MSC lung repair via lung-derived
microvesicles
Journal Club
2007.08.16.
Attila Csordas
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Alteration of Marrow Cell Gene Expression,
Protein Production and Engraftment into Lung
by Lung-derived Microvesicles: A Novel
Mechanism for Phenotype Modulation
Aliotta JM, Sanchez-Guijo FM, Dooner GJ, Johnson KW, Dooner MS,
Greer KA, Greer D, Pimentel J, Kolankiewicz LM, Puente N, Faradyan
S, Ferland P, Bearer EL, Passero MA, Adedi M, Colvin GA,
Quesenberry PJ.
Stem Cells. 2007 Jul 2
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Microvesicles in cell-cell
communication
circular membrane
fragments shedding from
surface membrane or
endosomal compartment
during cell activation,
hypoxia, irradiation,
oxidative stress
role in cancer, infection,
cardiovascular diseases
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BM contribution to lung
BM donor populations: WBM, mesenchymal, hematopoietic, side
population
injury models: radiation, bleomycin, elastase, monocrotaline
animal models: mice, NOD/SCID, parabiotic, newborn
high number (20%) of type II pneumocytes and pulmonary
fibroblasts are marrow derived, few (0.1% of all lung cells) type I
pneumocytes, airway epithelial cells
open question: epigenetic way to transfer the lung phenotype?
mRNA without the DNA level
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Methods
WBM, lung harvest
GFP+ C57BL/6 mice
co-cultures: Multivell plates, Milicell plate inserts with 0.4um
pores
immunohistochemistry
lung conditioned media harvesting (centrifugation 300g, 10min)
RNase treatment
microvesicles isolation by ultracentrifuge (28000g for 60 min)
isolation of microvesicles by flow cytometry
fluorescent microscopy (conventional and deconvolution)
electron microscopy
real time RT-PCR for gene expression
transplantion of WBM into lung
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Main variables
• lung for coculture with WBM, time of
sacrifice and extraction after TBI: 3hrs, 5
days, 14 days
• lung or lung conditioned media for coculture
• length of coculture: 48 hrs, 7 days
• strength of TBI: 500, 1200 cGy (centigray)
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Figure 1. Summary of
experimental designs.
(a) Lung, WBM co-culture (three experiments)
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(b) Lung conditioned media (LCM), WBM co-culture
five experiments: radiation-injured LCM,
three experiments: non-irradiated LCM)
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(c) RNase-treated LCM, WBM co-culture
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(d) transplantation of WBM co-cultured with lung
or no lung or uncultured WBM into irradiated mice
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coculture 48hrs coculture 7 days
coculture 48hrs coculture 7 days
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Fig 2.
1. all groups elevated pulmonary expression
compared to WRB without lung
2. 3hrs, 14 days: no sigificant difference in
expression in irradiated compared to non-
irradiated
3. 5 days: significant increase in genes in
radiated compared to non-irradiated lung
more potent stimulus
4. 500cGy was the highest level,
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1. increased expression in all groups in genes expressed in other cells (c-kit, Sca-1,
adhesion protein genes P-,L-sel compared to control WBM without lung
2. no significant difference in radiated compared to non-irradiated at either time
points
3. kidney coculture did not express pulmonary markers
4. kidney coculture: unaltered or decreased markers (CD34, c-kit,VEGFR1,
PECAM, CXCR4), small increase (2.5 fold) in Sca-1)
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500cGy, 7 days, no Pro-Sp-B labelling,
21 more days without lung with IL3,6,11 positive for Pro-Sp-B,
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LCM induces marrow cells to express lung specific mRNA
RNase treatment of LCM attenuates expression changes
70% less
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Microvesicles
Figure 5. Isolation, imaging of lung-derived microvesicles.
(a) Electron microscopy of the ultracentrifuged LCM pellet demonstrates numerous
100-250 nm membrane-bound vesicles (top, bar = 300 nm; bottom, panel of individual
vesicles, bar = 100 nm)
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Isolating functional microvesicles with FACS
(b) FACS-separated GFP+ /PKH26+ events (R2), (0.13% of all events)
Particles contain both cell membrane (PKH26) as well
as cytoplasm (GFP+) and contain RNA
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RT-PCR from equal amounts of RNA from irradiated
samples
(c) Pulmonary epithelial cell marker expression in LCM
and its derived components (one experiment).
higher levels in LCM and derivatives compared to irradiated lung
highest level in LCM pellet
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Particles enter 0.1% of nucleated cells upon coculture 48hrs
non-irradiated lung particles enter too
Figure 6. WBM cells cultured with lung-derived microvesicles.
Particles are visualized in WBM, (c) FITC, (d) Texas red, (e) DAPI, (b) all filters. Three-
dimensional (3D) view reveals co-localization of (g) GFP and (h) PKH26. (f) FITC/Texas red
filters. Red bar = 10μm (one experiment).
DAPI binds to DNA, mildly to RNA too
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The phenotype of the accepting marrow cell
Figure 6. WBM cells cultured with lung-derived microvesicles.
(a) FACS-separated WBM that consumed GFP+/PKH26+ particles in culture (R2)
Wright-Giemsa staining:
granulocytes (74%), 26% indeterminate mononuclear cells
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(d) transplantation of WBM co-cultured with lung
or no lung or uncultured WBM into irradiated mice
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Transplanted co-cultured marrow cells have a
greater propensity to engraft the radiation-injured lung
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(l) GFP+/pro-Sp-C+ cells, percent of DAPI+ cells from lungs
Lungs from mice that received WBM co-cultured with radiation-injured or non-
irradiated lung had a higher number prosurfactant C+ (pro-Sp-C) cells that were
donor (GFP+) WBM-derived (1.55 +/- 0.07% and 2.01 +/- 0.22% of all nucleated
cells, respectively) compared with those that received uncultured WBM cells (1.05
+/- 0.12%; t-test, p = 0.02, 0.003, respectively, vs. uncultured WBM cohort; Figure
7l).
There was no significant difference in the number of GFP+/pro-Sp-C+ cells in mice
that received WBM co-cultured with radiation-injured or non-irradiated lung (t- test,
p = 0.08).
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GFP+/ pro-Sp-C+ cells had morphological features consistent with
type II pneumocytes (Figure 7b-k).
(l) GFP+/pro-Sp-C+ cells, percent of DAPI+ cells. GFP+/pro-Sp-C + (solid, dashed white arrows), GFP+/ pro-
Sp-C - (asterix) and GFP-/ pro-Sp-C + (clear arrow) cells, (d,g) FITC, (c,f) Texas red, (b,e) both filters/DAPI.
(k) Hematoxylin/eosin. 3D view reveals co-localization of (j) GFP, (i) pro-Sp-C. (h) FITC/Texas red filters. Red
bar = 20μm.
Text
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These findings suggest that transplanted WBM co-cultured
with lung have a greater tendency to participate in the
production of type II pneumocytes, in vivo, in the radiation-
injured lung than transplanted uncultured WBM.
In summary, these studies are the first to demonstrate that a
lung phenotype can be transferred to marrow cells from injured
lung cells through lung cell-derived microvesicles. In addition,
they suggest a mechanism for the transfer of information from
injured cells to healthy cells and may provide a mechanism for
some forms of phenotypic modulation of stem cells and tissue
repair.
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