Crafting a UX Strategy for Wearables and the Mobile Mainframe
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Transcript of Microvesiclesslide
Journal Club2007.08.16.
Attila Csordas
MSC lung repair via lung-derived microvesicles
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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 ModulationAliotta 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 lungBM 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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MethodsWBM, lung harvestGFP+ C57BL/6 miceco-cultures: Multivell plates, Milicell plate inserts with 0.4um poresimmunohistochemistrylung conditioned media harvesting (centrifugation 300g, 10min)RNase treatmentmicrovesicles isolation by ultracentrifuge (28000g for 60 min)isolation of microvesicles by flow cytometryfluorescent microscopy (conventional and deconvolution)electron microscopyreal time RT-PCR for gene expressiontransplantion 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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five experiments: radiation-injured LCM, three experiments: non-irradiated LCM)
(b) Lung conditioned media (LCM), WBM co-culture
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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 48hrs
coculture 7 days
coculture 7 days
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Fig 2.
1. all groups elevated pulmonary expression compared to WRB without lung2. 3hrs, 14 days: no sigificant difference in expression in irradiated compared to non-irradiated3. 5 days: significant increase in genes in radiated compared to non-irradiated lungmore potent stimulus4. 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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70% lessRNase treatment of LCM attenuates expression changes
LCM induces marrow cells to express lung specific mRNA
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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)
Microvesicles
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(b) FACS-separated GFP+ /PKH26+ events (R2), (0.13% of all events)
Isolating functional microvesicles with FACS
Particles contain both cell membrane (PKH26) as well as cytoplasm (GFP+) and contain RNA
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(c) Pulmonary epithelial cell marker expression in LCM and its derived components (one experiment).
RT-PCR from equal amounts of RNA from irradiated samples
higher levels in LCM and derivatives compared to irradiated lunghighest level in LCM pellet
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non-irradiated lung particles enter tooFigure 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).
Particles enter 0.1% of nucleated cells upon coculture 48hrs
DAPI binds to DNA, mildly to RNA too19
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
The phenotype of the accepting marrow cell
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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). 23
(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.
GFP+/ pro-Sp-C+ cells had morphological features consistent with type II pneumocytes (Figure 7b-k).
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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