Protocol for the derivation and alveolar type 2 differentiation of late-stage lung tip progenitors from the developing human lungs

Abstract

Molecular and cellular mechanisms of human lung alveolar development are poorly understood due to a lack of in vitro model systems. This protocol details the isolation, derivation, and genetic modification of lung tip epithelial progenitors from human fetal lungs. It includes steps for isolating distal lung epithelial cells, expanding tip progenitor organoids, culturing tip organoids in vitro, and differentiating them into alveolar type 2 cells. This will aid in understanding alveolar differentiation mechanisms and neonatal diseases. For complete details on the use and execution of this protocol, please refer to Lim et al.¹.

Keywords: Cell Differentiation; Cell culture; Cell isolation; Cell separation/fractionation; Developmental biology; Flow Cytometry; Organoids.

Graphical abstract

Figure 1

Derivation and in vitro organoid culture of late-stage distal lung tip progenitors from human fetal lungs, at 19–21 pcw, and alveolar type 2 (AT2) cell fate differentiation (A) Experimental scheme describing the establishment of alveolar lineage-positive (Lin^POS) distal tip progenitor organoids and their AT2 cell fate differentiation by exposure to AT2 medium for 1 week. (B and C) Bright field (top panel; B) and immunostaining images (middle and low panels; C) of a mixture of distal lung tip epithelial organoids. Arrowheads and arrows indicate Lin^POS (SOX9⁺, SOX2⁺, SFTPC⁺; folded) and lineage-negative (Lin^NEG) tip organoids (SOX9⁺, SOX2⁺, SFTPC^-; cystic), respectively. (D) FACS-isolating CD36 positive cells from a mixture of distal lung tip epithelial organoids. Only CD36+ cell subpopulation was purified following single, live-cell gating. (E) Enrichment of alveolar fated, Lin^POS tip organoid (right) is achieved by growing the CD36+ fraction, while CD36- fraction gives rise to Lin^NEG and non-alveolar organoids (left). (F and G) Immunostaining images comparing self-renewing, alveolar-fated Lin^POS tip organoids (left panels) and AT2 organoids (right panels). Scale bars, 50 μm.

Figure 2

Isolation of distal lung epithelial cells from human lung tissues, steps 1–14 (A) Human lung tissues at 19 pcw kept in cold Hibernate-E medium. (B–E) Isolation of the edge of the lung tissues, followed by mincing into small pieces. (F and G) Enzymatic dissociation of the fragmented pieces into single cells. (H and I) The single-cell pellet before (H) and after (I) removal of red blood cells.

Figure 3

Enrichment of alveolar-fated, Lin^POS tip organoids (A) Bright-field images of the distal lung tip epithelial organoids at 3 weeks after the initial cell isolation, at the end of passage 1. (B) Lin^POS tip organoids enriched at 3 weeks after CD36+ sorting. The CD36+ cell fraction enriches to form the alveolar-fated, Lin^POS tip organoids at passage 2. Arrows, cystic, Lin^NEG tip organoids. Scale bars, 100 μm.

Figure 4

AT2 cell fate differentiation of Lin^POS tip progenitor organoids By switching medium conditions from SN medium (A) to AT2 medium (B) the Lin^POS tip organoids readily differentiate to an AT2 cell lineage within 1 week. See also Figure 5 and Lim et al. 2023. Scale bars, 100 μm.

Figure 5

Comparison of Lin^POS and Lin^NEG tip progenitor organoids and AT2 organoids (A) The Lin^POS tip organoids can differentiate to an AT2 cell lineage by switching the culture medium conditions from the SN to AT2 medium, whereas the Lin^NEG tip organoids fail AT2 fate differentiation. See also Figure 4 and Lim et al. 2023. (B) A list of marker genes/proteins that are differentially expressed in each organoid line. Alveolar lineage markers include NKX2.1, pro SFTPC, mature SFTPB, mature SFTPC, LAMP3, ABCA3, HOPX, and SLC34A2. Tip progenitor markers include SOX9 and SOX2.

Figure 6

Repeated emergence of Lin^NEG cystic organoids Bright-field images showing repeated emergence of Lin^NEG cystic organoids (arrows) after enrichment of the CD36+ cells. Scale bars, 100 μm.

Figure 7

Airway fated organoids Bright field images showing non-alveolar cell lineage organoids, relatively dark and spheroid in shape (arrows). Scale bars, 100 μm.