Assessment of the physiological capacity of proliferation and self-renewal of second-order pneumocytes and bronchoalveolar stem cells generated in response to lung damage

The applicant's current research is assessing the possibility of differentiation of non-haemalopoietic stem cells obtained from mouse bone marrow (Sca-1 + CD45-CXCR4 + SSEA1 +) into lung stem cells - pncumocytes II order (AT2). The possibility of such differentiation has already been proven (Kassmeret al., 2013), however, research is currently conducted on the model of induced lung injury. The animal model is SPC-KO/SPC-TK double transgenic mice lacking the expression of a surfactant protein C (SPC-KO) and containing an apoplosis-inducing gene linked to the promoter of the SPC gene (SPC-TK). The aim of the activity to be performed is to assess the physiological ability to proliferate and self-renew second order pneumocytes and bronchioalveolar stem cells (BASC) generated in response to lung damage. To achieve the intended purpose, the applicant will isolate AT2 and BASC cells from properly prepared lungs from two groups of SPC-KO SPC-TK mice. (experimental group subjected to ganciclovir and bleomycin induced lung injury, m = 10; control group with normal lung, n = 10). Organoid cultures will be established from both cell lines, harvested after 16 days, and then some will be fixed in the form of microscopic preparations, and the other part, after disintegration of the three-dimensional structure of the organoids, will be subjected to cytometric analysis for quantitative and qualitative analysis. The results of these analyzes will be compared between the groups and will be statistically analyzed. This particular animal model was designed for the planned research due to the fact that the lack of SPC protein, which is one of the main components of the surfactant, is the cause of many human diseases (Hong et al., 2017). In addition, the use of SPC-KO/SPC-TK mice enables "targeted partial dcplection" of second order pneumocytes using ganciclovir. The discussed topic significantly affects the problem of lung regeneration necessary in various pathological conditions. Second order pncumocytes as well as bronchoalveolar lung stem cells constitute a very small number of lung cell populations that are directly responsible for the regeneration of this organ. AT2 and BASC cells are susceptible to damage, which causes slow and often insufficient lung regeneration. Therefore, there is a need to develop an effective method of their isolation from damaged lungs, multiplication and their extracorporeal differentiation, and then implantation into the damaged organ. The results obtained as a result of the planned preliminary research will become a significant contribution to undertaking a much wider project, which will be a continuation of the undertaken topic. As reported in the literature, the proliferation potential and the ability of stem cells to differentiate into cells of a different type increases significantly in damaged organs. The planned research assumes that both second-order pneumocytes and bronchoalveolar stem cells isolated from damaged lungs of SPC-KO/SPC-TK mice will be much more active, which will translate into an increased amount of organoids grown in the experimental group. The tc organoids should have a higher total number of cells than in the control group. Depending on which cell line they arise from, they should contain a higher percentage of self-renewing stem cells (in the case of the AT2 cell line), and in the case of the BASC cell line a higher percentage of both self-renewed BASC cells and second-order pneumocytes. The tc results could support the hypothesis that lung injury stimulates second order pnemocytes and bronchoalveolar stem cells to increase their physiological ability to proliferate and self-renew. Obtaining the results confirming this hypothesis will directly contribute to the attempt of extracorporeal proliferation of lung stem cells, which may be used in the future to increase the regenerative potential of this organ.