Abstract
Induced Pluripotent Stem Cell‐Derived Basal Cells Provide A Novel Source of Multilineage Airway Epithelial Cells
The FASEB journal, Vol.33(S1), pp.847.1-847.1
04/2019
DOI: 10.1096/fasebj.2019.33.1_supplement.847.1
Abstract
Introduction
Gene‐corrected autologous cells provide one avenue for treating respiratory diseases, such as Cystic Fibrosis and Primary Ciliary Dyskinesia that harbor an underlying genetic defect. Basal cells are critical stem cells in the airways with the capacity to generate all cells of the respiratory epithelium. Use of these cells is limited due to inherent difficulties in gene editing and expansion in vitro. Efficient generation of basal cells from iPSCs has the potential to generate enough cells for both autologous cell therapy and for in vitro disease modelling. Building on our airway differentiation protocol, we have derived a strategy for the specification, isolation and expansion of iPSC‐derived basal cells (iBasal) improving the efficiency of generating respiratory epithelium from iPSC.
Methods
iPSC are cultured using a modified differentiation protocol pushing the formation of lung progenitor cells sorted on day 17 of differentiation for CD47hiCD26lo cells. These cells express transcription factor NKx2.1 and can be cultured in defined serum‐free media in the absence of wnt signaling. Over 4 passages we evaluated; 1) expression of basal cell markers, 2) clonality by a limited dilution clonogenic assay and by spheroid formation and 3) stem cell potential in spheroid and air‐liquid interface differentiation assays. Gene expression was compared to that of primary endogenous basal cells by bulk and single cell RNAseq, and validated by qPCR.
Results
Under our defined culture conditions, iBasal can be derived from NKx2.1 expressing lung progenitor cells. They are clonogenic forming colonies expressing cytokeratin 5 (krt5), p63 and integrin alpha 6 (IGTA6), acquiring more mature basal cell marker, nerve growth factor receptor (NGFR), expression after passaging when cultured on NIH3T3‐J2 feeders or in defined media. iBasal have the capacity for differentiation into a functional, tight junction forming epithelium containing both mucus secreting and motile multiciliated cells at the air liquid interface. In addition, they can form and maintain as spheroids which can also differentiate to generate airway epithelial cells. iBasal can be passaged and maintain a basal cell phenotype similar to that of paired passaged human airway basal cells. Single cell sequence data on iBasal and paired primary and cultured basal cells will provide the ultimate validation of iBasal.
Conclusion
We have generated iBasal from four independent iPSC lines with functional properties akin to that of primary basal cells. iBasal, therefore, provide the opportunity to generate sufficient functional and gene‐corrected autologous basal cells to develop patient specific, high throughput screening platforms overcoming the current limitations.
Support or Funding Information
Cystic Fibrosis Foundation FIRTH17XX0, FIRTH15XX1, NIH:NHLBI R01 HL139828‐01, Hastings Foundation
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Details
- Title: Subtitle
- Induced Pluripotent Stem Cell‐Derived Basal Cells Provide A Novel Source of Multilineage Airway Epithelial Cells
- Creators
- Amy L Ryan - MedicineUSCLos AngelesCAErik J Quiroz - MedicineUSCLos AngelesCAJenny Magallanes - MedicineUSCLos AngelesCABen A Calvert - MedicineUSCLos AngelesCASophia Petraki - MedicineUSCLos AngelesCAReem Elteriefi - MedicineUSCLos AngelesCAJinghui Gao - MedicineUSCLos AngelesCA
- Resource Type
- Abstract
- Publication Details
- The FASEB journal, Vol.33(S1), pp.847.1-847.1
- Publisher
- The Federation of American Societies for Experimental Biology
- DOI
- 10.1096/fasebj.2019.33.1_supplement.847.1
- ISSN
- 0892-6638
- eISSN
- 1530-6860
- Number of pages
- 1
- Grant note
- NIH:NHLBI (R01 HL139828‐01) Hastings Foundation Cystic Fibrosis Foundation (FIRTH17XX0; FIRTH15XX1)
- Language
- English
- Date published
- 04/2019
- Academic Unit
- Anatomy and Cell Biology
- Record Identifier
- 9984288855502771
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