Recapitulating human cardio-pulmonary co-development using simultaneous multilineage differentiation of pluripotent stem cells

  1. Wai Hoe Ng
  2. Elizabeth K Johnston
  3. Jun Jie Tan
  4. Jacqueline M Bliley
  5. Adam W Feinberg
  6. Donna B Stolz
  7. Ming Sun
  8. Piyumi Wijesekara
  9. Finn Hawkins
  10. Darrell N Kotton
  11. Xi Ren  Is a corresponding author
  1. Department of Biomedical Engineering, Carnegie Mellon University, United States
  2. Advanced Medical and Dental Institute, Universiti Sains Malaysia, Malaysia
  3. Department of Materials Science and Engineering, Carnegie Mellon University, United States
  4. Center for Biologic Imaging, University of Pittsburgh, United States
  5. Center for Regenerative Medicine of Boston University and Boston Medical Center, United States
6 figures, 2 videos, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
Mesoderm and endoderm co-induction from hiPSCs using CHIR.

(a) Diagram showing the experimental design (b) Cells following Stage-1 differentiation expressed MIXL1 (Mesendodermal lineage), SOX17 (definitive endoderm), and NCAM1 (mesoderm). Scale bar = 125 μm. (c) Majority of SOX17 cells were also FOXA2+. Scale bar = 62.5 μm. (d) Fold change of hiPSCs for FOXA2 (n = 3 each; 4 vs 7, p < 0.001; 7 vs 10, p < 0.001; 4 vs 10, p < 0.001), SOX17 (n = 3 each; 4 vs 7, p < 0.001; 7 vs 10, p < 0.001; 4 vs 10, p = 0.9978) and NCAM1 (n = 3 each; 4 vs 7, p < 0.001; 7 vs 10, p < 0.001; 4 vs 10, p < 0.001). All data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. ‘n’ refers to biological replicates. Diagram created using BioRender (http://biorender.com/).

Figure 1—figure supplement 1
Primitive streak induction from hiPSCs using CHIR.

(a) Diagram showing the experimental design. (b) Pluripotent marker (OCT4) expression on hiPSCs prior to induction with CHIR. (c) Cells following 48 hr CHIR treatment expressed T (primitive streak), MIXL1 (Mesendodermal lineage) but not pluripotent (OCT4) marker. FACS analysis of cells for (d) T and (e) MIXL1. (f) Schematic diagram showing the experimental design for flow cytometry analysis at the end of Stage-2 co-differentiation. (g) Flow cytometry analysis of endoderm marker (SOX17) and mesodermal marker (CD13) on hiPSCs, cells differentiated from standard protocol with or without Activin A. Scale bar = 125 μm for 20 X images. Diagram created using BioRender (http://biorender.com/).

Figure 2 with 1 supplement
Stepwise cardio-pulmonary co-differentiation from hiPSCs using chemical defined, growth factor-free protocol.

(a) Schematic diagram showing the overall differentiation strategy. (b) Immunofluorescence (IF) showing staining of lung (NKX2.1+) and cardiac (NKX2.5+). (c) IF (d,e) and quantitative PCR (qPCR) analysis of the induction of lung and cardiac progenitors on Day-15 of differentiation. (c–e) The effects of different CHIR concentrations during Stage-1 of differentiation. Fold change over hiPSCs (d) NKX2.1 (n = 3 each; 4 vs 7, p < 0.001; 7 vs 10, p < 0.001; 4 vs 10, p = 0.9993) and (e) NKX2.5 (n = 3 each; 4 vs 7, p< 0.001; 7 vs 10, p = 0.0053; 4 vs 10, p < 0.001). (f–h) The effects of different exposure time of CHIR (7 µM) treatment during the first 2 days of differentiation. qPCR analysis of (g) NKX2.1 (n = 3 each; 24 vs 48, p < 0.001; 48 vs 72, p < 0.001; 24 vs 72, p < 0.001) and (h) NKX2.5 (n = 3 each; 24 vs 48, p < 0.001; 48 vs 72, p = 0.1503; 24 vs 72, p < 0.001). Scale bar = 500 μm for whole well scan; Scale bar = 125 μm for 20 X images. All data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. ‘n’ refers to biological replicates. Diagram created using BioRender (http://biorender.com/).

Figure 2—figure supplement 1
Characterization of Day-15 cardio-pulmonary progenitors.

FACS analysis of Day-15 cells from (a) cardio-pulmonary, (b) pulmonary, and (c) cardiac differentiation protocol. (d) Day-15 cardio-pulmonary progenitors did not express p63, PAX8, TUJ1, PAX6 markers; NKX2.5 expression colocalized with cTnT, and partially with COUPTFII; and expression of NFATC, WT1 and MLC2v was not detected. Scale bar = 75 μm for 40 X images.

Figure 3 with 3 supplements
The effect of Nodal and BMP signaling during Stage-1 of co-differentiation on cardio-pulmonary induction.

IF (a,d) and qPCR (b,c,e,f) analysis of the induction of lung (NKX2.1+) and cardiac (NKX2.5+) progenitors on Day-15 of differentiation (a–c) The effects of exogenous nodal activation (Activin A, 20 ng/mL) or its inhibition (A8301, 1 µM). Fold change over hiPSCs for (b) NKX2.1 (n = 3 each; Activin A /A8301 vs. Activin A+ /A8301, p = 0.1939; Activin A /A8301 vs. Activin A /A8301+, p < 0.001; Activin A+ /A8301 vs. Activin A /A8301+, p < 0.001) and (c) NKX2.5 (n = 3 each; Activin A /A8301 vs. Activin A+ /A8301, p < 0.001; Activin A /A8301 vs. Activin A /A8301+, p < 0.001; Activin A+ /A8301 vs. Activin A /A8301+, p = 0.8649). (d-f) The effects of exogenous BMP4 (20 ng/mL) or BMP inhibitor (DMH1, 2 µM). qPCR analysis of (e) NKX2.1 (n = 3 each; BMP4 /DMH1 vs. BMP4+ /DMH1, p < 0.001; BMP4 /DMH1 vs. BMP4 /DMH1+, p < 0.001; BMP4+ /DMH1 vs. BMP4 /DMH1+, p < 0.001) and (f) NKX2.5 (n = 3 each; BMP4 /DMH1 vs. BMP4+ /DMH1, p < 0.001; BMP4 /DMH1 vs. BMP4 /DMH1+, p = 0.0044; BMP4+ /DMH1 vs. BMP4 /DMH1+, p < 0.001). Scale bar = 500 μm for whole well scan; Scale bar = 125 μm for 20 X images. All data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. ‘n’ refers to biological replicates. Diagram created using BioRender (http://biorender.com/).

Figure 3—figure supplement 1
Initial co-induction medium for CHIR-directed differentiation.

Cells were induced by CHIR in (a) mTESR1 (b) and RPMI-based medium, followed by representative IF staining of NKX2.1 and NKX2.5 following 15 days of differentiation. Scale bar = 500 μm for whole well scan; Scale bar = 125 μm for 20 X images. Diagram created using BioRender (http://biorender.com/).

Figure 3—figure supplement 2
Combination of TGF-β and WNT inhibition during Stage-2 of co-differentiation is required for cardio-pulmonary induction.

(a) Schematic diagram illustrating the experimental design. (b–d) IF staining showing NKX2.1 and NKX2.5 expression on Day-15 of differentiation (b), and the corresponding qPCR analysis of (c) NKX2.1 (n = 3 each; A8301+ /IWP4+ vs. A8301+ /IWP4, p < 0.001; A8301+ /IWP4+ vs. A8301 /IWP4+, p < 0.001; A8301+ /IWP4 vs. A8301 /IWP4+, p < 0.001) and (d) NKX2.5 (n = 3 each; A8301+ /IWP4+ vs. A8301+ /IWP4, p < 0.001; A8301+ /IWP4+ vs. A8301 /IWP4+, p < 0.001; A8301+ /IWP4 vs. A8301 /IWP4+, p = 0.9986). Scale bar = 500 μm for whole well scan; Scale bar = 125 μm for 20 X images. All data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. Diagram created using BioRender (http://biorender.com/).

Figure 3—figure supplement 3
Roles of BMP4 during Stage-3 of co-differentiation.

(a) Schematic diagram illustrating the experimental design. (b) IF staining showing NKX2.1 and NKX2.5 expression on Day-15 of differentiation, and the corresponding qPCR analysis of (c) NKX2.1 (n = 3 each; BMP4 /DMH1 vs. BMP4+ /DMH1, p = 0.9737; BMP4 /DMH1 vs. BMP4 /DMH1+, p < 0.01; BMP4+ /DMH1vs. BMP4 /DMH1+, p = 0.0128) and (d) NKX2.5 (n = 3 each; BMP4 /DMH1 vs. BMP4+ /DMH1, p = 0.3330; BMP4 /DMH1 vs. BMP4 /DMH1+, p < 0.001; BMP4+ /DMH1vs. BMP4 /DMH1+, p < 0.01). Scale bar = 500 μm for whole well scan; Scale bar = 125 μm for 20 X images. All data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. Diagram created using BioRender (http://biorender.com/).

Figure 4 with 6 supplements
3D suspension culture of cardio-pulmonary μTs expedites AT2 maturation.

(a) Schematic diagram illustrating the Stage-4 maturation protocol involving replating of Day-15 cardiac and pulmonary progenitors onto ultra-low adhesion plate (for 3D suspension culture) or the transwell insert (for ALI culture). (b) Whole mount staining of cardiopulmonary μT on Day-18, scale bar 75 μm. (c) Live μT imaging of NKX2.1GFP and SFTPCTdTomato reporter signals during the first 3 days of maturation (Day-16 – Day-18). (d-f) qPCR analysis of hiPSC control, Day-15 cells and Day-18 cells (from ALI or suspension culture) for (d) NKX2.1 (n = 3 each; Day-15 vs. ALI, p = 0.9998; Day-15 vs. Suspension, p = 0.0547; ALI vs. Suspension, p = 0.0486), (e) SFTPC (n = 3 each; Day-15 vs. ALI, p = 0.1896; Day-15 vs. Suspension, p < 0.001; ALI vs. Suspension, p < 0.01). (f) NKX2.5 (n = 3 each; Day-15 vs. ALI, p < 0.001; Day-15 vs. Suspension, p = 0.8367; ALI vs. Suspension, p < 0.001). Scale bar = 125 μm. (g–n) Schematic diagram showing the differentiation procedure without (g) and with (j) Activin A during Stage-1 of differentiation (h, k) Live μT imaging of NKX2.1GFP and SFTPCTdTomato reporter signals during Day-16 to Day-18. Scale bar = 125 μm for 10 X images. (i, l) Whole mount staining of μTs on Day-18. (m–n) qPCR analysis of hiPSC control, Day-15 cells and Day-18 cells (from Activin-free or Activin) for (m) NKX2.1 (n = 3 each; Day-15 (No Activin) vs. Day-18 (No Activin), p < 0.001; Day-15 (Activin) vs Day-18 (Activin), p = 0.0147; Day-15 (No Activin) vs. Day-15 (Activin), p = 0.1316), (n) SFTPC (n = 3 each; Day-15 (No Activin) vs. Day-18 (No Activin), p < 0.001; Day-15 (Activin) vs Day-18 (Activin), p = 0.2417; Day-18 (No Activin) vs. Day-18 (Activin), p < 0.001). All data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar = 125 μm. ‘n’ refers to biological replicates. Diagram created using BioRender (http://biorender.com/).

Figure 4—figure supplement 1
Co-maturation of Day-15 cardiac and pulmonary progenitors on ALI and 3D suspension culture platforms.

(a) Schematic diagram showing the experimental design. (b) Live cell μTs imaging on the NKX2.1GFP and SFTPCTdTomato reporter signal over time. Scale bar = 125 μm for 10 X images. (c) qPCR for SFTPC (n = 3 each; Day-15 vs. Day-18, p < 0.001; Day-15 vs. Day-22, p = 0.5569; Day-18 vs. Day-22, p < 0.001). (d) IF of NKX2.1 and NKX2.5 on ALI membrane. Scale bar = 125 μm for 20 X images. All data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. Diagram created using BioRender (http://biorender.com/).

Figure 4—figure supplement 2
Co-maturation of Day-15 cardiac and pulmonary progenitors on 2D submerged culture.

(a) Schematic diagram showing the experimental design. (b) Live cell imaging of the NKX2.1GFP and SFTPCTdTomato reporter signal over time. Scale bar = 125 μm for 10 X images. Diagram created using BioRender (http://biorender.com/).

Figure 4—figure supplement 3
Co-maturation of Day-15 cardiac and pulmonary progenitors in Matrigel Droplet and 3D suspension culture platforms.

(a) Schematic diagram showing the experimental design. (b) qPCR for NKX2.1 (n = 3 each; Day-18 Suspension vs. Day-18 Matrigel, p = 0.0781), SFTPC (n = 3 each; Day-18 Suspension vs. Day-18 Matrigel, p < 0.001), NKX2.5 (n = 3 each; Day-18 Suspension vs. Day-18 Matrigel, p < 0.001). All data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. Diagram created using BioRender (http://biorender.com/).

Figure 4—figure supplement 4
Maturation of pulmonary progenitors derived from Activin A-based protocol on 3D suspension culture.

(a) Schematic diagram showing the experimental design. (b) Live μT imaging on the NKX2.1GFP and SFTPCTdTomato reporter signal over time. Scale bar = 125 μm for 10 X images. Diagram created using BioRender (http://biorender.com/).

Figure 4—figure supplement 5
Verification of cardio-pulmonary co-differentiation protocol on BU1 hiPSCs.

(a) Schematic diagram illustrating the process of cardio-pulmonary co-differentiation, highlighting the adjustment of CHIR concentration during the first 2 days of differentiation. (b) IF staining of mesendoderm, endoderm and mesoderm after Stage-1 differentiation with 7 μM CHIR. (c,d) IF staining of NKX2.1 and NKX2.5 following 15 days of co-differentiation. Scale bar = 500 μm for whole well scan; Scale bar = 125 μm for 20 X images; (e) Confocal imaging of Day-18 μT. Scale bar = 125 μm for confocal images. Diagram created using BioRender (http://biorender.com/).

Figure 4—figure supplement 6
Comparing fluorescence of NKX2.1GFP and SFTPCTdTomato of BU3-NGST vs. non-reporter BU1.

No autofluorescence was observed in non-reporter line BU1 on Day-18. Scale bar = 125 μm.

Figure 5 with 1 supplement
Cardio-pulmonary segregation in the dual-lineage μT.

(a) Schematic diagram illustrating the timeline for the investigation. Scale bar = 125 μm (b) Histological analysis of cardio-pulmonary μTs at different stages of segregation. Scale bar = 125 μm (c) Diagram showing measurement of the total perimeter of GFP+ pulmonary compartment (red color) and its overlapping perimeter with non-GFP compartment (white color) using Image J. Scale bar = 125 μm (d) Box plot showing percentage overlapping region of GFP+ with non-GFP tissues on Day- 18 (n = 30 each;; CKDCI vs. KDCI, p = 0.3979; CKDCI vs. KDCI+ IWP4; p > 0.9999; CKDCI vs. KDCI+ NSC, p = 0.4293; KDCI vs. KDCI+ IWP4, p = 0.3979; KDCI vs. KDCI+ NSC, p > 0.9999; KDCI+ IWP4 vs. KDCI+ NSC, p = 0.4293), Day-22 (n = 30 each; CKDCI vs. KDCI, p = 0.0077; CKDCI vs. KDCI+ IWP4; p = 0.0112; CKDCI vs. KDCI+ NSC, p < 0.001; KDCI vs. KDCI+ IWP4, p = 0.9994; KDCI vs. KDCI+ NSC, p = 0.8318; KDCI+ IWP4 vs. KDCI+ NSC, p = 0.7674) and Day-25 (n = 30 each; CKDCI vs. KDCI, p < 0.001; CKDCI vs. KDCI+ IWP4; p < 0.001; CKDCI vs. KDCI+ NSC, p < 0.001; KDCI vs. KDCI+ IWP4, p = 0.4271; KDCI vs. KDCI+ NSC, p = 0.7275; KDCI+ IWP4 vs. KDCI+ NSC, p = 0.9623). (e) Histological analysis of cTnT expression on the segregated cardiac and pulmonary μTs, with co-staining of NKX2.5 and NKX2.1. Scale bar = 125 μm. All data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. ‘n’ refers to biological replicates. Diagram created using BioRender (http://biorender.com/).

Figure 5—figure supplement 1
Day-15 cells in CKDCI vs. KDCI for co-maturation.

(a) Schematic diagram showing the experimental design. (b) Live μT imaging on the NKX2.1GFP and SFTPCTdTomato reporter signal over time. Scale bar = 125 μm for 10 X images. (c) qPCR analysis for NKX2.1 (n = 3 each; Day-15 vs. Day-18 CKDCI, p = 0.1522; Day-15 vs. Day-18 KDCI, p = 0.2912; Day-18 CKDCI vs. Day-18 KDCI, p < 0.05), SFTPC (n = 3 each; Day-15 vs. Day-18 CKDCI, p < 0.001; Day-15 vs. Day-18 KDCI, p > 0.9999; Day-18 CKDCI vs. Day-18 KDCI, p < 0.001). (d) Time-lapse images of μTs cultured in KDCI medium from Day-16 to Day-23. Scale bar = 125 μm. (e) Percent overlapping of GFP vs. non-GFP (Day-16 vs. Day-20, p < 0.001; Day-16 vs. Day-23, p < 0.001; Day-20 vs. Day-23, p < 0.001). All data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. Diagram created using BioRender (http://biorender.com/).

Figure 6 with 1 supplement
Characterization of cardio-pulmonary μT maturation.

(a) Lamellar bodies found in cardio-pulmonary μT. Scale bar = 1 μm. Cardio-pulmonary μT expressing (b) Pro-SFTPC and HOPX. Cardio-pulmonary μT also exhibited striated pattern as indicated by (c) cTnT and (d) Sarcomeric Alpha Actinin. Scale bar = 125 μm. (e) Calcium imaging of cardiac μTs following treatment with Verapamil.

Figure 6—figure supplement 1
Characterization of cardio-pulmonary μT.

IF for (a) Pro-SFTPB and (b) HOPX gene expression (n = 3 each; Day-15 vs. Day-18 Suspension, P < 0.01). IF for (c) S100A4. (d) FOXJ1, (e) MUC5AC, and (f) p63. Scale bar = 125 μm.

Videos

Video 1
Contacting cardiac μT following 7 days after withdrawal of CHIR.
Video 2
Calcium influx capability of cardiac μT loaded with Cal-520.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)β-actinGenBank (NM_001101.3)Hs01060665_g1
Gene (Homo sapiens)NKX2.1GenBank (NM_001079668.2)Hs00968940_m1
Gene (Homo sapiens)FOXA2GenBank (NM_02178.4)Hs00232764_m1
Gene (Homo sapiens)SOX17GenBank (NM_022454.3)Hs00751752_s1
Gene (Homo sapiens)NKX2.5GenBank (NM_004387.3)Hs00231763_m1
Gene (Homo sapiens)SFTPCGenBank (NM_001172357.1)Hs00161628_m1
Gene (Homo sapiens)NCAM1GenBank (NM_000615.6)Hs00941821_m1
Gene (Homo sapiens)HOPXGenBank (NM_001145459.1)Hs04188695_m1
Cell line (Homo sapiens)BU3-NGSTBoston University (Kotton’s Lab)RRID:CVCL_WN82
Cell line (Homo sapiens)BU1Boston University (Kotton’s Lab)-
Cell line (Homo sapiens)Normal Human Bronchial Epithelial (NHBE) cellsLonzaCat# CC-2541; RRID:CVCL_S124
Antibodyanti-NKX2.1(Rabbit monoclonal)AbcamCat# ab76013;RRID:AB_13107841:500
Antibodyanti-NKX2.5 (Goat polyclonal)R&D SystemsCat# AF2444;RRID:AB_3552691:500
Antibodyanti-Pro-SFTPB (Rabbit polyclonal)Seven HillsCat# WRAB-55522RRID:AB_1:200
Antibodyanti-Pro-SFTPC (Rabbit polyclonal)Seven HillsCat# WRAB-9937; RRID:AB_4517211:500
Antibodyanti-HOPX (Mouse monoclonal)Santa CruzCat# Sc-398703; RRID:AB_26879661:100
Antibodyanti-cTnT (Mouse monoclonal)Thermo Fisher ScientificCat# MA5-12960; RRID:AB_110007421:200
Antibodyanti-Sarcomeric Alpha Actinin (Mouse Monoclonal)Thermo Fisher ScientificCat# MA1-22863; RRID:AB_5574261:200
Antibodyanti-p63 (Mouse monoclonal)Biocare MedicalCat# CM163A; RRID:AB_105827301:100
Antibodyanti-MUC5AC (Mouse monoclonal)Thermo Fisher ScientificCat# MA5-12178;RRID:AB_109780011:100
Antibodyanti-FOXJ1 (Mouse monoclonal)Thermo Fisher ScientificCat# 14-9965-80; RRID:AB_15488361:100
Antibodyanti-PAX8 (Mouse monoclonal)ThermoFisher ScientificCat# MA1-117RRID:AB_25368281:100
Antibodyanti-β-Tubulin III (Mouse monoclonal)Sigma-AldrichCat# T8578RRID:AB_18412281:100
Antibodyanti-PAX6 (Mouse monoclonal)BioLegendsCat# 862,001RRID:AB_28012371:100
Antibodyanti-COUPTFII (Mouse monoclonal)R&D SystemsCat# PP-H7147-00RRID:AB_19642141:100
Antibodyanti-MLC2v (Rabbit Polyclonal)ProteinTech GroupCat# 10906–1-APRRID:AB_21474531:100
Antibodyanti-NFATC (Mouse monoclonal)Thermo Fisher ScientificCat# MA3-024RRID:AB_22360371:100
Antibodyanti-WT1 (Mouse monoclonal)Novus BiologicalsCat# NBP2-44606RRID:AB_not found1:100
Antibodyanti-Brachyury (Goat polyclonal)R&D SystemsCat# AF2085; RRID:AB_22002351:50
Antibodyanti-MIXL1 (Rabbit polyclonal)Thermo Fisher ScientificCat# PA5-64903; RRID:AB_26647371:50
Antibodyanti-NCAM1 (Rabbit monoclonal)Cell Signaling TechnologiesCat# 99,746T; RRID:AB_28684901:50
Antibodyanti-FOXA2 (Mouse monoclonal)Santa Cruz TechnologyCat# Sc-271103; RRID:AB_106144961:50
Antibodyanti-SOX17 (Goat polyclonal)R&D SystemsCat# AF1924; RRID:AB_3550601:200
Antibodyanti-OCT4 (Mouse monoclonal)Santa CruzCat# sc-5279RRID:AB_6280511:100
Antibodyanti-CD13 APC-conjugatedBD BiosciencesCat# 557454; RRID:AB_3986241:10
AntibodyDonkey anti-mouse IgG (H + L), Alexa Fluor 488Thermo Fisher ScientificCat# A21202; RRID:AB_1416071:500
AntibodyDonkey anti-rabbit IgG (H + L), Alexa Fluor 488Thermo Fisher ScientificCat# A21206; RRID:AB_25357921:500
AntibodyDonkey anti-rabbit IgG (H + L), Alexa Fluor 568Thermo Fisher ScientificCat# A10042; RRID:AB_27575641:500
AntibodyDonkey anti-goat IgG (H + L), Alexa Fluor 647Thermo Fisher ScientificCat# A21447; RRID:AB_1418441:500
Recombinant DNA proteinActivin AR&D Systems338-AC-010
Recombinant DNA proteinRecombinant human BMP4R&D Systems314 BP
Recombinant DNA proteinRecombinant human KGFPeproTech100–19
Commercial assay, kitHigh-Capacity cDNA Reverse Transcription kitApplied Biosystems4368814
Commercial assay, kitTaqMan Fast Advanced Master MixThermo Fisher Scientific4444556
Commercial assay, kitFixable Violet Dead Cell Stain KitThermo Fisher ScientificL34955
Chemical compound, drugshESC-qualified Matrigel Basement Membrane MatrixCorning354,234
Chemical compound, drugsGrowth Factor Reduced Basement Membrane MatrixCorning354,230
Chemical compound, drugsmTESR PlusStem Cell Technologies05825
Chemical compound, drugsDulbecco’s Phosphate-Buffered Saline (DPBS)Corning45000–430
Chemical compound, drugsReLESRStem Cell Technologies05873
Chemical compound, drugsStemPro Accutase Cell Dissociation ReagentThermo Fisher ScientificA1110501
Chemical compound, drugsRPMI1640Corning10–040-CV
Chemical compound, drugsGlutaMAXThermo Fisher Scientific35050061
Chemical compound, drugsB-27 minus insulin SupplementThermo Fisher ScientificA1895601
Chemical compound, drugsB-27 Supplement (Complete)Thermo Fisher Scientific12587–010
Chemical compound, drugsTrypLE ExpressThermo Fisher Scientific12605028
Chemical compound, drugsHyclone FetalClone 1 Serum (U.S)GE HealthcareSH30080.03
Chemical compound, drugsY-27632 dihydrochlorideCayman Chemical1000558310
Chemical compound, drugsCHIR99021Reprocell04000402
Chemical compound, drugsA8301Sigma AldrichSSML1314-1MG
Chemical compound, drugsDMH-1Tocris4126/10
Chemical compound, drugsIWP4Tocris5214/10
Chemical compound, drugsAll-trans Retinoic AcidCayman11,017
Chemical compound, drugsDexamethasoneSigma AldrichD4902
Chemical compound, drugs8-bromoadenosine 3’,5’-cyclic monophosphate sodium salt (cAMP)Sigma AldrichB7880
Chemical compound, drugs3-Isobutyl-1-methylxanthine (IBMX)Sigma AldrichI5879
Chemical compound, drugsNSC668036Tocris5813/10
Chemical compound, drugsPneumaCult-ALI Basal MediumStemcell Technologies05002
Chemical compound, drugsPneumaCult-ALI Maintenance SupplementStemcell Technologies05006
Chemical compound, drugsTRIzol ReagentThermo Fisher Scientific15596018
Chemical compound, drugsChloroformSigma-AldrichC2432
Chemical compound, drugsGlycoblueThermo Fisher ScientificAM9516
Chemical compound, drugsIsopropanolACROS Organic327272500
Chemical compound, drugsEthanol 200 ProofPharmaco-AAPLDSP-C7-18
Chemical compound, drugsMethanolFisher ChemicalBPA412-1
Chemical compound, drugsParaformaldehydeSigma AldrichP6148-500G
Chemical compound, drugsTriton X-100Sigma AldrichX100-500ML
Chemical compound, drugsBovine Serum AlbuminFisher BioReagentsBP9706-100
Chemical compound, drugsPhosphate Buffer Saline 20 XGrowcellsMRGF-695–010 L
Chemical compound, drugsHistoclearGreat LakesGL-1100–01
Chemical compound, drugsAntigen Unmasking Solution, Citric Acid BasedVector LaboratoriesH-3300; RRID:AB_2336227
Chemical compound, drugsDAPI-Fluoromount-GSouthern Biotech0100–20
Chemical compound, drugsHoechst 33,342Thermo Fisher Scientific62,249
Chemical compound, drugsTrypLE Express EnzymeThermo Fisher Scientific12605010
SoftwareImage JVersion 1.8.0.182; RRID:SCR_003070
SoftwareFlowjoVersion 7.6.1; RRID:SCR_008520
OtherTranswell insert (0.4 μm)Greiner Bio-One662,641
OtherUltra-low adherence 24-well PlateGreiner Bio-One662,970
OtherUltra-low adherence 96-well PlateGreiner Bio-One650,979
OtherNanodrop 2000 SpectrophotometerThermo Fisher ScientificND2000CLAPTOP; RRID:SCR_018042
OtherEVOS FL Auto 2 Imaging SystemThermo Fisher ScientificAMAFD2000
OtherCFX96 Touch Real-Time PCR Detection SystemBio-Rad1855196; RRID:SCR_018064
OtherImmEdge Hydrophobic Barrier PAP PenVector LaboratoriesH-4000; RRID:AB_2336517
OtherHistoGel Specimen Processing GelRichard Allen Scientific11330057

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  1. Wai Hoe Ng
  2. Elizabeth K Johnston
  3. Jun Jie Tan
  4. Jacqueline M Bliley
  5. Adam W Feinberg
  6. Donna B Stolz
  7. Ming Sun
  8. Piyumi Wijesekara
  9. Finn Hawkins
  10. Darrell N Kotton
  11. Xi Ren
(2022)
Recapitulating human cardio-pulmonary co-development using simultaneous multilineage differentiation of pluripotent stem cells
eLife 11:e67872.
https://doi.org/10.7554/eLife.67872