part by Smad3 signaling, as studies have demonstrated that the
absence of Smad3 leads to improved epithelization and decrease in
inflammation
51
and Smad3 knock out animals were resistant to the chronic
effects of radiation.
31,32
In addition, Lee et al showed that the use of Smad3
siRNA prevents the delirious effects of radiation on the skin.
38
While our data did
not demonstrate significant changes in Smad3 expression by day 18, an
explanation for this finding is that since Smad3 is a downstream mediator of
TGF-β, it may be too early to see corresponding changes in Smad3 expression
at this time point. However it may indicate that non-canonical TGF-β signaling
pathways may also play a role in pathologic fibrosis, which has been suggestive

28
in other studies.
48

Interestingly our data demonstrate no significant acceleration of wound
healing in non-irradiated animals with cellular treatment. While there is clear
support that Mesenchymal Stem Cells (MSCs) therapy can aid in pathologic
wound healing, there is conflicting evidence in the literature of whether adipose
stem cell therapy has the same effects in physiologic wound healing. Lee et al
demonstrated that the wound healing effect of Adipose Derived Stem Cells was
significantly “enhanced” specifically under hypoxic conditions in a murine wound
model.
52
On the other hand, Ebrahimian et al demonstrated that ADSCs aid in
both pathologic and physiologic wound healing by accelerated wound closure,
increasing viscolesticity and promoting angiogenesis.
53
In addition other literature
supports the view that ADSCs proliferation and functions are enriched under
pathological conditions.
24,54
Therefore more investigation is warranted to further
deduce their utility under both conditions.
The immunomodulatory properties of ADSCs are now being elucidated and
it has recently been discovered that ADSCs can downregulate activating cell
surface marker on dendritic cells (DCs) and in turn inflammatory cytokine
secretion of IL-12 and TNF-α. In addition they can induce a secondary immune
response as ADSCs treated DCs can downregulate CD4+ T cells, thus inhibiting
IL-2 and IFN-γ secretion.
21
Others studies also support direct effects of ADSCs in
down-regulating of T helper cells.
22
Interestingly it has been demonstrated that
the immunosuppressive properties of ADSCs are stimulated in inflammatory
conditions, which in turn can downregulate expression of pro-fibrotic factors in a

29
mechanism that is not completely known.
23
While previous data demonstrated
that MSCs derived from other tissue could have direct effects on macrophages,
there is no published data investigating ADSCs effects on macrophage
function.
36,55
M2-macrophages are thought to have primary anti-inflammatory
properties.
56
Interestingly our data demonstrated that ADSCs leads to decreased
M2-macrophage recruitment as well as inflammation, which seems inconsistent.
However previous literature demonstrated that M2-macrophages leads to a pro-
fibrotic response and plays a role in pathologic pulmonary, cardiac and renal
fibrosis and inhibition can suppress these fibrotic processes.
48,57-61
M2-
macrophages are also potent secretors of TGF-β
58,62
as well as released TGF-β
further stimulates M2-macrophage recruitment which may further potentiate
secretion of fibrotic factors.
63
Our data demonstrates that ADSCs treated wounds
lead to down-regulation of pathologic TGF-β, as well as decreased M2-
macrophage recruitment which is suggestive that M2-macrophage may mediate
radiation-induced tissue fibrosis as well. Interestingly, Chiang et al demonstrated
that radiation-induced hypoxia creates a unique tumor microenvironment that
favors M2-macrophages.
64
In addition, M2-macrophages secrete arginase-I,
which lead to the production of ornithine and polyamines. These molecules are
precursors of collagen synthesis
62
, thus increased M2-macrophage presence in
the wound bed can result in over production of collagen leading to the fibrosis
that is seen in chronic radiation injury. However still the initiating signal mediating
TGF-β and M2 macrophage recruitment remains to be determined.
ADSCs hold promise in regenerative medicine and tissue engineering they also

30
proving to be valuable in modulating the immune response and it may prove to
be a more feasible and an efficient vehicle for stem cell therapy. While the
applications of ADSCs is a novel emerging field, it has already shown to aid in
improving cerebral ischemia
65
critical sized calvarial defects
66
, and hind limb
ischemia in animal models
67
. ADSCs have been already used in clinical trials for
treatment of diabetic wounds
68
, chronic fistulas in Crohn’s disease
69,70
, critical
limb ischemia and many others diseases.
33
Since hypertrophic scars and keloid
formation are in part due to the exaggerated expression of TGF-β and
inflammation, ADSCs therapy may play a useful role in these pathological
conditions as well, in which studies are yet to be initiated in these fields.
In conclusion, in this study we have established a reproducible pre-clinical
model of radiation-induced delayed wound healing in Balb/c mice. We have also
demonstrated that purified ADSCs delivered topically in a biologic matrix
significantly accelerated closure in wounds in chronically irradiated animals
compared to the controls in part by down regulating TGF-β, decreasing
inflammation, suppression M2-macrophage recruitment and releasing trophic
factors. However the exact signaling pathway leading to TGF-β overexpression
and M2-macrophage recruitment still needs to be defined. We are hopeful as we
collect more evidence, that this data will serve as an IRB-approved clinical trial in
patients with non-healing radiation induced wounds. This technique would be a
viable alternative to major surgical debridement and reconstructive procedures
that subject patients to unnecessary risk and morbidity.

31
CHAPTER 3
Future Directions
While there is convincing evidence that chronic radiation skin injury can be
in part rescued by ADSCs, the exact mechanism of chronic radiation skin injury
still needs to be elucidated. Radiation therapy is a standard mode of primary or
adjuvant treatment for various types of cancer and therefore bystander injury to
the skin poses a significant problem. As indicated earlier, there is literature
demonstrating a persistent up-regulation of inflammatory cytokines in chronic
radiation skin injury that clearly contribute to the disease process. An increased
inflammatory infiltrate is seen in these wounds thus indicating the mechanism of
injury is multi-factorial and not solely due to depletion of resident stem cells in the
microenvironment.
Radiation induces dysregulation of normal signaling pathways in cells. It
has been demonstrated that when these abnormal cells proliferate their progeny
acquire this abnormal phenotype as well
71
, indicating that these changes are
heritable and affect the genome of the cell. The heritable changes in the genome
can result from alteration of the DNA structure such as genomic mutations or
from modification in the methylation status of the DNA that do not alter the
genetic code but have influence on the phenotype. The last are known as
epigenetic modifications that although not altering the genetic sequence, have
the potential to modify gene expression. Numerous studies suggest that radiation
exposure can leads to epigenetic changes in cells, however this mechanism still
remains to be proven.
72,73
Thus, we hypothesize that epigenetic changes such as

32
hyper- or hypomethylation of CpG islands in promoter regions of DNA, in part
contributes to altered gene and protein expression that lead to the persistent
changes in radiation-induced chronic skin injury.
We have created a bank of irradiated and normal human skin samples
under an IRB approved protocol from patients requiring reconstructive surgery for
chronic wounds induced by radiation. Using established protocols, an Illumina
Infinium450k DNA methylation analysis was performed on paired skin samples
from radiation damaged and normal skin. Quantitative Real Time PCR (qRT-
PCR) on selected loci was performed to determine if altered methylation status
translated into changes in gene expression. We investigated candidate genes for
further analysis and western blot was performed on the skin samples to assess
whether gene expression changes correlated to differences in protein expression.
In addition, immunohistochemical analysis (IHC) was performed on the candidate
proteins in order to determine location of expression.
We identified 478 genes were differentially methylated in radiated versus
control skin. Of them, 30% were found in the promoter region of the DNA. qRT-
PCR analysis confirmed that the methylation changes in the promoter regions
corresponded to alteration of gene expression. We found 5 relevant genes that
had significant methylation changes in radiated vs. non-irradiated skin: HOX C4,
THBS1, CMYA5, EFS and JDP2. Western blot has confirmed that protein
expression corresponded to changes in gene expression. In addition we found
that expression of some of the dysregulated protein was localized to the basal
layer of keratinocytes.

33
Our preliminary data demonstrate that there are epigenetic modifications,
specifically methylation of promoter regions, in radiated compared to normal skin.
Interestingly, we found a specific histone deacetylase (HDAC) recruiter that was
found to be significantly over-expressed in radiated skin. Of note previous
literature has indicated that HDAC inhibitors are beneficial in the treatment of
chronic radiation-induced wounds.
74,75
Thus epigenetic changes may contribute
to the pathophysiology of chronic radiation skin injury and thus a potential
therapeutic target. We plan to continue characterizing these genes and
determine their role in radiation skin injury by gene knock out experiments as well
as transgenic knockout animals in the setting of our radiation model.

34

Figures:

Figure 1
A) Schematic diagram of the experimental outline.
B) The illuminated area represents the radiation field on the dorsal, lateral
surface of Balb/c mice.
C) Graphical representation of delayed wound healing after exposure to10Gy of
radiation.
D) Phenotypic appearance of healing wound in non-irradiated animals (a)
compared to radiated (b) animals at post-operative day 18.

A)
B)
C)
D)
a) b)
% Defect

35

Figure 2
A) Histological analysis of non-irradiated wounds at 10X a) and 20X
magnifications(c), compared to radiated wounds at 10X (b) and 20X (d)
magnifications.
B) Quantitative Real Time PCR (qRT-PCR) for TGF-β (a) and Smad-3 (b)
expression in skin from non-irradiated and radiated animals.

A)
B)
a) b)
c) d)
a) b)
Relative RNA Abundance
Relative RNA Abundance

36

Figure 3
A) Florescent Activated Cell Sorting (FACS) analysis for expression of two well-
established mouse mesenchymal stem cell surface markers, Sca-1 (a), CD 105
(b) or Sca-1 and CD105 double positive cells (c).
B) Immunohistochemical analysis for the presence of GFP positive ADSCs
incorporated into the wound bed after topical application; a) negative control, b)
POD7, c) POD18, d) POD26.

A)
B)
a) b)
c)
d)
a) b) c)

37

Figure 4
Quantification of wound closure at POD 18 A) and POD 26 B) with or without a
single application ADSCs.
C) Phenotypic appearance of healing wounds in radiated animals at POD18 with
no cells application (a) fibroblasts application (b) or ADSCs application(c).
D) Quantification of the percentage of wound closure in radiated wounds without
cell treatment compared to fibroblasts and ADSCs treated groups.

D)
B)
C)
A)
a) b) c)
%
Closure
%
Closure
%
Closure

38

Figure 5
A) Histological evaluation of the radiated wounds at POD 18 without cell
application at 10X (a) and at 20X (c) magnifications compared to radiated
wounds with ADSC application at 10X (b) and 20X (d) magnifications.
B) Keratin14 immunohistochemical staining of radiated wounds without ADSCs
(a) compared to with ADSCs (b)
C) CD31 immunohistochemical staining of radiated wounds without ADSCs (a)
compared to with ADSCs (b)
D) M2- macrophages immunohistochemical staining in radiated wounds without
ADSCs (a) compare to with ADSCs (b) at 40X magnification.
E) The averaged macrophage number counted per 40X high power field (HPF) in
radiated wounds without ADSCs (a) compared to with ADSCs treatment (b).

A)
B)
C)
D)
E)
a) b)
c) d)
a) b)
a) b)
a) b)
Average cells per 5 HPFs

39

Figure 6
qRT-PCR of TGF-b (A) and Smad3 (B) expression in radiated wounds at day 18
between the treatment groups.

A)
B)
Relative RNA Abundance Relative RNA Abundance

40

Figure 7
A) In vitro scratch assay to assay for the migration of irradiated fibroblasts upon
addition of 2% DMEM at time 0 (a) and 48hrs (b) compare to 2 % fibroblast
conditioned media at time 0 (c) and at 48hrs (d) and 2% ADSCs conditioned
media at time 0 (e) and at 48hrs (g).
B) Quantification of the scratch assay by measuring cell migration. Cell migration
was quantified at 0hr, 12hr, 24hr, 36hrs and 48hrs post addition of the control
and conditioned media.
C) Quantification of the proliferation capacity of irradiated fibroblasts upon
addition of 10% DMEM versus 10% FB and 10% ADSCs conditioned media.

% Closure
A)
B)
C)
a) b)
c) d)
e) f)
Time 0 Time 48
% Proliferating Cells

41

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Abstract (if available)

Abstract Recent literature supports the notion that Adipose Derived Stem Cells (ADSCs) in lipoaspirated fat could significantly improve radiation-induced wounds. However the mechanism of action is not completely understood which has limited clinical applicability of this technique. Therefore, we sought to develop a murine model of chronic radiation skin injury and to have a better understanding of how ADSCs exerts these beneficial effects. ❧ We generated a murine model of chronic radiation-induced skin injury in Balb/c mice. Animals were treated topically with ADSCs in a transglutaminase (Tg) gel matrix. Fibroblast (FB) cells in the Tg gel or Tg gel alone without cells were used as controls. Alterations in pathologic signaling molecules and immune responses were evaluated. In addition, in vitro analysis was performed to investigate paracrine influences. ❧ Wound closure was significantly impaired after radiation exposure and demonstrated pathological up-regulation of TGF-β, which is the hallmark finding of chronic radiation injury. Topical delivery of ADSCs to radiated wounds resulted in significantly accelerated wound closure compared to fibroblasts and no cells, respectively. Interestingly no difference in wound closure was observed after ADSCs treatment under physiologic or normal wound healing conditions. ADSCs application reduced pathologic expression of TGF-β in healing radiated wounds. In addition, ADSCs treatment lead to less recruitment of M2-macrophages in the wound bed of ADSCs treated irradiated wounds. In vitro analysis revealed that soluble factors also mediate beneficial effects in wound healing. ❧ Thus, purified ADSCs delivered topically in a biologic matrix significantly accelerated wound closure specifically in irradiated animals. Based on our data we propose that ADSCs, in part, exert their beneficial effects by suppressing the pathological TGF-β expression, decreasing M2-macrophage recruitment and releasing soluble mediators that aid in ameliorating the deleterious effects of radiation.

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Asset Metadata

Creator Davis, Gabrielle B. (author)

Core Title Topical adipose-derived stem cell therapy ameliorates radiation-induced delayed wound healing

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 07/30/2013

Defense Date 06/19/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag adipose-derived stem cells,OAI-PMH Harvest,radiation skin injury

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Maxson, Robert E., Jr. (committee chair), Ragina, Neli (committee member), Tokes, Zoltan A. (committee member)

Creator Email gabrielle.davis@med.usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-304435

Unique identifier UC11295000

Identifier etd-DavisGabri-1883.pdf (filename),usctheses-c3-304435 (legacy record id)

Legacy Identifier etd-DavisGabri-1883.pdf

Dmrecord 304435

Document Type Thesis

Format application/pdf (imt)

Rights Davis, Gabrielle B.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA