Figures
- FIGURE 1
Lung progenitor co-cultures yield lung epithelial organoids. a) Schematic of organoid co-culture system. Green represents the lung epithelial progenitor cells that give rise to organoids: GFP+Sca1+EpCAM+ cells isolated by fluorescence-activated cell sorting from β-actin-GFP mice are shown. Orange represents lung endothelial cells used in the co-culture to provide support to epithelial cells. Yellow represents Matrigel extracellular matrix mixture. Cells suspended in Matrigel in a transwell are submerged partially in the medium to achieve an air–liquid interface culture. b–d) Representative images of the three types of organoid that can be derived from Sca1+EpCAM+ lung epithelial progenitor cells in co-cultures with endothelial cells. b) Representative bronchiolar organoid containing cells positive for club cell secretory protein (CCSP) or bronchiolar cell markers; c) bronchioalveolar organoid containing bronchiolar cells and alveolar cells; d) alveolar organoid containing cells positive for surfactant protein C (SPC). All images were obtained from fluorescence imaging by co-staining for CCSP (red) and SPC (green), with 4′,6-diamidino-2-phenylindole (DAPI; blue). Images provided courtesy of Samuel P. Rowbotham (Boston Children's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA; and Harvard Stem Cell Institute, Cambridge, MA, USA).
- FIGURE 2
Lung tumour-propagating cells can be used in organoid cultures and in vivo transplantation. a) Schematic of organoid co-culture system for tumour cells. Colours are as shown in figure 1a, except that tumour cell subsets (e.g. Sca1+ tumour cells) are placed in co-culture instead of normal lung progenitors. b) Schematic of assay used to establish tumour-propagating cell activity in vivo. The Lox-stop-lox-Kras-G12D;p53-flox/flox mouse strain is infected intranasally with Adenovirus-Cre to initiate lung tumorigenesis. Lung adenocarcinomas arise and are dissected out for digestion to obtain single-cell suspensions. Tumour cell subpopulations are isolated by fluorescence-activated cell sorting (FACS) and transplanted by intratracheal delivery into the immunosuppressed nude mouse strain. Resulting tumours (referred to as secondary tumours) in the recipient nude mice are indicative of tumour-propagating cell function. Secondary tumours can be dissected and processed for single-cell suspensions in a reiterative process. Reproduced and modified from [28] with permission.
