1

Stem cell-based

approachesto prevent

hyperoxic lung injuryTim van Haaften, Juliana Rey-Parra, Roisin Byrne, Lavinia Coltan, Rajesh Anthuvan,

Farah Eaton, Bernard Thébaud

Department of Pediatrics, Northern Alberta Neonatal Intensive Care Program

Women and Children!s Health Research Institute

University of Alberta, Edmonton, Canada

Lung Development, Injury and Repair

(1509 or 1511-1553)

“Considérations Générales sur la

Nature des Acides” (1778)

"air" is responsible for combustion and the source

of acidity

- Oxygen (Greek for acid-former)

- Azote (Greek for no life)

- Hydrogen (Greek for water-former)

Antoine Laurent LavoisierChimiste français (1743-1794)

2

“For as a candle burns much faster

in dephlogisticated (oxygen-enriched) than in common air,

so we might live out too fast, and the animal powers

be too soon exhausted in this pure kind of air.

A moralist, at least, may say,

that the air which nature has provided for us

is as good as we deserve”

Priestley J: Experiments and Observations on Different Kinds of Air.

Second Edition. London, 1775, 101

History of assisted ventilation in newborns

"Many children, especiallythose prematurely born, diefrom inability to expandtheir lungs sufficientlywhen they take their firstbreath. I have no doubtthat in many of thosecases, lives could be savedby starting the respirationartificially by means ofapparatus operating in themanner described above.”

Alexander Graham Bell, 1889 Donald and Lord, 1953

3

www.embryology.ch

Histological stages of lung development

www.embryology.ch

Historical BPD originally described by Northway

Northway et al, NEJM 1967Survivors with BPD:

34 GA, 2234gNon-survivors:31 GA, 1660g

www.embryology.ch

Northway et al, NEJM 1967Survivors with BPD:

34 GA, 2234gNon-survivors:

31 GA, 1660g

“New BPD”Jobe A.

Pediatr Res 1999

The “New BPD”: An Arrest in Lung Development

Intrauterine

Cytokine

exposure

Initial and

Chronic

Ventilation

Oxygen

exposurePostnatal

Gluco-

corticoids

Postnatal

Infection Nutrition

Experimental Models mimicking BPD

Prematurity Chorioamnionitis Oxygen Ventilation Dex

-(born at

saccular stage)± +

(max 24 hrs*)+

± +(2 weeks)

++

(+)

± +(max 8 hours)

+-

± +(2 weeks)

++

4

Decreased Alveolarization in BPD:

Potential Long Term Consequences

Cutz E, Chiasson D. New Engl J Med 2008

12-year-old boy history of prematurity (26 wks-596g)

and BPD who died of an acute asthma attack.

Age-mached control

Wong PM et al. Eur Respir J 2008

Thin section CT of a 20 year old non-smoking male born 1355g at

28 weeks gestation, dependent upon supplementary oxygen to 321

days postpartum. There is severe emphysema and almost complete

replacement of the right upper lobe with a large bulla

Mechanisms of Lung Injury and Repair

Remain Incompletely Understood

Griffiths MJD et al. Lancet 2005

Alveolar Type 2 cell dedifferentiationBone Marrow-derived cell

homing and engraftment

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• A stem cell is a cell that has the ability to divide (self replicate) for

indefinite periods—often throughout the life of the organism.

• Under the right conditions, or given the right signals, stem cells can

differentiate into many different cell types. That is, stem cells have

the potential to develop into mature cells that have characteristic

shapes and functions, such as heart cells, skin cells, or nerve cells.

Stem Cell Potential

TotipotentZygote or fertilized egg

Embryo Pluripotent

Differentiated

Cells(© 2001 Terese Winslow, Caitlin Duckwall)

Bone Marrow-derived Hematopoietic Stem Cell:

the best characterized Stem Cell

(© 2001 Terese Winslow, Caitlin Duckwall)

Mesenchymal Stem Cells (MSC)

Bone Marrow-derived Mesenchymal Stem Cell:

a “high reparative potential” Stem Cell

Friedenstein AJ. Int Rev Cytol 1976

6

Bone Marrow-derived Mesenchymal Stem Cell:

a “high reparative potential” Stem Cell

<http://stemcells.nih.gov/info/basics/basics4>

Tim van Haaften, MSc

Experimental O2-induced “BPD” in Newborn Rats

Alveolar stage (P4-14)Birth (P0) P15

95% Oxygen

O2

x10 x20

Co

ntr

ol-

Ro

om

air

5µm

95

% O

2-

BP

D m

od

el

50µm

FITC lectin Barium AngiogramH&E stainingScanning Electron

Microscopy

Scanning Electron

Microscopy

50µm

Alveolar structures Lung vascular structures

Decreased lung resident MSC in O2-induced lung injury

CD44 CD54 CD31CD73 CD90 CD45

10 10 10 10 100 1 2 3 4

BM

Lu

ng

NL

un

g H

10 10 10 10 100 1 2 3 4

10 10 10 10 100 1 2 3 4

10 10 10 10100 1 2 3 4

10 10 10 10 100 1 2 3 4

10 10 10 10 100 1 2 3 4

10 10 10 10 100 1 2 3 4

10 10 10 10100 1 2 3 4

10 10 10 10 100 1 2 3 4

10 10 10 10 100 1 2 3 4

10 10 10 10100 1 2 3 4

10 10 10 10 100 1 2 3 4

10 10 10 10 100 1 2 3 4

10 10 10 10100 1 2 3 4

10 10 10 10 100 1 2 3 4

10 10 10 10 100 1 2 3 4

10 10 10 10100 1 2 3 4

10 10 10 10 100 1 2 3 4

A

Ch

on

dro

gen

icA

dip

og

en

icO

steo

gen

ic

BM Lung N Lung H

Co

ntr

ol

med

ia

BM-MSC migrate towards O2-injured lungs

B

4

8

12

16

RF

U

*

DM

EM

No

rmO

2L

un

g

Hy

per

O2

Lu

ng

0

Migration(8µm pore)

MSC

CF

U-F

nu

mb

er p

er 1

06No

rm

ox

iaH

yp

ero

xia

C0

0.5

1

1.5

2

2.5

BM

0

200

400

600

800

Lung

*

Normoxia Hyperoxia

7

DM

EM

SA

GM

SA

GM

+

O2-i

nju

red

lu

ng

DAPI Merge SP-C

In vitro BMSC co-cultured with oxygen-injured lung

adopt a lung alveolar epithelial cell phenotype

BMSC

0.4µm

pore

Co-culture2!!

Ct

18

S

0

.5

1

1.5

2

DM

EM

SAG

MLung

in S

AG

M

qRT-PCRSP-C mRNA

DMEM

SAGM

Lung

Fetal type II AEC

In vitro, BMSC co-cultured with injured lung adopt

morphological features of alveolar epithelial cells

BMSC + culture media

BMSC + injured lung

In vivo intra-tracheal BMSC therapy is feasible

and results in lung engraftment

Untreated Lung BMSCs Treated Lung

x25

Blue = DAPI, marks cell nuclei

Red = Surfactant protein C (SP-C) marks lung cells

Green = CFSE labeled BMSC

Engrafted BMSCs express the

alveolar epithelial cell marker SP-C

CFSE SP-C Merge

x150

Engrafted cells: 3.7% ± 2.9%

Conversion: 75.3%± 24.5%

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Airway delivery of BM-derived MSC improves

survival in O2-induced BPD in newborn rats

Time (Days)

% S

urv

ival

*

Exposure to 95% Oxygen0

20

40

60

80

100

0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

Normoxia

Hyperoxia +

BM-MSC

Hyperoxia

Hyperoxia +

PASMC

CFSE (green) labeled BM-MSC

Airway delivery of BM-derived MSC improves

exercise capacity in O2-induced BPD in newborn rats

Time (Days)

*P<0.001 vs

Hyperoxic

0

200

400

600

800

Distance (m)

*

Normoxia

Hyperoxia

Hyperoxia +

BM-MSC

Hyperoxia +

PASMC

Airway delivery of BM-derived MSC prevents

alveolar injury in O2-induced BPD in newborn rats

10X 20X

Normoxia Hyperoxia Hyperoxia + BM-MSC Hyperoxia + PASMC

Normoxia

Hyperoxia

Hyperoxia + BM-MSC

Hyperoxia + PASMC

Mean Linear Intercept (µm)

0

10

20

30

40

50

60

*

*

N=6/group

*P<0.05

No

rmo

xia

Hy

per

ox

iaH

yp

ero

xia

+B

MS

C`

Improved Vascular Morphometry

Airway delivery of BM-derived MSC attenuates

O2-induced lung vascular injury

Normoxia

Hyperoxia

Hyperoxia +

BMSC

Decreased Pulmonary Hypertension

0

.01

.02

.03

PA

AT

(se

c.)

*P<0.001 vs Normoxia

(Fisher’s PLSD) n = 10/group

*

RV

/LV

+S

*P<0.001 vs Hyperoxia

(Fisher’s PLSD) n = 10/group

0

.1

.2

.3

.4

.5

*

9

Normoxia

Hyperoxia

Hyperoxia + MSC

Hyperoxia + PASMC

0

50

100

150

200

250

300Vessels/High Powered Field (40x)

*

*P<0.001 vs Hyperoxic

(Fisher’s PLSD)

Normoxia Hyperoxia

+ MSC

Hyperoxia

+ PASMC

Hyperoxia

Airway delivery of BM-derived MSC prevents

capillary rarefaction in O2-induced BPDInterim Summary (1/3):

Adult BM-MSC prevent O2-induced lung injury

Bone Marrow-derived cell

homing and engraftment

Adult Bone Marrow-derived MSC:

!Migrate preferentially towardsinjured lung

!can be administeredintratracheally similar toroutinely used surfactant

! engraft into the injured lung

! adopt a lung phenotype

! improve survival

! prevent O2-induced lung injury

Harnessing the Therapeutic Potential ofHuman Umbilical Cord Blood derived cells

• Clinically relevant

• Discarded source of

potent stem cells

• Strong reparative

potential

• Devoid of ethical dilemma

Juliana Rey, MD, PhD

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Mean Linear Intercept (µm)

0

10

20

30

40

Control Hyperoxia Hyperoxia

+ UCB

*

N=5 each, *P<0.02 vs Hyperoxia

Hyperoxia+UCBHyperoxia (BPD model)Normoxic Control

O2 chamber

HUCB-derived cells attenuate O2-induced

Lung Injury in Newborn Nude RatsIdiopathic Pulmonary Fibrosis (PF)

• Progressive dyspnea and

abnormal lung function

• Characterized by epithelial

injury, fibroblast proliferation

• ECM growth, collagen

deposition

• No proven prevention or

treatment

• Life expectancy 3-5 years http://images.google.ca/imgres?imgurl=http://upload.wikimedia.org

/wikipedia/en/thumb/d/d1/PCP_CAP_CXR

BLM +HUCB cells n=7

BLM n=6

Control n=7

HUCB-cells improve Exercise Capacity

in Bleomycin (BLM)-induced PF in Mice

*p<0.05

*

*

5

10

15

20

25

30

35

40

45

Min

ute

s

Before 7days

after

treatment

14 days

after

treatment

21 days

after

treatment

Co

mp

lian

ce c

m/H

2O

.s/m

l

BLM +

HUCB

BLMControl

0.00

0.01

0.02

0.03

HUCB-cells improve Lung Function inBLM-induced PF in Mice

Ela

stan

cecm

/H2

O.s

/ml

BLM +

HUCB

BLMControl

0.00

10.0

30.0

50.0*

*

*p<0.05

Compliance Elastance

7 6 7 7 6 7

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HUCB-cells decrease BLM Lung Fibrosis

To

tal

coll

agen

(!

g/m

g)

Control BLM BLM +

HUCB

0

8

4

12

*

*

7 6 7

Control BLM BLM +

HUCB

Hy

dro

xy

pro

lin

e (!

g/m

g)

0.00

0.60

0.40

0.20

0.80

*

*

7 6 7

Collagen Content

Control BLM BLM + HUCB-MSC

Interim Summary (2/3):HUCB-derived cells prevent experimental lung injury

Bone Marrow-derived cell

homing and engraftment

Engrafted BMSCs express the

alveolar epithelial cell marker SP-C

CFSE SP-C Merge

x150

Engrafted cells: 3.7% ± 2.9%

Conversion: 75.3%± 24.5%

Roisin Byrne, MSc

12

BMSC-derived CdM decreases O2-induced

apoptosis of rat AEC2N

orm

ox

iaH

yp

ero

xia

DMEM BM-MSC

Conditioned media

Room

air

Hyp

eroxia

80%

DMEM

n = 10

BMSC

CdM

n = 9

% of Apoptotic AEC2

in hyperoxia

0102030405060708090

*

*P<0.05

BMSC-derived CdM improves AEC2

wound healing in vitro

BMSC-derived CdM enhances

lung endothelial cell cord formation in vitro

In vivo effect of BMSC-derived CdM in acute

LPS-induced lung injury

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BMSC-derived CdM reduces BAL total cell and

neutrophil count in LPS-induced lung injury

Interim Summary (3/3):

MSCs may act via a paracrine mechanism

Bone Marrow-derived cell

secretion of repair modulating factors

Bone Marrow-derived cell

homing and engraftment

Paracrine activity

Injury

Today in the Lab: HUCB derived cells

prevent lung injury

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Injury

Tomorrow in the NICU? Perspectives• To improve our understanding of lung development-injury-

repair

• To improve our understanding of stem cell biology (homing,

engraftment, plasticity…) and identify of the best “reparative”cell

• To carefully assess the short- and long-term safety (tumor

formation…?) and efficacy (structure and function) of stem cell-based therapies

• No single magic bullet for a multi-factorial disease

• Incremental improvement and combined use of availableand new therapeutic strategies

The Team

Gaia Weissmann

Juliana Rey Roisin Byrne

Lavinia Coltan

“Stem Cell ” Team

Collaborators:

- Jacques Galipeau, Montreal- Merv Yoder, U Indianapolis

- Jonathan Davis, Boston

- Fabio Mosca, Milano

- Lorenza Lazzari, Milano

- John Akabutu, U of Alberta

Christina Luong

Arul Vadivel

Rajesh Anthuvan

Paul Waszak

Bev Morgan

Farah Eaton

PPHN

Lung Progenitor Cell“Angiogenesis”

Team

“Tech” Team

Before Thomas Edison, it was evident that

we would always produce light with a candle

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Preparation of BMSC Conditioned Media CdM

Media CollectionSerum-Starve for

24 hours

• BMSCs freshly isolated from

adult Sprague Dawley rats or C57

mice.

• Supernatant replenished with

serum for in vitro studies and

concentrated and de-salted for in

vivo studies.

Stem Cells 80%

Confluent

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Experimental design

2. Wound Healing Assay with fetal rat Type II Alveolar Epithelial Cells

MATRIGEL

3. Rat lung microvascular endothelial cell cord formation assay

85% O2

36 hours

Apoptosis &

Proliferation

1. Oxygen-Induced Injury of fetal rat Type II Alveolar Epithelial Cells

The Decision to Go to the Moon: President John F. Kennedy's

"Special Message to the Congress on Urgent National Needs”

May 25, 1961

Patrick Bouvier Kennedy (August 7, 1963 - August 9,1963) was the younger son of United States PresidentJohn F. Kennedy and First Lady Jacqueline BouvierKennedy. He was born five and a half weeksprematurely by caesarean section at the Otis Air ForceBase Hospital, with a birth weight of 2,112 g…

… Kennedy was aware that Drs. Paul Swyer andMaria Delivoria had successfully ventilated an infantwith RDS in Toronto nearly two years earlier, whobecame one of the few survivors of neonatal positivepressure ventilation until that time. A decision wasmade to transfer the baby there for care, but politicalpressures intervened. At the last minute, the child wastransferred to Boston Children's Hospital, where hewas placed in a hyperbaric oxygen chamber, and diedtwo days later of hyaline membrane disease.

His obituary in The New York Times pointed out that,at that time, all that could be done "for a victim ofhyaline membrane disease is to monitor the infant'sblood chemistry and to try to keep it near normallevels. Thus, the battle for the Kennedy baby was lostonly because medical science has not yet advancedfar enough to accomplish as quickly as necessarywhat the body can do by itself in its own time".

More than any other single event, the death of thisinfant served to ignite public and medical awarenessto the need for neonatal intensive care and soon led tothe establishment of NICUs around the country.

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Monday 21st JULY 1969

“The still unnamed white

substance, Dr. Clements

said, is a surface active

agent normally secreted

by mammalian lungs to

coat the inside walls of

the alveoli and thus

prevent lung collapse…”

Alveoli in a Term infant

Hyaline Membrane Disease of Premature Infants

is due to Surfactant Deficiency

O2

CO2

The drug is a credit to microbiologists, physiologists and pathologistswho were the first to define the substance, which occurs naturally in thebody but is deficient in premature babies. Testing with laboratoryanimals yielded encouraging results. Trials with humans establishedsynthesized surfactant's efficacy, and it was approved for use in 1990!

Collapsed Airway in a Preterm Infant