292 Chapter 10 results from the interactions between the cells rather than an intrinsic defect in the alveolar epithelial cells. There are several potential reasons for why unfractionated organoids had lowered regenerative capacity, one of which being other cells in the supporting cell populations. Although there were no significant differences detected in the proportions of supporting cells, the screened cellular markers might have missed some of the other less common cell types that might have impact on epithelial cell regeneration. The potential differences in the survival of these cells in the organoid cultures can also contribute to the results generated. Another potential explanation for why the unfractionated cell population resulted in reduced regenerative capacity could be the differences between paracrine factors secreted from the supporting cells isolated from IPF lungs and control lungs, which was out of the scope of these initial observations. Lastly, the influence of potential imprinting of the native fibrotic ECM on the supporting cell populations could result in the reduced regenerative capacity in the unfractionated cultures. The supporting cell populations in lung parenchyma have been previously implied to be responsive to instructions coming from the fibrotic ECM [17]. While the ECM-mimicking substance used in this study during organoid culture was the same for both control and IPF groups, investigating how fibrotic ECM is involved in the interactions between the stromal cells and epithelial cells in IPF may reveal additional mechanisms of how epithelial repair is impaired in IPF. Considering the recent reports of transcriptomic profiling of epithelial cells which showed an ECM-producing gene signature in the epithelial cells [18], this newly produced ECM could be responsible for the feedback provided to the supporting cells, which in turn may reduce regenerative capacity of epithelial cells. In addition to the fibroblasts, which are well documented to contribute to the fibrotic microenvironment seen in IPF lungs [19], a certain subpopulation of endothelial cells has been found to be in close proximity to fibroblastic foci in IPF lungs and could thus also contribute [20] [21]. Similarly, profibrotic macrophages contribute to a more fibrotic microenvironment [22], which subsequently gives further feedback towards both epithelial cells and supporting cell populations. The overruling capacity of (fibrotic) ECM on delicate interactions between lung resident cells requires further attention to reveal its complete potential. In the last decade, the importance of the influence of lung microenvironment has been also reflected in the methods developed to mimic ECM in in vitro models. Generation of appropriate substitutes for ECM to be used in these models started with relatively simple models composed of one type of material. Models prepared with synthetic polymers provide greater control over properties such as stiffness, fiber organization and degradability natural polymers , while those models employing natural polymers have advantages in availability of bioactive cues and cell-binding
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