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
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