WS6 enables scalable ex vivo expansion and gene editing of human basal epithelial cells

WS6 enables scalable ex vivo expansion and gene editing of human basal epithelial cells | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results WS6 enables scalable ex vivo expansion and gene editing of human basal epithelial cells View ORCID Profile Jessica C. Orr , View ORCID Profile Elizabeth K. Haughey , View ORCID Profile Andrew S. Farr , View ORCID Profile David R. Pearce , View ORCID Profile Niamh A. McCarthy , Shalini Kamu Reddy , Maral J. Rouhani , View ORCID Profile Charlotte Percival , View ORCID Profile Isabelle Rose , Anna Straatman-Iwanowska , Rebecca Dale , Megan Guthrie , View ORCID Profile Giada Benedetti , View ORCID Profile Olivia R. Pape , View ORCID Profile Juan Moisés Ocampo-Godinez , View ORCID Profile Elizabeth F. Maughan , View ORCID Profile Colin R. Butler , View ORCID Profile Dale A. Moulding , View ORCID Profile Alexandra Y. Kreins , View ORCID Profile Giovanni Giuseppe Giobbe , View ORCID Profile Paolo De Coppi , View ORCID Profile William Grey , View ORCID Profile Antonella F. M. Dost , View ORCID Profile Robert A. Hirst , View ORCID Profile Deborah L. Baines , View ORCID Profile Yuki Ishii , View ORCID Profile Christopher O’Callaghan , View ORCID Profile Sam M. Janes , View ORCID Profile Robert E. Hynds doi: https://doi.org/10.1101/2025.09.15.675861 Jessica C. Orr 1 Epithelial Cell Biology in ENT Research Group, Developmental Biology and Cancer Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jessica C. Orr Elizabeth K. Haughey 2 Infection, Immunity, and Inflammation Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Elizabeth K. Haughey Andrew S. Farr 1 Epithelial Cell Biology in ENT Research Group, Developmental Biology and Cancer Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andrew S. Farr David R. Pearce 3 UCL Cancer Institute, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for David R. Pearce Niamh A. McCarthy 1 Epithelial Cell Biology in ENT Research Group, Developmental Biology and Cancer Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Niamh A. McCarthy Shalini Kamu Reddy 4 GOSH BRC CRISPR Service, Great Ormond Street Hospital For Children , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maral J. Rouhani 5 Lungs for Living Research Centre , UCL Respiratory, Division of Medicine, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Charlotte Percival 5 Lungs for Living Research Centre , UCL Respiratory, Division of Medicine, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Charlotte Percival Isabelle Rose 6 Institute of Infection and Immunity, School of Health and Medical Sciences , City St George’s, University of London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Isabelle Rose Anna Straatman-Iwanowska 7 Centre for PCD Diagnosis and Research, Division of Respiratory Sciences, University of Leicester , Leicester, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rebecca Dale 7 Centre for PCD Diagnosis and Research, Division of Respiratory Sciences, University of Leicester , Leicester, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Megan Guthrie 8 ProteoStem Laboratory, Centre for Blood Research, York Biomedical Research Institute, University of York , York, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Giada Benedetti 9 Stem Cells and Regenerative Medicine Section, Developmental Biology and Cancer Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Giada Benedetti Olivia R. Pape 1 Epithelial Cell Biology in ENT Research Group, Developmental Biology and Cancer Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Olivia R. Pape Juan Moisés Ocampo-Godinez 2 Infection, Immunity, and Inflammation Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK 10 Department of Immunology and Gene Therapy, Great Ormond Street Hospital for Children NHS Foundation Trust , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Juan Moisés Ocampo-Godinez Elizabeth F. Maughan 1 Epithelial Cell Biology in ENT Research Group, Developmental Biology and Cancer Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK 11 National Paediatric Tracheal Service, Great Ormond Street Hospital for Children NHS Foundation Trust , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Elizabeth F. Maughan Colin R. Butler 1 Epithelial Cell Biology in ENT Research Group, Developmental Biology and Cancer Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK 11 National Paediatric Tracheal Service, Great Ormond Street Hospital for Children NHS Foundation Trust , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Colin R. Butler Dale A. Moulding 12 Microscopy Core Facility, UCL Great Ormond Street Institute of Child Health, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dale A. Moulding Alexandra Y. Kreins 2 Infection, Immunity, and Inflammation Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK 10 Department of Immunology and Gene Therapy, Great Ormond Street Hospital for Children NHS Foundation Trust , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alexandra Y. Kreins Giovanni Giuseppe Giobbe 9 Stem Cells and Regenerative Medicine Section, Developmental Biology and Cancer Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Giovanni Giuseppe Giobbe Paolo De Coppi 9 Stem Cells and Regenerative Medicine Section, Developmental Biology and Cancer Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Paolo De Coppi William Grey 8 ProteoStem Laboratory, Centre for Blood Research, York Biomedical Research Institute, University of York , York, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for William Grey Antonella F. M. Dost 13 Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), University Medical Center , Utrecht, The Netherlands 14 Oncode Institute, Hubrecht Institute , Utrecht, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Antonella F. M. Dost Robert A. Hirst 7 Centre for PCD Diagnosis and Research, Division of Respiratory Sciences, University of Leicester , Leicester, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Robert A. Hirst Deborah L. Baines 6 Institute of Infection and Immunity, School of Health and Medical Sciences , City St George’s, University of London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Deborah L. Baines Yuki Ishii 5 Lungs for Living Research Centre , UCL Respiratory, Division of Medicine, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yuki Ishii Christopher O’Callaghan 2 Infection, Immunity, and Inflammation Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christopher O’Callaghan Sam M. Janes 5 Lungs for Living Research Centre , UCL Respiratory, Division of Medicine, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sam M. Janes Robert E. Hynds 1 Epithelial Cell Biology in ENT Research Group, Developmental Biology and Cancer Department, UCL Great Ormond Street Institute of Child Health, University College London , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Robert E. Hynds For correspondence: rob.hynds{at}ucl.ac.uk Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Modeling human epithelial diseases and developing cell-based therapies require robust methods to expand and manipulate epithelial stem and progenitor cells in vitro . Basal stem/progenitor cells from stratified epithelia can be expanded in 3T3-J2 fibroblast feeder cell co-culture systems, and the addition of the ROCK inhibitor Y-27632 enhances proliferation and culture longevity, a phenomenon described as ‘conditional reprogramming’. Here, we present a method incorporating the small molecule WS6 to further improve the proliferation and lifespan of cultured epithelial cells from multiple tissues, including airway, skin, and thymus. Cells maintained in this medium (‘EpMED’; FAD+Y+WS6) retain basal stem/progenitor cell identity and function, including the capacity to differentiate. We demonstrate their capacity to engraft in vivo in a tracheal transplantation model. In a second application, we generate clonal CRISPR-Cas9 genome edited nasal cultures, introducing targeted knockouts of DNAH5 or DNAI2 to create primary ciliary dyskinesia disease models. We anticipate that our method will have broad applications in epithelial cell biology, disease modeling, and regenerative medicine, while reducing reliance on immortalized or cancer cell lines and animal experimentation. Introduction Robust in vitro cell culture systems for primary human epithelial cells underpin key applications in disease modeling, functional genomics, and regenerative medicine. The discovery that mouse embryonic fibroblasts can act as feeder cells enabled primary epithelial cell culture for the first time 1 . The initial medium, which contained fetal calf serum and hydrocortisone, was later refined to additionally include adenine, cholera toxin, epidermal growth factor, insulin, and triiodothyronine (‘FAD medium’; reviewed in 2 ). This formulation supports the expansion of epithelial basal stem/progenitor cells from multiple tissues, including skin, cornea, esophagus, thymus, and respiratory tract 3 – 6 . The addition of the Rho kinase inhibitor Y-27632 to FAD enhances epithelial cell colony formation potential, proliferation, and longevity 7 – 10 . Despite improvements to the 2D culture of epithelial stem cells over recent decades, the ability to extensively expand primary human cells from clinically available biopsy samples remains challenging, particularly from patients with epithelial diseases that limit stem cell function 11 , 12 . A previous report described maintaining mouse totipotent stem cells with a small-molecule cocktail 13 . The cocktail included 1-azakenpaullone, a Wnt pathway activator that we previously found to promote airway basal cell proliferation 14 . It also contained TTNPB, a retinoic acid analogue, and WS6, which is reported to bind to Erb3 binding protein 1 15 and IKKε, a regulator of the NFkB pathway 16 . In this article, we investigate the potential of these small molecules to enhance epithelial cell culture. We demonstrate that the addition of WS6 to FAD medium, alone or in combination with Y-27632, enhances epithelial cell colony formation, proliferation, and culture duration. As a first application, we demonstrate that cells expanded in this combination of small molecules can engraft in tracheal transplantation assays in vivo . WS6 supplementation also enables advanced disease modeling, as we show by developing isogenic clonal CRISPR-Cas9 knockout primary cell cultures for DNAH5 or DNAI2 , genes in which pathogenic variants are known to cause primary ciliary dyskinesia. Results WS6 improves epithelial basal stem cell culture To explore the effects of the small molecule cocktail described by Hu et al. 13 on human epithelial cells, we assessed the individual components (1-azakenpaullone, TTNPB, and WS6) and their combinations on nasal epithelial cell proliferation. Of these, WS6 alone, or in combination with the other compounds, markedly increased cell numbers ( Fig. 1A ). We next asked whether WS6 and Y-27632 act synergistically. In both bronchial and skin cultures, each compound increased proliferation, but their combination was most effective, specifically at 1-10 μM Y-27632 with 0.1-1 μM WS6. At higher WS6 concentrations (>1 μM), toxicity was observed ( Fig. 1B ). Download figure Open in new tab Figure 1: WS6 promotes epithelial basal cell proliferation in 3T3-J2 co-culture A) Six-day CellTiter-Glo assay to examine the effect of combinations of the small molecules (2.5 µM 1-azakenpaullone, 0.2 µM TTNPB and 0.5 µM WS6) identified to induce totipotency in mouse embryonic stem cells by Hu et al. 2023 13 on epithelial basal cell proliferation (n = 6 passage four primary human nasal basal cell donors, indicated by shapes). One-way ANOVA with Dunnett’s multiple comparison test vs DMSO control wells. B) Four-day CellTiter-Glo assays to assess the effect of varying concentrations of Y-27632 and WS6 on cell proliferation in passage three bronchial (left, mean of five donors is shown) and skin (right, mean of two independent cultures from one donor) basal cells previously grown in FAD medium. C) Colony formation assays to assess the effect of Y-27632 (Y), WS6, and the combination of both on colony forming potential of human epithelial cells from different tissues (n = 1 donor per tissue; tissue type indicated by shape; passages 1-3). One-way ANOVA with Tukey’s multiple comparisons test. D) Colony formation assays to assess the effect of Y-27632 (Y), and the combination of Y and WS6 on colony forming potential of FACS-sorted EPCAM + human tracheal (n = 4 donors) or nasal (n = 3 donors) epithelial cells directly from biopsies. Paired t-test per sample location. Colony formation assays provide a functional readout of basal stem/progenitor cell potential and Y-27632 is known to enhance colony formation 17 , 18 . To assess the effect of WS6, WS6 alone or in combination with Y-27632 was added at the time of assay seeding to nasal basal cells that had been established on 3T3-J2 feeder layers in FAD medium. Alone, WS6 produced a similar effect to Y-27632, while the combination further increased colony formation ( Supplementary Fig. 1 ). Prompted by these findings, we assessed colony forming efficiency in cell cultures derived from a variety of epithelia, finding that Y-27632, WS6 and the combination increased colony formation of epithelial cells derived from the skin, thymus, nose, trachea, bronchus, or small airways ( Fig. 1C ). We also tested whether WS6 could enhance the initial culture efficiency of epithelial cells from primary human tissue. We dissociated tracheal and nasal biopsies into single cells and sorted EPCAM⁺ cells individually onto feeder layers. For both cell types, WS6 increased the number of cells capable of forming colonies in vitro ( Fig. 1D ). Download figure Open in new tab Supplementary Figure 1: WS6 increases colony formation in cultured nasal basal cells Representative well images and quantification of colony formation assays to assess the effect of Y-27632 (Y), WS6 and the combination of both on colony forming potential of nasal basal cells that had been isolated in FAD medium for one passage (n = 9 primary human nasal basal cell donors, indicated by shape). One-way ANOVA with Tukey’s multiple comparisons test. To examine the effect of WS6 beyond primary cells, we assessed its effect on proliferation in commonly used cell lines. WS6 did not induce proliferation in A549 cells (a lung adenocarcinoma cell line), H520 cells (a lung squamous cell carcinoma cell line), or HBEC3-KT cells 19 (an airway basal cell line immortalized by transduction with cyclin-dependent kinase 4, CDK4, and human telomerase reverse transcriptase, hTERT). However, WS6 did induce proliferation of nasal basal cells transduced with BMI1 20 ( Supplementary Fig. 2 ). Download figure Open in new tab Supplementary Figure 2: WS6 increases proliferation in BMI1 -transduced cells but not other immortalized or cancer cell lines Four-day CellTiter-Glo assays to assess the effect of varying concentrations of Y-27632 and WS6 on cell proliferation of epithelial cell lines (A549, lung adenocarcinoma; H520, lung squamous cell carcinoma; HBEC3-KT, CDK4- and hTERT- transduced bronchial epithelial basal cells; and BMI1 -transduced nasal epithelial basal cells). Each data point represents the mean of technical triplicates for n = 3 experimental repeats. One-way ANOVA with Dunnett’s multiple comparison test vs DMSO control wells. Long-term culture of epithelial cells in WS6 To assess long-term cell expansion in WS6-containing medium, cells from six nasal brush biopsies were divided between three conditions and cultured in parallel: FAD medium alone, FAD plus 5 μM Y-27632 (FAD+Y), or FAD with 5 μM Y-27632 and 100 nM WS6 (FAD+Y+WS6). All three conditions supported early passage expansion of cells with characteristic epithelial cell cobblestone morphology ( Fig. 2A ). Over time, however, cells in FAD alone became enlarged, and developed bright, refractile colony centers consistent with the onset of stratification ( Supplementary Fig. 3 ). Addition of Y-27632 delayed this process but FAD+Y+WS6 cultures maintained consistent morphology ( Supplementary Fig. 3 ). Across all conditions, cells maintained cytoplasmic expression of keratin 5 (KRT5), a basal cell intermediate filament protein 21 , and nuclear expression of TP63, a transcription factor central to epithelial basal cell identity 22 , 23 ( Fig. 2B and Supplementary Fig. 4 ). Cells in FAD+Y+WS6 expanded more rapidly and could be maintained for extended culture durations, with one donor culture exceeding 50 passages ( Fig. 2C ). Colony formation was consistently higher in FAD+Y and FAD+Y+WS6 cultures than in FAD alone ( Fig. 2D ), and at higher population doublings, more colonies were formed by FAD+Y+WS6 cultures than FAD+Y cultures ( Fig. 2D ), indicating preservation of functional stem cell potential during prolonged culture. Download figure Open in new tab Supplementary Figure 3: The morphology of epithelial basal cells cultured with WS6 remains consistent over long-term passage Phase-contrast microscopy images show cultures in FAD, FAD+Y-27632(Y) or FAD+Y+WS6 at the indicated passages (n = 1 donor). X represents passages at which imaging was not possible due to culture failure. Scale bars = 100 µm. Download figure Open in new tab Supplementary Figure 4: Epithelial basal cells retain KRT5 and TP63 expression during culture Immunofluorescence staining of nasal basal cell cultures in FAD, FAD+Y-27632(Y) or FAD+Y+WS6 at the indicated passages (n= 1 donor). Cells were fixed on day three of culture (TP63, magenta; KRT5, cyan; DAPI, blue). X represents passages at which imaging was not possible due to culture failure. Scale bars = 100 µm. Download figure Open in new tab Figure 2: WS6 extends the culture duration of epithelial basal cells A) Phase-contrast images of nasal epithelial basal cells cultured in FAD (left), FAD+Y-27632 (Y, center) or FAD+Y+WS6 (right) at indicated passages. Scale bar = 100 µm. B) Immunofluorescence staining of passage three nasal epithelial basal cells fixed at day three of culture (TP63, magenta; KRT5, cyan; DAPI, blue). Scale bar = 100 µm. C) Population doublings for donor-matched nasal epithelial basal cells cultured in FAD, FAD+Y or FAD+Y+WS6 (n = 6 donors). D) Colony formation assays for donor-matched nasal epithelial basal cells cultured in FAD, FAD+Y or FAD+Y+WS6 at passages three, six, and nine. X indicates that FAD cultures had undergone replicative senescence prior to passage nine. Representative images from a single donor are shown (left) and colony number quantified (right; n = 6 donors, passages three and six = one-way ANOVA with Tukey’s multiple comparison, passage nine = paired t-test). Previous studies have shown improved lentiviral transduction of epithelial basal cells with Y-27632 24 , 25 . We therefore investigated the effect of WS6 on lentiviral transduction with a fluorescent reporter. Y-27632 improved transduction as expected, but WS6 conferred no benefit, alone or in combination with Y-27632 ( Supplementary Fig. 5 ). Download figure Open in new tab Supplementary Figure 5: Y-27632 but not WS6 improves lentiviral transduction of primary epithelial cells A) Bronchial basal cells previously cultured without Y-27632 (Y) or WS6 were spin transduced with a lentiviral vector carrying eGFP. B) Example flow cytometry plots demonstrating ITGA6 staining of epithelial cells (but not 3T3-J2 feeder cells). C) Flow cytometry analysis of transduction efficiency in control medium (FAD), FAD+Y, FAD+WS6 or FAD+Y+WS6. Each data point represents the mean of technical triplicates from one donor (n = 4 donors, one-way ANOVA with Dunnett’s multiple comparisons test comparing each condition to FAD). D) Bronchial and tracheal basal cells previously cultured without Y-27632 or WS6 were spin transduced with a lentiviral vector carrying ZsGreen. E) Example flow cytometry plots demonstrating EPCAM staining of epithelial cells (but not 3T3-J2 feeder cells). F) Flow cytometry analysis of transduction efficiency in control medium (FAD), FAD+Y, FAD+WS6 or FAD+Y+WS6. Each data point represents the mean of technical duplicates from one donor (n = 3 donors, one-way ANOVA with Dunnett’s multiple comparisons test comparing each condition to FAD). To assess the oncogenic risk associated with prolonged culture, we performed low-pass whole-genome sequencing (0.4x coverage) on nasal basal cells from seven donors expanded in FAD+Y+WS6 for 9-10 passages. All samples remained diploid without evidence of chromosomal rearrangements ( Fig. 3A ). We also transplanted cells expanded for 12-14 passages in FAD+Y+WS6 into NSG mice subcutaneously in Matrigel. After 12 week, no tumors were observed. In 3/6 mice, we were able to recover the injected Matrigel which contained epithelial structures resembling differentiated airway tissue, confirming the retention of differentiation capacity in vivo and suggesting that the cells remained committed to the airway lineage ( Fig. 3B ). Download figure Open in new tab Figure 3: Cells cultured in WS6 are genomically stable and do not form tumors in mice A) A representative copy number profile from low-pass whole-genome sequencing of nasal epithelial cells expanded in FAD+Y-27632(Y)+WS6 (n = 6 donors at passage nine, n = 1 donor at passage ten). Bin-level copy numbers were estimated with QDNAseq, converted to absolute scale, and low-quality bins (<99.9999% bases or <70% mappability) were excluded. Genome-wide plots show unflagged bins (points) and segmented copy number per chromosome (purple horizontal lines). B) Subcutaneous injection of nasal epithelial basal cells expanded in FAD+Y+WS6 for 12-14 passages. Tumors formed in 0/6 mice. Hematoxylin and eosin staining (upper) and immunofluorescence staining (lower; KRT5, cyan; MUC5AC, grey; acetylated tubulin, ACT, magenta; DAPI, blue) representative of 3/6 mice in which epithelial structures could be found within the extracellular matrix recovered 12 wk after injection. Scale bar lengths are indicated within the figure panel. For in vitro and in vivo applications, epithelial basal cells must retain the capacity for tissue-appropriate differentiation 26 . When induced to differentiate in air-liquid interface (ALI) cultures after four passages in FAD medium, FAD+Y or FAD+Y+WS6 ( Fig. 4A ), nasal epithelial cells formed a pseudostratified epithelium containing both muco-secretory and ciliated cells ( Fig. 4B-C and Supplementary Fig. 6A ). At either passage 4 or 10, FAD+Y cultures and FAD+Y+WS6 cultures produced comparable numbers of ciliated cells ( Fig. 4D ). By contrast, passage 10 cultures in these conditions consistently produced a higher proportion of MUC5AC⁺ muco-secretory cells compared to passage 4 ( Fig. 4D ). Similarly, FAD+Y+WS6 cells were able to differentiate in 3D organoid assays throughout long-term culture ( Supplementary Fig. 6B-D ), with one donor retaining differentiation capacity after 38 passages ( Supplementary Fig. 6E ). These findings confirm that basal cells retain multilineage differentiation capacity even after extended passaging in WS6. Download figure Open in new tab Supplementary Figure 6: Nasal epithelial cells cultured in FAD+Y+WS6 retain the capacity for multipotent differentiation in air-liquid interface cultures and organoid assays A) Immunofluorescence staining of passage four and ten ALI cultures of nasal epithelial basal cells from a single donor from FAD (left), FAD+Y (center) or FAD+Y+WS6 (right) cultures. (ACT, magenta; MUC5AC, grey; DAPI, blue). Scale bars = 20 µm. B) Schematic diagram of nasal epithelial basal cell differentiation in organoid cultures with representative hematoxylin and eosin staining of formalin-fixed, paraffin-embedded organoid culture from a passage three FAD+Y+WS6 nasal epithelial basal cell culture. Scale bar = 100 µm. C) Whole-well phase-contrast images (upper left, scale bar = 1000 µm) at day 21/22 of organoid cultures derived from passage three, six or nine nasal epithelial basal cells from FAD, FAD+Y or FAD+Y+WS6 cultures. Images were analyzed using the OrgaQuant script to detect organoids (upper right, scale bar = 100 µm). Mean predicted organoid volume per condition is shown (lower). (n = 6 donors, passage 3 = one-way ANOVA with Tukey’s multiple comparison, passage 6 and 9 = paired t-test). D) Immunofluorescence staining of passage three or passage nine organoid cultures of nasal epithelial basal cells from FAD (left), FAD+Y (center) or FAD+Y+WS6 (right) cultures. (upper: KRT5, cyan; KRT8, magenta; lower: ACT, magenta; MUC5AC, grey; DAPI, blue). Scale bars = 50 µm. E) Immunofluorescence staining of passage 38 organoid cultures of nasal epithelial basal cells from FAD+Y+WS6 culture. (left: KRT5, cyan; KRT8, magenta; right: ACT, magenta; MUC5AC, grey; DAPI, blue). Scale bars = 50 µm. X represents passages at which assessment was not possible due to culture failure. F) Transepithelial electrical resistance (TEER) measurements of passage four, seven and ten air-liquid interface (ALI) cultures derived from nasal epithelial basal cells from FAD, FAD+Y-27632(Y) or FAD+Y+WS6 cultures. (n = 6 donors, passage 4 = one-way ANOVA with Tukey’s multiple comparison, passage 7 and 10 = paired t-test). Download figure Open in new tab Figure 4: Nasal epithelial cells retain the capacity for multipotent differentiation during extended culture A) Schematic diagram of nasal epithelial basal cell differentiation in air-liquid interface (ALI) cultures. B) Hematoxylin and eosin staining of formalin-fixed, paraffin-embedded ALI cultures from passage four (P4, left) and P10 (right) cells previously expanded in FAD+Y-27632(Y)+WS6. Scale bar = 100 µm C) Whole-mount immunofluorescence staining of ALI cultures from P4 (left) and P10 (right) cells previously cultured in FAD+Y+WS6 (acetylated tubulin, ACT, magenta; MUC5AC, grey). Scale bar = 20 µm. D) Proportions of epithelial cell types from P4 and P10 ALI cultures of nasal epithelial basal cells from flow cytometry. X indicates that FAD cultures had undergone replicative senescence prior to P10 and thus could not be used for ALI experiments (n = 5-6 donors, P4 = one-way ANOVA with Tukey’s multiple comparison, P10 = paired t-test). E) Ciliary beat frequency analysis of differentiated ALI cultures from P4, P7 and P10 cells previously expanded in FAD, FAD+Y or FAD+Y+WS6. Coverage and beat frequency of moving cilia are displayed for representative fields of view of FAD (P4; top left) and FAD+Y+WS6 (P4; bottom left) conditions. Mean cilia beat frequency is quantified (right). Each data point represents the average frequency of all moving cilia from five fields of view in three technical replicate wells (n = 6 donors; indicated by shape). F) Ussing chamber analysis of ion transport in ALI cultures derived from FAD+Y+WS6 cultures. Representative short circuit current (Isc) trace from a single donor after addition of amiloride (100 µM, apical), forskolin (10 µM, bilateral), CFTRinh-172 (10 µM, apical), ATP (100 µM, apical) and ouabain (1 mM basolateral) as indicated (left). Change in Isc (ΔIsc) of day 30 ALI cultures in response to amiloride, forskolin, CFTRinh-172 and ATP (points represent the mean of triplicate wells per donor, n = 4 donors). Given the appropriate cellular composition of the differentiated ALI cultures, we next investigated the functionality of the epithelium. First, across all passages and medium compositions, there were no significant differences in the transepithelial electrical resistance (TEER) measurements, suggesting high epithelial integrity ( Supplementary Fig. 6F ). Second, the ciliary beat frequency of ciliated cells within FAD+Y+WS6 ALI cultures was within the expected normal range for nasal cells 27 , 28 ( Fig. 4E ). Third, ion channel function was preserved as FAD+Y+WS6 ALI cultures responded appropriately to pharmacological stimuli: amiloride, indicating activity of the amiloride-sensitive epithelial Na⁺ channel (ENaC); forskolin and CFTRinh-172, indicating cAMP-regulated CFTR activity; and ATP, indicating calcium-activated Cl⁻ channel (CaCC) activity. Additional ATP-evoked conductances included an ouabain-sensitive component, consistent with Na⁺/K⁺-ATPase involvement ( Fig. 4F ). We next asked whether WS6 influences primary human cell culture systems beyond 2D stratified epithelia. In small airway organoids, 1 μM WS6 increased proliferation while the same concentration reduced proliferation of alveolar organoids ( Supplementary Fig. 7 ). Gastric organoid growth was unaffected by WS6 ( Supplementary Fig. 7 ). In hematopoietic stem and progenitor cell cultures, WS6 decreased the total nucleated cell output in a dose-dependent manner, with a modest enrichment of HSCs attributable to preferential loss of more differentiated cell populations ( Supplementary Fig. 8A-8G ). Colony forming unit (CFU) assays showed that WS6 did not alter hematopoietic progenitor lineage output, with similar proportions of CFU-macrophage (CFU-M), CFU-granulocyte (CFU-G), CFU-granulocyte macrophage (CFU-GM), and CFU-granulocyte erythrocyte macrophage megakaryocyte (CFU-GEMM) colonies across all conditions ( Supplementary Fig. 8H ). Taken together, these findings show that WS6 stimulates the proliferation of human primary cells in a context-dependent and cell type-specific manner. Download figure Open in new tab Supplementary Figure 7: The effect of WS6 on primary human respiratory and gastric organoid cultures Upper: Phase-contrast images of organoids treated with DMSO control, 10 nM, 100 nM, or 1 µM WS6. Scale bars = 500 µm. Lower: CellTiter-Glo endpoint/initial luminescence ratio to assess the effect of varying concentrations of WS6 on primary epithelial organoid cultures (airway and alveolar organoids: n = 3 biological replicates; gastric organoids: n = 2 donors, one with independent body and fundus organoid cultures, one with independent antrum and fundus organoid cultures; points represent the mean of triplicate wells per organoid culture). One-way ANOVA with Dunnett’s multiple comparison test vs DMSO control wells. Download figure Open in new tab Supplementary Figure 8: The effect of WS6 on primary human hematopoietic stem and progenitor cell cultures 5,000 CD34 + cord blood stem and progenitor cells were cultured for 7 d in medium containing DMSO (control), 10 nM WS6, 100 nM WS6, or 1 µM WS6 before analysis by flow cytometry. A) Fold change in total nucleated cells. B) Fold change in CD34+ cells. C) Absolute number of hematopoietic stem cells (HSCs; CD34 + , CD45RA - , CD90 + , EPCR + ). D) Proportion of CD34 + cells. E) Proportion of CD34+CD45RA - cells. F) Proportion of HSCs. G) Representative flow cytometry plots showing CD34 and CD45RA expression. H) Colony forming unit (CFU) assay. CFU-macrophage (CFU-M), CFU-granulocyte (CFU-G), CFU-granulocyte macrophage (CFU-GM), and CFU-granulocyte erythrocyte macrophage megakaryocyte (CFU-GEMM). In all panels, one-way ANOVAs were performed with Dunnett’s multiple comparisons test comparing each condition to the control. In vivo transplantation of WS6-expanded epithelial basal cells To assess in vivo regenerative potential, we transplanted human nasal basal cells isolated and expanded in FAD+Y+WS6 into the tracheas of immunodeficient NOD scid gamma (NSG) mice. Cells were transduced with a ZsGreen-luciferase lentiviral vector for tracking. Tracheas were preconditioned by oropharyngeal polidocanol instillation, a detergent that denudes the airway epithelium 29 , before delivery of cells 5 hr later 30 ( Fig. 5A ). Engraftment was detected in all 11 recipients by IVIS imaging at day 7, though signal intensity varied between recipients ( Fig. 5B ). Luciferase activity persisted throughout the 28 d experiment, with a gradual reduction over time ( Fig. 5B ), and was localized specifically to the trachea in post-mortem imaging ( Fig. 5C ). At the 28-day end-point, whole-mount ( Fig. 5D ) and histological analyses ( Fig. 5E ) confirmed engraftment of human basal cells and their differentiation to muco-secretory and ciliated cells in vivo ( Fig. 5E ). Download figure Open in new tab Figure 5: Tracheal transplantation of basal stem cells expanded in FAD+Y+WS6 A) Schematic diagram of ZsGreen-luciferase transduced nasal epithelial basal cell transplantation in the trachea of immunodeficient NSG mice and monitoring by in vivo luciferase bioluminescence imaging (IVIS). B) Quantification of IVIS signal over 28 d following cell engraftment (n = 11 mice). C) IVIS imaging of a living mouse 7 d after transplantation with human basal cells (left) and the same mouse post-mortem, demonstrating the tracheal specificity of cell engraftment. D) Whole-mount imaging of ZsGreen in a freshly excised trachea 28 d following basal cell transplantation. Scale bar = 1 mm. E) Hematoxylin and eosin staining of sections from a mouse trachea 28 d after basal cell transplantation (upper) and immunofluorescence staining demonstrating ciliated cell differentiation (acetylated tubulin, ACT, ciliated cells, magenta) and human origin (STEM121, grey). Scale bar = 50 µm. Clonal epithelial basal cell culture and gene editing Many gene editing studies in epithelial cells rely on immortalized lines rather than primary cultures 31 . While clonal basal cell cultures have enabled insights into genetic heterogeneity 32 , 33 , their expansion and manipulation remain inefficient. In most cell culture systems, clonal cells are difficult to passage, cryopreserve, and differentiate. As a result, in cases where primary cells are used, most approaches involve bulk populations that contain a mixture of edited and unedited cells 34 – 37 . Others use lentiviral systems with selectable markers, but these often result in persistent expression of Cas9 and guide RNAs 38 , limiting their suitability for therapeutic applications. To evaluate whether culture in FAD+Y+WS6 allows clonal cell expansion after gene editing, we used CRISPR–Cas9 genome editing to develop primary ciliary dyskinesia (PCD) disease models ( Fig. 6A ). First, we targeted DNAH5 , the most commonly mutated gene in PCD (15–21% of cases 39 ), which disrupts outer dynein arm (ODA) function and produces a well-defined phenotype of ciliary immotility 40 . Nucleofection of CRISPR-Cas9-sgRNA ribonucleoprotein complexes introduced gene edits in bulk nasal basal cell cultures. In parallel GFP plasmid nucleofections, an 89% efficiency was achieved ( Supplementary Fig. 9A ). As expected, edited bulk cultures displayed a mixture of beating and static cilia ( Supplementary Video 1 ). Consistent with this, both normal and ODA-defective cilia were present within individual ALI cultures by transmission electron microscopy (TEM; Supplementary Fig. 9B ). Download figure Open in new tab Supplementary Figure 9: CRISPR-Cas9-mediated DNAH5 -knockout in primary airway basal cells expanded in FAD+Y+WS6 A) Flow cytometry quantification of EPCAM⁺ epithelial cells expressing GFP following plasmid nucleofection, representing approximate nucleofection efficiency for single donor used for DNAH5 -knockout (KO) experiment. B) Transmission electron microscopy of ciliary cross-section from the bulk population containing both DNAH5 -KO and wild-type (WT) cells. Blue arrows indicate normal outer dynein arms (ODA) in WT cilia and purple indicate loss of ODA in DNAH5 -KO. (40,000× magnification, scale bar = 100 nm). C) Transepithelial electrical resistance (TEER) measurements from air-liquid interface (ALI) cultures derived from DNAH5- KO clones, corresponding WT clones, healthy basal cells and primary ciliary dyskinesia patient basal cells (n = 1 non-PCD and PCD patient donors with triplicate wells; n = 4 WT and DNAH5 -KO clones from a single donor). Download figure Open in new tab Figure 6: WS6 allows clonal basal cell culture in primary ciliary dyskinesia disease modeling A) Schematic representation of the workflow for epithelial basal cell expansion in FAD+Y-27632(Y)+WS6, CRISPR-Cas9 genome editing and differentiation in air-liquid interface (ALI) cultures. B) Quantification of the percentage of basal cell clones that reached 60% confluence from 285 wells and were passaged at each stage of expansion between 96-, 24- and 6-well plates (representative of three clonal culture experiments with this cell donor). C) Quantification of Sanger sequencing results showing the proportions of DNAH5- knockout (KO) or wild-type (WT)/heterozygous clones. D) Representative immunofluorescence staining of a basal cell clone (passage six, four passages after clonal cell sorting) after differentiation at ALI (acetylated tubulin, ACT, magenta; MUC5AC, grey). Scale bar = 20 µm. E) Representative immunofluorescence staining of individual ciliated cells from WT and DNAH5 -KO clones differentiated at ALI (acetylated tubulin, ACT, magenta; DNAH5, cyan; DAPI, blue; n = 3 WT clones, n = 3 KO clones; passage seven, five passages after clonal cell sorting). Scale bar = 10 µm. F) Transmission electron microscopy of ciliary cross-section from WT and DNAH5 -KO clones differentiated at ALI (passage seven, five passages after clonal cell sorting). Arrows show normal outer dynein arms in a WT clone and loss of ODA in DNAH5 -KO clones (40,000× magnification, scale bar = 100 nm, n = 1 WT clone, n = 3 KO clones). G) Ciliary beat frequency analysis of differentiated ALI cultures derived from WT clones, the bulk population containing both DNAH5 -KO and unedited cells, and DNAH5 -KO basal cell clones (n = 3 WT and DNAH5 -KO clones, and n = 3 experimental replicates of the bulk population). Coverage and beat frequency of moving cilia are displayed for representative fields of view (top) and frequency distributions between the 4-20 Hz analysis cut-offs (bottom). Clones were isolated by single-cell sorting and transferred through 96-, 24-, and 6-well formats, with some attrition at each stage ( Fig. 6B ). Overall, 7% of cells expanded in 6-well plates, and 14 of 19 (73.7%) of these were DNAH5 knockouts ( Fig. 6C ). Clonal cultures retained the capacity to differentiate into both muco-secretory and ciliated cell lineages ( Fig. 6D ). Their epithelial integrity and barrier function, assessed by TEER, were comparable to non-PCD and PCD ALI cultures ( Supplementary Fig. 9C ). In clonal knockout ALI cultures, DNAH5 expression was undetectable by immunofluorescence ( Fig. 6E ). TEM confirmed ODA defects in all cilia assessed from clonal knockout cell cultures (100% ODA defects based on 50 cilia cross sections for DNAH5 KO clone). ( Fig. 6F ). Ciliary beat frequency (CBF) measurements confirmed complete loss of motility, faithfully recapitulating the PCD phenotype for DNAH5 mutation ( Fig. 6G ). A second experiment targeting the DNAI2 gene had similar results: 66% of cells were nucleofected with GFP ( Supplementary Fig. 10A ) and analysis of clonal cultures revealed that 22 of 37 (59.5%) clones were DNAI2 knockouts ( Supplementary Fig. 10B ). TEER readings confirmed that their epithelial integrity and barrier function were comparable to non-PCD and PCD ALI cultures ( Supplementary Fig. 10C ). TEM again showed that only a subset of cilia were defective in bulk cultures ( Supplementary Fig. 10D ), while clones had either consistently intact or consistently defective ciliary ultrastructure ( Supplementary Fig. 10E ). Western blot confirmed loss of DNAI2 in knockout clonal cultures ( Supplementary Fig. 10F ). Following clonal expansion, neither wild-type or DNAI2 -edited cells showed chromosomal abnormalities ( Supplementary Fig. 10G ). Download figure Open in new tab Supplementary Figure 10: CRISPR-Cas9-mediated DNAI2 -knockout in primary airway basal cells expanded in FAD+Y+WS6 A) Flow cytometry quantification of EPCAM⁺ epithelial cells expressing GFP following plasmid nucleofection, representing approximate nucleofection efficiency for single donor used for DNAI2- knockout (KO) experiment. B) Quantification of Sanger sequencing results showing the proportions of DNAI2- KO and wild-type (WT)/heterozygous clones. C) Transepithelial electrical resistance (TEER) measurements from air-liquid interface (ALI) cultures derived from DNAI2- KO clones, corresponding WT clones, healthy basal cells and primary ciliary dyskinesia patient basal cells (n = 1 non-PCD and PCD patient donors with triplicate wells, n = 3 WT and DNAI2 -KO clones; non-PCD and PCD control data are repeated from Supplementary Fig. 9C ). D) Transmission electron microscopy (TEM) of ciliary cross-section from the bulk population containing both DNAI2- KO and unedited cells. Blue arrows indicate normal outer dynein arms (ODA) in WT cilia and purple indicate loss of ODA in DNAI2 -KO in D and E. (40,000× magnification, scale bar = 200 nm). E) TEM images showing ciliary cross-sections from WT or DNAI2 -KO clones differentiated in ALI cultures (n = 1 WT, n = 2 KO clones; scale bars = 200 nm). F) Western blot analysis of DNAI2 expression in WT clones (n = 3) and DNAI2 -KO clones (n = 3). G) Copy number profiles from low-pass whole-genome sequencing of clonal nasal cell cultures expanded in FAD+Y-27632(Y)+WS6 (n = 1 WT clone, n = 3 DNAI2 -KO clones). Bin-level copy numbers were estimated with QDNAseq, converted to absolute scale, and low-quality bins (<99.9999% bases or <70% mappability) were excluded. Genome-wide plots show unflagged bins (points) and segmented copy number per chromosome (green horizontal lines). Discussion Our novel epithelial cell culture medium, EpMED (FAD+Y+WS6), provides a straightforward approach for the rapid expansion of primary human epithelial basal cells from limited clinical material. This scalability in a 2D culture format contrasts with organoid-based systems, which typically expand more slowly and at a smaller scale. Although WS6 modestly increased the size of airway organoids, its principal advantage lies in supporting high-efficiency, sustained expansion in 2D co-culture with 3T3-J2 feeder cells. Cells expanded in WS6 retained key functional features of their tissue of origin. Basal cells maintained the capacity for multilineage differentiation and functional ion transport, without evidence of reprogramming to a more primitive or embryonic-like state. For example, passage 38 nasal cultures formed ciliated epithelium in a 3D differentiation assay. Moreover, all donor lines ultimately underwent replicative senescence with extended passaging suggesting that cells were not immortalized. High-passage cultures showed no signs of transformation or chromosomal instability, and cells did not form tumors in immunodeficient mice. The ability to maintain and differentiate primary epithelial basal cells at scale supports the replacement of cancer cell lines and immortalized cells in epithelial studies. The proliferative effect of WS6 was restricted to primary basal cells and BMI1 -transduced derivatives, with no enhancement of proliferation observed in cancer cell lines or in hTERT/CDK4-immortalized basal cells. Cultured basal cells tolerate genetic manipulation well, including lentiviral transduction and CRISPR-Cas9 editing. A key advantage of the WS6-containing system over Y-27632 alone is its support of clonal culture. This enabled the derivation of isogenic gene-edited clones from primary basal cells, as demonstrated in our cell culture models of primary ciliary dyskinesia. Prior approaches have relied on bulk cultures containing mixed populations of edited and unedited cells. The ability to isolate and expand clonal knockouts opens the way for dissection of gene function in human epithelium. Our approach will be transferable to a range of monogenic disorders, such as ichthyoses, cystic fibrosis and epidermolysis bullosa. We anticipate that the method will also be useful in a number of other disease modeling applications. The extended lifespan of epithelial basal cells under these conditions will enable more extensive experimental use of material from individual donors, including those with epithelial disorders. Improved culture success also facilitates the use of primary cells from donor groups traditionally underrepresented in vitro , including older individuals and patients with disorders in which stem cell exhaustion or fragility has previously limited functional investigation. Additionally, viral or bacterial infection studies have relied on non-human (e.g. Vero E6 cells in SARS-CoV-2 research), cancer-derived (e.g. A549 cells) or immortalized cell lines (e.g. MDCK, 16HBE14o− cells). When combined with recent platforms for high-throughput differentiation of airway epithelium in multiwell formats 41 , this method presents an opportunity to study infection in the human context. Similarly, extensive basal cell expansion supports cancer modeling strategies in which sequential genomic modifications are introduced into a defined background 42 . We have not attempted to expand primary human tumor material with WS6, but previous studies using Y-27632 alone have reported mixed results with low efficiency and preferential expansion of normal epithelial stem/progenitor cells 43 , 44 . Accordingly, tumor-directed applications should include enrichment for malignant cells and verification of expected genomic alterations early in and throughout culture. Epithelial basal cells have been used successfully in clinical settings for conditions such as severe epidermal burns 45 and limbal stem cell deficiency 46 . In these settings, the ability to rapidly generate transplantable material improves feasibility and reduces the time and cost associated with manufacturing. The enhanced expansion afforded by this method has the potential to extend epithelial cell therapy to additional indications, particularly those where access to sufficient autologous tissue is a constraint. Starting with small nasal brush biopsies, our method would allow us to generate 70 million basal stem cells, estimated to be the requirement for whole-organ tracheal bioengineering 10 , within 14.4 (range 12-18) d. The system also permits efficient gene editing of epithelial cells, broadening its potential use in combined cell and gene therapy strategies. Proof-of-concept has been established in the genetic skin disease epidermolysis bullosa, where transplantation with autologous epidermal cells that underwent ex vivo retroviral gene addition was life-saving 47 . The ability to derive clonal cultures may offer further safety advantages by enabling preclinical characterization of viral integration sites prior to transplantation 48 . There remain some limitations of this method for basal cell expansion. Cell expansion was robust in all donors studied but inter-individual variation was seen in the duration of cultures. This may reflect an intrinsic donor characteristic, the potency of stem cells present in the initial biopsy sample, or on variation in the extent to which these culture conditions provide ideal conditions for a given donor’s cells. In addition, the continued reliance on 3T3-J2 mouse fibroblast feeder cells also complicates clinical translation as production of GMP-grade feeder cells is time-consuming and costly. The molecular mechanisms by which 3T3-J2 fibroblast feeder cells, Y-27632 and WS6 improve epithelial cell culture are only partially understood. 3T3-J2 cells form the foundation of the only epithelial cell culture system shown to support the maintenance of basal cells capable of supporting long-term regeneration in vivo . However, it is unclear which secreted factors, cell-cell or cell-matrix interactions are responsible. Previous work suggests roles for secreted growth factors and extracellular matrix components 49 , 50 , but no medium composition has yet proven equivalent to co-culture for long-term human epithelial cell culture, with proven stem cell maintenance through in vivo transplantation. Y-27632 was discovered as a ROCK1/2 inhibitor that inhibits smooth muscle contraction 51 . Y-27632 inhibits anoikis (apoptosis in response to cell dissociation) in stem cell culture applications 52 , in addition to increasing the proliferation rate of cultured epithelial cells. However, Y-27632 still promotes proliferation in ROCK1/2 -knockout cells 53 , indicating the involvement of additional targets beyond the Rho-ROCK pathway. WS6 was identified in high-throughput compound screening through its ability to induce pancreatic beta cell proliferation 16 . WS6 has multiple cellular targets, including Erb3 binding protein-1 (EBP1, PA2G4), a cell cycle-regulated protein that has two isoforms, with the p48 isoform promoting proliferation and p42 inhibiting it 54 , and the IκB kinase IKKε, a component of the NF-κB signaling pathway. As such, it is unclear which of these targets play roles in basal cell expansion, or indeed whether multiple pathways are affected by WS6-treatment. Further work will be needed to clarify the individual and synergistic contributions of these components, and to define the molecular circuits that govern epithelial stem cell expansion in this method. Methods Cell culture 3T3-J2 fibroblast culture and feeder layer preparation 3T3-J2 mouse embryonic fibroblasts (Kerafast) were maintained in DMEM with sodium pyruvate (Gibco 41966) supplemented with 7% bovine serum (Hyclone, SH30072.04; lot AE29427271), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, P0781-100ML). Cultures were maintained at 37 °C in a humidified 7.5% CO₂ incubator, with medium replaced three times per week. To prepare feeder layers, cells were mitotically inactivated by treatment with 4 μg/mL mitomycin C (Sigma-Aldrich, M4287, 0.4 mg/mL stock in PBS) for 3 h. After washing and trypsinization, cells were seeded at 20,000-30,000 cells/cm² in fresh 3T3-J2 cell culture medium. Epithelial cells were added the following day. Primary epithelial 2D cell culture Human basal epithelial cells were obtained from nasal, tracheal, bronchial, lung and skin tissues with patient consent (Table S1). Ethical approval was obtained through National Research Ethics Committees (Living Airways Biobank, REC 24/NW/0168, nasal cells; REC 18/SC/0514, tracheal, bronchial, small airway cells; REC 11/LO/1522, skin and gastric cells; REC 07/Q0508/43, thymus cells). Tissues were transported to the laboratory in transport medium consisting of αMEM (Gibco, 22571020) supplemented with 100 U/mL penicillin, and 100 μg/mL streptomycin, 10 μg/mL gentamicin (15710-049, Gibco) and 250 ng/mL amphotericin B (Fisher, 10346503). Nasal brushings were performed using cytology brushes (Medical Packaging Corporation, CYB-1). To generate single cell suspensions, the brushes were incubated in 0.1% trypsin/EDTA (Sigma-Aldrich, 59418C) in RPMI (Gibco, 21875) for 20 min at 37°C with vigorous shaking every 2 min. The cell suspensions were transferred to new 15 mL tubes and the brushes washed with RPMI medium. After this, the trypsin was neutralized with fetal bovine serum (FBS; Gibco, A5256701; lot 2575638). The cell suspensions were centrifuged at 300 × g for 5 min and resuspended in red blood cell lysis buffer (Life Technologies, 00-4333-57) for 10 min at room temperature. The cells were centrifuged again, washed with RPMI, centrifuged and resuspended in relevant medium for cell seeding. Lung tissue was minced using a sterile scalpel for 5 min, this tissue and tracheal and bronchial biopsies were incubated in 16 U/mL dispase (Corning, 354235) in RPMI for 20 min at room temperature and then mechanically disrupted using sterile forceps. This was followed by incubation in 0.1% trypsin/EDTA for 30 min at 37°C and neutralisation with FBS. The cells were filtered through a 100 µm strainer (Falcon, 352360) and single cell suspensions were centrifuged at 300 × g for five min and resuspended in the relevant medium for cell seeding. Epidermis was explanted onto 3T3-J2 feeder cells in FAD+Y medium and expanded in FAD+Y for two passages prior to cryopreservation. Cells were then thawed in FAD medium for the experiments reported here. FAD medium was prepared by combining 500 mL DMEM without pyruvate (Gibco, 41965062), 50 mL FBS, and 5 mL penicillin-streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin in 0.9% NaCl). 175 mL of this medium was removed and 125 mL Ham’s F-12 (Gibco, 21765029) added. The medium was then supplemented with the following: 5 mL adenine (Sigma-Aldrich, A3159-5G; 17.9 mM stock in 0.1 M HCl, 179 μM final), 0.5 mL gentamicin (Life Technologies, 15710049; 10 mg/mL stock, 10 μg/mL final), 40 μL hydrocortisone (Sigma-Aldrich, H0888-1G; 6.25 mg/mL in ethanol, 0.5 μg/mL final), 0.5 mL triiodothyronine (Sigma-Aldrich, T6397-100MG; 2.02 μM stock in PBS, 2 nM final), 0.5 mL insulin (Merck, I0516; 5 mg/mL in ∼12.5 mM HEPES, 5 μg/mL final), 4.3 μL cholera toxin (Sigma-Aldrich, C8052-1MG; 11.9 μM stock in water, 0.1 nM final), and 50 μL EGF (PeproTech, AF-100-15-500ug; 100 μg/mL stock in 0.1% BSA/PBS, 10 ng/mL final). Where indicated, 5 μM Y-27632 (Tebu Bio, T1725; 500 μL of 5 mM stock in distilled water) and/or 100 nM WS6 (Stratech, B4880-APE; 50 μL of 1 mM stock in DMSO) were also added. This epithelial cell medium (‘EpMED’; FAD containing 5 μM Y-27632 and 100 nM WS6) is now our routine cell culture medium for establishing and maintaining basal epithelial cell cultures. Epithelial cells were cultured at 37 ° C and 5% CO 2 with three medium changes per week. Epithelial cells can be enriched from co-cultures using differential trypsinization; 3T3-J2 feeder cells have a greater sensitivity to trypsin than primary basal cells allowing a first trypsin-wash cycle to remove only the 3T3-J2 cells, and a subsequent trypsin step to remove the more firmly attached epithelial cells. Epithelial cells were counted following differential trypsinization and population doublings (PD) were calculated as PD = 3.32 × [log(cells harvested/cells seeded)]. Primary small airway and alveolar organoid culture Human small airway 55 and alveolar organoid 56 cultures were derived from distal lung tissue (Diakonessen Hospital Utrecht) as previously described. Cells were plated as single cell suspensions at 500 cells/µL (small airway) or 300 cells/µL (alveolar) in 10 µL 80% Basement Membrane Extract (R&D Systems, 3533-005-02) at the bottom of a 96-well organoid culture plate (STEMCELL Technologies, 200-0562). Primary gastric organoid culture Endoscopic mucosal biopsies taken from the fundus, body and antrum of the stomach were dissociated to single cells in TrypLE Express (Thermo Fisher Scientific, 12605010) for 5 min and cultured in 12-well plates (Thermo Fisher Scientific, 10098870) in 30 µL droplets of Matrigel (Corning, 354230). Gastric organoid medium consisted of Advanced DMEM F-12 (Thermo Fisher Scientific, 12634) supplemented with 10 mM HEPES (Thermo Fisher Scientific, 15630080), 2 mM Glutamax (Thermo Fisher Scientific, 35050061), 1% penicillin-streptomycin, 100 µg/mL primocin (Thermo Fisher Scientific, NC9392943), 1× B-27 supplement minus vitamin A (Thermo Fisher Scientific, 12587010), 1.25 mM n-acetylcysteine (Sigma-Aldrich, A9165), 100 ng/mL Wnt-3A (Peprotech, 315-20), 500 ng/mL R-spondin 1 (Peprotech, 120-38), 100 ng/mL noggin (Peprotech, 120-10C), 50 ng/mL human EGF (Thermo Fisher Scientific, PMG8043), 10 nM gastrin (Sigma-Aldrich, G9020), 3 µM CHIR99021 (Tocris, 4423), 5 µM A83-01 (Sigma-Aldrich, SML0788) and 200 ng/mL FGF10 (Peprotech, 100-26). This medium was supplemented with 10 µg/mL Y-27632 (Tocris, 1254) for the first 24 h. Organoids were passaged by TrypLE and manual disaggregation with a P1000 pipette and split 1:3-15. Primary hematopoietic stem and progenitor cell culture Human cord blood CD34+ cells (Anthony Nolan) were cultured in SFEM II (STEMCELL Technologies, 09655) supplemented with 150 ng/mL human stem cell factor (Peprotech, 300-07-10UG), 150 ng/mL human FLT3-ligand (Peprotech, 300-19-10UG), 20 ng/mL human thrombopoietin (Peprotech, 300-18-10UG) and 1% penicillin-streptomycin. Where indicated, the medium was supplemented with 10 nM, 100 nM or 1 μM WS6. Cells were cultured at 37°C and 5% CO 2 for 7 d with a medium change on day four. Cell line culture HBEC3-KT cultures were maintained in Keratinocyte-SFM (Gibco, 17005042); A549 and H520 cells were maintained in RPMI 1640 (Gibco, 21875034) supplemented with 10% FBS, 1 mM sodium pyruvate (Gibco, 11360070), 100 U/mL penicillin, and 100 μg/mL streptomycin. BMI1 -transduced cells 57 were maintained in PneumaCult-Ex Medium (STEMCELL Technologies, 05008) on tissue culture plastic coated with 1% PureCol (Advanced BioMatrix, 5005) in PBS for 30 min at room temperature and left to air dry for 5 min. All cultures were maintained at 37°C and 5% CO 2 , with medium changes three times per week. CellTiter-Glo assays For primary 2D cell cultures, compounds and DMSO were added to the black-walled 384-well assay plates (Thermo Fisher Scientific, 142761) using an ECHO 550 liquid handler (Labcyte) or in 5 µL FAD medium. 2000 cells/well were then added in FAD medium using a microplate washer/dispenser (BioTek EL406, Agilent) or using a multichannel pipette under sterile conditions. After 4 or 6 d of culture, the medium was removed and the cell number was determined using the CellTiter-Glo Luminescent Cell Viability protocol (Promega). After 20 min, bioluminescence was measured using a Envision II plate reader (PerkinElmer) or a VANTAstar plate reader (BMG LABTECH, 1 s integration time). For cell lines, 1000 cells/well were seeded in black-walled 96-well plates (Corning, 3603), after 48 h compounds were added in cell culture medium (0.1% DMSO), after 48 h the medium was removed and the CellTiter-Glo Luminescent Cell Viability protocol was performed as above. For small airway and alveolar organoids, 100 µL organoid medium was supplemented with 10 nM, 100 nM, 1 µM WS6 or DMSO as a vehicle control (small airway; 1%, alveolar; 0.1%) and the medium was refreshed every 2-4 d. Images were taken at 14 d and the CellTiter-Glo 3D Cell Viability Assay (Promega, G9681) was performed according to the manufacturer’s protocol. 100 µL CellTiter-Glo reagent was added to the wells and the plate was put on a Thermo Fisher Scientific Multidrop Combi+ plate shaker for 15 min at room temperature. After an additional incubation time of 15 min at room temperature, 100 µL of the solution was transferred to a black bottom/black wall plate (Greiner Bio-One, 655076) and luminescence was measured (Tecan Spark, 500 ms integration time). Measurements from each well were normalized to the mean of measurements of three replicate wells taken in the same way on day 0, immediately after plating the organoid lines. For gastric organoids, 500 µL organoid medium was supplemented with 10 nM, 100 nM, 1 µM WS6 or 0.1% DMSO as a vehicle control and the medium was refreshed every 3 d. Images were taken at 7 d, the organoid medium was removed and replaced with 30 µL ice-cold RPMI. 100 µL CellTiter-Glo reagent was added and the plate was shaken at 60 rpm on a see-saw shaker (Stuart) for 15 min. 100 µL of the solution was transferred to a black wall plate (Corning, 3603) and luminescence was measured on a VANTAstar plate reader (1 s integration time). Measurements from each well were normalized to the mean of measurements of three replicate wells taken in the same way on day 0, immediately after plating the organoid lines. Colony formation assays To assess colony forming efficiency (CFE), epithelial basal cells were seeded at 1,000 cells per well with technical triplicates into six-well plates that had been seeded with 3T3-J2 feeder cells the day prior. After 7 d, plates were fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature and stained with 1% crystal violet solution (Sigma-Aldrich) for a further 10 min. Plates were washed in water and air-dried overnight. Colonies were counted manually under a brightfield microscope, classifying all coherent colonies of more than 10 cells as a colony. CFE was calculated as: (number of colonies / number of seeded cells) x 100. Plates were imaged using an Epson Perfection V600 flatbed scanner. Histology processing Chamber slides were fixed in 4% PFA for 10 min, washed with PBS and stored in PBS prior to staining. Formalin-fixed, histogel-embedded organoids and ALI cell cultures, and formalin-fixed tissues were processed in a Leica TP1050 tissue processor. Samples were embedded in type 6 paraffin wax (Epredia, 8336) using an embedding station (Sakura Tissue-TEK TEC) and 5 µm sections were cut on a Microm HM 325 microtome and stored at 4°C before use. Hematoxylin and eosin (H&E) staining was performed on sections using an automated staining system (Sakura TissueTek DRS) and imaged on a Nanozoomer Digital Pathology (Hamamatsu). Histology images were collated and presented from whole-slide image files using PATHOverview 58 (available on GitHub https://github.com/EpiCENTR-Lab/ PATHOverview). Immunofluorescence staining Slides were dewaxed using an automated protocol, washed in PBS, and a hydrophobic ring was drawn around the sample using an ImmEdge pen (Vector Laboratories, H-4000). Sections were blocked with 1% bovine serum albumin (BSA; Merck, 1.12018.0100), 5% normal goat serum (NGS; Gibco, 16120064) and 0.1% Triton X-100 (Sigma-Aldrich, X-100) in PBS for 1 h at room temperature. Primary antibodies (Table S2) were diluted in blocking buffer and applied to slides overnight at 4°C. Slides were washed twice in PBS. Species appropriate secondary antibodies conjugated to Alexa Fluor dyes (Table S3) were diluted 1:1000 in 5% NGS, 0.1% Triton X-100 in PBS and applied to slides for 3 h at room temperature in the dark. 200 ng/mL DAPI (Sigma-Aldrich, D9542) in PBS was applied to the slides for 20 min. Slides were washed twice in PBS and a coverslip was applied manually with Immu-Mount (Thermo Fisher Scientific, 9990402). Whole-mount ALI cultures were transferred to 1.5 mL Eppendorf tubes and blocked in PBS containing 2% BSA, 5% NGS, and 0.1% Triton X-100 for 1 h at room temperature at 20 rpm on a see-saw shaker. Primary antibodies (Table S2) were diluted in blocking buffer and incubated overnight at 4°C at 60 rpm on a mini orbital shaker (Stuart, SSM1). After three 5-min PBS washes on the see-saw shaker, secondary antibodies (Table S3) were diluted 1:1000 in 2% BSA, 5% NGS, 0.1% Triton X-100 in PBS and applied for 3 h at room temperature in the dark on the see-saw shaker. DAPI (Sigma-Aldrich, D9542) was applied at 100 ng/mL for 20 min in the dark on the see-saw shaker. Inserts were washed twice in PBS and once in distilled water, then mounted with Immu-Mount within imaging spacers (Grace Bio-Labs, 654006). All images were acquired using a Nikon Ti2 fluorescence microscope. Air-liquid interface cultures Semi-permeable membrane supports (Pore size 0.4 µm; Corning, 3470 or Sarstedt Ltd, 83.3932.041) were coated with 50 µg/mL rat tail collagen I (Corning, 354236) in 0.02N acetic acid (Sigma-Aldrich, 1.00063.1011). After 1 h at room temperature, the liquid was removed and the membranes washed twice with PBS. They were left to air dry for 15 min. To establish ALI cultures, airway basal cells were seeded onto the collagen-coated semi-permeable membrane supports (2 x 10 5 cells in 200 µL epithelial growth medium, Fig. 6 and Supplementary Fig. 7 ; 3 x 10 5 cells in 250 µL epithelial growth medium, Fig. 4 and Supplementary Fig. 5 ). 500 µL epithelial growth medium was added to the basolateral side. After 24 h (at confluence), medium was removed from the apical side and replaced basolaterally with PneumaCult-ALI medium (STEMCELL Technologies, 05001) supplemented with 4 µg/mL heparin (Sigma-Aldrich, H4784) and 0.48 µg/mL hydrocortisone (Sigma-Aldrich,H0888). Medium was refreshed three times per week. Mucus was removed by gentle apical washing with PBS once a week. ALI cultures were maintained for 28-35 d before analyses. Transepithelial electrical resistance (TEER) was measured using an EVOM manual resistance meter (World Precision Instruments). Mucus was removed by gentle apical washing with PBS and measurements taken with 200 ul of PBS (pH 7.2, Ca-Mg-, Gibco, 20012-019) added to the apical compartment. Three readings were taken from each insert once resistance had stabilized (5-10 s) and triplicate inserts were measured for each condition. Transepithelial ion transport To establish ALI cultures for transepithelial ion transport analysis, 1 x 10 6 airway basal cells were seeded onto collagen-coated Snapwell membranes (Corning, 3407) in 300 µL epithelial growth medium. ALI cell culture was carried out as above with 1.5 mL medium in the basolateral side. At 30 d, transepithelial ion transport was measured using the Ussing chamber technique with symmetrical bicarbonate buffered salt solution (in mM): NaCl, 117; NaHCO 3 , 25; KCl, 4.7; MgSO 4 , 1.2; KH 2 PO 4 , 1.2; CaCl 2 , 2.5; D-Glucose, 11 (equilibrated with 5% CO 2 to pH 7.3-7.4). The solution was maintained at 37°C, bubbled with 21% O 2 /5% CO 2 gas mix. The following drugs were applied: amiloride (apical, 100 μM) to inhibit the epithelial sodium channel (ENaC); forskolin (bilaterally, 10 μM) to stimulate CFTR; CFTRinh172 (apical, 10 μM) to inhibit CFTR; ATP (apical, 100 μM) to stimulate calcium-activated chloride channels and ouabain (basolateral, 1 mM) as previously described 59 – 61 . All drugs/chemicals were obtained from Sigma-Aldrich. Data were collected and analyzed using LabChart 7 software (ADI Instruments). Functional analysis of cilia For top-down ciliary beat frequency (CBF) analysis, ALI cultures were left to stabilize at 37°C for 30 min after PBS washing/TEER measurements. Fast time-lapse videos were acquired at 37°C with 5% CO₂, using a Nikon Eclipse Ti-E inverted microscope (Nikon, Japan) equipped with a Prime BSI Express camera and a Nikon Super Plan Fluor ELWD 20XC PH objective. For CBF analysis, we captured 512 frames at a frame rate of 86.95 frames per s. Cilia beat frequency and active area were quantified using the CiliaFreqMap ImageJ macro ( https://doi.org/10.5281/zenodo.17107574 ) with the range 4-20 Hz. To assess ciliary motility, differentiated epithelium was scraped from transwells using 200 µL of HEPES-buffered Medium 199 (Gibco, 22340-020), gently dissociated by pipetting, and transferred onto a glass slide for side-on visualization of the epithelium using the above Nikon Eclipse Ti-E inverted microscope. The remaining cell suspension was transferred onto a glass slide within a hydrophobic ring and allowed to air dry at room temperature. The slides were then fixed in 4% PFA for 15 min and immunofluorescence staining was performed as described above using 0.1% Triton X-100 (Sigma, X100-500ML) in PBS as wash buffer and 5% milk powder (Marvel) in 0.1% Triton X-100 PBS as blocking buffer. Slides were mounted with ProLong Gold antifade reagent with DAPI (Thermo Fisher Scientific, P36935). Transmission electron microscopy For transmission electron microscopy (TEM) of ciliary axenomes, the ALI cultures were scraped from transwells using 200 µL of HEPES-buffered Medium 199 (Gibco, 22340-020) and fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M Sorensen’s buffer (pH 7.3) for 24 h. The cells were postfixed in 1% aqueous solution of osmium tetroxide. Following several washes, the tissue was dehydrated through a graded ethanol series and then propylene oxide (Merck). The epithelium was embedded in TAAB812 resin (TAAB Laboratory Equipment) and sections, 70 nm in thickness, were cut using a Leica UC7 Ultramicrotome (Leica Microsystems). These sections were then mounted on copper mesh grids and contrasted for 2 min with 4% uranyl acetate solution in methanol (VWR), followed by 2 min in lead citrate (Reynolds’ solution). Imaging was performed using a JEOL JEM-1400 transmission electron microscope (JEOL) operated at 120 kV, and digital images were acquired using a Xarosa camera and Radius software (EMSIS). Western Blotting Differentiated epithelium from ALI cultures was scraped from transwells in 200 µL PBS and centrifuged at 400 × g for 5 min. Cell pellets were resuspended in RIPA lysis buffer (Thermo Fisher Scientific, 89901) containing protease and phosphatase inhibitors (Thermo Fisher Scientific, 10025743) on ice. Lysates were stored at −20°C and thawed on ice before use. Protein concentration was quantified using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, 23225), following the manufacturer’s microplate protocol. The assay was performed with triplicate technical replicates, using a 1:10 dilution of protein samples. Protein samples were denatured in Laemmli buffer (Sigma-Aldrich, S3401-1VL) with a 10 min incubation at 95°C. Denatured samples were loaded into gels and electrophoresed at 120 V for 75 min (DNAH5 Figure 6E : NuPAGE 3-8% Tris-Acetate gels (Invitrogen, EA0375BOX) and NuPAGE Tris-Acetate SDS Running Buffer (Invitrogen, LA0041); DNAI2 Supplementary Figure 7J: 4-12% Bis-Tris gels (Invitrogen, NW04125BOX) and MOPS SDS running buffer (Invitrogen, B0001). Protein was dry-transferred onto a nitrocellulose membrane (Invitrogen, IB33002) with an Invitrogen iBlot3 machine following manufacturer recommended transfer protocol based on target protein size. Blots were blocked for 1 hr at room temperature in 5% milk powder (VWR Chemicals, 84615.0500) in tris-buffered saline (TBS; Cell Signaling Technology, 12498S) with 0.1% TWEEN20 in TBS (TBST; Sigma-Aldrich, P9416-100ML), before overnight incubation with primary antibody (Table S4) at 4°C with rotation. Blots were washed in TBST and transferred to HRP-conjugated secondary antibody (Table S4) for 2 h at room temperature. Blots were imaged after a 2 min incubation in Crescendo HRP substrate (Millipore, WBLUR0100) and chemiluminescence imaging on a Bio-Rad ChemiDoc MP imaging system. Fluorescence-activated cell sorting for clonal cell culture Single cell suspensions from digested bronchial biopsies and nasal brushings were stained in sorting buffer (1× PBS, 1% FBS, 25 mM HEPES (Gibco, 15630-049) and 1 mM EDTA (Invitrogen, AM9260G) with anti-CD45-PE (1:20 dilution; BD Pharmingen 555483), anti-CD31-PE (1:20 dilution, BD Pharmingen 555446), anti-EPCAM-APC (1:50 dilution, BioLegend 324208) antibodies for 15 min at 4°C. Cells were washed and resuspended in sorting buffer containing 1 μg/mL DAPI. Single DAPI - CD45 - CD31 - EPCAM + cells were sorted (BD FACS Aria Fusion) into wells of a 96-well plate, precoated with 50 µg/mL rat tail collagen I and mitotically inactivated 3T3-J2 feeder cells with 50 μL epithelial conditioned medium. Epithelial cell-conditioned medium was a 50:50 mix of fresh epithelial medium and sterile filtered medium (0.22 µm filtered; SLS, B2B06412) that had been collected from proliferating epithelial cells (50-90% confluency) for 24 h and supplemented with 10 ng/mL EGF, 5 μM Y-27632 and 100 nM WS6 where indicated. An additional 50 μL epithelial cell-conditioned medium was added 3 d post-sort. After 7 d and 10 d, medium was replaced with fresh epithelial medium. At 14 d, whole-well images were taken on a Leica Dmi8 microscope and the number of wells with a single colony were quantified. Flow cytometry Fully differentiated air–liquid interface cultures were harvested for flow cytometry to quantify basal, ciliated, and muco-secretory cell populations. Transwells were washed twice with pre-warmed PBS for 5 min at 37°C, excised, and transferred to 1.5 mL tubes containing 1 mL Triple E dissociation buffer (Thermo Fisher Scientific, 12604013). Tissues were incubated at 37°C for 8 min, briefly vortexed, and returned to the incubator. This cycle was repeated once. Dissociated cells were transferred to DMEM supplemented with 10% FBS to neutralize enzymatic activity, centrifuged at 400 × g for 5 min at 4°C, and resuspended in PBS for counting and downstream processing. A total of 1-2 x 10 5 cells were pooled from all samples and divided into unstained, live/dead stained, and heat-shocked (65°C, 10 min) live/dead stained control conditions. Cells were stained with LIVE/DEAD Fixable Dye (Thermo Fisher Scientific, L34992; 1:1000 in PBS) for 20 min at 4°C, washed, and resuspended in FACS buffer (PBS, 1% BSA, 1 mM EDTA (Invitrogen, 15575-038). A third of these cells were distributed between fluorescence minus one (FMO) control wells. Following centrifugation at 400 × g for 5 min at 4°C, cells were stained with antibodies targeting cell surface antigens (Table S5) in 50 μL FACS buffer for 30 min at 4°C in the dark. After washing, cells were fixed in 100 μL 4% PFA for 20 min at 4°C, permeabilized in 150 μL permeabilization buffer (1:10 dilution in PBS; eBioscience, 00-8333-56), and stained with 50 μL intracellular antibodies in permeabilization buffer for 30 min at 4°C. Cells were washed twice in FACS buffer, resuspended in 100 μL FACS buffer, and analyzed on a CytoFLEX flow cytometer (Beckman Coulter). Compensation was performed using UltraComp plus eBeads (Invitrogen, 01-3333-41) and live/dead-stained cells. Nasal organoid differentiation assay To generate 3D nasal airway organoids, basal cells were enzymatically dissociated and counted. Organoid medium consisted of 50% BEBM (Lonza, 3171) and 50% DMEM (Gibco, 41966) supplemented with BEGM additives (Lonza, 4175; excluding triiodothyronine, gentamicin, amphotericin B, and retinoic acid). Retinoic acid (100 nM; Sigma-Aldrich, R2625, stock 10 mM in ethanol) was freshly added before use. 5 μM Y-27632 was added to the organoid medium for cell seeding. Ultra-low attachment 96-well plates (Corning) were pre-coated with 30 μL of 25% Matrigel (Corning, 354230; growth factor–reduced) in organoid medium and incubated at 37°C for 30 min. Then, 2,500 basal cells were seeded per well in 65 μL of 5% Matrigel. Cultures were fed by adding 70 μL of fresh medium on days 3, 10, and 17. At 21 d, whole-well images were taken at 10× magnification and tracheosphere size was quantified using OrgaQuant 62 . R was used to remove aberrant detection, including objects within 110 pixels of the image edges. Predicted organoid volume was calculated as ($ = (4/3) ∗ - ∗ .³) and the mean was taken from all organoids detected per condition. Organoids were harvested with ice-cold PBS, and centrifuged at 300 × g for 5 min. Organoids were fixed in 4% PFA for 30 min on ice, washed in PBS, and embedded in HistoGel TM (Epredia, HG-4000-012) for paraffin processing. CRISPR-Cas9 gene editing and clonal epithelial cell culture Primary nasal basal cells were isolated from fresh nasal brushings or thawed from frozen stocks and expanded in WS6-containing medium on mitotically-inactivated 3T3-J2 fibroblasts. For nucleofection, epithelial cells were harvested at ∼80% confluency by differential trypsinization to remove feeder cells. Cells were counted and resuspended at 2 x 10⁵ per reaction. Ribonucleoprotein (RNP) complexes were assembled by incubating 0.5 µL (30.5 pmol) Cas9 (IDT) and 0.5 µL (50 pmol) sgRNA (Table S6) at room temperature for 10 min. For DNAH5 knockout, 1 µL of Cas9 electroporation enhancer (IDT) was added to improve nucleofection efficiency. For DNAI2 , 1.5 µL of 100 µM donor template oligonucleotide introducing a premature stop codon was added; this ssODN served as both the HDR template and as an electroporation enhancer, yielding an HDR-mediated knockout. Nucleofection was performed using the 4D-Nucleofector System (Lonza) with the P3 Primary Cell kit. Each reaction included 2 x 10⁵ cells resuspended in 20 µL of P3 buffer (16.25 µl nucleofector solution + 3.75 µl supplement 1), mixed with the pre-formed RNP complex, and transferred to a single well of a 16-well cuvette. Electroporation was carried out using the program CA137. Immediately following nucleofection, 100 µL of culture medium was added, and cells were transferred to feeder cell-containing 6-well plates in epithelial cell-conditioned medium (prepared as per the Fluorescence-activated cell sorting for clonal cell culture section). Plates were incubated at 37°C and 5% CO₂ for 72 h. To generate clonal cultures, epithelial cells were stained with anti-EPCAM-BV421 (BioLegend, 324219; 1:100 dilution) for 30 min at 4°C in FACS buffer, washed, and resuspended at 1 x 10⁶ cells/mL in cell culture medium. EPCAM⁺ single cells were sorted into individual wells of feeder cell-containing 96-well plates using a BD FACSAria Fusion or BD FACSAria III. Epithelial cell-conditioned medium was supplemented as above and added to each well (100 µL per well). An additional 50 µL of medium was added every 2–3 d until 7 d, after which full medium changes were performed. Confluent clones were sequentially expanded into 24-well and 6-well plates prior to downstream differentiation or analysis. In parallel, nucleofected epithelial cells were seeded directly onto transwells for differentiation at the air–liquid interface (ALI) and referred to as ‘bulk cultures’, as they contained mixed edited and unedited populations. Genomic DNA was isolated from expanded clones using the AllPrep DNA/RNA Mini Kit (Qiagen, 80004) and quantified by NanoDrop (Thermo Fisher Scientific). Targeted genomic loci ( DNAH5 and DNAI2 ) were PCR-amplified using Q5 High-Fidelity DNA Polymerase (NEB) with primers listed in Table S7. 50 µl reactions contained 1× Q5 buffer, 0.02 U/µL polymerase, 50 ng genomic DNA, 200 µM dNTPs, and 0.5 µM of each primer. Thermocycling was performed on a ProFlex thermocycler (Thermo Fisher Scientific) with an initial denaturation at 98°C for 30 s; 35 cycles of 98°C for 10 s, 50–72°C for 30 s, and 72°C for 30 s; followed by a final extension at 72°C for 2 min. A single amplification product was confirmed using 1% agarose gel, purified using ExoSAP-IT PCR Product Cleanup (Thermo Fisher Scientific, 75001.1.ML) and sent for Sanger sequencing (Source Bioscience). Lentivirus production and cell transduction The lentiviral vectors pHIV-Luc-ZsGreen (a gift from Bryan Welm; Addgene plasmid 39196, http://n2t.net/addgene:39196 , RRID:Addgene_39196) or pCCL-CMV-EGFP (previously described 63 , 64 ) were produced in HEK293T cells by co-transfection with pMD2.G (a gift from Didier Trono; Addgene plasmid 12259, http://n2t.net/addgene:12259 , RRID:Addgene_12259) and pCMVR8.74 (a gift from Didier Trono; Addgene plasmid 22036, http://n2t.net/addgene:22036 , RRID:Addgene_22036) in jetPEI transfection reagent (Polyplus). Viral particles were collected 48 h after transfection and concentrated by combining the supernatants with PEGit concentrator (5×; System Biosciences, LV810A-1) overnight at 4°C. After centrifugation at 1,500 × g for 45 min, the supernatant was removed and the pellet was resuspended in 1/10th of the original volume of PBS. Concentrated supernatants were stored at −80°C until use. Virus was titrated by transduction of HEK293T cells and flow cytometry. To spin-transduce primary epithelial cells, cells in suspension equivalent to 15,000-16,000 cells/cm 2 , epithelial medium containing 4 or 8 μg/mL polybrene and small molecules as indicated in Supplementary Fig. 5 were combined with virus particles at the indicated MOI. Cells were centrifuged at 920 × g for 1 h at 30°C and then incubated at 37°C, 5% CO 2 for 5-7 h. After, medium was refreshed to remove the polybrene and the viral particles, and 3T3-J2 fibroblast feeder cells were added at 20,000/cm 2 . Cells were expanded for 48 or 72 h before analysis by flow cytometry. After trypsinization in 0.05% trypsin/EDTA, cells were stained with either anti-EPCAM/CD326 (BV421; BioLegend, 324219) or anti-ITGA6/CD49f (PE-Dazzle; BioLegend, 313626) antibodies to allow exclusion of 3T3-J2 cells during analysis. Flow cytometry was performed on a BD LSRII cell analyzer (BD Biosciences). ZsGreen+ flow sorting was performed on a BD FACSMelody TM Cell Sorter. Whole-genome sequencing karyotyping Low-pass whole-genome sequencing was performed by GENEWIZ (Azenta Life Sciences). Demultiplexing and FASTQ generation were carried out by UCL Genomics, followed by read alignment and bin-level copy number estimation using QDNAseq. The resulting QDNAseqCopyNumbers objects were processed in R. Raw bin copy numbers were converted to absolute values, and segmented copy numbers were taken directly from QDNAseq output. Bin quality metrics from QDNAseq annotations (the proportion of uniquely mappable bases and number of covered bases per bin) were used to flag low-quality regions. Bins with <99.9999% ‘bases’ or <70% ‘mappability’ were excluded. Genome-wide plots were generated with ggplot2, using only unflagged bins. Chromosome offsets were computed from the QDNAseq annotation to ensure consistent genome-wide coordinates. For visualization, points represent individual bins, and horizontal lines indicate segmented copy number. In vivo experiments Animal studies were approved by the University College London Biological Services Ethical Review Committee and licensed under UK Home Office regulations (project licence PP2060881). For subcutaneous cell transplantations, basal cells expanded in FAD+Y+WS6 for 12-14 passages were separated from 3T3-J2 feeder cells by differential trypsinization. 2 x 10⁶ cells were resuspended in 400 μL growth factor–reduced Matrigel and kept on ice. Under 2–3% isoflurane anesthesia, 200 μL of the cell suspension was injected subcutaneously into each flank of NSG mice. After 3 months, mice were culled, and Matrigel plugs were excised and processed for histology. For tracheal transplantation, basal cells transduced with the pHIV-Luc-ZsGreen lentiviral vector expanded in FAD+Y+WS6 for 6 passages were differentially trypsinized to remove 3T3-J2 feeder cells and counted. Mice were pre-conditioned with 10 μL 2% (w/v) polidocanol in PBS by oropharyngeal delivery to disrupt the tracheal epithelium 65 . 5-6 h later, 3.5 x 10⁶ cells were introduced in 25 μL epithelial cell culture medium by oropharyngeal instillation under 2-3% isofluorane anesthesia. Cell engraftment was monitored once weekly by IVIS imaging of luciferase activity under 2-3% isofluorane anesthesia. Mice were killed under anesthesia following IVIS imaging at 28 d, tracheas were removed and opened longitudinally along the ventral surface. The trachea was flattened under a microscopy slide in a small volume of PBS and ZsGreen was visualized using a Leica MZFLIII stereoscope and imaged with iDS uEye software (2× magnification). Brightness and contrast were adjusted using ImageJ. Tracheas were fixed in 4% PFA overnight at 4°C and paraffin embedded for histological analyses. IVIS analysis was performed in Aura software; the background measurement was obtained by taking an average of a control reading from the abdomen of each mouse analyzed. Data visualization and statistics The schematic diagrams in Figures 1B , 1D, 3B, 4A, 5A, 6A, ED5A, ED5D and ED6C were created using BioRender.com . Analyses and data visualization were performed in GraphPad Prism v10.6.0 and in RStudio [v4.2.2] with the tidyverse [v2.0.0] packages: dplyr ( https://dplyr.tidyverse.org/ v1.1.4), ggplot2 ( https://ggplot2.tidyverse.org/ v3.5.1), ggpubr ( https://rpkgs.datanovia.com/ggpubr/ v0.6.0), rstatix ( https://rpkgs.datanovia.com/rstatix/ v0.7.2), tidyr ( https://tidyr.tidyverse.org/ v1.3.1), stringr ( https://stringr.tidyverse.org/ v1.5.1), tibble ( https://tibble.tidyverse.org/ v3.2.1). FlowJo (v10.8.1) was used for flow cytometry data. All statistical tests are reported in figure legends. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. Funding This work was funded by the UK Regenerative Medicine Platform (UKRMP2) Engineered Cell Environment Hub (Medical Research Council (MRC); MR/R015635/1), the Longfonds BREATH lung regeneration consortium, a project grant jointly funded by DEBRA UK and Cure EB (GR000070), a BBSRC/AstraZeneca CASE PhD Studentship (EKH), a UCL Child Health Research PhD studentship (ASF), a MRC-DTP PhD studentship (NAM) and Rosetrees Trust PhD Plus Awards (supporting JCO and ASF; PhD2022\100046 and PhD2025\100016). REH’s work was additionally supported by a National Institute for Health and Care Research (NIHR) Great Ormond Street Hospital Biomedical Research Centre Catalyst Fellowship, Great Ormond Street Hospital Charity (V4322), The Royal Society (RG\R1\241421) and the CRUK Lung Cancer Centre of Excellence (C11496/A30025). SMJ was supported by a Cancer Research UK (CRUK) programme grant (EDDCPGM\100002), and an MRC Programme grant (MR/W025051/1). SMJ received support from the CRUK Lung Cancer Centre of Excellence (C11496/A30025) and the CRUK City of London Centre, the Rosetrees Trust, the Roy Castle Lung Cancer foundation, the Garfield Weston Trust and University College London Hospitals Charitable Foundation. SMJ’s work was supported by a Stand Up To Cancer (SU2C)-LUNGevity Foundation American Lung Association Lung Cancer Interception Dream Team Translational Research Grant and Johnson and Johnson (SU2C-AACR-DT23-17 to SM Dubinett and AE Spira). SU2C is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C. MJR was supported by a CRUK City of London Centre Clinical Academic Training Fellowship (BCCG1C8R). RAH was supported by the Leicester Institute of Precision Health. AYK’s work was supported by the Wellcome Trust (222096/Z/20/Z). This work was partly undertaken at UCL ICH/GOSH (REH), partly at UCL/UCLH (SMJ) and partly at the University of Leicester (RAH), who received a proportion of funding from the Department of Health’s NIHR Biomedical Research Centre’s funding scheme. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. Author contributions JCO, YI, SMJ and REH conceived the study. JCO, ASF and NAM performed cell culture experiments, analyzed data and visualized data. EKH performed PCD disease modeling studies using CRISPR-Cas9 editing, analyzed data and visualized data. DRP and REH performed cell transplantation experiments. SKR assisted with the design of CRISPR-Cas9 editing experiments. JCO, MJR and CP performed biopsy flow sorting and colony formation assays. IR performed Ussing chamber experiments under the supervision of DLB. AS-I and RD performed transmission electron microscopy and immunofluorescence analysis of ciliated epithelial cells under the supervision of RAH. MG performed HSC experiments under the supervision of WG. GB performed gastric organoid experiments under the supervision of GGG and PDC. OP counted colony formation assays. JMO-G and AYK provided thymic epithelial cells. EFM and CRB performed nasal brush biopsies. DM wrote a macro for and assisted with analysis of ciliary beat frequency. AFMD performed airway and lung organoid experiments. YI connected kenpaullone with WS6 and performed initial optimization experiments. REH performed lentiviral transduction experiments, analyzed data, visualized data and wrote the first manuscript draft. CO’C, SMJ and REH acquired funding for the project. All authors reviewed the manuscript and provided comments. Declaration of interests SMJ has received fees for advisory board membership from BARD1 Life Sciences. He has received grant income from GRAIL Inc. and is an unpaid member of a GRAIL advisory board. SMJ has received lecture fees for academic meetings from Chiesi and AstraZeneca and his wife works for AstraZeneca. REH has received speaker fees from AstraZeneca and REH and DRP have received royalties as inventors on licensed intellectual property licensed to AstraZeneca that is unrelated to the work in this manuscript. The remaining authors declare no conflicts of interest. Acknowledgements We thank Kyle O’Sullivan and Rebecca Towns (University College London Biological Services, UK) for technical assistance, Ayad Eddaoudi and Panagiota Constandinou for help with flow cytometry and cell sorting, and Stephen Hart (University College London, UK) for providing the BMI1 -transduced human airway basal cells used in Supplementary Fig. 2 . We thank Aleksandra Szejn (University of Leicester, UK) for assistance with TEM quantification. We are also grateful to Denise R. Bairros de Pilger (The Francis Crick Institute, UK) for access to an ECHO 550 liquid handler, and to the Francis Crick Institute Cell Services Facility for A549 and H520 lung cancer cell lines. Finally, we thank Sian Goldsworthy and UCL Genomics (University College London, UK) for running FASTQ and QDNAseq on low-pass whole-genome sequencing data. Funder Information Declared Medical Research Council, https://ror.org/03×94j517 , MR/R015635/1 , MR/W025051/1 Debra , GR000070 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982 Rosetrees Trust NIHR Great Ormond Street Hospital Biomedical Research Centre Royal Society , RG\R1\241421 Great Ormond Street Hospital Children's Charity Cancer Research UK References 1. ↵ Rheinwald , J. G. & Green , H . Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells . Cell 6 , 331 – 343 ( 1975 ). OpenUrl CrossRef PubMed Web of Science 2. ↵ Hynds , R. 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