Embedded is my classical style (no design, based on the figure section, Powerpoint instead of Keynote) Journal Club presentation on the following paper with the help of SlideShare: Alteration of Marrow Cell Gene Expression, Protein Production and Engraftment into Lung by Lung-derived Microvesicles: A Novel Mechanism for Phenotype Modulation by 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 Thanks for the permission, Jason Aliotta. After the abstract you can find some critical points we digged out during our journal club answered by the first author, Jason Aliotta himself.
Abstract: Numerous animal studies have demonstrated that adult marrow-derived cells can contribute to the cellular component of the lung. Lung injury is a major variable in this process; however, the mechanism remains unknown. We hypothesize that injured lung is capable of inducing epigenetic modifications of marrow cells, influencing them to assume phenotypic characteristics of lung cells. We report that, under certain conditions, radiation injured lung induced expression of pulmonary epithelial cell-specific genes and prosurfactant B protein in cocultured whole bone marrow cells separated by a cell-impermeable membrane. Lung conditioned medium had a similar effect on cocultured whole bone marrow cells and was found to contain pulmonary epithelial cell-specific RNA-filled microvesicles that entered whole bone marrow cells in culture. Also, whole bone marrow cells cocultured with lung had a greater propensity to produce type II pneumocytes after transplantation into irradiated mice. These findings demonstrate alterations of marrow cell phenotype by lung-derived microvesicles and suggest a novel mechanism for marrow cell directed repair of injured tissue.
a., What was the control in case of Figure 5c, (c) Pulmonary epithelial cell marker expression in LCM
and its derived components (one experiment)? What really surprises us was that on that figure, the dark first bar is the WBM cocultured with lung, while the elevated levels are from LCM coculture experiments. What would be good to know is the level of gene expression in the normal lung and set it as a control to know the fold differences.
Jason Aliotta: The control was cultured whole bone marrow cells NOT exposed to lung. This figure is a bit confusing. The black bar represents gene expression in irradiated lung not marrow cells exposed to irradiated lung in co culture. The next bar represents gene expression in lung conditioned media made from irradiated lung followed by the ultracentrifuged pellet derived from lung conditioned media made from irradiated lung followed by sorted particles isolated from the lung conditioned media made from irradiated lung. These were not co-culture experiments. The level of expression is expressed as a fold difference compared to cultured whole bone marrow cells. This comparison group was chosen becasue we wanted to keep the presentation of our data consistent and this had been used as our comparison group throughout the paper. I think it would have been better to used normal lung as a comparison group.
b., Another problem was Figure 6B-H, if it is one cell with the microparticles in it, then why is the particle distribution so unevent and why there isn’t any PKH26 membrane signal in the cell membrane? Are the particles packaged into outer cell membrane before entering into the cell? What could be the mechanism?
Jason Aliotta: I wish I had a good answer for this. Microvesicles tend to clump together, prior to entering cells and even after entering cells. This might explain the uneven distribution but the mechanism for this is unknown (at least to me). We’ve observed that some marrow cells only have one or a few microvesicles after culture, others have many more (as seen in this picture). Again, I’m not sure why this is the case. It may have something to do with the phenotype of the accepting cell. These are all issues that we’re looking into but I just don’t have a great answer. My understanding of microvesicular uptake is that there are may ways that they enter cells. It is possible that since the marrow cell membrane is not PKH+ that these microvesicles are being engulfed by endocytosis rather than passing through the accepting cell membrane and leaving behind some of its own PKH26+ membrane. Again, this is all speculation and I haven’t yet looked into this in more detail.