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Method Article
Methods of generating human pluripotent stem cell-derived alveolar stem cells and their expansion
Shimpei Gotoh
Gotoh's group, Kyoto University
Corresponding Author
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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
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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This is a preprint. It has not completed peer review.
Method Article
Methods of generating human pluripotent stem cell-derived alveolar stem cells and their expansion
Shimpei Gotoh
Gotoh's group, Kyoto University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
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).