Fig. 5

ATIICs express exosome miR-371b-5p in response to injury. a Schematic strategy of co-cultures of hiPSC-ATIICs or hATIICs with hiPSC-ATICs in six-well transwell plates using exosome-depleted DM with and without BLM. b Western blotting of TSG101, CD63 and HSP70 in exosomes derived from cultured A549 (1), hiPSC-ATIICs (2) and hATIICs (3). QRT-PCR analysis of expression levels of cellular and exosome miR-371b-5p in the cultures of ATIIC phenotypes treated with various doses of BLM (c) as well as the level of exosome miR-371b-5p in the lavage samples isolated from BLM-treated mouse lungs transplanted with hiPSC-ATIICs or hmonos (d) by using ABM primer. The expression levels were normalized to 18 s and presented as percentage of the cellular or exosome miR-371b-5p level of A549 cells. e Schematic structures to show the sequences of miR-371b-5p (top), in which two underlined nucleotides are not identical in miR-292b-5p (bottom), as well as the sequences of miR-371b-5p-specific reverse primer (R-primer-I) and miR-292b-5p-specific reverse primer (R-primer-II). f QRT-PCR analysis of expression levels of exosome miR-371b-5p and miR-292b-5p in BLM-treated lungs with or without transplantation by using R-primer-I or R-primer-II. The expression levels were normalized to 18 s and presented as a ratio to the ABM primer detected ‘miR-371b-5p’ expression level as indicated. g Immunofluorescent staining of representative lung sections from BLM-challenged mice with or without transplanted hiPSC-ATIICs or hmonos by using a mouse anti-human nuclei antibody and rabbit anti-human proSPC antibody with DAPI counterstaining. The human nuclei antibody recognizes human cells (red) in mouse lungs; the anti-human proSPC stains both mouse and human SPC-expressing ATIICs (green). Many nuclei^-SPC⁺mouse ATIICs (indicated by white arrows) were identified in control and BLM-treated lungs transplanted with hiPSC-ATIICs, but not in the BLM-treated lungs receiving hmonos or saline. The nuclei⁺SPC⁺ human ATIICs (indicated by green arrows) were found only in the BLM-treated lungs transplanted with hiPSC-ATIICs. Some nuclei⁺ cells were SPC negative (indicated by red arrows), suggesting that they were ATICs that had been differentiated from the transplanted hiPSC-ATIICs. A magnified view of a mouse ATIIC (indicated by the white arrowhead), a human ATIIC (indicated by the green arrowhead), or an ATIC (indicated by red arrowhead) is shown in the corresponding image. BLM bleomycin, DAPI 4′,6-diamidino-2-phenylindole, DM differentiation medium, hATIICs human primary ATIICs, hiPSC-ATIICs human induced pluripotent stem cell-derived ATIICs, hmonos human peripheral blood monocytes, SPC surfactant protein C