Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.
Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by “reprogramming” somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-ß and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.
Chronic pulmonary diseases account for a large burden of morbidity and mortality worldwide1. Conditions that affect the conducting airways, such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral infections represent both common and rarer diseases, acquired and genetic, that contribute to this worldwide burden. The major functions of the conducting airways are to: 1) act as a conduit for the laminar flow of air, and 2) provide mucociliary clearance of pathogens and debris. Secretory, multiciliated, and basal cells represent the major epithelial cell types of the conducting airway. Rarer epithelial cell types include ionocytes, tuft cells, and neuroendocrine cells, and have been reviewed elsewhere2.
Broadly, a major barrier to understanding mechanisms of disease and advancing therapeutic approaches has been a consistent lack of human primary tissue for the use in preclinical models. HBECs are generally considered the gold-standard in vitro model of human airway epithelial biology and have played key roles in CF research in particular3. However, they are typically isolated either from bronchoscopic biopsies, from lung tissue after surgery, or from lungs rejected for transplantation purposes. Access to HBECs from individuals with genetic diseases, including the research priorities of CF and PCD, is infrequent and unpredictable. Overcoming this bottleneck to patient-derived airway epithelial cells is needed. iPSCs are readily generated from any individual, they retain the unique genetic background of the donor, and have a remarkable ability to proliferate and survive long term in cell culture conditions4,5,6. By recapitulating key embryologic steps, iPSCs undergo "directed-differentiation" into organ-specific cell types. We and others have developed protocols to generate iPSC-derived lung progenitors, alveolar type 2 cells7,8 and the cells of the conducting airway including airway basal cells9,10,11,12.
The airway basal cell is the stem cell of the conducting airway13. In previously published work, our group has generated iPSC-derived airway epithelial cells, including a subset (iBCs) that expresses canonical basal cell markers including TP63, KRT5, and NGFR. Following cues from adult basal cell biology, inhibition of dual SMAD pathways (TGF-β and BMP) leads to upregulation of NGFR+ iBCs11,14. NGFR+ iBCs are resuspended as single cells in droplets of extracellular matrix and cultured in a basal cell medium. They self-renew to maintain an iBC population and form epithelial spheroids; however, a proportion also differentiates into secretory cells in this format (referred to as 3-D culture). In the following protocol, we detail the steps to maintain and expand these iBCs in 3-D culture, as well as the steps required to generate a functional 2-D mucociliary ALI culture.
The initial steps of this differentiation protocol have been published previously and will not be reviewed here11,12. This manuscript will center on the expansion and purification of iBCs and their subsequent differentiation into functional secretory and multiciliated cells in ALI culture.
Each of the following steps should be performed using sterile technique in a biosafety level 2 laminar flow hood. All media should be warmed to room temperature (22 °C) prior to being added to cells, except when specifically mentioned otherwise. Each centrifugation step should be performed at room temperature (approximately 22 °C). Figure 1 outlines the schematics of the protocol.
1. Preparation of required media
NOTE: See Supplementary File 1 for details.
2. Thawing of cryopreserved iBCs
3. Dissociation of spheroids and expansion of iBCs in 3-D culture
4. Evaluation and purification of NGFR+ iBCs
5. Generation of mucociliary ALI cultures
6. Optional (but recommended) cryopreservation of iBCs
Following this protocol, 200,000 cryopreserved iBCs (previously confirmed to have a normal 46XY karyotype)11 were thawed and expanded in 3-D culture. Five days later, the resulting spheroids were dissociated, counted, and passaged again for further expansion. Approximately 480,000 cells were obtained and re-suspended in 3-D matrix (12 x 50 µL droplets, density 400 cells/µL). Fresh Basal Cell Medium was applied every 2-3 days. Ten days later, the cells were once again dissociated and counted. A total of 19.7 x 106 cells were harvested and prepared for FACS. 106 cells were stained with the APC-conjugated IgG1κ isotype control and the remaining 18.7 x 106 cells were stained with the APC-conjugated anti-NGFR antibody for 30 min protected from light (Figure 3).
NGFR+ gating was performed after comparison to the isotype control cells and was set purposely to collect the highest expressing NGFR+ cells (Figure 3). With this gating technique, 28% of live, single cells were NGFR+. While more cells were available, 750,000 cells were collected for downstream culture. Sorted cells were resuspended in Basal Cell Medium at a concentration of 50,000 cells/100 µL. 50,000 cells were then seeded onto each 6.5 mm porous membrane insert, which had been coated with human recombinant laminin-521 (2 µg/200 µL) per the manufacturer's guidelines. 500 µL Basal Cell Medium was added to the basolateral chamber of each insert and the plate was placed in a 37 °C humidified incubator. Three days later, the media in the apical chamber was aspirated and the cells were ~90% confluent by light microscopy (Figure 4B). The media from the basolateral chamber was aspirated; ALI Differentiation Medium was added to the apical (100 µL) and basolateral (500 µL) chambers. The next day, the media from the apical chamber was aspirated.
Over the following 21 days, the cells were evaluated by light microscopy periodically and fed with fresh ALI Differentiation Medium (basolateral chamber only) every 2-3 days. Individual cells were initially easily identifiable (Figure 4A), had an elongated and spindle-shaped appearance, and formed a loosely packed monolayer (Figure 4B). Over the subsequent days to weeks, the cells formed a tightly packed, highly cellular, epithelial layer, and after 7-10 days there was the clear emergence of beating cilia and mucus production. TEER of the samples were calculated and similar to primary cell controls (range from 700-1600 Ω x cm2)11. Subsequent fixation (day 21-28) with paraformaldehyde and immunolabeling for canonical airway epithelial cell markers was performed for MUC5AC and acetylated-α-tubulin, among others (Figure 4C). Overall, with our observation of motile cilia, mucus production as well as confirmatory immunostaining of multiciliated and secretory cells which is similar to that of primary HBECs, we concluded that we successfully generated airway epithelial cells from induced pluripotent stem cells.
Figure 1: Overall schematic of protocol. Cryopreserved iBCs are thawed, expanded, and FACS purified prior to plating on porous membrane inserts, where they differentiate into a functional mucociliary epithelium. Please click here to view a larger version of this figure.
Figure 2: Representative phase contrast images. Representative phase contrast images demonstrating usual appearance of iBCs in 3-D culture after (A) 1 day, (B) 4 days, (C) 8 days, and (D) 14 days (just pre-NGFR sort). Scale bars represent 500 µm. Please click here to view a larger version of this figure.
Figure 3: Representative FACS plots. Representative FACS plots for non-reporter and fluorescent-reporter iBCs. Examples of isotype controls are shown and were used to select for the highest expressing NGFR+ cells. Fluorescent reporter-containing iPSC lines are "triple sorted" for NKX2-1GFP+ TP63tdTomato+ NGFR+ cells. Please click here to view a larger version of this figure.
Figure 4: Representative images of iBC cultures on porous membranes. Phase contrast images are shown (A) 1 day and (B) 3 days after plating. Representative immunolabeling of mucociliary cultures shown in (C); acetylated-alpha tubulin (green) and MUC5AC (red). Scale bars represent 25 µm. Please click here to view a larger version of this figure.
Supplementary File 1: Media component table. Please click here to download this File.
HBECs are the gold-standard cell-type to study diseases of the airway epithelium. Due to their limitations (including accessibility and difficulty to genetically manipulate), we have generated a protocol for the derivation of iBCs and ALI cultures. These cells can be derived from any donor and retain their unique genetic background, thus allowing for basic developmental studies, disease modeling, and novel therapeutic development.
While each individual step of the described protocol is necessary, there are several that deserve extra mention. Firstly, at each step that requires dissociation of spheroids into single cells, it is critical to avoid excessive pipetting; enzymatic digestion (with dispase or trypsin) promotes better cell survival, while excessive pipetting leads to significant cell death. Secondly, when purifying NGFR+ iBCs, utilization of the isotype control is crucial and we recommend selecting for the highest expressing NGFR+ cells with FACS (Figure 3). This approach results in optimal ALI cultures with appropriate mucociliary differentiation. Finally, the preparation and seeding of porous membranes precisely as documented in the protocol is fundamental for ALI culture survival. While we typically seed 30,000-60,000 cells per insert, we have had success with as few as 20,000 cells. While successful cultures can be generated using the human embryonic stem cell-qualified matrix coating, we have more recently used human recombinant laminin-521 with a significantly higher durability of the ALI cultures.
Very rarely, iPSC differentiations may fail to upregulate an adequate percentage of NGFR+ iBCs. In this instance, serial passaging of iBCs (in 3-D culture) can result in an increased NGFR frequency over time. Limitations of this protocol include the time, cost, and expertise needed to generate these cells. Additionally, we recognize that many researchers are interested in the less common cell types of the airway epithelium (e.g., ionocytes, neuroendocrine cells). While we have detected some of these rarer cell types, as in primary HBEC cultures, they are not reproducibly identified, likely due to incomplete knowledge regarding the developmental cues required to generate these cells.
As it is written, the above protocol begins with the thawing of already cryopreserved iBCs. The details prior to this cryopreservation are previously described and beyond the scope of this manuscript11,12.
Our airway epithelial ALI culture method allows for the generation of functional airway epithelial cells from nearly any donor. This greatly increases the accessibility to precious genetically controlled airway epithelial cells that may be used for disease modeling, drug screening, future cell-based therapies, as well as to improve the understanding of the developmental patterning within the airway epithelium.
The authors have nothing to disclose.
We thank the members of the Hawkins, Kotton, and Davis laboratories for their helpful input over the years regarding this and other projects. We also are indebted to Brian Tilton (BU Cell Sorting Director) for his dedication and technical expertise, and we are grateful to Greg Miller and Marianne James of the Boston University Center for Regenerative Medicine (CReM) for their support and technical expertise in maintenance and characterization of patient-specific iPSCs, supported by NIH grants NO1 75N92020C00005 and U01TR001810. This work was supported by NIH grants U01HL148692, U01HL134745, U01HL134766, and R01HL095993 to D.N.K, R01HL139876 to B.R.D, R01 HL139799 to F.H., and Cystic Fibrosis Foundation (CFF) grants CFF 00987G220 and CFF WANG20GO to D.N.K, CFF DAVIS15XX1, DAVIS17XX0, DAVIS19XX0 to B.R.D, CFF SUZUKI19XX0 to S.S, CFF BERICA2010 to A.B., and CFF HAWKIN20XX2 to F.H.
12-well tissue culture treated plate | Corning | 3515 | flat bottom |
3-D growth factor reduced Matrigel | Corning | 356230 | |
A83-01 | ThermoFisher Scientific | 293910 | |
Cell culture inserts (Transwell) | Corning | 3470 | 6.5mm diameter; 0.4μm pore size |
Dimethyl sulfoxide (DMSO) | Sigma | D8418 | |
Dispase II, Powder | ThermoFisher Scientific | 17105-041 | |
DMH1 | ThermoFisher Scientific | 412610 | |
Dulbecco's Modified Eagle Medium/Nutrient Mixture (DMEM):F-12 | ThermoFisher Scientific | 11330-032 | |
Dulbecco's Phosphate Buffered Saline (PBS) (1X) | Gibco | 14190-144 | |
Ethylenediaminetetraacetic acid disodium salt solution (EDTA) | Sigma | E7889 | |
Fetal Bovine Serum (FBS) | Fisher | SH3007003E | |
Hanks' Balanced Salt Solution (HBSS) | ThermoFisher Scientific | 14175-079 | |
HEPES | Sigma | H3375 | |
hESC-qualified Matrigel | Corning | 354277 | |
Mouse IgG1kappa isotype control, APC-conjugated | Biolegend | 400122 | |
Mouse monoclonal anti-human CD271/NGFR, APC-conjugated | Biolegend | 345108 | |
PneumaCult-ALI Medium (ALI Differentiation Medium) | StemCell Technologies | 05001 | |
PneumaCult-Ex Plus Medium (Basal Cell Base Medium) | StemCell Technologies | 05040 | |
Primocin | InvivoGen | ant-pm-2 | |
rhLaminin-521 | ThermoFisher Scientific | A29248 | Final Concentration: 10ug/mL |
Trypsin-EDTA Solution, 0.05% | Invitrogen | T3924 | |
Y-27632 | Tocris | 1254 |