STEM CELL THERAPY in

LUNG INJURY and ASTHMA 가톨릭대학교 의과대학

서울성모병원 소아청소년과

윤 종 서

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INTRODUCTION

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STEM CELL

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DEFINITION of STEM CELL

C: differentiated cell

B: progenitor cell

A: stem cell • Biological cells

– divide (through mitosis)

– differentiate into diverse

specialized cell types

– self-renew to produce more

stem cells

www.wikipedia.org 4

POTENCY DEFINITIONS

Stem cells Potency

Totipotent •Omnipotent

Pluripotent •Differentiate into nearly all cells

•(except i.e. placenta)

Multipotent •Differentiate into a number of cells,

•But only a closely related family of cells

Oligopotent •Differentiate into only a few cells,

•i.e. myeloid progenitor

Unipotent •Produce only one cell type, their own

•Have the property of self-renewal 5

STEM CELLS

www.wikipedia.org 6

STEM CELL

• Progenitor cell: unipotent, sometimes oligopotent

• Adult stem cell

• Embryonic stem cell

• Locations – Bone marrow stem cell

– Cord blood stem cell

– Amniotic fluid stem cell

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STEM CELL

• Hematopoietic stem cell

• Mesenchymal stem cell

= Marrow stromal cell

= Multipotent stromal cell

• Induced pluripotent stem cell (iPS cells, iPSC)

• Resident/Endogenous stem cell

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STEM CELL THERAPY

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www.wikipedia.org

STEM CELL THERAPY

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Korean FDA Approved Stem Cell Therapies

1. Hearticellgram AMI

- Autologous bone marrow-derived MSC

- Acute myocardial infarction

2. Cartistem

- Allogeneic umbilical cord blood derived MSC

- Osteoarthritis

3. Cupistem

- Autologous adipose derived MSC

- Crohn’s disease

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STEM CELL THERAPY in RESPIRATORY DISEASE : LESS DEVELOPED

Kotton, Am J Respir Crit Care Med, 2012

1. Complex structure of lung (vs cartilage)

2. Many kinds of cells (more than 20)

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CLINICAL TRIALS

in STEM CELL THERAPY

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CLINICAL TRIALS of STEM CELL THERAPY for LUNG INJURY (published)

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CLINICAL TRIALS (Unpublished)

A. Phase II

Human Adult Stem Cells (MSCs) for the

Treatment of Moderate to Severe COPD

B. Phase I:

Human Umbilical Cord-derived MSCs for BPD

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Human Adult Stem Cells (MCSs) for the Treatment of Moderate to Severe COPD

healthy adult donors

IV

Phase II: Unpublished

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Human Adult Stem Cells (MCSs) for the Treatment of Moderate to Severe COPD

• Safe

• ↓circulating CRP

• Not improved

– The 6-minute walk test

– Pulmonary function

Phase II: Unpublished 17

HUMAN STUDIES in STEM

CELL THERAPY:

LESS DEVELOPED!

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STEM CELL THERAPY in LUNG INURY

ANIMAL

STUDIES

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ANIMAL STUDIES in STEM CELL THERAPY for LUNG INJURY

• MSC

• Hematopoietic stem cell

• Induced pluripotent stem cell

• Amniotic fluid stem cell

• others

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Amniotic fluid stem cells

1. Amniotic fluid stem cells (AFSCs)

2. Embryonic stem cells

• Derived from blastocysts

• Pluripotent

• Limitations

– Ethical concern

– Potential to develop teratoma

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Blastocyst

AMNIOTIC FLUID STEM CELL

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AMNIOTIC FLUID STEM CELL

Magnetic activated cell sorting (MACS)

C-kit positive cells

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Methods

Isolation, culture, and labeling of human amniotic fluid stem cells (hAFSC)

Nude mouse with hyperoxic lung

injury

hAFSC

1. uptake and

integration into the

lung

2. Differentiation into

cells having type II

pneumocyte marker

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Results (1) hAFSC distribute mainly in the lung and liver, sometimes in the head

Red color: hAFSC luciferase bioluminescence

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Results (2)

hAFSC engrafted in the alveolar walls

Red color and purple dot: hAFSC fluorescence marker

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Hypothesis

Pulmonary fibrosis mouse

Murine amniotic fluid stem

cells

Cytokine 에 변화

“No” pulmonary fibrosis mouse

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METHODS Aminotic fluid stem

cells

C57BL/6J mouse

C57BL/6J mouse

IT bleomycin

Pulmonary fibrosis

IV injection

Outcomes

1. Lung tissue

2. PFTs

3. Cytokines

4. Others

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Bleomycin-induced Pulmonary Fibrosis Model

• Female C57BL/6J mice

• 10-12 weeks of age

• Bleomycin IT

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Mouse Amniotic Fluid Stell Cells

Maintain at 70–80% of confluency

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Aminocentesis

at 12-15 days

of gestation

Amniotic Fluid Stem Cells Transplant

• Two hours after IT bleomycin

• AFSC from male labeled with

CM-Dil

• Injected via tail vein

• 1X106 of AFSCs

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Results

Amniotic Stem Cells on Pulmonary Fibrosis

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Characterization of pre-transplant murine AFSCs

: mAFSCs do not reveal type II pneumocytes marker

(+) control :Red,

immunostaining for pro-surfactant

protein-C Murine AFSCs

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Murine Amniotic Fluid Stem Cells Intergrated and Acquired Type II Pneumocyte Phenotype

Green=SPC=type 2 pneumocyte phenotype

Red=CM-Dil=mAFSC

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Murine Amniotic Fluid Stem Cells Intergrated and Acquired Type II Pneumocyte Phenotype

Red=SPC=type 2 pneumocyte phenotype

Green dot = Y chromosome = mAFSC

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Effect of mAFSC on Pulmonary Fibrosis

• Histology

Control Bleomycin

Interstitial thickening, inflammation, alveolar collapse, and cystic air spaces

21 days after bleomycin

Bleomycin + mAFSC

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Histological Fibrotic Assessment

0

1

2

3

4

5

6

Bleomycin Bleomycin +

mAFSC day 0

Bleomycin +

mAFSC day 14

Ash

croft

sco

re

P<0.05

P<0.05

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Collagen Content

0

500

1000

1500

Bleomycin Bleomycin +

mAFSC day 0

Bleomycin +

mAFSC day 14

Collagen (

ug/w

hole

lung)

P<0.05

P<0.05

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Differential counts of cells in BAL fluid

0

1

2

3

4

5

6

7

Bleomycin Bleomycin + mAFSC

day 0

Bleomycin + mAFSC

day 14

No. of

cells

(x10

5)

Total cells

0

0.5

1

1.5

2

2.5

3

3.5

Bleomycin Bleomycin + mAFSC

day 0

Bleomycin + mAFSC

day 14

No. of

cells

(X10

5)

Lymphocytes

0

0.2

0.4

0.6

0.8

Bleomycin Bleomycin +

mAFSC day 0

Bleomycin +

mAFSC day 14

No. of

neutr

ophils

(X10

5)

Neutrophils

P<0.05

P<0.05

P<0.05

P<0.05

P<0.05

P<0.05

P<0.05

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Effect of mAFSC on Pulmonary Fibrosis

• PFTs (H, lung tissure elastance)

21 days after bleomycin & mAFSC

0

20

40

60

80

100

120

Control Bleomycin Bleomycin +

mAFSC

H (cm

H2O

/mL)

C57BL/6J mice (n=6)

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Cytokines profiles, whole lung, day 3

C5a, CCL2, ICAM-1, M-CSF, TIMP-1 변화를 보임!

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Cytokines profiles, BAL

0

10

20

30

40

50

60

70

pg/m

L

Control

Bleomycin

Bleomycin+mAFSC

*

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CCL2 [C-C motif ligand 2] = monocyte chemotactic protein-1 [MCP-1]

• The role of CCL2 has been described in asthmatic

lungs

• CCL2 was also identified as an important profibrotic

mediator

Sun et al. AJPLCMP 2011

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Hematopoietic Stem

Cell Therapy

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Hematopoietic stem cells (HSCs)

• In contrast to mesenchymal stem cells,

hematopoietic progenitor cells

– Better and more uniformly characterized,

– More easily isolated

– An excellent and long-standing safety record

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Hematopoietic stem cells (HSCs) Analysis of engraftment of intranasally delivered CB-CD34 cells

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Hematopoietic stem cells (HSCs)

More human DNA in injured lungs

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Hematopoietic stem cells (HSCs)

Figure 3. Analysis of epithelial differentiation of engrafted CB-CD34 cells

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Hematopoietic stem cells (HSCs)

Proliferative activity of engrafted CB-derived cells at 8 weeks after inoculation.

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Hematopoietic stem cells (HSCs) in lung injury animal: similar results as shown in MSCs

• Longterm pulmonary engraftment, replication, clonal

expansion, and reconstitution of injured respiratory

epithelium by fusion-independent mechanisms.

• Cord blood–derived surfactant-positive epithelial cells

appear to act as progenitors of the distal respiratory

unit, analogous to resident type II cells.

• Graft proliferation and alveolar epithelial differentiation

are promoted by lung injury

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Induced pluripotent stem cells (iPS cells)

&

Embryonic stem cells (ES cells)

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Induced pluripotent stem cells (iPS cells) & embryonic stem cells (ES cells)

• ES cells : ethical problem

• iPS cells -> tumorgenicity

• Inactivate or remove the oncogenes

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Induced Pluripotent Stem Cells (iPSs)

Kotton, Am J Respir Crit Care Med, 2012 53

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STEM CELL THERAPY in

ASTHMA

STEM CELL THERAPY in ASTHMA

• No human trial.

• Some animal studies.

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ANIMAL STUDIES on ASTHMA

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Adipose-derived stromal cells

•OVA challenged

•TDI-induced

Bone marrow derived MSCs

iPSC-MSCs (from human fibroblast)

ANIMAL STUDIES on ASTHMA

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1. Exogenous SCs migrate to sites of inflammation

2. Lung inflammation↓

• Airway eosinophilia/neutrophilia↓

• Goblet cells↓

• Airway mucus production↓

3. Lung injury/remodeling↓

• Collagen deposit↓

• Smooth muscle thickening↓

4. Airway hyperresponsiveness↓

5. IL-4, IL-5, and TGF-β1 levels in the BALF↓

6. Via modulating CD4+ T cells ?

CONCLUSION

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1. Supplementation of local resident stem cells 2. Fusion and transdifferentiation 3. Support of local stem cell niches 4. Immunomodulation thru cytokines (paracrine)

FUTURE STUDIES

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Stem cells

1. Characterization of surviving cells

• Phenotypically

• Functionally

2. Autologous vs allogeneic ?

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The END Thank you!