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 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CLINICAL TRIALS (Unpublished)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 ?