In Vivo Models and Cell Delivery for Lung Indications

Marlowe Eldridge MD

Department of Pediatrics and Biomedical Engineering University of Wisconsin School of Medicine and

Public Health

NO DISCLOSURES

Manabezho Falls, Upper Peninsula of Michigan

� What is the primary question? Safety? Efficacy?

� How closely does the animal model translate to the human disease and stage of disease?

� What are the technical challenges of the model development?

� Does the model allow measurement of relevant physiologic information?

� Does the model allow mimicking of the planned human dosing schedule?

FACTORS TO CONSIDER FOR SELECTING THE APPROPRIATE ANIMAL MODEL

� Hyperoxia

� Mechanical ventilation

� Hyperoxia and mechanical ventilation

CURRENT MODELS FOR BPD

� Percentage of inspired oxygen (FIO2)

� Duration of administration

� Timing of administration

RAT MODEL OF BRONCHOPULMONARY

DYSPLASIA

BRONCHOPULMONARY DYSPLASIA

� Typically seen in the following population: � Premature babies

� <1500 g birth weight

� Mechanical ventilation

� Oxygen therapy

� 20-30% of ventilated newborns develop BPD

� Defined by: � Disrupted distal lung growth

� Requirement of supplemental oxygen for hypoxemia

� Abnormal chest radiograph at 36 weeks

� Persistent respiratory signs and symptoms

BDP DISEASE ETIOLOGY AND AT-RISK POPULATION

Kinsella, J.P., Greenough, A., and Abman, S.H. 2006. Bronchopulmonary dysplasia. Lancet 367:1421-1431.

� Mimic the disease as closely as possible � Bronchopulmonary dysplasia

� Decreased alveolarization and septation of alveoli � Decreased pulmonary vascularity

� Access to animals, cost, housing (ease)

� Rats born with lungs developed to point of pre-term human

� Ability to achieve endpoints in your model � Comparability of animal to human

CHOOSING THE ANIMAL MODEL

� Vasculature:

� High pulmonary vascular resistance

� Fewer arteries

� Decreased angiogenesis

� Lower surface area

� Abnormal vasoreactivity

BPD FEATURES

� Airways:

� Fewer and simplified alveoli

� Increased smooth muscle in airways

� Cardiovascular:

� Right and left heart hypertrophy and dysfunction

� Prominent systemic-to-pulmonary collateral vessels (some large)

http://www.embryology.ch/anglais/rrespiratory/phasen07.html

LUNG DEVELOPMENT - STAGES

LUNG DEVELOPMENT - STAGES

Seaborn et al., Vol 21, Issue 12 December 2010, Pages 729–738

ALVEOLAR GROWTH AND PULMONARY CAPILLARITY IN NEONATAL LUNG INJURY

IN RATS

Van Haaften et. al., Am. J. Respir. Crit. Care. Med; 2009 vol. 180 no. 11 1131-1142

� Exposed to 95%O2 PND 1-14 � Dams rotated � Studied P14 or P21

P14

ISOLATION AND DIFFERENTIATION OF BONE MARROW STROMAL CELL CULTURES

AND EXPERIMENTAL DESIGN

Aslam et. al., Am. J. Respir. Crit. Care. Med; 2009 vol. 180 no. 11 1122-1130

TREATMENT WITH CONDITIONED MEDIA OF BMSC, NOT PASMC, PREVENTED ALVEOLAR

LOSS

Aslam et. al., Am. J. Respir. Crit. Care. Med; 2009 vol. 180 no. 11 1122-1130

BMSC TREATMENT MODESTLY IMPROVED THE VOLUME DENSIT Y OF ALVEOLAR WALL TISSUE IN

HYPEROXIA-TREATED MICE

Aslam et. al., Am. J. Respir. Crit. Care. Med; 2009 vol. 180 no. 11 1122-1130

MOUSE GENETIC MODEL OF BRONCHOPULMONARY

DYSPLASIA

Benjamin et al., 2007.

FGF SIGNALING IS REDUCED IN BPD LUNG

FGFR3;FGFR4 MUTANTS SHOW SIMPLIFIED ALVEOLI

Fgfr

3-;F

gfr4

- C

ontr

ol

P8 P5

ETV MUTANT LUNGS EXHIBIT SIMPLIFIED ALVEOLI

John Herriges

MOUSE MODEL OF ACUTE LUNG INJURY

AND SUBSEQUENT FIBROSIS

OmentumStromal Cell IP Delivery

BLEOMYCIN 2: INTRATRACHEAL INSTILLATION OF BLEOMYCIN INDUCES INFLAMMATION AND

FIBROSIS

Interstitial inflammation

020040060080010001200140016001800

Airway inflammation C

ell n

umbe

r x

10

-3

AIRWAY EPITHELIAL THICKNESS

Blm Blm + OSC Sal Ctrl

Day 3

μm

* *

BLM BLM +OSC

Sal Ctrl

50 μm BAL Cytokines/Chemokines

* * *

Day 3

Con

cent

ratio

n (p

g/m

l)

IL-6 IL-12 G-CSF CCL-2

PRELIMINARY RESULTS B

AL

Cel

l Num

ber

Day 7

Airway inflammation

*Volume Density

of Lesion

Blm Blm + OSC

Blm OSC

Interstitial inflammation

BA

L C

ell N

umbe

r

*

EXPERIMENTAL SETUP

Intraperitoneal injection of sterile polyacrylamide slurry

Surgical removal of omentum

Transfer of purified cells 4x106 (Intraperitoneal

injection)

BLM Instillation

(0.04 U/mouse in 30 μl saline, 30G needle; Aerosolized with high pressure

syringe)

Prepare cell suspension OSC Analysis

Bronchoalveolar Lavage Histology

Airway Epithelium

e)

HISTOLOGY OF OMENTUM AFTER BEAD INJECTION

Day 1 Day 3 Day 2

Bleo Ctrl 7 d

bleo ctrl

Lung

MIGRATION OF IP INJECTED OSCS

MOUSE GENETIC MODEL OF CONGENITAL

DIAPHRAGMATIC HERNIA

CONGENITAL DIAPHRAGMATIC HERNIA

� CDH has an incidence of 1 in 2500 births

� Pulmonary hypoplasia and hypertension is a important clinical feature of CDH

� Persistent Pulmonary hypertension accounts for 95% mortalities inn CDH

CONGENITAL DIAPHRAGMATIC HERNIA

� Recent studies have focused on identifying genes that control diaphragm development

� These genes may play important roles in pulmonary and pulmonary vascular development

� A recent study identified Pbx1 as an important gene involved in diaphragm development ((Russel l et al , 2012)

� Do Pbx genes play an important role in pulmonary or pulmonary vascular development? � Lung mesenchyme specific knockout of Pbx1

WHY DO CERTAIN PATIENTS WITH CDH HAVE PERSISTENT RESPIRATORY DISTRESS AND PULMONARY HYPERTENSION DESPITE SURGICAL CORRECTION OF THE DIAPHRAGMATIC

HERNIA?

Suggests obstruction to blood flow leaving the right heart, in the lungs, or returning to the left heart

RV

LV

RA

LAMPA

RV

LV

RA LAMPA

WT CKO

TTBX4 CRE PBX CKO MICE HAVE HYPERTROPHIED RIGHT VENTRICLE AND

RIGHT ATRIAL ENLARGEMENT AT P28

P28

WT14.7g

CKO10.3g

RV

LV

RA

LA

RVLV

RA LA

PPBX MUTANT MICE HAVE HYPERTROPHIED RIGHT VENTRICLE AND RIGHT ATRIAL

ENLARGEMENT AT P28

P10

WT H&E

P10

WT H&EP10

CKO H&E

P10

CKO H&E10x 10x

10x 10x

LLUNGS OF PBX MUTANT MICE ARE SIMPLIFIED AT P10