[PPT] - Outline Hematopoeitic stem cells (HSCs) 1. Mesenchymal Stem PowerPoint Presentation

SLIDE 1

1

Stem Cell-Based Therapy in Pulmonary Vascular Disease

Stella Kourembanas, MD

Harvard Division of Newborn Medicine

Boston Children’s Hospital Harvard Medical School

6th International Conference on Neonatal & Childhood Pulmonary Vascular Disease

No Disclosures:

Outline

1. Mesenchymal Stem Cell-based therapies

for diseases of the lung

Bronchopulmonary Dysplasia (BPD)
Pulmonary Hypertension (PH)
2. Mechanism of Mesenchymal Stem Cell

action

Hematopoeitic stem cells (HSCs) Endothelial progenitor cells (EPCs) Mesenchymal stromal (stem) cells or bone marrow stromal cells (MSCs, BMSCs)

Bone Marrow-Derived Stem Cells

SLIDE 2

2

Mesenchymal Stromal (Stem) Cells

Derived from bone marrow (BMSC); blood;

and other tissues

Immunodepletion by FACS Analysis Using

ISCT Guidelines

Can differentiate into connective tissue

lineages

MSC Differentiation

MSCs

Teeth

Preclinical & Clinical Uses of MSCs

Clinical trials on autologous and allogeneic

MSCs, including

Crohn’s, Multiple Sclerosis, End stage liver

disease, GVHD, ventricular dysfunction, MI, refractory SLE, Diabetes, COPD

ProchymalTM, commercial MSC prep (Osiris

Therapeutics Inc, Columbia, MD)

FDA approved for phase III trial for Crohn’s Disease

http://clinicaltrials.gov

Therapeutic Effects of MSCs

Animal models of lung injury

IPF, ALI, BPD

Animal models of Pulmonary Hypertension

SLIDE 3

3

Bronchopulmonary Dysplasia (BPD)

Prolonged lung

inflammation

Alveolar

simplification; thickened alveolar septae; & dysmorphic vascular growth

PH (RVH and lung

vascular remodeling)

BPD

Mesenchymal Stem Cells for Lung Injury

Hyperoxia-induced neonatal lung injury model: BPD

MSCs and MSC-Conditioned Media (CM) Prevent PH in Hyperoxia-BPD Model

Aslam et al., Am J Respir Crit Care Med. 2009;180:1122-30

21% Oxygen 75% Oxygen

BMSC PASMC

BMSC-CM (cell-free) but NOT BMSCs Prevent Alveolar Injury

Cells Media

SLIDE 4

4

BPD Rescue: Experimental Design

(BAL cell count) (CT Angio)

Late MSC-CM Treatment Reduces Lung Inflammation and Reverses RVH in BPD Model

MSC-CM MLF-CM Normoxia

0.0 0.1 0.2 0.3 0.4 0.5

*

Fulton’s Index (RVH) 75% Oxygen Macrophages/BAL

0.0 200000.0 400000.0 600000.0 800000.0

*

#

75% Oxygen

MSC-CM MLF-CM Normoxia

MSC-CM Reverse PA Pruning in Hyperoxia-BPD Model

Hansmann, G., Fernandez-Gonzalez, A., et al., Pulm. Circ. 2012;2:170-81

MSC-CM Normalize Long-Term Lung Function in Hyperoxia-BPD Model

Hansmann, G., Fernandez-Gonzalez, A., et al., Pulm. Circ. 2012;2:170-81

SLIDE 5

5

MSCs & MSC-CM Treatment of Experimental BPD

Prevent lung injury, inflammation, and PH Reverse lung fibrosis and PA devascularization Partially reverse alveolar injury Normalized long term lung function Reversed PH and RVH MSC-CM are more effective than MSCs on preventing and reversing lung disease

Mesenchymal Stem Cells & PH– Hypoxic Mouse Model

Effect of MSCs on reversing established PH? Gene delivery in vivo: Heme Oxygenase-1

Enzymatic Activity of Heme Oxygenase (HO)

Fe cGMP GTP HO-1,2 NADPH Heme CO Biliverdin-IX Bilirubin-IX O

2

Guanylyl cyclase Vascular smooth muscle cell Biliverdin reductase Inflammation Fibrinolysis Antioxidant Platelets Relaxation Adult WT and H0-1 KO Mice in hypoxia (8.5 % O2) Functional and morphological analysis: RVSP, RV/LV+S and PA remodeling PBS or MLFs or PASMCs WT-MSCs H01-MSCs

5 7

week

Experimental Design

week

injection IV

SLIDE 6

6

HO-1-MSCs Reverse Hypoxic PH

0.1 0.2 0.3 0.4 Normoxia PBS Control SHO1- BMSC WT-FVB- BMSC

Normal RVSP in H0-1-MSC- Treated Hypoxic WT Mice Normal RV Weight in H0-1- MSC-Treated Hypoxic WT Mice

RV/LV+S RVSP (mm Hg)

(10) (8) (10) (13) (8) (8) (13) (8)

Hypoxia x 7 weeks

* *

§

Liang et al., Stem Cells 2011; 29:99-107

H0-1-MSC Transplantation Prevents RV Failure/Thrombus in Hypoxic HO-1 Null Mice

10 20 30 40 50 60 70

PBS H0-1-MSC Animals with thrombus (%)

**

RV injury

HO-1 +/- HO-1 -/- Normoxia 7 wks Hypoxia Liang et al., Stem Cells 2011; 29:99-107

1. Transdifferentiation into lung cells
2. Stimulation of endogenous lung

progenitors cells to repair lung injury

3. Paracrine:Immunomodulation;anti-

inflammatory effects

Mechanisms of MSC Action:

Rapid Clearance of Donor MSCs

500 1000 1500 2000 2500 3000 3500 2 4 6 8 10 12 14 16 Days post injection

Normoxia Hypoxia PBS Control

Donor MSC per 106 Recipient Lung Cells

SLIDE 7

7

Transplanted MSCs express HO-1

20 40 60 80 100 120 Day 1 Day 2 Day 7 Day 14

hHO1 mRNA per resident MSC

(arbitrary units)

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Specimen Groups

Ave No. BASC/TB

* *

MSC-CM Increase Endogenous Lung Progenitor Cell Number In Vivo

Bronchioalveolar stem cells (BASCs)

Hrxa + PASMC-CM Hrxa + MSC Hrxa + MSC-CM

Tropea et al., Am J Physiol Lung Cell Mol Physiol. 2012;302:L829-37.

Mechanisms of MSC Action: Immunomodulation

HO-1 CO

Modified from: Iyer & Rojas, Expert Opinion on Biological Therapy, 2007

Conditioned Media Proteomics

0% 10% 20% 30% 40% 50% 60%

Proliferation & Apoptosis Cell-Cell Interactions & Cell Motility Immune Response & Inflammation Respiratory Disease

Among Proteins Detected:

CD63, CD81, moesin,

HSP90, HSP 70

Fraction of BMSC-CM Proteome

SLIDE 8

8

Exosome Release by MSCs

Paracrine Factors Exosome Recipient Cell Recipient Cell Multivesicular Body

MSC

Exosomes as mediators of paracrine effect Conventional view of paracrine secretion of soluble proteins Modified from Lai et al, Regen Med 2011

Size Fractionation of MSC-CM and EM Analysis

MEX: MSC-derived exosomes FEX: Fibroblast- derived exosomes Lee et al., Circulation. 2012;126:2601-11.

Hypoxia

Injection, JV or TV Vascular Remodeling RV Systolic Pressure RV Hypertrophy MEX

2, 4, 7 days, 3 weeks

FEX Inflammation

Experimental Design

MEX Mediate the Anti-inflammatory Effects of MSCs on the Lung

x 48 hrs

Lee et al., Circulation. 2012;126:2601-11.

SLIDE 9

9

Hypoxia Induces an Early Peak of Lung Inflammation in Mice

1 2 3 4 5 6 7 8 9 10 2d 4d 7d 9d 11d 3w

BAL Macrophage # (x 105 / ml)

Hypoxia

*

Vergadi et al., Circulation. 2011;123:1986-95.

Dose-Dependent Effects of MEX

n Hypoxic Lung Inflammation (I)

Days in Hypoxia

MEX Low dose

Lee et al., Circulation. 2012;126:2601-11.

Days in Hypoxia MEX Low dose MEX High dose Alveolar MΦs (x103) Hypoxia, 7 days High dose

Dose-Dependent Effects of MEX

n Hypoxic Lung Inflammation (II)

Lee et al., Circulation. 2012;126:2601-11.

Systemic Infusion of MEX Inhibits Chronic HPH

x 3 weeks

SLIDE 10

10

Systemic Infusion of MEX Inhibits Hypoxic Vascular Remodeling

For 3 weeks

* *

0.2 0.4 0.6 0.8 1 1.2

+

+ + + +

Relative Proliferation

*

FBS (5%) 125 62.5 31.5 16

(ng/ml)

ns mMEX

MEX Inhibit PASMC Proliferation

Human & Mouse MEX Suppress the

Hypoxic Activation of STAT-3

In Vivo Lung Cultured hPAECs

Lee et al., Circulation. 2012;126:2601-11.

SLIDE 11

11

MEX Treatment Modulates Lung miR-204 Levels

Hypoxia 48 h Relative levels of miRNA-204

Regulation of Hypoxic Lung Signaling Pathways by MSC-derived Exosomes (MEX)

Summary

Exosomes mRNA, miRNA, proteins, etc.

Structural & Functional Repair

[Immunomodulation; resident progenitor cell stimulation]

Mesenchymal stem cells

Biomarkers of health and disease Therapeutic agents in lung/CVR disease; Gene

delivery Mechanism of action

Paracrine

Release of anti-inflammatory cytokines

Acknowledgments

Changjin Lee Kostis Sdrimas Georg Hansmann Angeles Fernandez-

Gonzalez

Sally Vitali Olin Liang Muhammad Aslam Georgios Konstantinou Rajiv Baveja

S. Alex Mitsialis

Xianlan Liu Helen Christou Tom Martin Mark Perrella Carla Kim Kristen Tropea-Leman