Bronchioalveolar stem cells derived from mouse-induced pluripotent stem cells promote airway epithelium regeneration

Abstract

Background: Bronchioalveolar stem cells (BASCs) located at the bronchioalveolar-duct junction (BADJ) are stem cells residing in alveoli and terminal bronchioles that can self-renew and differentiate into alveolar type (AT)-1 cells, AT-2 cells, club cells, and ciliated cells. Following terminal-bronchiole injury, BASCs increase in number and promote repair. However, whether BASCs can be differentiated from mouse-induced pluripotent stem cells (iPSCs) remains unreported, and the therapeutic potential of such cells is unclear. We therefore sought to differentiate BASCs from iPSCs and examine their potential for use in the treatment of epithelial injury in terminal bronchioles.

Methods: BASCs were induced using a modified protocol for differentiating mouse iPSCs into AT-2 cells. Differentiated iPSCs were intratracheally transplanted into naphthalene-treated mice. The engraftment of BASCs into the BADJ and their subsequent ability to promote repair of injury to the airway epithelium were evaluated.

Results: Flow cytometric analysis revealed that BASCs represented ~ 7% of the cells obtained. Additionally, ultrastructural analysis of these iPSC-derived BASCs via transmission electron microscopy showed that the cells containing secretory granules harboured microvilli, as well as small and immature lamellar body-like structures. When the differentiated iPSCs were intratracheally transplanted in naphthalene-induced airway epithelium injury, transplanted BASCs were found to be engrafted in the BADJ epithelium and alveolar spaces for 14 days after transplantation and to maintain the BASC phenotype. Notably, repair of the terminal-bronchiole epithelium was markedly promoted after transplantation of the differentiated iPSCs.

Conclusions: Mouse iPSCs could be differentiated in vitro into cells that display a similar phenotype to BASCs. Given that the differentiated iPSCs promoted epithelial repair in the mouse model of naphthalene-induced airway epithelium injury, this method may serve as a basis for the development of treatments for terminal-bronchiole/alveolar-region disorders.

Keywords: Induced pluripotent stem cells; Progenitor cells; Stem cell transplantation; Tissue regeneration.

Fig. 1

Differentiation of iPSCs into BASCs. a Schema of iPSC differentiation procedure: iPSCs were differentiated for 24 days through hanging-drop-based formation of embryoid bodies (EBs); the BM was supplemented from 0 to 24 days with 20 ng/mL keratinocyte growth factor (KGF) and DCI (① d10–d24 or ② d14–d24). EBs were induced using the hanging-drop method for the first 3 days, and the obtained EBs were transferred at 3 days to super-low-adherent culture dishes and then at 5 days to adherent culture dishes. Cells were cultured until 24 days in the medium. b Pluripotency of undifferentiated iPSCs (0 days) at passage 25. Immunofluorescence labelling of mouse iPSCs for the stem cell markers OCT4, SOX2, and SSEA-1. The GFP gene was knocked-in under the Nanog promoter, which allowed detection of GFP (green) in undifferentiated cells. Scale bar = 100 μm. c Flow cytometry analysis for BASC identification. Comparison of protocols ① d10–d24 DCI and ② d14–d24 DCI revealed that BASC differentiation efficiency did not differ significantly between the protocols (P > 0.05, Student’s t test); horizontal line inside indicates median and whiskers indicate min to max values. DCI, 10 nM dexamethasone plus 0.1 mM 8-bromoadenosine 3′5′-cyclic monophosphate sodium salt and 0.1 mM 3-isobutyl-1-methylxanthine; iPSCs, induced pluripotent stem cells; BASCs, bronchioalveolar stem cells; GFP, green fluorescent protein

Fig. 2

Identification of BASCs. Transmission electron micrographs of iPSC-derived BASCs. a Immunofluorescence labelling of iPSC-derived differentiated cells. Yellow scale bar = 100 μm; white scale bar = 20 μm. b A single BASC is shown containing microvilli (red arrowhead), immature lamellar body-like structures (black arrowhead), and secretory granules (red arrow); black arrow, mitochondria; N, nucleus. Scale bar = 1 μm. iPSCs, induced pluripotent stem cells; BASCs, bronchioalveolar stem cells; CCSP, club cell secretory protein; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; SPC, surfactant protein C

Fig. 3

Establishment of mouse distal airway injury model. a Schematic showing experimental design. Corn oil or naphthalene was injected intraperitoneally into mice at 0 days. At 1 days, DMEM and differentiated iPSCs were intratracheally administered in the control and iPS groups, respectively. Mice were sacrificed at 5 days (n = 4 for all groups) and 15 days (n = 4 for NA group and iPS group, n = 5 for control group). b Preparation of naphthalene-treatment model. Corn oil and NA groups are shown from the second day after treatment. In the NA group, CCSP-positive club cells (green) were completely detached. NA, naphthalene treatment; iPSCs, induced pluripotent stem cells; IT, intratracheal injection; BW, body weight; H&E, haematoxylin and eosin; CCSP, club cell secretory protein; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride

Fig. 4

Quantification of CCSP-positive club cells at BADJs on day 5 and 15 after naphthalene treatment. a Immunofluorescence labelling of club cell marker CCSP (green) in each group. Nuclei were counterstained with DAPI (blue). Scale bar = 100 μm. b Dot-and-whisker plot showing numbers of CCSP-positive cells at BADJs. Horizontal line represents median and whiskers show min and max values. *P < 0.01, **P < 0.05 vs all other groups; one-way ANOVA followed by Tukey’s multiple-comparison test. CCSP, club cell secretory protein; BADJs, bronchioalveolar-duct junctions; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride

Fig. 5

CCSP and SPC immunofluorescence in lung sections 5 days after intratracheal transplantation of differentiated iPSCs. Differentiated iPSCs were labelled with PKH26 cell tracker (yellow). Nuclei were counterstained with DAPI (blue). PKH26-positive, CCSP-positive (green), and SPC-positive (red) cells (PKH^pos/CCSP^pos/SPC^pos cells, white arrow), which are BASCs, were present around BADJs. PKH^pos/CCSP^neg/SPC^neg cells (white dotted arrow) were also detected. Bottom panels: magnified view of area a in top panel. PKH^pos/CCSP^pos/SPC^pos cells formed clumps. Yellow scale bar = 100 μm; white scale bar = 20 μm. CCSP, club cell secretory protein; SPC, surfactant protein C; iPSCs, induced pluripotent stem cells; BADJs, bronchioalveolar-duct junctions; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride

Fig. 6

CCSP and SPC immunofluorescence in lung sections 15 days after intratracheal transplantation of differentiated iPSCs. Differentiated iPSCs were labelled with PKH26 cell tracker (yellow). Nuclei were counterstained with DAPI (blue). PKH26-positive, CCSP-positive (green), and SPC-positive (red) cells (PKH^pos/CCSP^pos/SPC^pos cells, white arrow), which are BASCs, were found as part of the epithelium of the BADJ. PKH^pos/CCSP^pos/SPC^neg cells (white arrowhead), which are club cells, were scattered. Middle and bottom panels: magnified view of areas a and b in top panel, respectively. CCSP, club cell secretory protein; SPC, surfactant protein C; iPSCs, induced pluripotent stem cells; BADJs, bronchioalveolar-duct junctions; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride