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Method Article
Shimpei Gotoh
Gotoh's group, Kyoto University
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Although the methods of generating human lung epithelial cells have been studied1-8, there have been no reports for efficient generation and expanding alveolar type II cells as tissue stem cells producing pulmonary surfactant. Here we show a novel protocol for efficient generation and long-term expansion of SFTPC+ alveolar stem cells derived from human pluripotent stem cells (hPSCs). Although we previously developed a method of 3D co-culture differentiation of CPM+ cells with fetal human lung fibroblasts to induce alveolar stem cells6, the induction efficiency had been < 20% and it was difficult to maintain the alveolar stem cells for a long term. In this protocol, we added a step for preconditioning NKX2.1+ cells toward alveolar stem cell differentiation and established a method of their passages. Moreover, we also developed a method of fibroblast-free induction of SFTPC+ alveolar stem cells in order to avoid the usage of human fetal lung fibroblasts. This protocol accompanies Yamamoto et al (10.1038/nmeth.4448), Nature Methods, published online October 2, 2017.
Corrections have been made to this protocol since it was published
In the version of this protocol initially published, the concentration of 8-Br-cAMP, 3-isobutyl-1-methylxanthine and KGF in “Alveolarization medium” described in “E-2” section of “Procedure” was incorrectly typed;
8-Br-cAMP (it was 100 nM; the correct units are μM), 3-isobutyl-1-methylxanthine (it was 100 nM; the correct units are μM) and KGF (it was 100 ng/ml; the correct concentration is 10 ng/ml).
Keywords
Pluripotent stem cell, alveolar epithelial cell differentiation, long-term culture, organoid
Kyoto University is a patent applicant related to the method of alveolar differentiation from iPS cells.
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.pdf
Supplementary Protocol Figure 2 Induction and expansion of alveolar organoids.
- supplement0.docx
Table Troubleshooting
- supplement0.pdf
Supplementary Protocol Figure 1 Phase contrast images of iPSC-derived stepwise differentiated cells toward preconditioned CPM^high^ progenitor cells.
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Associated Publications
Method Article
Shimpei Gotoh
Gotoh's group, Kyoto University
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 03 Oct, 2017
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Although the methods of generating human lung epithelial cells have been studied1-8, there have been no reports for efficient generation and expanding alveolar type II cells as tissue stem cells producing pulmonary surfactant. Here we show a novel protocol for efficient generation and long-term expansion of SFTPC+ alveolar stem cells derived from human pluripotent stem cells (hPSCs). Although we previously developed a method of 3D co-culture differentiation of CPM+ cells with fetal human lung fibroblasts to induce alveolar stem cells6, the induction efficiency had been < 20% and it was difficult to maintain the alveolar stem cells for a long term. In this protocol, we added a step for preconditioning NKX2.1+ cells toward alveolar stem cell differentiation and established a method of their passages. Moreover, we also developed a method of fibroblast-free induction of SFTPC+ alveolar stem cells in order to avoid the usage of human fetal lung fibroblasts. This protocol accompanies Yamamoto et al (10.1038/nmeth.4448), Nature Methods, published online October 2, 2017.
Corrections have been made to this protocol since it was published
In the version of this protocol initially published, the concentration of 8-Br-cAMP, 3-isobutyl-1-methylxanthine and KGF in “Alveolarization medium” described in “E-2” section of “Procedure” was incorrectly typed;
8-Br-cAMP (it was 100 nM; the correct units are μM), 3-isobutyl-1-methylxanthine (it was 100 nM; the correct units are μM) and KGF (it was 100 ng/ml; the correct concentration is 10 ng/ml).
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