Figure 3. HH-induced ventral foregut spheroids give rise to lung organoids.

(A) hESCs were differentiated into foregut spheroids by treating cells with 4 days of ACTA and then an additional 4–6 days of NOG+SB+FGF4+Ch with the addition of the HH agonist SAG. Representative whole mount images of spheroids in a matrigel droplet are shown at low (left, scale bar 200 µm) and high magnification (right, scale bar 100 µm). (B) The addition of SAG to the NOG+SB+FGF4+Ch spheres caused a reduction in SOX2 and CDX2 transcripts (top panel) and a significant increase of NKX2.1 transcript (bottom panel) compared to NOG+SB+FGF4+Ch spheres (without SAG). Other foregut lineages (PAX8, PDX1, HHEX) were not significantly different when SAG was added. (C) The majority of the cells in NOG+SB+FGF4+Ch+SAG spheres expressed FOXA2, SOX2 and NKX2.1 protein. Scale bars represent 50 µm. (D) Timeline showing NOG+SB+FGF4+Ch+SAG induced foregut spheroids grown and maintained in FGF10. Note that Day 1 is the day spheroids were plated in Matrigel. The scale bar represents 100 µm. (E) Organoids express lung markers in a manner consistent with mouse lung development. All expression is shown relative to undifferentiated pluripotent stem cells (hPSC), and adult human lung is shown as a reference. Lung progenitor markers NMYC and ID2 were very low in adult lung, and were expressed at high levels in early organoid cultures, but were reduced over time (D = Days in culture), whereas NKX2.1 expression remained relatively constant. In contrast, SFTPC is known to be expressed at low levels in distal lung progenitors, but increases and is highly expressed in AECII cells. Consistently, SFTPC is highly expressed in adult human lungs and increases over time in organoid cultures and the AECI marker HOPX is also highly expressed in adult human lung and increases over time in organoids. *p < 0.05. All error bars represent SEM.

DOI: http://dx.doi.org/10.7554/eLife.05098.010

Figure 3—figure supplement 1. Overview of conditions tested to generate human lung organoids.

hPSCs are OCT4 and NANOG positive. After 4 days of 100 ng/ml Activin A, definitive endoderm (FOXA2 and SOX17 positive) was generated and then treated with two different conditions. In the top branch, NOG+SB+FGF4+Ch spheroids were generated, and different conditions were tested to promote lung organoid differentiation. In the bottom branch, NOG+SB+FGF4+Ch+SAG spheroids were generated, and different conditions were tested to promote lung organoid differentiation. Ultimately, we determined that spheroids generated with NOG+SB+FGF4+Ch+SAG and that were subsequently embedded in Matrigel and expanded in FGF10 gave rise to ‘Human Lung Organoids’ (HLOs).

DOI: http://dx.doi.org/10.7554/eLife.05098.011

Figure 3—figure supplement 2. FGF-low culture conditions cause a loss of organoid epithelium over time.

(A) NOG+SB+F+Ch foregut spheroids were generated and then cultured in SAG+SU for 10 days followed by 1% FBS ± FGF10. Timeline images show organoids cultured in 1% FBS. By day 20, 3D structures appeared ‘fuzzy’, which indicates an outgrowth of mesenchymal tissue. Scale bar represents 200 µm. (B) NOG+SB+F+Ch foregut spheroids treated with SAG+SU and maintained in 1% FBS showed an increase in Vimentin (VIM, green) immunofluorescence over time. Scale bar represents 50 µM. (C) NOG+SB+F+Ch foregut spheroids treated with SAG+SU and maintained in 1% FBS (upper panel) or 1% FBS+FGF10 (lower panel) had a significant increase of VIM expression starting at day 20 (D20) compared to hPSCs and showed very weak E-CADHERIN (CDH1) expression compared to D20 HLOS (optimized conditions, as described in Figure 3). Lastly, both conditions appeared to lose NKX2.1 expression over time. (D) NOG+SB+F+Ch+SAG spheroids maintained in 1% FBS (basal media) also appear to lose epithelial structures over time. Scale bar represents 200 µm. (E) By day 20 (D20) the tissue had very few epithelial structures expressing ECAD (white, left panel) and there was robust VIM expression (green, right panel) at both time points. Scale bar represents 50 µm. HLO *p < 0.05. All error bars represent SEM.

DOI: http://dx.doi.org/10.7554/eLife.05098.012

Figure 3—figure supplement 3. Foregut spheroids express lung and foregut specific markers.

(A) NOG/SB/FGF4/Ch/SAG spheroids coexpress NKX2.1 (green) and the endoderm marker FOXA2 (red). (B) The majority of the cells in the spheroid co-expresses SOX2 (green) and FOXA2 (red). Scale bars represent 50 µM.

DOI: http://dx.doi.org/10.7554/eLife.05098.013

Figure 3—figure supplement 4. Ventral foregut spheroids do not express appreciable levels of PAX8 protein.

Although NOG+SB+FGF4+Ch+SAG ventral foregut spheroids expressed PAX8 mRNA (Figure 3B), we did not detect PAX8 protein in spheroids using immunofluorescence, whereas PAX8 protein in FGF2 8 day treated foregut monolayers (ACTA followed by NOG/SB) was readily detectable. Left panel: scale bar represents 50 µm. Right panel: scale bar represents 200 µm, inset scale bar represents 100 µm.

DOI: http://dx.doi.org/10.7554/eLife.05098.014

Figure 3—figure supplement 5. Foregut spheroids consist of both epithelial and mesenchymal cells.

NOG/SB/FGF4/Ch/SAG spheroids have a minor population of Vimentin (VIM, white) positive mesenchymal cells, while the majority of cells are epithelial and express ECAD (red). Scale bar represents 50 µM.

DOI: http://dx.doi.org/10.7554/eLife.05098.015

Figure 3—figure supplement 6. Lung organoids contain both proximal and distal domains.

NOG/SB/FGF4/Ch/SAG spheroids cultured for 15 days with FGF10 express the distal lung epithelium marker SOX9 (green) and proximal marker SOX2 (white) as separate domains in the epithelium labeled by ECAD (red). Z-stack images are shown every 40 µm sections through the HLO. Scale bar represents 200 µm.

DOI: http://dx.doi.org/10.7554/eLife.05098.016