Leseprobe
iii
ABSTRACT
Conventional medical treatments fail to address the underlying problems
associated with the damage inflicted by a coronary event. Thus, the long-term
prognosis of patients admitted for heart failure is disheartening, with reported
survival rates of 25 percent. Recent advances in stem cell research highlight the
potential benefits of autologous stem cell transplantation for stimulating repair in
heart tissue. However, a majority of those suffering from cardiovascular diseases
are older adults whose autologous cells no longer possess optimum functional
capacity. Additional work is needed to identify the optimal cell types or conditions
that will promote cardiovascular regeneration across all age groups. A
pretreatment, such as short-term hypoxia, and concurrent implementation of a
novel progenitor, such as those that co-express Isl-1 and c-Kit, may enhance the
results reported in clinical trials completed to date. However, the effects of short-
term hypoxia in this novel cell type are unknown and warrant investigation in
vitro.
Cloned adult and neonatal Isl-1+ c-Kit+ human cardiovascular progenitor
cells were characterized and expanded for study. Populations from both age
groups were preconditioned using short-term hypoxia (1% O
2
for six hours) and,
to identify shifts in gene expression, compared to their respective control (21%
O
2
at 37 °C) via qRT-PCR. Flow cytometry and western blot analysis was utilized
to measure phosphorylation of Akt. Progression through the cell cycle was also
iv
analyzed by flow cytometry. Cellular function was evaluated by the use of a
TUNEL assay and Transwell® invasion assay.
Hypoxia-mediated alterations of a genetic or functional nature in Isl-1+ c-
Kit+ human cardiac progenitors are clearly age-dependent. Although both age
groups accrued benefit, the neonatal progenitors procured significantly greater
improvements. Short-term hypoxia significantly elevated Akt phosphorylation in
neonatal Isl-1+ c-Kit+ human cardiac progenitors. Benefits afforded to both age
groups by hypoxic pretreatment included significant upregulation of pro-survival
transcripts, and enhanced invasion capabilities in vitro.
Therefore, prior to transplantation, hypoxic preconditioning may improve
the ability of transplanted stem cells to home towards damaged areas of the
heart and support cardiac regeneration in vivo.
v
ACKNOWLEDGEMENTS
I would like to first thank my committee for their constructive comments,
insightful review of my work, and for their dedication to academia. I would like to
thank Dr. Bournias-Vardiabasis for her guidance, support, and for taking notice of
my potential. I would like to thank Dr. Kearns-Jonker for opening her laboratory to
me, for fostering a supportive work environment, and for being an exemplary
mentor. I would like to thank Dr. Thompson for his helpful comments, and for
promoting academic research at CSUSB. I am also grateful to CIRM for
establishing the Bridges Program, and for funding this work.
I am very thankful to have received instruction from the faculty of the
Biology Department at CSUSB. I would like to thank them all for nurturing my
scientific curiosity. I am especially grateful to Dave Coffey for his helpful advice
and for his help in honing my technical skills.
I am also appreciative of the support received throughout my time at Loma
Linda University. I would like to thank Nancy Appleby for going above and
beyond to assist me, for her invaluable instruction, and for being an absolute
pleasure to work with. I also wish to thank Jonathan Baio and Tania Fuentes for
their assistance, and for bringing cheer to the laboratory.
I would like to thank my friends and family, especially my parents for their
unfailing patience and support. I would also like to thank my sisters for imparting
me with the gift of brotherhood, inspiring me to serve as a positive role model,
and for shaping me into the person I am today. I dedicate this work to them.
vi
TABLE OF CONTENTS
ABSTRACT ... iii
ACKNOWLEDGEMENTS ... v
LIST OF TABLES ... vii
CHAPTER ONE: INTRODUCTION ... 1
Background ... 1
Definition of Terms ... 9
CHAPTER TWO: MATERIALS AND METHODS ... 10
In Vivo Components and Experiments ... 10
In Vitro Assays ... 12
Flow Cytometry ... 15
Statistical Analysis ... 17
CHAPTER THREE: RESULTS ... 18
Akt Activation in Isl-1+ c-Kit+ hCPCs ... 18
Isl-1+ c-Kit+ hCPC Function After Exposure to Short-Term Hypoxia ... 20
CHAPTER FOUR: DISCUSSION ... 24
Conclusions ... 30
APPENDIX A: FIGURES ... 32
REFERENCES ... 42
vii
LIST OF TABLES
Table 1. Primer Sequences Used for qRT-PCR
...13
1
CHAPTER ONE
INTRODUCTION
Background
Etiology and Global Impact of Cardiovascular Diseases
Cardiovascular diseases (CVDs) encompass a suite of diseases that
affect the heart and its vessels, the most common disease being coronary artery
disease (CAD). CAD is typified by atherogenesis (plaque accretion) and
subsequent lesion formation, both stenotic and non-stenotic, within the coronary
artery. Stenotic lesions innately restrict blood flow by growing towards the lumen,
but tend to have smaller lipid cores and thick fibrous caps that are not very
susceptible to rupture (Libby & Theroux, 2005). On the other hand, a non-
stenotic lesion develops abluminally and, therefore, does not inherently affect
coronary circulation until the constitutive lipid-rich core and thin fibrous cap are
disturbed, initiating coronary thrombosis and occlusion (Libby & Theroux, 2005).
Despite our comprehensive understanding regarding the etiology of CVDs,
they remain the number one cause of death across the globe
accounting for
approximately one out of every three deaths worldwide (Deaton et al., 2011;
Mozaffarian et al., 2015). Additionally, patients hospitalized for heart failure
typically develop permanent scarring of the heart and significant loss of
cardiovascular function. Traditional treatments fail to address this issue and, as a
2
result, the long-term outlook of these patients is abysmal, with five-year survival
rates lower than that of most cancer patients (Stewart, MacIntyre, Hole,
Capewell, & McMurray, 2001). Public health efforts to curb the prevalence of
CVDs have been largely focused on raising awareness of the many risk factors
that are strongly correlated to the development of CVDs. Indeed, CVDs are
highly preventable. Eliminating major CVD risk factors, which are mainly a result
of poor lifestyle behaviors, results in up to a 90 percent lower lifetime CVD risk
(McGill, McMahan, & Gidding, 2008). Unsurprisingly, as public awareness and
use of evidence-based medical therapies for secondary prevention increased,
the number of deaths in the United States attributed to CVDs (from 2001 to 2011)
decreased by 30.8 percent, yet remained alarmingly high at approximately
600,000 deaths per year (Mozaffarian et al., 2015). Therefore, while much
progress has been made through raising of public awareness, the extreme
burden of CVDs on worldwide human health will likely continue to persist lest
novel treatments are developed that can repair the damaged caused by infarction
and significantly lessen the associated mortality.
Cardiovascular Stem Cell-based Therapy for the Injured Heart
Although cardiac stem cells were known to be prevalent within the
neonate heart, the notion that these cells steadily diminish and senesce into
adulthood was widely accepted and, thus, the adult human heart was designated
a terminally differentiated organ with no regenerative capabilities. With little
evidence to suggest otherwise, early clinical trials for the treatment of myocardial
3
injuries primarily focused on bone marrow-derived cells for stimulating
cardiovascular repair. However, the discovery that the adult human heart indeed
houses populations of stem cells that support its regeneration (Beltrami et al.,
2003), prompted investigators to shift their focus onto transplantation of cardiac
progenitor cells (CPCs) for the treatment of myocardial infarction.
The potential benefits of endogenous CPC transplantation as a means of
stimulating repair in heart tissue are currently under investigation with
encouraging results in clinical trials (Bolli et al., 2011; Gerbin & Murry, 2015;
Makkar et al., 2012). Phase I clinical research studies completed to date,
examining the safety and feasibility of CPC transplantation, have noted little to no
adverse effects resulting from cardiovascular infusion of stem cells, and, thus,
the procedure is considered safe by clinicians. Most notably, investigators have
reported significant reductions in total scar tissue and significant improvements in
left ventricular ejection fraction
progressing even after 12 months post-op (Bolli
et al., 2011; Makkar et al., 2012). However, it has been demonstrated that,
although transplanted CPCs alleviate some cardiovascular dysfunction, they fail
to engraft and differentiate into new myocardium (Hong et al., 2014). Therefore, it
is believed that the observed reductions in scar size and improvements in cardiac
function
typically measured using left ventricular ejection fraction
are a result
of paracrine signaling stemming from growth factors secreted by newly
transplanted cells (Barile et al., 2014). Furthermore, autologous stem cells
4
derived from adult patients are known to lack the functional potency of their
neonatal counterparts (T. I. Fuentes et al., 2013).
Due to the correlation between incidence of CVDs and age, older adults,
by way of sheer numbers, are the group most in need of cell-based cardiac
therapy. If adult autologous stem cells are unable to impart significant functional
benefits after transplantation, then the overall application of autologous stem cell-
based therapies for treatment of cardiovascular injury may be drastically limited
to treating neonatal heart diseases. Even so, stem cell-based therapies for the
treatment of congenital heart diseases (CHDs) are of significant interest to
clinicians. While the number of afflicted patients is relatively small in comparison
to those suffering from adult CVD, CHDs occur in roughly one percent of live
births worldwide and CHD incidence has increased in recent decades (Marelli,
Mackie, Ionescu-Ittu, Rahme, & Pilote, 2007). Indeed, the mortality rate for CHDs
is declining and more of those born with CHD are surviving into adulthood.
However, data gathered between 2007 and 2010 reveals that CHD-related
mortality for neonates is still a cause for concern at 10.1%. In the year 2011
alone, mortality related to CHD in the U.S. was 4900 (Mozaffarian et al., 2015).
Thus, CHDs remain responsible for more deaths than any other congenital birth
defect. All too often, neonates suffering from a particularly severe heart
malformation are in need of a heart transplant. Complete organ transplants are
costly, invasive, and, most importantly, limited by the availability of donor organs.
Hence, there has been a growing interest in stem cell-based therapies for
5
treating CHDs. Clinical trials completed to date have established the safety of
stem cell transplantation for the treatment of hypoplastic left heart syndrome and
other CHDs (Ishigami et al., 2015). However, much like the clinical trials that
established the safety of stem cell therapy for CVDs in adults, further work is
needed to improve post-operative outcomes.
The effectiveness of cell-based treatments for the heart may be
significantly improved by use of a novel cell type or pretreatment method, such
as hypoxic exposure, that alters the function of adult-derived CPCs to mirror that
of the more capable neonatal stem cells. Any benefits afforded to adult CPCs
would also likely be procured by neonatal CPCs. However, additional work is
needed to identify these optimal cell types or conditions that will promote
autologous stem cell-mediated cardiovascular regeneration in the injured adult
and neonatal human heart. To date, several potential preconditioning methods
have been evaluated for their ability to augment cellular function. For instance, it
has been demonstrated that pretreatment with growth factors enhances the
therapeutic efficacy of mesenchymal stem cells for myocardial infarction (Hahn et
al., 2008). Additionally, cobalt protoporphyrin pretreatment protects human
embryonic stem cell-derived cardiomyocytes from oxidative stress (Luo et al.,
2014). However, the benefits afforded by any one preconditioning method may
vary depending upon the characteristics of the cell type chosen for therapy.
Interestingly, there is a growing body of evidence in support of a master heart
progenitor that gives rise to all the cells in the developed adult heart (Kattman,
6
Huber, & Keller, 2006; Laugwitz, Moretti, Caron, Nakano, & Chien, 2007; Wu et
al., 2006). The discovery, characterization, and subsequent implementation of
this primordial heart cell would undoubtedly lead to improved patient outcomes
following cell therapy. To date, several populations of resident CPCs have been
identified within the human heart and are promising candidates for use in future
studies (T. Fuentes & Kearns-Jonker, 2013).
Optimizing Cell Type
CPCs are capable of self-renewal, expansion, and differentiation into all
three major cell types of the heart (Bu et al., 2009; Moretti et al., 2006). The Isl-
1+ c-Kit+ population represents one type of CPC that was initially found
exclusively within fetal progenitor populations (Simpson et al., 2012). However,
Isl-1+ c-Kit+ hCPCs were subsequently identified within endogenous progenitor
populations isolated from human neonatal and adult cardiac tissue, as well as
from sheep cardiac tissue, as reported by the Kearns-Jonker laboratory at Loma
Linda (T. I. Fuentes et al., 2013; Hou et al., 2012). Isl-1 is a transcription factor
that is required early in development for the survival, proliferation, and migration
of CPCs into the primordial heart and, as a result, the developed heart is largely
a product of Isl-1+
cells (Cai et al., 2003). Specifically, Isl-1+ CPCs play a critical
role in early heart formation by contributing to the outflow tract, right atrium, right
ventricle, and septum (Cai et al., 2003; Dodou, Verzi, Anderson, Xu, & Black,
2004; Yang et al., 2013). One of the most widely studied progenitor cell types,
however, is the c-Kit+ CPC. While current clinical trials using c-Kit+ cells show
7
promise, the role of c-Kit+
cells in the development and regeneration of the heart
remains controversial (Cheng et al., 2014; Ferreira-Martins et al., 2012; Kubo et
al., 2008; van Berlo et al., 2014; Zaruba, Soonpaa, Reuter, & Field, 2010). Isl-1+
c-Kit+ CPC populations, by virtue of their distinct protein fingerprint, may be
inherently superior to those CPC populations expressing only Isl-1 or only c-Kit,
and may be better suited for transplantation into the hypoxic heart. Moreover, this
double-positive CPC population may react more strongly to preconditioning
methods (aimed at enhancing cell function) when compared to single-positive
CPCs. Thus, in order to surpass the results attributed to paracrine effects, and
reach the desired levels of cardiovascular repair by direct engraftment, further
investigation of Isl-1+ c-Kit+
CPCs is warranted.
Hypoxic Preconditioning
Using other models, previous studies have established that hypoxia
treatment can boost cellular function through intracellular signaling pathways
(Filippi et al., 2014; Hu et al., 2014; Studer et al., 2000; van Oorschot, Smits,
Pardali, Doevendans, & Goumans, 2011; Yan et al., 2012). However, the effects
of low oxygen tension on Isl-1+ c-Kit+
hCPC function have yet to be elucidated.
Nonetheless, it is well understood that the partial pressure of oxygen in the
tissues where CPCs reside, is much lower than in atmospheric air. Theoretically,
the maximum amount of oxygen that can be bound by hemoglobin in the alveolar
capillaries is approximately 21 percent by volume (mL O2/100 mL blood) or 159
torr. However, because the rate at which hemoglobin binds and releases oxygen
8
is limiting, the maximum amount that can be bound per milliliter of blood is much
lower, approximately 105 torr
. Additionally, Fick's law of diffusion applies at every
point of gas exchange, thus further limiting the amount of oxygen that makes its
way to the deep tissues of the heart per unit of time. Because of these limitations,
by the time oxygen reaches the heart, its partial pressure can be as low as 30
torr (Ivanovic, 2009). Furthermore, the heart houses small microenvironments, or
niches, that support cardiac stem cells and play vital roles in the regulation of
stem cell function (Li & Xie, 2005). While the tissues of the heart may experience
a gradient of partial pressures, the innermost stem cell niches of the heart and
their constituent CPCs are consistently hypoxic
with oxygen levels as low as
1.0 percent, approximately 7.6 torr (Kimura & Sadek, 2012; Sanada et al., 2014;
Tan et al., 2016).
Accordingly, the impact of short-term hypoxia (six hours at 1.0 percent O
2
)
on the in vitro biology of clonal Isl-1+ c-Kit+ hCPCs must be evaluated for
pertinent information that will help optimize future transplant studies within animal
models. Historically, in other cell types, Protein kinase B (Akt) expression
increases in response to short-term hypoxia (Beitner-Johnson, Rust, Hsieh, &
Millhorn, 2001) and is well-known to play vital roles in numerous cell functions
including cell survival, proliferation, and chemotaxis (Manning & Cantley, 2007).
An improvement in just one of these cellular processes may have a substantial
effect on the overall efficacy of stem cell transplantation for cardiovascular
therapy. Therefore, the hypothesis that short-term hypoxia upregulates Akt
9
phosphorylation in Isl-1+ c-Kit+ hCPCs and is correlated with enhanced cell
function in vitro was tested. The combined benefits of using other
progenitor cell
types, such as those characterized by Isl-1, and pretreatment to prepare these
transplanted cells for the hypoxic environment of the damaged heart, may allow
for improved cardiac regeneration in vivo.
Definition of Terms
CVD: cardiovascular disease; CAD: coronary artery disease; CPC: cardiac
progenitor cell; CHD: congenital heart disease; MEM NEAA: Minimum essential
medium non-
essential amino acids; DPBS: Dulbecco's phosphate
-buffered
saline; qRT-PCR: Quantitative reverse transcription polymerase chain reaction;
SDF-
1: Stromal cell
-derived factor-
1 ; SDS
-PAGE: Sodium dodecyl sulfate
polyacrylamide gel electrophoresis; TUNEL: Terminal deoxynucleotidyl
transferase deoxyuridine triphosphate nick-end labeling; PIK3CA:
Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit
; HSP: Heat
shock protein; MW: Molecular weight marker; ROS: Reactive oxygen species.
10
CHAPTER TWO
MATERIALS AND METHODS
In Vivo Components and Experiments
Isolation and Culture of Isl-1+ c-Kit+ hCPCs
The Institutional Review Board of Loma Linda University approved the
protocol for use of tissue that was discarded during cardiovascular surgery,
without identifiable private information, for this study with a waiver of informed
consent. Isl-1+ c-Kit+
hCPCs were isolated from cardiac tissue as previously
described (T. I. Fuentes et al., 2013). Briefly, atrial tissue was cut into small
clumps (approximately 1.0 mm
3
) then enzymatically digested using collagenase
(Roche, Indianapolis, IN) at a working concentration of 1.0 mg/mL. The resulting
solution was then passed through a 40-µm cell strainer. Cells were then cloned in
a 96-well plate by limiting dilution to a final concentration of 0.8 cells per well to
create populations for expansion. Twelve hCPC clones, derived from six distinct
donor samples, were used in this study. Clonal hCPC cultures were
supplemented with growth media comprised of 10% fetal bovine serum (Thermo
Scientific, Waltham, MA), 100 µg/mL Penicillin-Streptomycin (Life Technologies,
Carlsbad, CA), 1.0% minimum essential medium non-essential amino acids
solution (MEM NEAA, Cat no. 11120052, Life Technologies, Carlsbad, CA), and
11
20% endothelial cell growth media (Lonza, Basel, Switzerland) in Medium 199
(Life Technologies, Carlsbad, CA).
Hypoxic Preconditioning
The day before hypoxic pretreatment, Isl-1+ c-Kit+ hCPCs received fresh
culture media and, if necessary, were passaged to achieve 80% confluency
within 24 hours. Experimental hCPCs were then placed in a HeracellTM 150 tri
-
gas incubator (Thermo Scientific, Waltham, MA) set to 1.0% O
2
, 5.0% CO
2
, and
94% N
2
for six hours at 37 °C. Control hCPC conditions were 21% O
2
, 5.0% CO
2
,
and 74% N
2
at 37 °C. Cells were then immediately processed for analysis to
avoid prolonged exposure to normoxic conditions.
Transwell® Invasion Assay
Cultrex® basement membrane extract (Trevigen, Gaithersburg, MD) was
applied to the upper chamber of a Corning HTS Transwell® plate (8.0-µm pore
size, Venlo, Limburg). Isl-1+ c-Kit+ hCPCs were suspended in starvation media
composed of 98.5% Iscove's Modified Dulbecco's Medium with GlutaMAXTM (Life
Technologies, Carlsbad, CA), 1.0% insulin-transferrin-selenium (Life
Technologies, Carlsbad, CA), and 0.5% fetal bovine serum (Thermo Scientific,
Waltham, MA) then plated onto the coated wells at a density of 50,000 cells per
well. Stromal cell-derived factor-
1 (SDF
-
1, Life Technologies, Carlsbad, CA), a
chemoattractant, was diluted with growth media to a final concentration of 100
ng/mL and administered to the lower chamber. After 48 hours of incubation at 37
°C, the cells in the lower chamber were dissociated, stained with calcein AM (BD
12
Biosciences, San Jose, CA), and analyzed using an FLx800TM microplate
fluorescence reader (BioTek Instruments, Winooski, VT).
In Vitro Assays
Quantitative Reverse Transcription PCR
Isl-1+ c-
Kit+ hCPCs were washed with Dulbecco's Phosphate
-Buffered
Saline (DPBS), then lysed using TRIzol® reagent (Life Technologies, Carlsbad,
CA). Total RNA was isolated using the RNeasy® Mini Kit (Qiagen, Venlo,
Limburg) and cDNA was prepared with superscript III (Life Technologies,
Carlsbad, CA). Quantitative reverse transcription polymerase chain reaction
(qRT-PCR) was performed in triplicate using Go-Taq® qPCR Mastermix
(Promega, Madison, WI). Measurements were recorded using the iCycler iQTM5
PCR Thermal Cycler (Bio-Rad, Hercules, CA). Cycler settings were set to 94 °C
for 10 minutes, 94 °C for 15 seconds, 52 - 56 °C (depending on the primer) for 60
seconds, and 72 °C for 30 seconds for a total of 45 cycles. Human primers were
created using the National Center for Biotechnology Information Primer-BLAST
program as listed in Table 1. Relative gene expression data was analyzed using
the comparative C
T
method (Schmittgen & Livak, 2008).
13
Table 1. Primer Sequences Used for qRT-PCR.
PRIMER
SEQUENCE
ACTNB FWD
TTT GAA TGA GCC TTC GTC CCC
ACTNB REV
GGT CTC AAG TCA GTG TAC AGG TAA GC
BCL2 FWD
TGC ACC TGA CGC CCT TCA C
BCL2 REV
AGA CAG CCA GGA GAA ATC AAA CAG
T (Brachyury) FWD
ACT GGA TGA AGG CTC CCG TCT CCT T
T (Brachyury) REV
CCA AGG CTG GAC CAA TTG TCA TGG G
HMOX1 FWD
CTC TCG AGC GTC CTC A
HMOX1 REV
TTG AGC ACC TGG CCC CCA GA
HSP40 FWD
TTT TCG GAG GGT CCA ACC CCT
HSP40 REV
TCT TGT TTG AGG CGG GAT GGC C
HSP70 FWD
TGA CCA AGA TGA AGG AGA TCG
HSP70 REV
GTC AAA GAT GAG CAC GTT GC
HSP90 FWD
GGA TTT GAG GGG AAG A
HSP90 REV
TGA GCT TTC ATG ATT C
POU5F1 (Oct-4) FWD AAC CTG GAG TTT GTG CCA GGG TTT
POU5F1 (Oct-4) REV TGA ACT TCA CCT TCC CTC CAA CCA
PDGFRA FWD
GCG CAA TCT GGA CAC TGG GA
PDGFRA REV
ATG GGG TAC TGC CAG CTC AC
PIK3CA FWD
AAC AAT GCC TCC ACG ACC AT
PIK3CA REV
TCA CGG TTG CCT ACT GGT TC
RELA FWD
GCG AGA GGA GCA CAG ATA CC
RELA REV
GGG GTT GTT GTT GGT CTG GA
Western Blotting
Protein immunoblots for Akt and phosphorylated Akt were prepared using
protein from normoxic hCPCs and hCPCs exposed to 6 hours of hypoxia (1.0%
O
2
). Additionally, blots
for
-Actin and p-Akt were prepared using samples
obtained from untreated normoxic controls, normoxic hCPCs treated with stromal
cell-derived factor-
1 (SDF
-
1), and hCPCs treated
with both SDF-
1 and
hypoxia. Following 18 hours of serum deprivation, hCPCs were stimulated using
14
10 µg/mL of SDF-
1 (Biolegend, San Diego, CA). Hypoxic groups were exposed
to low oxygen conditions during the final six hours of the 18-hour starvation
period. All protein lysates were loaded into a 12% Tris-glycine pre-cast gel
(Thermo Scientific, Waltham, MA), separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a
nitrocellulose membrane (Bio-Rad, Hercules, CA). After blocking with 5% bovine
serum albumin (BSA) in Tris-buffered saline with Tween-20 (TBST), membranes
were labeled with mouse anti-
human
-Actin antibody (2F1-1) (1:500 dilution,
Biolegend, San Diego, CA), rabbit anti-human Akt monoclonal antibody (pan)
(C67E7) (1:500 dilution Cell Signaling Technology, Danvers, MA), or rabbit anti-
human Phospho-Akt monoclonal antibody (S473) (D9E) (1:300 dilution, Cell
Signaling Technology, Danvers, MA) overnight at 4 °C with agitation. The
following day, membranes were washed and labeled using either an IRDye®
680RD-conjugated goat anti-mouse antibody (1:5000 dilution, LI-COR, Lincoln,
NE) or an IRDye® 800CW-conjugated goat anti-rabbit antibody (1:5000 dilution,
LI-COR, Lincoln, NE) in 5.0% BSA in TBST for 60 minutes at room temperature.
Final protein levels were visualized using an Odyssey® infrared imaging system
(LI-COR, Lincoln, NE) model 9120. Resulting protein bands were analyzed using
ImageJ software.
15
Flow Cytometry
Progenitor cell populations were fluorescently labeled with antibodies as
recommended by their respective manufacturers then analyzed using a
MACSQuant® analyzer (Miltenyi Biotec, Auburn, CA). Quantification of data was
performed using FlowJo software (Ashland, OR). Small particulate matter, dead
cells, and gas-bubbles were excluded from final analysis using forward-scatter
and side-scatter data.
Antibodies Used in Cytometry Experiments
Antibodies used for cytometric analysis include: Anti-Isl-1 (1H9) mouse
monoclonal antibody, (1:50 dilution, Abcam, Cambridge, MA), Anti-c-Kit Rat
IgG2b Kappa monoclonal antibody (2B8) conjugated to Dylight 650 (0.5 mg/mL,
Novus Biologicals, Littleton, CO), Anti-Akt phospho (Serine 473) rabbit Ig
polyclonal antibody (0.23 mg/mL, Biolegend, San Diego, CA), Fluorescein-anti-
BrdU (PRB-1) monoclonal antibody (1:20 dilution, Phoenix Flow Systems, San
Diego, CA), FITC goat anti-mouse IgG polyclonal antibody (1:25 dilution,
Southern Biotech, Birmingham, AL), PE goat anti-mouse IgG polyclonal antibody
(1:100 dilution, Southern Biotech, Birmingham, AL), and FITC goat anti-rabbit
IgG polyclonal antibody (1:50 dilution, BD Biosciences, San Jose, CA).
Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling
Isl-1+ c-Kit+ hCPCs were rinsed with DPBS and treated with a solution of
0.5 mM H
2
O
2
in culture media for 21 hours to induce cell death (Clément,
Ponton, & Pervaiz, 1998). Terminal deoxynucleotidyl transferase deoxyuridine
16
triphosphate nick-end labeling (TUNEL) assay was performed following the
manufacturer's recommendations. Briefly, cells were counted using trypan blue
and concentrated to 10
6
cells/mL. The concentrated progenitor cell solution was
then re-suspended in 1.0 mL of 1% paraformaldehyde in DPBS, placed on ice for
30 minutes, then reconstituted into 1.0 mL of 70% ethanol for overnight
incubation at -20 °C. The next day, the cells were labeled with Br-dUTP (Phoenix
Flow Systems, San Diego, CA) and re-suspended in antibody solution containing
the Fluorescein anti-BrdU antibody (Phoenix Flow Systems, San Diego, CA).
Population analysis was performed using flow cytometry.
Cell Cycle Analysis
Isl-1+ c-Kit+ hCPCs at 80% confluency were trypsinized, counted, and
concentrated to 2.5 X 10
5
cells per 0.3 mL DPBS. Ice-cold 70% ethanol (0.7 mL)
was added drop-wise to fix the cells then stored at -20 °C overnight. The
following day, cells were incubated at 37 °C for 1 hour with RNase A (0.5 mg/mL,
Life Technologies, Carlsbad, CA). Propidium Iodide solution (0.5 mg/mL) was
added, and the resulting cell solution was analyzed using a MACSQuant®
analyzer (Miltenyi Biotec, Auburn, CA). Cytometer data was quantified using
FlowJo software (Ashland, OR).
17
Statistical Analysis
Data was reported as mean +/- standard error. Error bars were designed
using propagation of error and the Cousineau method (Morey, 2008). P values <
0.05 were deemed significant.
18
CHAPTER THREE
RESULTS
Akt Activation in Isl-1+ c-Kit+ hCPCs
Hypoxic Preconditioning Stimulates the Akt Pathway
in Isl-1+ c-Kit+ hCPCs
Akt, a master regulator of numerous genes their corresponding proteins,
oversees various critical cell processes such as apoptosis, proliferation, and
chemotaxis (Manning & Cantley, 2007). To determine the effect of short-term
hypoxia on the Akt pathway within Isl-1+ c-Kit+ hCPCs, clonal populations
previously characterized by my laboratory colleagues (T. I. Fuentes et al., 2013),
expressing Isl-1, c-Kit, Brachyury, Oct-4, and Platelet-derived growth factor
receptor-
(PDGFRA,
Fig.1A, B), were selected for experimentation. Enhanced
Akt phosphorylation is of main interest for its potential benefit in autologous
hCPC-based therapy for myocardial infarction in older adults. To determine
whether the hypoxia-mediated activation of Akt is significantly influenced by the
age of the cell donor, expression of phosphatidylinositol-4,5-bisphosphate 3-
kinase catalytic subunit (
PIK3CA), which is known to directly activate Akt (Baba
et al., 2011), was measured via qRT-PCR in both control and experimental cells.
Data analysis revealed that pretreated cells, regardless of age group, expressed
significantly higher levels of PIK3CA mRNA when compared to non-treated,
normoxic, Isl-1+ c-Kit+ hCPCs. Moreover, the data also shows that neonatal
19
hCPCs more strongly upregulate PIK3CA mRNA than do adult hCPCs and may
lead to superior Akt activation in neonatal populations (Fig. 2A). Additionally,
antibody labeling of activated Akt was measured by flow cytometry in adult and
neonatal hCPC clones (Fig. 2B). Comparative analysis of the flow cytometry data
gathered against phosphorylated Akt further highlights the distinction between
the hypoxia-mediated activation of Akt in adult and neonatal Isl-1+ c-Kit+ hCPCs.
While p-Akt expression indeed trends upwards in preconditioned adult clones,
only the neonatal clones procured statistically significant elevations in Akt
activation after preconditioning (Fig. 2C). However, the promise of short-term
hypoxia preconditioning in adult Isl-1+ c-Kit+ hCPCs is illustrated via protein
immunoblots (Fig. 3A). Quantification of the imaged protein from a single
representative adult clone shows that phosphorylation of Akt can be promoted by
exposure to short-term hypoxia (Fig. 3B). Moreover, serum starved adult Isl-1+ c-
Kit+ hCPCs show a significant decrease in phosphorylated Akt after stimulation
with SDF-
1
(Fig. 3C, D). However, when serum deprivation is conducted in
tandem with short-term hypoxia and followed up with SDF-
1 treatme
nt, adult Isl-
1+ c-Kit+ hCPC p-Akt protein levels are rescued to levels at or above normoxic
control, but not to a statistically significant degree (Fig. 3D).
20
Isl-1+ c-Kit+ hCPC Function After Exposure to Short-Term Hypoxia
Isl-1+ c-Kit+ hCPCs Invade More Readily After Exposure
to Short-term Hypoxia
Hypoxia has also been shown to influence cellular motility (Filippi et al.,
2014; van Oorschot et al., 2011; Yan et al., 2012). To determine if short-term
hypoxia has any effect on Isl-1+ c-Kit+ hCPC motility, and to validate the
selection of the six-hour exposure time, the chemotactic response to SDF-
1
amongst non-treated and pretreated hCPCs was compared using a Transwell®
invasion assay. A time course experiment was performed in which a
representative clone was pretreated with hypoxia for 3 hours, 6 hours, and 18
hours for comparison of invasion capacities with normoxic control. Over the same
48-hour incubation period, the six-hour hypoxia-pretreated group most efficiently
invaded through the basement membrane layer and into the receiver well (Fig.
4A). Using the six-hour time point across 10 different biological replicates, a
statistically significant difference was noted between non-treated hCPCs and
pretreated hCPCs. The hCPCs preconditioned with six hours of hypoxia invaded
through the basement membrane extract and transwell pores in significantly
greater numbers than the non-treated hCPCs (Fig. 4B). However, when the data
is analyzed by age group, it is clear the adult hCPCs do not respond as
vigorously to the hypoxia pretreatment. A trend towards enhanced invasion is
present, but this trend is not significant. Only in the neonatal hCPCs did we
observe a statistically significant hypoxia-mediated improvement in chemotactic
response to SDF-
1 (Fig. 4C).
21
Short-term Hypoxia Triggers a Pro-survival Response
in Isl-1+ c-Kit+ hCPCs
In other cell types, hypoxic pretreatment has been shown to improve
survival (Hu et al., 2014; Yan et al., 2012). To determine the effect of short-term
hypoxia on Isl-1+ c-Kit+ hCPCs, the expression profiles of several heat-shock
proteins in control and experimental populations were examined using qRT-PCR.
Data analysis confirmed that pretreated hCPCs indeed express higher levels of
select heat-shock protein (HSP) mRNAs (Fig. 5A, B). However, only HSP70 is
significantly upregulated by hypoxic preconditioning and only in neonatal clones
(Fig. 5B). Subsequent electrophoresis of PCR products confirmed the
upregulation of HSP70 in neonatal Isl-1+ c-Kit+ hCPCs via hypoxia
preconditioning (Fig. 5C). Further gene expression analysis indicated that short-
term hypoxia not only induces a stress response but also significantly
upregulates the transcription of genes associated with cell survival (Fig. 6A, B).
However, only the neonatal group exhibited a significant change
an
approximate twofold increase in the RELA gene transcript, known for its role in
the regulation of apoptosis (Beg & Baltimore, 1996). If the data for both age
groups is pooled, a threefold increase of Hemoxygenase 1 (HMOX1) is also
noted (p = 0.04, n = 6). HMOX1 plays a role in protecting the cells from oxidative
damage (Poss & Tonegawa, 1997). Within a six-hour timeframe, we found that
the hypoxia-induced modifications to the pro-survival gene program resulted in
fewer apoptotic cells in response to oxidative stress (Fig. 7A) However,
quantification of results obtained from three independent adult clones revealed
22
little change in apoptotic response (Fig. 7B). TUNEL assay results from three
individual neonatal hCPC populations reveal a downward trend in the number of
apoptotic cells in response to H
2
O
2
(Fig. 7C).
Hypoxia-pretreated Isl-1+ c-Kit+ hCPCs Remain Undifferentiated
To evaluate the effects of short-term hypoxia on differentiation in Isl-1+ c-
Kit+ hCPCs, the expression of several differentiation markers in control and
experimental hCPCs was evaluated using qRT-PCR. Quantification of data
revealed no significant difference between pretreated and non-treated adult
hCPCs in two out of three markers examined (Fig. 8A). Only PDGFRA, present
in sub-populations of progenitors with superior regenerative capacity (Hidaka et
al., 2010; J. Kim et al., 2010) was significantly altered, upregulated 1.4-fold (p =
0.004). In neonatal clones, a significant downregulation of Brachyury and
upregulation of PDGFRA was noted (Fig. 8B). The Brachyury gene transcript is
directly linked to the regulation of MESP1 (Robert David et al., 2011) a protein
coding gene that promotes cardiovascular differentiation (R. David et al., 2008).
Additionally, it is important to note that expression of the Oct-4 transcription
factor remains unchanged in both age groups. Altogether, these findings suggest
that short-term hypoxia stimulates neither differentiation nor de-differentiation in
Isl-1+ c-Kit+ hCPCs.
Hypoxia Pretreatment Does Not Alter Normal Cell Cycle Progression
Hypoxia is also known to influence the cell cycle (Gardner et al., 2001;
Grayson, Zhao, Bunnell, & Ma, 2007; Kook et al., 2008; Koshiji et al., 2004;
23
Studer et al., 2000; van Oorschot et al., 2011). A more proliferative stem cell
population would undoubtedly be beneficial for the repair of damaged
myocardium. To determine the effect of short-term hypoxia in regards to Isl-1+ c-
Kit+ hCPC proliferation, cell cycle analysis was performed on non-treated and
pretreated hCPC populations of both adult and neonatal origin. No significant
difference was identified between control and experimental groups (Fig. 9A).
Moreover, there was no apparent difference between normoxic and hypoxic
groups, whether adult and neonatal, in their progression through the cell cycle
(Fig. 9B, C). These results support the conclusion that short-term hypoxia does
not alter normal cell cycle progression in Isl-1+ c-Kit+ hCPCs.
24
CHAPTER FOUR
DISCUSSION
In this study, Isl-1+ c-Kit+ hCPCs were expanded as clonal populations
and used as a model to test the hypothesis that short-term hypoxia exposure
enhances Isl-1+ c-Kit+ hCPC function in vitro. The results presented here show
that short-term hypoxia is a feasible and practical pretreatment that benefits
neonatal hCPC invasive capabilities in vitro.
Recent studies lend credence to the efficacy of cardiac stem cell
transplants for the amelioration of cardiac dysfunction in mouse models (Hong et
al., 2014; Matsuura et al., 2009). Furthermore, human trials have not only
established the safety of autologous cardiac stem cell transplantation but also
produced some very encouraging results (Bolli et al., 2011; Makkar et al., 2012).
However, in both mice and human studies, improvements in cardiac function
typically measured using left ventricular ejection fraction
are thought to be a
result of paracrine signaling stemming from the growth factors secreted by newly
transplanted cells (Barile et al., 2014; Hong et al., 2014). Additionally, the
functional capabilities of hCPCs are known to vary greatly between age groups
with neonatal hCPCs consistently outperforming their adult counterparts (T. I.
Fuentes et al., 2013). However, the incidence of cardiovascular disease is most
prevalent within older adult hearts that are populated by functionally inferior
25
hCPC populations. Hence, administration of autologous hCPCs for treatment of
the majority of cardiac injuries is limited, stimulates repair primarily by means
other than direct engraftment, and leaves much to be desired. Cell-based
therapies for the human heart
of all ages
may be augmented via the use of a
novel cell type or pretreatment method, such as hypoxic exposure. If
preconditioning indeed improves hCPC function, behavior of adult-derived clones
could be selectively improved to reflect that of their functionally superior neonatal
counterparts. However, CPC populations express distinct protein signatures that
play an important role in overall cellular function and merit serious consideration
when selecting cell populations for therapy. While the c-Kit+ CPC remains the
most widely studied and implemented, the application of a novel Isl-1+ c-Kit+
hCPC population may aid in the effort to optimize autologous cell-based
therapies for regeneration of the heart.
The idea that preconditioning cells prior to transplantation may benefit
donor cell function in vivo, has gained a significant following in recent years
(Hahn et al., 2008; Hu et al., 2014; Luo et al., 2014; Rosenblum et al., 2014; van
Oorschot et al., 2011; Yan et al., 2012). However, although this research is
gaining momentum, the genetic analysis and functional assays presented in this
study have never been performed on Isl-1+ c-Kit+ hCPCs. Using other models,
previous studies have established that hypoxia induces a persistent increase in
phosphorylation of Akt for up to 24 hours with the effect peaking at six hours
(Beitner-Johnson et al., 2001). Activation of Akt, a versatile kinase that regulates
26
several cellular functions, may result in hCPC functional improvements and
enhanced cardiac repair. In the present study, enhanced Akt phosphorylation
was indeed observed after only six hours of hypoxia, thus demonstrating that Isl-
1+ c-Kit+ hCPCs exhibit similar behavior in response to hypoxia when compared
to other cell types. However, age seems to play a significant role as the
magnitude of hypoxia-mediated Akt activation was markedly reserved in adult
clones. While both age groups displayed increased phosphorylation of Akt after
the six-hour time point, only the neonatal group data was statistically significant.
However, it is important to note that the adult clones are notoriously difficult to
stimulate and, in response to SDF-
1 after starvation, levels of p
-Akt significantly
decrease. Short-term hypoxic preconditioning, on the other hand, rescued
previous levels of phosphorylated Akt observed prior to serum deprivation in
adult Isl-1+ c-Kit+ hCPCs. As hypothesized, this hypoxia-mediated activation of
Akt in Isl-1+ c-Kit+ hCPCs was indeed correlated with elevated expression of
PIK3CA, a protein that plays an essential role in the upstream activation of Akt
(Baba et al., 2011). Experimental neonatal Isl-1+ c-Kit+ hCPCs exhibited a
remarkable 43-fold significant increase of PIK3CA mRNA when compared to
their respective controls. Adult Isl-1+ c-Kit+ hCPCs, on the other hand, only
expressed a 1.8-fold increase in PIK3CA mRNA due to hypoxic preconditioning.
The data here suggests that neonatal Isl-1+ c-Kit+ hCPCs react more strongly to
the hypoxic pretreatment and transcribe more of the upstream activator of Akt.
27
Altogether, these findings confirm that six hours of hypoxia exposure significantly
increases Akt activation in neonatal Isl-1+ c-Kit+ hCPCs.
Nonetheless, hypoxia is a stressor that, as demonstrated here, triggers a
physiological response in hCPCs. As oxygen levels decline, the mitochondria
within a cell increase the production of reactive oxygen species (ROS) (Chandel
et al., 2000; Guzy et al., 2005). The accumulation of ROS after short periods of
hypoxia has been shown to confer resistance against future oxygen
shortages
(Hoek, Becker, Shao, Li, & Schumacker, 1998). However, longer periods of
hypoxia coincide with excessive accumulation of ROS that are known to promote
cell death via caspase activation and DNA damage (Filomeni, De Zio, & Cecconi,
2015; Kamata et al., 2005; J.-Y. Kim & Park, 2003; Moungjaroen et al., 2006).
Altogether, chronic oxidative stress has the potential to impair the functional
capacity and overall health of a population of cells (van Oorschot et al., 2011).
The extent of this stress response in Isl-1+ c-Kit+ hCPCs was evaluated by
measuring the impact of short-term hypoxia on the expression of several HSP
mRNAs. The induction of the heat-shock pathway during hypoxia has been well
documented in other cell types (Baird, Turnbull, & Johnson, 2006). Genetic
analysis of adult and neonatal Isl-1+ c-Kit+ hCPCs revealed a trend towards
hypoxia-mediated activation of transcripts for several heat shock proteins.
However, only the upregulation of HSP70 mRNA was statistically significant and
was found only in the neonatal group. HSP70 is known to play a role in stabilizing
Akt (Koren et al., 2010) and in the inhibition of apoptosis (Jiang et al., 2009;
28
Powers, Clarke, & Workman, 2008). Under hypoxia, Akt and other proteins are at
risk of degradation, thus, the survival response likely includes upregulation of
HSP mRNAs as an attempt to promote stabilization of proteins that are needed
for cell survival.
Accordingly, pro-survival gene expression was also found to be
upregulated in hypoxic Isl-1+ c-Kit+ hCPCs. Preconditioned hCPCs displayed a
dramatic increase in transcripts encoding HMOX1, a pro-survival gene that
becomes upregulated in response to hypoxia and affords protection against
future oxidative damage (Poss & Tonegawa, 1997). Furthermore, the transcript
encoding the NF-
B p65 subunit (
RELA), a known Akt downstream effector that
inhibits programmed cell death (Beg & Baltimore, 1996; Madrid, Mayo, Reuther,
& Baldwin, 2001), was significantly upregulated, but only in the pretreated
neonatal group. Although a significant difference in apoptosis was not observed,
hypoxia-preconditioned hCPCs indeed exhibited significantly enhanced invasion
capabilities when compared to their normoxic counterparts. Moreover, when
additional time points were tested, the six-hour time point yielded the greatest
number of cells that successfully invaded through the transwell membrane.
Applied to multiple biological replicates, the six-hour pretreatment of hCPCs
resulted in significantly improved chemotaxis in response to SDF-
1, which is in
parallel to what has been observed in other models (Filippi et al., 2014; van
Oorschot et al., 2011; Yan et al., 2012). Not surprisingly, however, these results
were significant only in neonatal hCPCs. According to the PCR data, the
29
pretreated adult hCPCs did not upregulate PIK3CA mRNA transcripts to the
same extent as the pretreated neonatal hCPCs. This suggests that the Akt
pathway is not sufficiently activated by short-term hypoxia in adult hCPCs and
may explain why only the neonatal clones displayed significantly elevated p-Akt
and enhanced invasion capacity.
Furthermore, depending on the cell type, hypoxic exposure may either
enhance proliferation (Grayson et al., 2007; Kook et al., 2008; Studer et al.,
2000; van Oorschot et al., 2011), lead to G1 arrest (Gardner et al., 2001; Koshiji
et al., 2004; Utting et al., 2006), or influence the differentiation process (Lin, Lee,
& Yun, 2006; Studer et al., 2000; Utting et al., 2006). After treatment with
hypoxia, human mesenchymal stem cells acquire enhanced proliferative abilities
(Grayson et al., 2007) while, on the other hand, murine embryonic fibroblasts
encounter G1 arrest (Gardner et al., 2001). Ideally, in the early stages after
transplantation, donor cells must survive, continue to divide, migrate to the
damaged myocardium, and remain multipotent as they engraft. Isl-1+ c-Kit+
hCPCs indeed migrate more readily, progress normally through the cell cycle,
and retain expression of pluripotency markers after short-term hypoxic treatment.
The results reported here using Isl-1+ c-Kit+ hCPC clones are in line with those
of other cardiovascular progenitors (Hu et al., 2014; van Oorschot et al., 2011),
suggesting that pretreatment with short-term hypoxia will enhance functional
efficacy. Short-term hypoxia yielded mild improvements in adult CPCs, procured
significant benefit to neonatal CPCs, and therefore, is a promising method for
30
improving cellular function. However, it is important to acknowledge that the most
significant results were obtained in the neonatal group
enhanced Akt activation,
upregulated pro-survival transcripts (RELA, HSP70), and improved invasion
capabilities. Additional work is required to maximize the stimulation of adult
hCPCs to mirror the function of neonatal CPCs and, in the process, optimize
autologous adult CPCs for superior transplantation. Nevertheless, the results
presented here demonstrate that outcomes of surgical procedures involving
neonatal CPCs
for the treatment of CHDs or for transplant in HLA-matched
adult patients
may be improved by preconditioning donor cells using short-term
hypoxia.
Conclusions
Short-term hypoxia, as a pretreatment, is a viable approach for supporting
cell survival and enhancing migratory capabilities in neonatal Isl-1+ c-Kit+
hCPCs. While the benefits accrued by adult Isl-1+ c-Kit+ hCPCs via
preconditioning were reserved in comparison to neonatal clones, the applicability
of neonatal CPCs in the clinical setting is significant and, therefore, hypoxia-
preconditioned Isl-1+ c-Kit+ hCPCs warrant further investigation in animal
models. The positive effects of short-term hypoxia include: 1) enhanced
chemotaxis, which would render the cells more likely to reach damaged tissues
and successfully engraft, and 2) elevated levels of PIK3CA, HSP70, RELA, and
HMOX1 mRNA transcripts, which are important for cellular signaling and survival.
31
These findings, if implemented in vivo, may improve cardiac repair after
infarction. Thus, in order to validate the efficacy of short-term hypoxia as an
effective pretreatment strategy to optimize cell-based repair, future in vivo
experiments comparing the performance of hypoxia-preconditioned CPCs to non-
treated CPCs are currently in the design phase.
32
APPENDIX A
FIGURES
33
Figure 1. Expression of early progenitor markers within a representative clonal hCPC population.
(A) Isl-1 and c-Kit expression within a representative clonal hCPC population as measured by
flow cytometry. Dotted lines indicate isotype controls and solid lines indicate Isl-1 or c-Kit
antibody-labeled hCPC. Double-labeled cells are shown in dot-plot. (B) Expression of stem cell
markers in a representative Isl-1+ c-Kit+ hCPC clone is shown here by electrophoresis of PCR
products. MW = molecular weight marker.
c-Kit
Cell Numb
e
r
Fluorescence Intensity
APC
c-Kit
PE
Isl
-1
PE
Is
l-1
APC
c-Kit
Negative Control
Isl-1
A
B
MW
P
DG
F
RA
B
rac
hy
ury
Oct
-4
123 bp
394 bp
496 bp
34
Figure 2. Short-term hypoxia upregulates phosphorylation of Akt in Isl-1+ c-Kit+ hCPCs. (A) Adult
and neonatal Isl-1+ c-Kit+ hCPCs exposed to short-term hypoxia were compared to their
respective normoxic control via qRT-PCR (n = 8). Quantification of results revealed that hypoxia
preconditioning yields significant upregulation of PIK3CA transcripts within both age groups.
Additionally, electrophoresis of PIK3CA primer products confirms PCR amplification of the target
gene segment. Subsequently, phosphorylation of Akt was then measured in preconditioned Isl-1+
c-Kit+ hCPCs and their respective controls by flow cytometry. (B) Representative histogram of
increased p-Akt monoclonal antibody binding after exposure to six hours of hypoxia. (C)
Quantification of seven independent hCPCs revealed significantly increased (15.4%, p = 0.019)
Akt phosphorylation in hypoxia-pretreated neonatal groups. Adult clones displayed modest Akt
activation, but was deemed non-significant after further investigation.
N
0
20
40
60
PIK3CA
Normoxic
Neonate
Hypoxic
Neonate
0
1
2
3
PIK3CA
Fo
ld
Cha
n
ge
Normoxic
Adult
Hypoxic
Adult
0
500
1000
1500
2000
Fl
u
o
resce
n
ce
In
te
n
sity
Normoxic
Hypoxic
Neonate
Adult
p-Akt Fluorescence
Intensity
Ce
ll Nu
m
b
e
r
Normoxic
Hypoxic
B
C
A
*
*
MW
H
N
*
Neonate
Adult
330 bp
Actin
MW
H
35
Figure 3. Hypoxia-induced Akt activation in adult Isl-1+ c-Kit+ hCPCs. (A) Protein immunoblots
depicting Akt and p-Akt protein bands from normoxic and hypoxic representative adult hCPC
samples. (B) Percentage of phosphorylated Akt in total Akt of non-treated and pretreated adult
hCPC protein
quantified using ImageJ.
(C) Western blots illustrating
-Actin and p-Akt protein
from a representative normoxic adult clone, a SDF-
1
-treated normoxic adult clone, and an adult
clone treated with both SDF-
1 and hypoxia. (D) Fold change of phosphorylated Akt, relative to
-Actin, was quantified using ImageJ.
0
1
2
3
1
Fo
ld
Cha
n
ge
p-Akt
Control
SDF
SDF+H
*
B
Akt
p-Akt
MW N H CTR MW N H CTR
0%
5%
10%
15%
20%
Normoxic Hypoxic
p
-A
kt/
A
kt
(%)
-Actin
p-Akt
SDF
Control
SDF &
Hypoxia
SDF
Treated
Untreated
Control
C
D
A
36
Figure 4. Invasion capabilities of Isl-1+ c-Kit+ hCPCs in response to SDF-
1. (A) Quantification of
hCPC performance in Transwell® invasion assay as influenced by duration of hypoxic exposure.
Cell numbers were measured by calcein AM in quadruplicate using a representative Isl-1+ c-Kit+
hCPC. A significant increase in cell number was noted for each time point, with six hours of
hypoxia yielding the greatest improvement. (B) Pooled data from both adult and neonate Isl-1+ c-
Kit+ hCPCs, after six hours of hypoxia, show that hypoxia-pretreated hCPCs exhibit significantly
improved invasion (n = 10, p = 0.017). (C) Invasion assay results organized by age group reveals
that benefits afforded by hypoxia are age dependent. Adult hCPCs exhibited slight enhancements
of their invasion capabilities, but only neonatal hCPCs procured statistically significant
improvements.
0
5000
10000
15000
20000
25000
30000
35000
Cell Num
b
e
r
Normoxic
3-hr Hypoxia
6-hr Hypoxia
18-hr Hypoxia
0
5000
10000
15000
20000
25000
30000
Normoxic
Hypoxic
*
B
*
*
*
*
*
A
C
0
10000
20000
30000
40000
50000
Normoxic
Adult
Hypoxic
Adult
Normoxic
Neonate
Hypoxic
Neonate
Cell Num
b
e
r
*
Cell Numb
e
r
37
Figure 5. Transcription of heat shock proteins (HSPs) in response to short-term hypoxia. (A) Fold
change of HSP mRNAs in adult Isl-1+ c-Kit+ hCPCs resulting from pretreatment with short-term
hypoxia (n = 4). (B) Neonatal Isl-1+ c-Kit+ hCPC HSP mRNA expression reveals a significant
two-fold elevation of HSP70 in response to short-term hypoxia (p = 0.022, n = 5). (C)
PCR
products of HSP mRNAs upregulated in adult and neonatal hCPCs, as visualized by agarose gel
electrophoresis, validate the significant increase of HSP70 in neonatal hCPCs after short-term
hypoxic preconditioning. MW = molecular weight marker, N = normoxic, H = hypoxic.
0
1
2
3
HSP40
HSP70
HSP90
Fo
ld
Cha
n
ge
Normoxic Adult
Hypoxic Adult
0
1
2
3
4
HSP40
HSP70
HSP90
Fo
ld
Cha
n
ge
Normoxic Neonate
Hypoxic Neonate
*
A
B
C
Adult Neo Adult Neo Adult Neo
HSP40 HSP70 HSP90
MW N H N H N H N H MW N H N H
38
Figure 6. Select genes associated with cell survival are elevated in response to short-term
hypoxia. (A) Expression of mRNA transcripts associated with cell survival in adult Isl-1+ c-Kit+
hCPCs and relative fold changes after hypoxic exposure. BCL2 and HMOX1 are strongly
upregulated in response to hypoxia, but the changes are not statistically significant (n = 3). (B)
Neonatal Isl-1+ c-Kit+ hCPC pro-survival gene expression in response to short-term hypoxia.
RELA and HMOX1 are elevated in response to short-term hypoxia, but only RELA is significant (p
= 0.02, n = 3).
A
B
0
1
2
3
4
5
6
7
8
BCL2
HMOX1
RELA
Fo
ld
Cha
n
ge
Normoxic Adult
Hypoxic Adult
0
1
2
3
4
BCL2
HMOX1
RELA
Fo
ld
Cha
n
ge
Normoxic Neonate
Hypoxic Neonate
*
39
Figure 7. Programmed cell death in response to oxidative damage is not significantly reduced.
Using 0.5 mM H
2
O
2
, apoptosis was induced in non-treated and preconditioned Isl-1+ c-Kit+
hCPCs of both adult and neonatal origin. Relative DNA fragmentation was measured using Brd-U
and anti-BrdU antibody. (A) Cytometric analysis of BrdU-DNA binding within a representative
clone is shown here, with and without hypoxic pretreatment. The group treated with short-term
hypoxia exhibited an approximate 50% decrease in apoptotic cells after induction of cellular death
by 0.5 mM H
2
O
2
. (B) Quantification of TUNEL assay results obtained using three independent
adult clones are pictured here, showing that apoptosis is not significantly reduced (p = 0.89). (C)
Pooled results of TUNEL assay for three neonatal clones with and without hypoxic pretreatment.
Apoptosis in response to oxidative stress trends downward in the hypoxia preconditioned group
but not to a significant degree (p = 0.34).
Negative Control
Normoxic
B
rdU Fluo
resce
n
ce
In
te
n
sity
A
Hypoxic
Propidium Iodide Fluorescence Intensity
0
2
4
6
8
10
12
14
16
Normoxic
Neonate
Hypoxic
Neonate
A
p
o
p
to
tic
Cells (
%)
C
B
0
5
10
15
20
25
Normoxic
Adult
Hypoxic
Adult
A
p
o
p
to
tic
Cells (
%)
40
Figure 8. Isl-1+ c-Kit+ hCPCs maintain expression of early differentiation markers after hypoxic
exposure. Quantitative RT-PCR was used to examine the effects of short-term hypoxia on the
expression of differentiation markers present in cardiovascular progenitors. (A) Pooled PCR
results from three individual adult hCPC clones reveals no significant change in differentiation
markers with the exception of PDGFRA (1.4-fold increase, p = 0.004). (B) Quantification of PCR
data from four independent neonatal clones reveals a similar upregulation of PDGFRA (1.8-fold, p
= 0.0003), as well as a decrease in Brachyury transcripts (0.58-fold, p = 0.004).
A
0
0.5
1
1.5
2
2.5
Brachyury
Oct-4
PDGFRA
Fo
ld
Cha
n
ge
Normoxic Adult
Hypoxic Adult
*
B
0
0.5
1
1.5
2
2.5
Brachyury
Oct-4
PDGFRA
Fo
ld
Cha
n
ge
Normoxic Neonate
Hypoxic Neonate
*
*
41
Figure 9. Short-term hypoxia does not alter normal cell cycle progression. Preconditioned Isl-1+
c-Kit+ hCPCs, of both neonate and adult origin, were stained using propidium iodide and
compared to their respective normoxic controls via flow cytometry. (A) Flow cytometry histogram
of cell cycle analysis in a representative clonal population, both with and without hypoxic
pretreatment. (B) Quantification of nine individual results from three independent adult clones
reveals that short-term hypoxia does not significantly affect normal cell cycle progression in adult
Isl-1+ c-Kit+ hCPCs. (C) Quantification of data from six technical replicates using three
independent neonatal clones confirms that normal cell cycle progression is also unaffected by
short-term hypoxia in neonatal Isl-1+ c-Kit+ hCPCs.
Hypoxic
Normoxic
Propidium Iodide Fluorescence Intensity
Cell Numb
e
r
A
B
0
20
40
60
80
100
G1
S
G2
Cell Fr
e
qu
e
n
cy
Normoxic Adult
Hypoxic Adult
C
0
20
40
60
80
100
G1
S
G2
C
ell Fr
eq
ue
ncy
Normoxic Neonate
Hypoxic Neonate
42
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