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% O2 for six hours) and, to identify shifts in gene expression, compared to their respective control (21% O2 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 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.

Excerpt

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

for six hours) and,

to identify shifts in gene expression, compared to their respective control (21%

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

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.

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.

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

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

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

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

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

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,

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

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

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

)

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

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.

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

) 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

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

, 5.0% CO

, and

94% N

for six hours at 37 °C. Control hCPC conditions were 21% O

, 5.0% CO

and 74% N

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

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

method (Schmittgen & Livak, 2008).

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%

). 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

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.

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

in culture media for 21 hours to induce cell death (Clément,

Ponton, & Pervaiz, 1998). Terminal deoxynucleotidyl transferase deoxyuridine

triphosphate nick-end labeling (TUNEL) assay was performed following the

manufacturer's recommendations. Briefly, cells were counted using trypan blue

and concentrated to 10

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

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).

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.

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

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-

(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).

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-

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).

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

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

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

(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;

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.

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

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

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.

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;

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

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

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.

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.

APPENDIX A

FIGURES

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

Fluorescence Intensity

APC

c-Kit

Isl

-1

l-1

APC

c-Kit

Negative Control

Isl-1

rac

ury

Oct

-4

123 bp

394 bp

496 bp

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.

PIK3CA

Normoxic

Neonate

Hypoxic

Neonate

PIK3CA

Cha

Normoxic

Adult

Hypoxic

Adult

500

1000

1500

2000

resce

sity

Normoxic

Hypoxic

Neonate

Adult

p-Akt Fluorescence

Intensity

ll Nu

Normoxic

Hypoxic

Neonate

Adult

330 bp

Actin

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.

-Actin and p-Akt protein

from a representative normoxic adult clone, a SDF-

-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.

Cha

p-Akt

Control

SDF

SDF+H

Akt

p-Akt

MW N H CTR MW N H CTR

10%

15%

20%

Normoxic Hypoxic

-A

kt/

(%)

-Actin

p-Akt

SDF

Control

SDF &

Hypoxia

SDF

Treated

Untreated

Control

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.

5000

10000

15000

20000

25000

30000

35000

Cell Num

Normoxic

3-hr Hypoxia

6-hr Hypoxia

18-hr Hypoxia

5000

10000

15000

20000

25000

30000

Normoxic

Hypoxic

10000

20000

30000

40000

50000

Normoxic

Adult

Hypoxic

Adult

Normoxic

Neonate

Hypoxic

Neonate

Cell Num

Cell Numb

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.

HSP40

HSP70

HSP90

Cha

Normoxic Adult

Hypoxic Adult

HSP40

HSP70

HSP90

Cha

Normoxic Neonate

Hypoxic Neonate

Adult Neo Adult Neo Adult Neo

HSP40 HSP70 HSP90

MW N H N H N H N H MW N H N H

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).

BCL2

HMOX1

RELA

Cha

Normoxic Adult

Hypoxic Adult

BCL2

HMOX1

RELA

Cha

Normoxic Neonate

Hypoxic Neonate

Figure 7. Programmed cell death in response to oxidative damage is not significantly reduced.

Using 0.5 mM H

, 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

. (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

rdU Fluo

resce

sity

Hypoxic

Propidium Iodide Fluorescence Intensity

Normoxic

Neonate

Hypoxic

Neonate

tic

Cells (

Normoxic

Adult

Hypoxic

Adult

tic

Cells (

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).

0.5

1.5

2.5

Brachyury

Oct-4

PDGFRA

Cha

Normoxic Adult

Hypoxic Adult

0.5

1.5

2.5

Brachyury

Oct-4

PDGFRA

Cha

Normoxic Neonate

Hypoxic Neonate

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

100

Cell Fr

Normoxic Adult

Hypoxic Adult

100

ell Fr

ncy

Normoxic Neonate

Hypoxic Neonate

REFERENCES

Baba, Y., Nosho, K., Shima, K., Hayashi, M., Meyerhardt, J. A., Chan, A. T., . . .

Ogino, S. (2011). Phosphorylated AKT expression is associated with

PIK3CA mutation, low stage, and favorable outcome in 717 colorectal

cancers. Cancer, 117(7), 1399-1408.

Baird, N. A., Turnbull, D. W., & Johnson, E. A. (2006). Induction of the heat

shock pathway during hypoxia requires regulation of heat shock factor by

hypoxia-inducible factor-1. Journal of Biological Chemistry, 281(50),

38675-38681.

Barile, L., Lionetti, V., Cervio, E., Matteucci, M., Gherghiceanu, M., Popescu, L.

M., . . . Vassalli, G. (2014). Extracellular vesicles from human cardiac

progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac

function after myocardial infarction. Cardiovascular Research, 103(4),

530-541.

Beg, A. A., & Baltimore, D. (1996). An essential role for NF-

B in preventing

TNF-

-induced cell death. Science, 274(5288), 782-784.

Beitner-Johnson, D., Rust, R. T., Hsieh, T. C., & Millhorn, D. E. (2001). Hypoxia

activates Akt and induces phosphorylation of GSK-3 in PC12 cells.

Cellular Signalling, 13(1), 23-27.

Beltrami, A. P., Barlucchi, L., Torella, D., Baker, M., Limana, F., Chimenti, S., . . .

Urbanek, K. (2003). Adult cardiac stem cells are multipotent and support

myocardial regeneration. Cell, 114(6), 763-776.

Bolli, R., Chugh, A. R., D'Amario, D., Loughran, J. H., Stoddard, M. F., Ikram, S.,

. . . Hosoda, T. (2011). Cardiac stem cells in patients with ischaemic

cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial. The

Lancet, 378(9806), 1847-1857.

Bu, L., Jiang, X., Martin-Puig, S., Caron, L., Zhu, S., Shao, Y., . . . Chien, K. R.

(2009). Human ISL1 heart progenitors generate diverse multipotent

cardiovascular cell lineages. Nature, 460(7251), 113-117.

Cai, C.-L., Liang, X., Shi, Y., Chu, P.-H., Pfaff, S. L., Chen, J., & Evans, S.

(2003). Isl1 identifies a cardiac progenitor population that proliferates prior

to differentiation and contributes a majority of cells to the heart.

Developmental Cell, 5(6), 877-889.

Chandel, N. S., McClintock, D. S., Feliciano, C. E., Wood, T. M., Melendez, J. A.,

Rodriguez, A. M., & Schumacker, P. T. (2000). Reactive oxygen species

generated at mitochondrial complex III stabilize hypoxia-inducible factor-

1 during hypoxia: a

mechanism of O2 sensing. Journal of Biological

Chemistry, 275(33), 25130-25138.

Cheng, K., Ibrahim, A., Hensley, M. T., Shen, D., Sun, B., Middleton, R., . . .

Marban, E. (2014). Relative roles of CD90 and c-kit to the regenerative

efficacy of cardiosphere-derived cells in humans and in a mouse model of

myocardial infarction. J Am Heart Assoc, 3(5), e001260.

Clément, M.-V., Ponton, A., & Pervaiz, S. (1998). Apoptosis induced by hydrogen

peroxide is mediated by decreased superoxide anion concentration and

reduction of intracellular milieu. FEBS letters, 440(1

2), 13-18.

David, R., Brenner, C., Stieber, J., Schwarz, F., Brunner, S., Vollmer, M., . . .

Franz, W. M. (2008). MesP1 drives vertebrate cardiovascular

differentiation through Dkk-1-mediated blockade of Wnt-signalling. Nature

Cell Biology, 10(3), 338-345.

David, R., Jarsch, V. B., Schwarz, F., Nathan, P., Gegg, M., Lickert, H., & Franz,

W.-M. (2011). Induction of MesP1 by Brachyury(T) generates the common

multipotent cardiovascular stem cell. Cardiovascular Research, 92(1),

115-122.

Deaton, C., Froelicher, E. S., Wu, L. H., Ho, C., Shishani, K., & Jaarsma, T.

(2011). The global burden of cardiovascular disease. European Journal of

Cardiovascular Nursing, 10(2 Suppl), S5-S13.

Dodou, E., Verzi, M. P., Anderson, J. P., Xu, S. M., & Black, B. L. (2004). Mef2c

is a direct transcriptional target of ISL1 and GATA factors in the anterior

heart field during mouse embryonic development. Development, 131(16),

3931-3942.

Ferreira-Martins, J., Ogorek, B., Cappetta, D., Matsuda, A., Signore, S.,

D'Amario, D., . . . Rota, M. (2012). Cardiomyogenesis in the developing

heart is regulated by c-kit-positive cardiac stem cells. Circ Res, 110(5),

701-715.

Filippi, I., Morena, E., Aldinucci, C., Carraro, F., Sozzani, S., & Naldini, A. (2014).

Short

term hypoxia enhances the migratory capability of dendritic cell

through HIF

1 and PI3K/Akt pathway.

Journal of Cellular Physiology,

229(12), 2067-2076.

Filomeni, G., De Zio, D., & Cecconi, F. (2015). Oxidative stress and autophagy:

the clash between damage and metabolic needs. Cell Death and

Differentiation, 22(3), 377-388.

Fuentes, T., & Kearns-Jonker, M. (2013). Endogenous cardiac stem cells for the

treatment of heart failure. Stem Cells and Cloning: Advances and

Applications, 6, 1-12.

Fuentes, T. I., Appleby, N., Tsay, E., Martinez, J. J., Bailey, L., Hasaniya, N., &

Kearns-Jonker, M. (2013). Human neonatal cardiovascular progenitors:

unlocking the secret to regenerative ability. PloS One, 8(10), e77464.

Gardner, L. B., Li, Q., Park, M. S., Flanagan, W. M., Semenza, G. L., & Dang, C.

V. (2001). Hypoxia inhibits G1/S transition through regulation of p27

expression. Journal of Biological Chemistry, 276(11), 7919-7926.

Gerbin, K. A., & Murry, C. E. (2015). The winding road to regenerating the human

heart. Cardiovascular Pathology, 24(3), 133-140.

Grayson, W. L., Zhao, F., Bunnell, B., & Ma, T. (2007). Hypoxia enhances

proliferation and tissue formation of human mesenchymal stem cells.

Biochemical and Biophysical Research Communications, 358(3), 948-953.

Guzy, R. D., Hoyos, B., Robin, E., Chen, H., Liu, L., Mansfield, K. D., . . .

Schumacker, P. T. (2005). Mitochondrial complex III is required for

hypoxia-induced ROS production and cellular oxygen sensing. Cell

Metabolism, 1(6), 401-408.

Hahn, J.-Y., Cho, H.-J., Kang, H.-J., Kim, T.-S., Kim, M.-H., Chung, J.-H., . . .

Kim, H.-S. (2008). Pre-treatment of mesenchymal stem cells with a

combination of growth factors enhances gap junction formation,

cytoprotective effect on cardiomyocytes, and therapeutic efficacy for

myocardial infarction. Journal of the American College of Cardiology,

51(9), 933-943.

Hidaka, K., Shirai, M., Lee, J. K., Wakayama, T., Kodama, I., Schneider, M. D., &

Morisaki, T. (2010). The cellular prion protein identifies bipotential

cardiomyogenic progenitors. Circ Res, 106(1), 111-119.

Hoek, T. L. V., Becker, L. B., Shao, Z., Li, C., & Schumacker, P. T. (1998).

Reactive oxygen species released from mitochondria during brief hypoxia

induce preconditioning in cardiomyocytes. Journal of Biological Chemistry,

273(29), 18092-18098.

Hong, K. U., Guo, Y., Li, Q.-H., Cao, P., Al-Maqtari, T., Vajravelu, B. N., . . .

Nong, Y. (2014). c-kit+ Cardiac stem cells alleviate post-myocardial

infarction left ventricular dysfunction despite poor engraftment and

negligible retention in the recipient heart. PloS One, 9(5), e96725.

Hou, X., Appleby, N., Fuentes, T., Longo, L. D., Bailey, L. L., Hasaniya, N., &

Kearns-Jonker, M. (2012). Isolation, characterization, and spatial

distribution of cardiac progenitor cells in the sheep heart. Journal of

Clinical & Experimental Cardiology, Suppl 6, 004.

Hu, S., Yan, G., Xu, H., He, W., Liu, Z., & Ma, G. (2014). Hypoxic preconditioning

increases survival of cardiac progenitor cells via the pim-1 kinase-

mediated anti-apoptotic effect. Circulation Journal, 78(3), 724-731.

Ishigami, S., Ohtsuki, S., Tarui, S., Ousaka, D., Eitoku, T., Kondo, M., . . . Oh, H.

(2015). Intracoronary autologous cardiac progenitor cell transfer in

patients with hypoplastic left heart syndrome: the TICAP prospective

phase 1 controlled trial. Circ Res, 116(4), 653-664.

Ivanovic, Z. (2009). Hypoxia or in situ normoxia: The stem cell paradigm. Journal

of Cellular Physiology, 219(2), 271-275.

Jiang, B., Wang, K., Liang, P., Xiao, W., Wang, H., & Xiao, X. (2009). ATP-

binding domain of heat shock protein 70 is essential for its effects on the

inhibition of the release of the second mitochondria-derived activator of

caspase and apoptosis in C2C12 cells. FEBS Journal, 276(9), 2615-2624.

Kamata, H., Honda, S.-i., Maeda, S., Chang, L., Hirata, H., & Karin, M. (2005).

Reactive oxygen species promote TNF

-induced death and sustained

JNK activation by inhibiting MAP kinase phosphatases. Cell, 120(5), 649-

661.

Kattman, S. J., Huber, T. L., & Keller, Gordon M. (2006). Multipotent Flk-1+

cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial,

and vascular smooth muscle lineages. Developmental Cell, 11(5), 723-

732.

Kim, J.-Y., & Park, J.-H. (2003). ROS-dependent caspase-9 activation in hypoxic

cell death. FEBS letters, 549(1), 94-98.

Kim, J., Wu, Q., Zhang, Y., Wiens, K. M., Huang, Y., Rubin, N., . . . Lien, C.-L.

(2010). PDGF signaling is required for epicardial function and blood vessel

formation in regenerating zebrafish hearts. Proceedings of the National

Academy of Sciences, 107(40), 17206-17210.

Kimura, W., & Sadek, H. A. (2012). The cardiac hypoxic niche: emerging role of

hypoxic microenvironment in cardiac progenitors. Cardiovascular

Diagnosis and Therapy, 2(4), 278-289.

Kook, S.-H., Son, Y.-O., Lee, K.-Y., Lee, H.-J., Chung, W.-T., Choi, K.-C., & Lee,

J.-C. (2008). Hypoxia affects positively the proliferation of bovine satellite

cells and their myogenic differentiation through up-regulation of MyoD.

Cell Biology International, 32(8), 871-878.

Koren, J., Jinwal, U. K., Jin, Y., O'Leary, J., Jones, J. R., Johnson, A. G., . . .

Dickey, C. A. (2010). Facilitating Akt clearance via manipulation of Hsp70

activity and levels. The Journal of Biological Chemistry, 285(4), 2498-

2505.

Koshiji, M., Kageyama, Y., Pete, E. A., Horikawa, I., Barrett, J. C., & Huang, L. E.

(2004). HIF

1 induces cell cycle arrest by functionally counteracting Myc.

The EMBO Journal, 23(9), 1949-1956.

Kubo, H., Jaleel, N., Kumarapeli, A., Berretta, R. M., Bratinov, G., Shan, X., . . .

Margulies, K. B. (2008). Increased cardiac myocyte progenitors in failing

human hearts. Circulation, 118(6), 649-657.

Laugwitz, K.-L., Moretti, A., Caron, L., Nakano, A., & Chien, K. R. (2007). Islet1

cardiovascular progenitors: a single source for heart lineages?

Development, 135(2), 193-205.

Li, L., & Xie, T. (2005). Stem cell niche: Structure and function. Annual Review of

Cell and Developmental Biology, 21(1), 605-631.

Libby, P., & Theroux, P. (2005). Pathophysiology of coronary artery disease.

Circulation, 111(25), 3481-3488.

Lin, Q., Lee, Y.-J., & Yun, Z. (2006). Differentiation arrest by hypoxia. Journal of

Biological Chemistry, 281(41), 30678-30683.

Luo, J., Weaver, M. S., Cao, B., Dennis, J. E., Van Biber, B., Laflamme, M. A., &

Allen, M. D. (2014). Cobalt protoporphyrin pretreatment protects human

embryonic stem cell-derived cardiomyocytes from hypoxia/reoxygenation

injury in vitro and increases graft size and vascularization in vivo. Stem

Cells Translational Medicine, 3(6), 734.

Madrid, L. V., Mayo, M. W., Reuther, J. Y., & Baldwin, A. S. (2001). Akt

stimulates the transactivation potential of the RelA/p65 subunit of NF-

through utilization of the IB kinase a

nd activation of the mitogen-activated

protein kinase p38. Journal of Biological Chemistry, 276(22), 18934-

18940.

Makkar, R. R., Smith, R. R., Cheng, K., Malliaras, K., Thomson, L. E., Berman,

D., . . . Johnston, P. V. (2012). Intracoronary cardiosphere-derived cells

for heart regeneration after myocardial infarction (CADUCEUS): a

prospective, randomised phase 1 trial. The Lancet, 379(9819), 895-904.

Manning, B. D., & Cantley, L. C. (2007). AKT/PKB signaling: navigating

downstream. Cell, 129(7), 1261-1274.

Marelli, A. J., Mackie, A. S., Ionescu-Ittu, R., Rahme, E., & Pilote, L. (2007).

Congenital heart disease in the general population changing prevalence

and age distribution. Circulation, 115(2), 163-172.

Matsuura, K., Honda, A., Nagai, T., Fukushima, N., Iwanaga, K., Tokunaga, M., .

. . Hagiwara, N. (2009). Transplantation of cardiac progenitor cells

ameliorates cardiac dysfunction after myocardial infarction in mice. The

Journal of Clinical Investigation, 119(8), 2204.

McGill, H. C., McMahan, C. A., & Gidding, S. S. (2008). Preventing heart disease

in the 21st century: Implications of the pathobiological determinants of

atherosclerosis in youth (PDAY) study. Circulation, 117(9), 1216-1227.

Moretti, A., Caron, L., Nakano, A., Lam, J. T., Bernshausen, A., Chen, Y., . . .

Martin-Puig, S. (2006). Multipotent embryonic isl1+ progenitor cells lead to

cardiac, smooth muscle, and endothelial cell diversification. Cell, 127(6),

1151-1165.

Morey, R. D. (2008). Confidence intervals from normalized data: A correction to

Cousineau (2005). The Quantitative Methods for Psychology, 4(2), 61-64.

Moungjaroen, J., Nimmannit, U., Callery, P. S., Wang, L., Azad, N., Lipipun, V., .

. . Rojanasakul, Y. (2006). Reactive oxygen species mediate caspase

activation and apoptosis induced by lipoic acid in human lung epithelial

cancer cells through Bcl-2 down-regulation. Journal of Pharmacology and

Experimental Therapeutics, 319(3), 1062-1069.

Mozaffarian, D., Benjamin, E. J., Go, A. S., Arnett, D. K., Blaha, M. J., Cushman,

M., . . . Stroke Statistics, S. (2015). Heart disease and stroke statistics -

2015 update: A report from the American Heart Association. Circulation,

131(4), e29-322.

Poss, K. D., & Tonegawa, S. (1997). Reduced stress defense in heme

oxygenase 1-deficient cells. Proceedings of the National Academy of

Sciences, 94(20), 10925-10930.

Powers, M. V., Clarke, P. A., & Workman, P. (2008). Dual targeting of HSC70

and HSP72 inhibits HSP90 function and induces tumor-specific apoptosis.

Cancer Cell, 14(3), 250-262.

Rosenblum, S., Smith, T. N., Wang, N., Chua, J. Y., Westbroek, E., Wang, K., &

Guzman, R. (2014). BDNF pretreatment of human embryonic-derived

neural stem cells improves cell survival and functional recovery after

transplantation in hypoxic-ischemic stroke. Cell Transplantation, 24(12),

2499-2461.

Sanada, F., Kim, J., Czarna, A., Chan, N. Y.-K., Signore, S., Ogórek, B., . . . Leri,

A. (2014). c-kit-positive cardiac stem cells nested in hypoxic niches are

activated by stem cell factor reversing the aging myopathy. Circ Res,

114(1), 41-55.

Schmittgen, T. D., & Livak, K. J. (2008). Analyzing real-time PCR data by the

comparative CT method. Nature Protocols, 3(6), 1101-1108.

Simpson, D. L., Mishra, R., Sharma, S., Goh, S. K., Deshmukh, S., & Kaushal, S.

(2012). A strong regenerative ability of cardiac stem cells derived from

neonatal hearts. Circulation, 126(11 Suppl 1), S46-S53.

Stewart, S., MacIntyre, K., Hole, D. J., Capewell, S., & McMurray, J. J. (2001).

More `malignant' than cancer? Five

year survival following a first

admission for heart failure. European Journal of Heart Failure, 3(3), 315-

322.

Studer, L., Csete, M., Lee, S.-H., Kabbani, N., Walikonis, J., Wold, B., & McKay,

R. (2000). Enhanced proliferation, survival, and dopaminergic

differentiation of CNS precursors in lowered oxygen. The Journal of

Neuroscience, 20(19), 7377-7383.

Tan, S. C., Gomes, R. S. M., Yeoh, K. K., Perbellini, F., Malandraki-Miller, S.,

Ambrose, L., . . . Carr, C. A. (2016). Preconditioning of cardiosphere-

derived cells with hypoxia or prolyl-4-hydroxylase inhibitors increases

stemness and decreases reliance on oxidative metabolism. Cell

Transplantation, 25(1), 35-53.

Utting, J., Robins, S., Brandao-Burch, A., Orriss, I., Behar, J., & Arnett, T. (2006).

Hypoxia inhibits the growth, differentiation and bone-forming capacity of

rat osteoblasts. Experimental Cell Research, 312(10), 1693-1702.

van Berlo, J. H., Kanisicak, O., Maillet, M., Vagnozzi, R. J., Karch, J., Lin, S.-C.

J., . . . Molkentin, J. D. (2014). c-kit+ cells minimally contribute

cardiomyocytes to the heart. Nature, 509(7500), 337-341.

van Oorschot, A. A., Smits, A. M., Pardali, E., Doevendans, P. A., & Goumans,

M. J. (2011). Low oxygen tension positively influences cardiomyocyte

progenitor cell function. Journal of Cellular and Molecular Medicine,

15(12), 2723-2734.

Wu, S. M., Fujiwara, Y., Cibulsky, S. M., Clapham, D. E., Lien, C.-l., Schultheiss,

T. M., & Orkin, S. H. (2006). Developmental origin of a bipotential

myocardial and smooth muscle cell precursor in the mammalian heart.

Cell, 127(6), 1137-1150.

Yan, F., Yao, Y., Chen, L., Li, Y., Sheng, Z., & Ma, G. (2012). Hypoxic

preconditioning improves survival of cardiac progenitor cells: role of

stromal cell derived factor-

CXCR4 axis. PloS One, 7(7), e37948.

Yang, Y.-P., Li, H.-R., Cao, X.-M., Wang, Q.-X., Qiao, C.-J., & Ya, J. (2013).

Second heart field and the development of the outflow tract in human

embryonic heart. Development, Growth & Differentiation, 55(3), 359-367.

Zaruba, M. M., Soonpaa, M., Reuter, S., & Field, L. J. (2010). Cardiomyogenic

potential of C-kit(+)-expressing cells derived from neonatal and adult

mouse hearts. Circulation, 121(18), 1992-2000.

Frequently asked questions

What is the abstract about?

The abstract discusses the limitations of conventional medical treatments for coronary events and the potential benefits of stem cell transplantation for heart tissue repair. It highlights the challenge of using autologous cells from older adults due to their diminished functional capacity. The abstract proposes that a pretreatment, like short-term hypoxia, combined with novel progenitors (Isl-1 and c-Kit co-expressing cells), may improve cardiovascular regeneration outcomes. The effects of short-term hypoxia on this novel cell type warrant further investigation in vitro.

What is the purpose of the acknowledgements section?

This is thanking the comittee, Dr. Bournias-Vardiabasis, Dr. Kearns-Jonker, Dr. Thompson, CIRM, the faculty of the Biology Department at CSUSB, Dave Coffey, Nancy Appleby, Jonathan Baio, Tania Fuentes, friends, and family for their support. It is dedicated to the author's sisters.

What does the table of contents include?

The table of contents outlines the document's structure, including sections for the abstract, acknowledgements, list of tables, introduction, materials and methods, results, discussion, appendix A (figures), and references. The introduction includes a background and definition of terms. The materials and methods cover in vivo components and experiments, in vitro assays, flow cytometry, and statistical analysis.

What does the list of tables include?

The list of tables contains only one entry, "Table 1. Primer Sequences Used for qRT-PCR" and specifies that it can be found on page 13.

What does Chapter One (Introduction) include?

Chapter One provides background information on cardiovascular diseases (CVDs), discussing their etiology, global impact, and the need for novel treatments. It covers cardiovascular stem cell-based therapy for the injured heart, including the use of cardiac progenitor cells (CPCs). The chapter also explores optimizing cell type, highlighting Isl-1+ c-Kit+ CPCs, and hypoxic preconditioning as a potential strategy to enhance cell function. It concludes with definitions of terms used throughout the document.

What are the key points of the section on Cardiovascular Stem Cell-based Therapy for the Injured Heart?

This section discusses the shift in focus from bone marrow-derived cells to cardiac progenitor cells (CPCs) for stimulating repair in heart tissue, and the encouraging results of recent clinical trials. However, transplanted CPCs often fail to engraft and differentiate into new myocardium, indicating that improvements are a result of paracrine signaling. Autologous stem cells from adult patients lack the functional potency of neonatal cells. Thus, new novel cell types or pre-treatment methods are needed.

What are the objectives of the section Optimizing Cell Type?

This section describes cardiac progenitor cells (CPCs) and their ability to self-renew, expand, and differentiate. Particular focus is on the Isl-1+ c-Kit+ population and its role in early heart formation. It compares this population to single positive cells and emphasizes the need for further investigation of Isl-1+ c-Kit+ CPCs.

What are the objectives of the Hypoxic Preconditioning?

This section explains hypoxia treatment's potential to boost cellular function through intracellular signaling pathways. It discusses oxygen levels in the body and heart tissues, suggesting the heart's stem cell niches and constituent CPCs are consistently hypoxic. In vitro evaluations of clonal Isl-1+ c-Kit+ hCPCs under short-term hypoxia (six hours at 1.0 percent O2) are warranted. This section tests the hypothesis that short-term hypoxia upregulates Akt phosphorylation and leads to enhanced cell function.

What does Chapter Two (Materials and Methods) describe?

Chapter Two details the materials and methods used in the study. It covers the isolation and culture of Isl-1+ c-Kit+ hCPCs, hypoxic preconditioning procedures, the Transwell® invasion assay, quantitative reverse transcription PCR (qRT-PCR), Western blotting techniques, and flow cytometry protocols. It also includes specific antibodies used and a description of cell cycle analysis, TUNEL assays, and statistical analysis methods. The study involved cloning Isl-1+ c-Kit+ hCPCs, preconditioning them with short-term hypoxia, and evaluating their function.

What are the main points of the Western blotting procedures mentioned in Chapter Two?

The Westen blotting procedure focuses on the effects of short-term hypoxia. The protein immunoblots are prepared for Akt and phosphorylated Akt. -Actin and p-Akt were prepped using serum starved hCPCs that were treated with SDF-1 and hypoxia.

What does the section on Antibodies Used in Cytometry Experiments include?

This section includes antibodies used for cytometric analysis like: Anti-Isl-1, Anti-c-Kit, Anti-Akt phospho (Serine 473), Fluorescein-anti-BrdU, FITC goat anti-mouse IgG, PE goat anti-mouse IgG, and FITC goat anti-rabbit IgG.

What is in chapter three results section?

This chapter mainly talks about the results of Akt Activation in Isl-1+ c-Kit+ hCPCs. Some results include that hypoxic preconditioning stimulates the Akt Pathway and that neonatal hCPCs more strongly upregulate PIK3CA mRNA than do adult hCPCs. In addition, the Isl-1+ c-Kit+ hCPCs are shown to Invade More Readily After Exposure to Short-term Hypoxia and to Trigger a Pro-survival Response.

What is discussed in Chapter Four's Conclusion section?

Short-term hypoxia is a viable pretreatment option for supporting cellular survival and enhancing migratory capabilities in neonatal Isl-1+ c-Kit+ hCPCs. It was mentioned the most significant results were found in the neonatal group and short-term preconditioning could improve surgical procedures.

Excerpt out of 60 pages - scroll top

Details

Title: Priming cardiovascular stem cells for transplantation using short-term hypoxia
College: California State University San Bernardino
Author: Ivan Hernandez (Author)
Publication Year: 2016
Pages: 60
Catalog Number: V336311
ISBN (eBook): 9783668272712
ISBN (Book): 9783668272729
Language: English
Tags: hypoxia stem cells cardiac progenitors cardiovascular disease
Product Safety: GRIN Publishing GmbH

Quote paper: Ivan Hernandez (Author), 2016, Priming cardiovascular stem cells for transplantation using short-term hypoxia, Munich, GRIN Verlag, https://www.grin.com/document/336311

Priming cardiovascular stem cells for transplantation using short-term hypoxia

Excerpt

Frequently asked questions

What is the abstract about?

What is the purpose of the acknowledgements section?

What does the table of contents include?

What does the list of tables include?

What does Chapter One (Introduction) include?

What are the key points of the section on Cardiovascular Stem Cell-based Therapy for the Injured Heart?

What are the objectives of the section Optimizing Cell Type?

What are the objectives of the Hypoxic Preconditioning?

What does Chapter Two (Materials and Methods) describe?

What are the main points of the Western blotting procedures mentioned in Chapter Two?

What does the section on Antibodies Used in Cytometry Experiments include?

What is in chapter three results section?

What is discussed in Chapter Four's Conclusion section?

Details