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| Status |
Public on Oct 14, 2020 |
| Title |
Derivation of airway basal stem cells from human pluripotent stem cells |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
The derivation of self-renewing tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would shorten the time needed to engineer mature cell types in vitro and would have broad reaching implications for the field of regenerative medicine. Here we report the directed differentiation of human iPSCs into putative airway basal cells (“iBCs”), a population resembling the epithelial stem cell of lung airways. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato) we track and purify these cells over time, as they first emerge from iPSC-derived foregut endoderm as developmentally immature NKX2-1GFP+ lung progenitors which then augment a TP63 program during subsequent proximal airway epithelial patterning. These cells clonally proliferate, initially as NKX2-1GFP+/ TP63tdTomato+ immature airway progenitors that lack expression of the adult basal cell surface marker, NGFR. However, in response to primary basal cell media, NKX2-1GFP+/ TP63tdTomato+ cells upregulate NGFR and display the molecular and functional phenotype of airway basal stem cells, including the capacity to clonally self-renew or undergo multilineage ciliated and secretory epithelial differentiation in air-liquid interface cultures. iBCs and their differentiated progeny recapitulate several fundamental physiologic features of normal primary airway epithelial cells and model perturbations that characterize acquired and genetic airway diseases. In an asthma model of mucus metaplasia, the inflammatory cytokine IL-13 induced an increase in MUC5AC+ cells similar to primary cells. CFTR-dependent chloride flux in airway epithelium generated from cystic fibrosis iBCs or their syngeneic CFTR-corrected controls exhibited a pattern consistent with the flux measured in primary diseased and normal human airway epithelium, respectively. Finally, multiciliated cells generated from an individual with primary ciliary dyskinesia recapitulated the ciliary beat and ultrastructural defects observed in the donor. Thus, we demonstrate the successful de novo generation of a tissue-resident stem cell-like population in vitro from iPSCs, an approach which should facilitate disease modeling and future regenerative therapies for a variety of diseases affecting the lung airways.Single-cell RNA-Sequencing profiling of human iPSC-derived basal cells, airway epithelium compared to primary human basal cells and airway epithelium.
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| Overall design |
We analyzed (1) iPSC-derived immature airway progenitors, (2) iPSC-derived BCs (iBCs) (3) iBC-derived air-liquid interface(ALI) cultures, (4) primary human basal cells, and (5) primary human basal cells differentiationed in ALI culture. For (1) iPSC-derived immature airway progenitors we differentiated a dual fluorescence iPSC reporter line (NKX2-1GFP;TP63tdTomato), hereaftr BU3 NGPT, in our previously publisehd airway differeniation protocol in imedia containing FGF2, FGF10, dexamethasone, cyclic AMP, 3-isobutyl-1-methylxanthine and Y-27632, hereafter “FGF2+10+DCI+Y” (McCauley et al., Cell Stem Cell, 2017) (2) iBCs were genereated from the same BU3 NGPT line but cultured in Pneumacult Ex-Plus media supplemented with SMAD inhibitors and Y-27632. Both samples were analyzed by sc-RNA-Seq on day 46 of differentiation. iBCs (GFP+/tdTomato+/NGFR+) from the BU3 NGPT iPSC line were sorted and plated in ALI conditions to form a pseudostratified airway epithelium on Transwell filters. Freshly isolated, P(0), primary human epithelial cells (basal cells) were cultured in BEGM media and analyzed by Sc-RNA-Seq but also differentiated in air-liquid interface culture in bronchial epithelial growth medium to form a mucociliary epithelium.
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| Contributor(s) |
Hawkins F, Villacorta C, Kotton D |
| Citation(s) |
34355203 |
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| Submission date |
Dec 18, 2019 |
| Last update date |
Aug 11, 2021 |
| Contact name |
Carlos Villacorta-Martin |
| Organization name |
Center for Regenerative Medicine at Boston University
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| Street address |
670 Albany St
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02118 |
| Country |
USA |
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| Platforms (1) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (7)
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| GSM4223672 |
CBRA-PNEX-DUAL-SMAD: BU3 NGPT Basal Cell Medium_1 |
| GSM4223673 |
CFKBRA-F210DCIY: BU3 NGPT FGF2+10+DCI+Y_2 |
| GSM4223674 |
CFKBRA-PNEX-DUAL-SMAD: BU3 NGPT Basal Cell Medium_2 |
| GSM4223675 |
BU3NGPT-ALI: BU3 NGPT ALI |
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| Relations |
| BioProject |
PRJNA596381 |
| SRA |
SRP238066 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE142246_RAW.tar |
104.5 Mb |
(http)(custom) |
TAR (of H5) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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