Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Microvesiclesslide

1,366 views

Published on

Journal Club presentation of the peer reviewed paper: 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







Published in: Technology, Health & Medicine
  • Be the first to comment

  • Be the first to like this

Microvesiclesslide

  1. 1. MSC lung repair via lung-derived microvesicles Journal Club 2007.08.16. Attila Csordas 1
  2. 2. 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 2
  3. 3. 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 3
  4. 4. 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 4
  5. 5. 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 5
  6. 6. 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) 6
  7. 7. Figure 1. Summary of experimental designs. (a) Lung, WBM co-culture (three experiments) 7
  8. 8. (b) Lung conditioned media (LCM), WBM co-culture five experiments: radiation-injured LCM, three experiments: non-irradiated LCM) 8
  9. 9. (c) RNase-treated LCM, WBM co-culture 9
  10. 10. (d) transplantation of WBM co-cultured with lung or no lung or uncultured WBM into irradiated mice 10
  11. 11. coculture 48hrs coculture 7 days coculture 48hrs coculture 7 days 11
  12. 12. 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, 12
  13. 13. 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) 13
  14. 14. 500cGy, 7 days, no Pro-Sp-B labelling, 21 more days without lung with IL3,6,11 positive for Pro-Sp-B, 14
  15. 15. LCM induces marrow cells to express lung specific mRNA RNase treatment of LCM attenuates expression changes 70% less 15
  16. 16. 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) 16
  17. 17. 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 17
  18. 18. 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 18
  19. 19. 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 19
  20. 20. 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 20
  21. 21. (d) transplantation of WBM co-cultured with lung or no lung or uncultured WBM into irradiated mice 21
  22. 22. Transplanted co-cultured marrow cells have a greater propensity to engraft the radiation-injured lung 22
  23. 23. (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
  24. 24. 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 24
  25. 25. 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. 25

×