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. Author manuscript; available in PMC: 2017 Aug 4.
Published in final edited form as: Cell Stem Cell. 2016 Jun 16;19(2):217–231. doi: 10.1016/j.stem.2016.05.012

Dual SMAD signaling inhibition enables long-term expansion of diverse epithelial basal cells

Hongmei Mou 1,2,3, Vladimir Vinarsky 1,2,4, Purushothama Rao Tata 1,2, Karissa Brazauskas 1,3, Soon H Choi 5, Adrianne K Crooke 5, Bing Zhang 6, George M Solomon 7,10, Brett Turner 8, Hermann Bihler 11, Jan Harrington 11, Allen Lapey 3, Colleen Channick 4, Colleen Keyes 4, Adam Freund 12, Steven Artandi 13, Martin Mense 11, Steven Rowe 7,8,9,10, John F Engelhardt 5, Ya-Chieh Hsu 6, Jayaraj Rajagopal 1,2,3,4,14
PMCID: PMC4975684  NIHMSID: NIHMS787427  PMID: 27320041

SUMMARY

Functional modeling of many adult epithelia is limited by the difficulty of maintaining relevant stem cell populations in culture. Here, we show that dual inhibition of SMAD signaling pathways enables robust expansion of primary epithelial basal cell populations. We found that TGFβ/BMP/SMAD pathway signaling is strongly activated in luminal and suprabasal cells of several epithelia, but suppressed in p63+ basal cells. In airway epithelium, SMAD signaling promotes differentiation, and its inhibition leads to stem cell hyperplasia. Using dual SMAD inhibition in a feeder-free culture system we were able to expand airway basal stem cells from multiple species. Expanded cells can produce functional airway epithelium that is physiologically responsive to clinically relevant drugs such as CFTR modulators. This approach is effective for clonal expansion of single human cells and for basal cell populations from epithelial tissues from all three germ layers, and may therefore be broadly applicable for modeling of epithelia.

Keywords: TGFβ/BMP4/SMAD signaling, dual SMAD signaling inhibition, p63+, basal cells, stemness, differentiation, dedifferentiation, replicative exhaustion, senescence, epithelial basal and stems cells, telomeres

Graphical abstract

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eTOC

Mou et al. show that small molecule-mediated SMAD signaling inhibition allows prolonged feeder-free culture of diverse functional epithelial basal stem cells in a 2D format. This methodology provides a facile patient-specific epithelial disease modeling platform, as shown by expanding airway epithelium from non-invasively obtained specimens from cystic fibrosis patients.

INTRODUCTION

Many epithelia are maintained by the regenerative capacity of adult stem cells. In stratified epithelia, basement membrane-resident p63+ basal cells can self-renew and often act as stem cells that give rise to differentiated progeny (Van Keymeulen and Blanpain, 2012; Melino et al., 2015; Mills et al., 1999; Yang et al., 1999). In some instances, adult basal cells function as uni-potent stem cells (Choi et al., 2012; Van Keymeulen et al., 2011), although these basal cells demonstrate a bi-potent stem cell plasticity upon transplantation (Van Keymeulen et al., 2011; Prater et al., 2014). Epithelial stem cell culture relying upon co-culture with irradiated fibroblasts was pioneered by Green and colleagues and remains the standard method for long-term expansion of human keratinocytes (Rheinwald and Green, 1975). The use of Rho-associated protein kinase (ROCK) inhibition has further improved epithelial culture systems (Liu et al., 2012) and was recently used to culture many organ-specific progenitor cells (Wang et al., 2015).

Despite these remarkable achievements, the prolonged expansion of adult epithelial stem cells remains challenging, and for many stratified and pseudostratified tissues there are no adequate culture systems, and importantly no methodology to clone cells. Furthermore, feeders, as a contaminating cell population, may interfere with the mechanistic interpretation of the effects of genetic and pharmacologic manipulation. Organoid culture models offer an alternative to fibroblast co-culture systems allowing stem cells from many organs propagated (Barcellos-Hoff et al., 1989; Rock et al., 2009; Sato et al., 2009; Tadokoro et al., 2016; Yin et al., 2014). However, after decades of epithelial stem cell culture research, the ability to expand and manipulate pure adult stratified and pseudo-stratified epithelial stem cells remains limited.

The transforming growth factor-β superfamily signaling have been implicated in regulating hematopoietic, hair follicle, melanocyte stem cell, and neural stem cell quiescence and activation (Genander et al., 2014; Kandasamy et al., 2014; Kobielak et al., 2007; Mira et al., 2010; Nishimura et al., 2010; Oshimori and Fuchs, 2012; Yamazaki et al., 2009). Additionally, BMP and TGFβ signaling balances stem cell proliferation, differentiation, and reversible cell cycle exit, and thereby regulates the maintenance and dynamic behavior of normal reservoirs of stem cells that are capable of responding to tissue injury (Guasch et al., 2007; He et al., 2004; Kobielak et al., 2007; Oshimori and Fuchs, 2012). Indeed, BMP/TGFβ signaling antagonists have already been used to facilitate the colon and mouse tracheal organoid culture (Sato et al., 2009; Tadokoro et al., 2016).

Given their roles in diverse stem cell systems, we assessed the role of BMP and TGFβ signaling in epithelia at large. We demonstrate that BMP and TGFβ signaling activity is relatively suppressed in p63+ basal cells, but is highly active in luminal and suprabasal cells which can represent both differentiated epithelial cells or unipotent non-basal progenitor cells (Choi et al., 2012; Van Keymeulen et al., 2011). We exploited the use of dual SMAD signaling inhibition to overcome the growth arrest and differentiation encountered in the culture of primary adult basal cells. The expanded murine and human cells maintain their ability to differentiate into functional tissues. Thus, we describe a culture system that may allow for the generation of non-invasive clinically-relevant disease models across many organ systems. Additionally, the methodology allows for the cloning of human stem cells. This in turn provides the key to being able to reproducibly develop clonally derived genetically modified human epithelia. Airway epithelia derived in this manner do lose their physiologic properties over time despite preserved cell differentiation. Future work will be needed to resolve this limitation.

RESULTS

SMAD signaling is highly active in the luminal epithelial compartment in airway

We used the mouse trachea as a model system to understand how TGFβ/BMP/SMAD signaling regulates basal stem cell behavior. The mouse trachea contains p63+/KRT5+ basal stem cells, which can self-renew and give rise to luminal (KRT8+) secretory club cells and ciliated cells (Rock et al., 2009). We note that both p-SMAD1/5/8 and p-SMAD2/3 staining (indicators of BMP and TGFβ signaling activity) are strongly positive in KRT8+ luminal cells (Figure 1A–B). In contrast, p63+ basal stem cells display weak or negative p-SMAD protein expression (Figure 1A–B). Furthermore, co-staining of p-SMAD with lineage-specific makers revealed that 92+/−8% of FOXJ1+ ciliated cells and 88+/−7% of SSEA1+ secretory cells are positive for p-SMAD1/5/8, while 94+/−10% of ciliated cells and 93+/−9% of secretory cells are positive for p-SMAD2/3 (Figure 1F), reflecting the dual activation of BMP and TGFβ signaling in luminal epithelia. In p63+ basal stem cells, while the vast majority of cells stain negative for p-SMAD, a small fraction of p63+ cells are clearly positive for p-SMAD1/5/8 (9+/−4%) and p-SMAD2/3 (10+/−4%) (Figure 1F). We speculate that this population represents differentiating basal cells. In fact, these p63+p-SMADlow cells also express a low level of KRT8, consistent with the onset of differentiation. We also demonstrate that nearly all Ki67+ replicating cells are negative or only weakly positive for p-SMAD1/5/8 and p-SMAD2/3 (Figure 1F and G), suggesting an inverse relationship between proliferation and SMAD signaling activity. The p-SMAD1/5/8 and p-SMAD2/3 staining pattern in the human bronchus mimics the pattern seen in the mouse trachea (Figure 1C–E and G).

Figure 1. SMAD signaling is active in differentiated cells but suppressed in basal stem cells of the airway epithelium.

Figure 1

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A and B. Co-staining of p-SMAD1/5/8 (A) or p-SMAD2/3 (B) with basal stem cell marker p63, differentiation maker KRT8, ciliated cell marker FOXJ1, club cell marker SSEA1 and proliferation marker Ki67 on mouse tracheal sections. C-D. Co-staining of p-SMAD1/5/8 with KRT5 and KRT8 (C) and with p63, FOXJ1, and Ki67 (D) on human bronchial sections. E. Co-staining of p-SMAD2/3 with p63, KRT8, FOXJ1, and Ki67 on human bronchial sections. For all, scale bar, 20 µm. F and G. Quantification of the percentage of p-SMAD1/5/8+ and p-SMAD2/3+ cells of the indicated cell types in mouse trachea (F) and human bronchus (G), n=3.

SMAD signaling is highly active in luminal and suprabasal cells, but suppressed in basal cells, in epithelial tissues of all 3 germ layers

We next examined p-SMAD status in diverse mouse epithelia that are developmentally derived from each of three germ layers (Figure 2A), including ectoderm-derived (skin, mammary gland), endoderm-derived (vocal fold, esophagus, prostate), and mesoderm-derived (epididymis) tissues. They are either stratified squamous (skin, vocal fold, esophagus) or pseudostratified (trachea, prostate, mammary gland, epididymis) epithelial tissues. All of these epithelial tissues possess p63+ basal cells and p63− luminal and suprabasal cells that are the progeny of basal cells (such as in skin, vocal fold, esophagus, airway, and epididymis) or that self-sustain (such as in mammary gland and prostate). We find that in all of these tissues p-SMAD1/5/8 and p-SMAD2/3 proteins are largely absent or weakly positive in p63+ basal cells and Ki67+ replicative cells, but are strongly positive in the p63− luminal and suprabasal cells (Figure 2B-2G). This consistent staining pattern suggests that the TGFβ/BMP/SMAD signaling pathways play a common role in diverse epithelia.

Figure 2. SMAD signaling is active in luminal and suprabasal cells, but suppressed in basal cells, in epithelial tissues derived from all 3 germ layers.

Figure 2

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A. Schematic of the analyzed p63+ basal cell-containing murine tissues derived from each germ layer. B-G. Co-staining of p-SMAD1/5/8 and p-SMAD2/3 with p63, Ki67, and a luminal and suprabasal marker KRT10 (B) or KRT13 (D and E) or KRT8 (C, F and G). Scale bar, 20 µm.

Activation of SMAD signaling occurs in differentiating luminal cells following injury

Since p-SMAD expression is primarily present in the differentiated luminal cells of airway, we assessed whether the phosphorylation of SMAD is associated with the progression of luminal differentiation during the early course of regeneration after sulfur dioxide (SO2) injury (Pardo-Saganta et al., 2015; Tata et al., 2013). We find that 8 hours post the injury (8 hpi), the majority of CK8+p-SMAD+ luminal cells in the proximal airway are sloughed off or loosely attached to the trachea, leaving behind a single layer of p63+SMAD− basal stem cells (Figure 3A–B and Figure S1A). At 24 hpi, we observe massive proliferation and expansion of basal cells (Figure S1B–C). These replicating cells, which are p63+ but KRT8− are negative for p-SMAD (Figure 3A–B and Figure S1A–C). Recently, we demonstrated that c-MYB-expressing basal cells and N2ICD-expressing basal cells start to segregate as early as 6 hours after SO2 injury, fully segregated by 24 hours post injury, and differentiate into ciliated and secretory cells, respectively (Pardo-Saganta et al., 2015). This suggests that the phosphorylation of SMAD is not involved in the cell fate segregation of basal stem cells during regeneration, but rather is associated with the actual process of luminal cell formation, as marked by KRT8 expression. Indeed, luminal cells at 48 hpi begin to express KRT8, coincident with increasing SMAD phosphorylation (Figure 3A–B and Figure S1A–C). By 72 hpi, the luminal cells further differentiate and begin to express mature lineage-specific markers (Pardo-Saganta et al., 2015; Tata et al., 2013). Consistently, these luminal cells are now strongly positive for p-SMAD, while basal stem cells remain p-SMAD-weak or negative (Figure 3A–B and Figure S1A–C).

Figure 3. Activation of SMAD signaling is associated with the progressive mucociliary differentiation.

Figure 3

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A. C57B6 mice were exposed to SO2. At 8, 24, 48 and 72 hours post SO2 injury, the mice were sacrificed and the trachea were collected for co-staining of p-SMAD1/5/8 with p63 and KRT8. The dashed line separates the sloughed luminal cell debris from the attached residual basal cell layer. B. Schematic of SMAD signaling activation changes along the course of mucocilary differentiation. C. Tracheal stem cells isolated from wild type, BMPRIAf/f, TGFβRIIf/f and SMAD4f/f mice were treated with Ad-Cre-GFP virus. The GFP+ cells were sorted to purity 1–2 days post infection. The cells were expanded briefly and then subjected to ALI differentiation. Left graph: quantification of ciliated cells and club cells in various conditions (mean ± s.d. n=3; **p ≤ 0.001). Right panel: whole mount staining of CCSP+ club cells and AcTub+ ciliated cells on ALI membranes. D. Immunostaining for KRT5, p63 and KRT8 of transverse sections of ALI membranes. The arrows indicate representative transitional differentiating cells. E. Tracheal stem cells were isolated from WT mice and differentiated on ALI for 14 days. At day 0, 1 µM DMH-1 and/or 1 µM A-83–01 were added to the lower chamber and upper chamber (as a thin layer) to inhibit the corresponding SMAD signaling activity. Left graph: quantification of ciliated cells and club cells in various conditions (mean ± s.d. n=5; **p ≤ 0.001, *p ≤ 0.01). Right panel: whole mount staining of CCSP+ club cells and AcTub+ ciliated cells on ALI membranes. F. Immunostaining for indicated markers of transverse sections of ALI membranes. The arrows indicate representative transitional differentiating cells. For all, scale bar, 20 µm. (See also Figure S1)

We also examined SMAD phosphorylation during human airway stem cell mucociliary differentiation in air-liquid interface (ALI) culture (Figure S1D). Twelve hours after ALI initiation (day 0.5), stem cells remain p63+KRT8− and also negative for both p-SMAD1/5/8 and p-SMAD2/3 (Figure S1E). The following day (Day 1.5), the cells begin to stratify to form a differentiated second layer of KRT8+p-SMAD+ cells (Figure S1E). Some p63+ cells are also positive for p-SMAD, perhaps suggesting that they are actively differentiating (Figure S1E). At day 2.5 and 3.5, more KRT8+p-SMAD+ differentiated cells appear, while the p63+ basal cells are now entirely p-SMAD negative or weak (Figure S1E). At day 13.5 when the luminal cells have already matured into ciliated cells and secretory cells, the phosphorylation of SMAD remain strongly positive in KRT8+ luminal cells, but is largely absent or weak in KRT5+ basal cells (Figure S1E). Thus, activation of SMAD signaling correlates with the appearance of KRT8+ luminal cells in both mouse and human airway epithelial differentiation (Figure 3B). Furthermore, phosphorylation of SMAD is persistent in mature airway luminal cells.

Inhibition of SMAD signaling compromises mucociliary differentiation

We next tested whether SMAD signaling blockade inhibits mucociliary differentiation. We isolated GSIβ4+EpCAM+ murine airway basal stem cells (Zhao et al., 2014) from the trachea of wild-type, BMPRIAf/f, TGFβRIIf/f and SMAD4f/f mice and infected them with Adeno-CreGFP virus to delete the various signaling pathway components (Figure 3C and Figure S1F). Mucociliary differentiation from the airway stem cells was performed using standard ALI culture. Two weeks after initiation of ALI, a mature airway epithelium generated from WT infected cells consists of ciliated cells and secretory cells. Deletion of BMPRIA, TGFβRII and SMAD4 results in significantly fewer differentiated cells (Figure 3C). Consistent with this finding, the loss of BMPRIA, TGFβRII and SMAD4 all resulted in airway epithelia with increased stratification and basal cells co-labeled with KRT8 (Figure 3D), suggesting that many basal stem cells are trapped in a transitional state as they are differentiating into KRT8+ luminal cells. Such differentiating cells are rarely found in the normal airway epithelium.

We also used BMP and TGFβ antagonists to examine mucociliary differentiation when BMP signaling or/and TGFβ signaling is blocked (Figure 3E). In controls, airway basal cells differentiated into airway epithelium with high efficiency after 2 weeks. In the presence of BMP antagonist (DMH-1) or TGFβ antagonist (A-83-01), fewer ciliated cells and club cells were generated. When both antagonists were used, the effect was more pronounced, with a >95% decrease in the production of differentiated cells (Figure 3E). Similarly, the treatment with BMP and TGFβ antagonists induced basal cell hyperplasia with hyper-proliferative KRT5+/p63+ cells and transitional KRT5+/KRT8+ cells lining the luminal compartment (Figure 3F). Of note, ciliated cells and club cells do appear after prolonged ALI culture even in the presence of the dual BMP/TGFβ inhibitors (Figure S1G). However, those ciliated and club cells that escape inactivation are positive for p-SMAD staining (data not shown) supporting the tight association of differentiation with p-SMAD status. In summary, both genetic deletion and pharmacological inhibition abrogate efficient generation of secretory and ciliated cells, suggesting that SMAD activation promotes continued differentiation of early committed cells to terminal maturation.

Blockade of SMAD signaling also compromises mucociliary differentiation in human cells. Differentiation was significantly suppressed when SMAD4 expression was reduced (Figure S1H–I). Only a small number of ciliated cells with truncated cilia were detected and club cells were nearly absent after 2 weeks of ALI culture (Figure S1I). Again, prolonged differentiation was associated with some mucociliary differentiation (Figure 1J). Consistently, the epithelia derived from SMAD4 shRNA-treated cells have numerous KRT5+KRT8+ and p63+KRT8+ cells trapped in transitional state. We also observed more Ki67+ cells in the SMAD4 shRNA epithelia compared to controls, suggesting that SMAD loss leads to increased proliferation (Figure S1J). These data indicate that there is cross-species conservation in the requirement for SMAD signaling to execute epithelial differentiation.

Dual SMAD signaling inhibition enables long-term airway stem cell expansion

Based on our observations that SMAD signaling is highly activated in non-cycling and differentiated luminal cells, we hypothesized that SMAD pathway inhibition would promote airway stem cell growth in vitro. Thus, we cultured human airway stem cells in various conditions (Figure 4A). Both BMP4 (50 ng/ml) and TGFβ (10 ng/ml) strongly suppressed cell growth. ROCK inhibitor (10 µM) alone modestly promoted cell expansion. However, both DMH-1 (1 µM) and A-83-01 (1 µM) greatly stimulated cell expansion. Three inhibitors together had the most profound effect (Figure 4A). The resulted cells were homogenous, small, and tightly packed as expected for stem cells. All cells were positive for KRT5 and p63, and >85% of cells were positive for Ki67 (Figure 4A). We also examined commonly used SMAD signaling inhibitors and found that they all similarly promoted cell growth (Figure S2A–B). Furthermore, we tested multiple previously reported culture media, including BEGM (Fulcher et al., 2005) and HTEK (You and Brody, 2013) (Figure S2C). Again, dual TGFβ/BMP inhibitors greatly enhanced cell proliferation in each of these media formulations. BMP and TGFβ are known to act through pathways other than the canonical SMAD pathway, including the MEK/MAPK, PI3K, and PKC-related cascades. Therefore, we tested the growth of human airway stem cells in the presence of their corresponding inhibitors. These compounds either had no effect or a detrimental effect on cell growth suggesting that BMP and TGFβ inhibitors are indeed acting through SMAD (Figure S2D).

Figure 4. Dual SMAD signaling inhibition facilitates long-term expansion of human airway stem cells.

Figure 4

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A. Human airway stem cells were expanded for 4 days in varying culture conditions. The cells were fixed and stained for p63, KRT5 and Ki67. Scale bar: upper two rows, 50 µm; lower two rows, 20 µm. B. Serial expansion of human airway stem cells in the control medium or the same medium with addition of TGFβ/BMP inhibitors. Left panel: phase contrast images of the cells at various passages. Right panel: the average population doubling times (mean ± s.d. n=10; ***p ≤ 0.0001) and a plot of number of doublings vs. the culture times of the human airway stem cells cultured in various media (from one representative culture). C. Mouse tracheal stem cells were expanded for 4 days in varying culture conditions. The cells were fixed and stained for p63/KRT5 and Ki67/SOX2. Scale bar: upper panel, 100 µm, bottom panel, 50 µm. D. Serial expansion of mouse tracheal stem cells in the control medium or the same medium with addition of TGFβ/BMP inhibitors. Top panel: phase contrast images of the mouse tracheal stem cells at various passages. Bottom panel: Left, the plot of number of doublings vs. the culture times of mouse tracheal stem cells cultured in various conditions (from one representative culture). Right, the staining of cell fate markers on mouse tracheal stem cells at passage 20. Scale bar, 20 µm. (See also Figure S2 and S5)

Next, we examined whether TGFβ/BMP inhibitors allows long-term expansion of human airway stem cells without fibroblast feeders. In the control medium, cells can only be passaged to P6 (~20 doublings at 1:10 splitting ratio, Figure 4B). Cells grown in the presence of TGFβ or BMP4 do not expand beyond P1. With dual TGFβ/BMP inhibition, however, cells are able to expand to P18–25 without a loss of replicative potential (~60–80 doublings, n=9, Figure 4B). They divide faster and are more homogenous in size in contrast to controls (Figure 4B). Nearly all cells express the stem cell markers as well as the transcription factors that define airway epithelial cell identity (Figure S2E–F). We did not detect KRT8 expression in the replicative p63+ cells (data not shown), suggesting that dual TGFβ/BMP inhibitors completely block differentiation in 2D culture. Furthermore, TGFβ/BMP inhibition enables single cell cloning. Human airway stem cells from 2 healthy subjects were sorted as singles and expanded individually to test their proliferation potential and mucociliary differentiation capacity following cloning. All clones examined had full capacity for significant expansion and multipotent differentiation into ciliated cells and secretory cells (Figure S2G). We also assessed our ability to expand bronchial stem cells from 2 patients harboring the ΔF508/ΔF508-CFTR mutation (Figure S2H). With control medium, the cells reached an expected expansion barrier at P4 and P5, whereas in the presence of TGFβ/BMP inhibitors, these cell lines expand to P12 and P17 and remained proliferative (Figure S2H). A similar effect is seen in mouse tracheal stem cell culture. Dual TGFβ/BMP inhibition strongly enhances cell proliferation (Figure 4C). As a result, mouse tracheal stem cells can be expanded in a feeder-free culture system for a considerably larger number of passages than previously possible while maintaining normal cell morphology and preserved stem cell identity (Figure 4D).

Loss of TGFβ signaling has been previously noted to lead to tumor formation in stratified epithelia (Guasch et al., 2007). To assess whether serial passaging is associated with tumorigenesis, we transplanted expanded human and mouse airway stem cells and human lung cancer A549 cells subcutaneously into NSG mice (Figure S2I). A549 cells robustly generate tumors in all cases after 2–8 weeks, with a latency determined by the number of injected cells (Figure S2I). We did not retrieve a single tissue outgrowth 8 months after injection of expanded human airway stem cells. Transplanted expanded mouse tracheal stem cells formed cystic structures, and immunofluorescence demonstrated that they were not tumors but rather comprised of differentiated ciliated and club cells (Figure S2J). The expanded cells remain genomically stable with no significant DNA alterations (no focal deletion, insertion, diploidy, or aneuploidy) when compared to early passage HUES8 and HUES9 cells (Figure S2K). Furthermore, transcriptome analysis of airway stem cells at early and late passages did not reveal any changes in tumor-associated gene expression (Figure S2L, GSE80408).

Expanded airway stem cells maintain the potential to differentiate into functional airway epithelia

We then tested the differentiation capacity of expanded human airway stem cells from early to late passages. The airway stem cells cultured in conventional medium can undergo efficient mucociliary differentiation only at early passages (Figure 5A). In contrast, human airway stem cells expanded in TGFβ/BMP inhibitors can generate airway epithelium with extremely high efficiency through passage 12, representing approximately 40 doublings (Figure 5A–B). Mucociliary differentiation remains robust through later passages, although the efficiency gradually declines. Secretory cell production appears to be more stable than ciliogenesis, as airway stem cells at P25 can still generate club cells (Figure 5A–B). Mouse airway stem cells also expand and differentiate through passage 12 (Figure S3A–B).

Figure 5. Expanded airway stem cells preserve the potential to differentiate into functional airway epithelia.

Figure 5

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A. Expanded human airway stem cells at different passages were differentiated on ALI for 16–20 days. The resulting membranes were fixed and stained for CCSP+ club cells and AcTub+ ciliated cells. Scale bar, 50 µm. B. Mucociliary differentiation efficiency at various passages (normalized to the efficiency of stem cell differentiation at P1 with TGFβ/BMP inhibitors) (mean ± s.d. n=3; **p≤0.001, *p ≤ 0.01). C. Immunofluorescence staining for markers of differentiation using wholemount (Scale bar, 100 µm) and histology sections (Scale bar, 20 µm) of P7 ALI cultures. Bottom, H&E staining of an ALI section (Scale bar, 50 µm). D. Staining for MUC5AC on human ALI cultures after treatment with IL-13. Scale bar, 50 µm. E. Area under the curve (AUC) ratios for CFTR correctors (Cmpd/Veh) measured on ΔF508/ΔF508 human bronchial ALI cultures (P4), (mean ± s.d. n=4; *p≤0.01). F. Chloride current density gradually declines with increased passages. Up to passage 8, the response to C18 remains at ~3-fold the vehicle response; at P10 and P12 with the decrease in currents, compound effects can no longer be resolved (mean ± s.d. n=3; *p ≤0.01). G. The expansion and differentiation of airway stem cells isolated from human tracheal biopsies (n = 30). (See also Figure S3, S4, and S5)

Expanded airway stem cells not only maintain their differentiation potential, but the resulting epithelia preserve their physiologic functions. For example, differentiated airway epithelial cells respond to IL-13 by induction of MUC5AC+ goblet cells in both human and murine epithelia (Figure 5D and Figure S3C). Additionally, airway epithelia maintain the ion channel physiology expected for their CFTR genotypes. Epithelium derived from CFTR-ΔF508 stem cells (P4) responds effectively to benzamil, an inhibitor of the epithelial sodium channel (ENaC) (Figure S3D). CFTR chloride ion transport activity is readily stimulated following addition of the cAMP agonist forskolin in combination with the VX770 potentiator. Pre-incubation with various CFTR-ΔF508 trafficking correctors (VX-809, VX-661, and C18) produced the anticipated salutary effects on chloride transport (Figure 5E and Figure S3D). Chloride currents also appropriately diminish with the addition of the specific CFTR inhibitor, CFTRinh-172 (Figure S3D). Importantly, Na+-currents and CFTR-mediated transmembrane Cl−-conductance declined over serial passages in both normal and CFTR-ΔF508 epithelia, while transepithelial resistance was preserved (Figure 5F and Figure S3E–F).

Given the ability to expand airway stem cells, we proceeded to test whether we can generate a large number of patient-specific airway stem cells from small clinically-relevant tissue biopsies. We performed mucosal biopsy with clinical biopsy forceps on discarded human trachea and bronchi. Thirty biopsies (10 biopsies from each of 3 donors) were taken throughout the proximal upper airways. We obtained 100 to 2000 viable airway basal stem cells per biopsy, and these cells proliferate robustly in the media with dual TGFβ/BMP inhibitor. Within 1 week, these cells expand to near 1 million cells, and we can passage at a 1:10 splitting ratio to ~1 ×1015 cells by P10 within 50 days. Differentiation analysis at P10 demonstrated that the cells are capable of forming polarized differentiated airway epithelia (Figure 5G).

Bronchoscopy with transbronchial biopsy is an invasive procedure that is associated with complications. Thus, we tested the possibility to expand airway stem cells from bronchoalveolar lavage (BAL) or simply from an induced sputum. We demonstrate that by using media with dual TGFβ/BMP inhibitors, a small number of cells (< 2000 cells) from sputum or discarded BAL can be expanded to 109–10 cells within a month and still remain proliferative (Figure S4A–B). Of patient samples, only one airway cell line was established from total 5 sputum samples. In the case of lavage, we produced 3 patient-specific lines out of 3 samples. The expanded cells were positive for the characteristic airway stem cell markers and are capable of forming polarized mucociliary airway epithelia (Figure S4A–B). Thus, large amounts of airway epithelium can be obtained from patients entirely non-invasively. We also tested dual TGFβ/BMP inhibition in pig and ferret, species for which clinically relevant animal models of cystic fibrosis have been generated (Rogers et al., 2008; Sun et al., 2010). Our culture approach robustly expands functional airway basal stem cells from the pig and the ferret (Figure S4C–E). Short-circuit current (Isc) measurements of differentiated ferret (P18) and pig epithelial cells (P7) demonstrate that, although the chloride current density has diminished, the CFTR activity is still intact at the indicated passage numbers (Figure S4F).

SMAD signaling inhibition promotes stem cell self-renewal by inhibiting differentiation, independent of telomere crisis-induced cellular senescence

Cellular senescence, defined as an irreversible cessation of cell division, has been proposed as the major obstacle preventing the long-term expansion of adult epithelial stem cells (Kiyono et al., 1998; Walters et al., 2013). However, we find that expression of the senescence marker p16ink4a is not correlated with growth arrest of human airway epithelial cells cultured in conventional medium (Figure S5A). On the other hand, we find that the majority of growth arrested cells are positive for KRT8, p-SMAD and display a downregulation of p63 (Figure S5A). This staining pattern also occurs when early passage stem cells (P0-P1) are exposed to TGFβ and BMP4 (Figure S5B–C). Indeed, TGFβ and BMP4-exposed cells stop proliferating, turn on KRT8, but do not alter p16ink4a expression (Figure S5B–C). Therefore, dual TGFβ/BMP inhibition contributes to sustained long term stem cell expansion primarily by suppressing differentiation.

Telomere crisis has been suggested as the mechanism for growth arrest (Kiyono et al., 1998). Thus, we measured telomerase activity and telomere length in human airway stem cells during serial passaging. We did not detect any endogenous telomerase activity in human airway stem cells (Figure S5D), even at P0. As a result, telomeres gradually shorten during successive cell divisions (Figure S5E). In control cultures, proliferation of airway stem cells ceases after passage 5–6, a barrier that has been noted previously (Fulcher et al., 2005; Walters et al., 2013). However, telomere lengths of cells in control culture are similar to the telomere lengths of cells cultured in TGFβ/BMP inhibitors at the same passage (Figure S5E). Thus, the observed growth arrest in control medium is not caused by telomere shortening-induced crisis as we predicted before. Furthermore, dual TGFβ/BMP inhibition does not stabilize the telomeres, as the telomeres continue to shorten over passaging (Figure S5E). After P25–30 (depending on the donor), airway stem cells stop proliferating abruptly. This ultimate growth arrest correlates precisely with complete erosion of telomeres. Telomere length likely demarcates the intrinsic proliferative potential of primary cells, while TGFβ/BMP dual inhibition facilitates stem cell self-renewal until eventually they reach a bona fide telomere crisis-induced growth arrest. To test this hypothesis, we overexpressed hTERT in airway stem cells at early passage (P3) in order to stabilize the telomeres, and we see a corresponding increase in the number of passages tolerated (Figure S5F–G). As predicted, cells with hTERT-stabilized telomeres remain acutely sensitive to differentiation induction triggered by withdrawal of the TGFβ/BMP inhibitors and the addition of TFGβ and BMP (Figure S5H). This data in aggregate demonstrates that precocious differentiation is the mechanism explaining the decreased cell replication and growth arrest that occurs in conventional cultures.

We next examined the effect of dual TGFβ/BMP inhibition on differentiated cells. Our laboratory has reported that airway club cells can undergo dedifferentiation into basal stem cells (Tata et al., 2013). By using CCSP-YFP club cells, we demonstrate that inhibition of SMAD signaling enhances the dedifferentiation of club cells into p63+ basal cells, while forced activation of SMAD signaling prevents it (Figure S5I–L).

Dual SMAD signaling inhibition enables the in vitro expansion of functional keratinocytes

We hypothesize that dual SMAD signaling inhibition can be used to expand many diverse epithelial basal cell types. We first tested this hypothesis with epidermal keratinocytes as they were historically the first extensively cultured basal stem cells (Rheinwald and Green, 1975). Our data indicated that human keratinocytes cannot be expanded without the use of feeders cells, consistent with the published literature (Rheinwald and Green, 1975, 1977) (Figure 6A). With feeder cells, keratinocytes form holoclones and can be passaged for many generations as previously described (Rheinwald and Green, 1977), but with the expected long doubling time (experiments were terminated at P8) (Figure 6A). In the presence of TGFβ/BMP inhibitors, keratinocyte growth is no longer dependent on feeder cells (Figure 6A). Indeed, the cells show faster growth rates, with a 2–3-fold decrease in the population doubling time compared to growth on feeders. Interestingly, in the presence of TGFβ/BMP inhibitors, the addition of feeder cells does not further promote keratinocyte proliferation (Figure 6A). Human keratinocytes grown in inhibitor media are uniformly positive for typical stem cell markers (Figure 6B). When differentiated on ALI, expanded human keratinocytes generate stratified epithelial cells with the appropriate cellular architecture and markers akin to their in vivo counterparts (Figure 6C).

Figure 6. Dual SMAD signaling inhibition enables long-term expansion of functional keratinocytes.

Figure 6

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A. Doubling time of adult human keratinocytes cultured in four different conditions: (i) Standard media, (ii) Standard media + J2 feeder cells, (iii) Inhibitor media, (iv) Inhibitor media + J2 feeder cells. (mean ± s.d. n=3; **p≤0.001). B. Phase contrast images and marker staining of human keratinocytes at indicated passages expanded in a feeder-free mode in the medium containing TGFβ/BMP inhibitors. Scale bar: 100 µm. C. Section immunofluorescence of various basal and differentiation markers on ALI culture (P4 and P12) and on human skin. Scale bar: 20 µm. D. Isolated mouse keratinocytes were seeded at 3% cell density and expanded for 4 days in various culture conditions. The cells were then fixed and stained for p63, KRT5, and Ki67. Scale bar: 100 µm. E. Phase contrast images of mouse skin keratinocytes at indicated passages. Scale bar: 50 µm. F. Differentiation of expanded mouse skin keratinocytes (P10) on ALI. The derived stratified epithelia were positive for KRT10 and involucrin.

Mouse keratinocyte expansion also depends on a feeder layer at the start of the culture, although unlike human keratinocytes, mouse keratinocytes can be cultured off feeders after several generations. We cultured and expanded murine skin keratinocytes in feeder-free manner with and without TGFβ/BMP4, as well as with their inhibitors (Figure 6D–E). Murine skin keratinocytes fail to grow in the presence of TGFβ or BMP4. In control media, the cells grow slowly and cannot be expanded to higher passages (Figure 6D–E). However, TGFβ/BMP inhibitors robustly stimulate cell proliferation and enable prolonged cell expansion (Figure 6D–E). Similarly, the expanded mouse skin keratinocytes produce a stratified epithelium when differentiated on ALI (Figure 6F).

Dual SMAD signaling inhibition enables in vitro expansion of a very diverse set of epithelial basal cells

In order to further explore the potential generalized use of dual SMAD signaling inhibition in promoting epithelial basal cell expansion, we isolated murine basal cells from representative epithelial tissues from each of the three germ layers. We demonstrate that epithelial basal cells isolated from esophagus (Figure 7A), epididymis (Figure 7D–E), larynx/vocal fold (Figure S6A–B), forestomach (Figure S6D–E) and mammary gland (Figure S7A–B) can be expanded to high passage numbers, while still maintaining their replicative potential, as well as normal cell morphology and appropriate stem cell markers.

Figure 7. Dual SMAD signaling inhibition enables in vitro expansion of a diverse set of organ-specific basal cells.

Figure 7

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A. Phase contrast images and marker staining of mouse esophageal basal cells at indicated passages expanded in the medium containing TGFβ/BMP4 inhibitors. Scale bar: 100 µm (Phase contrast images) & 50 µm (immunofluorescence). B-C. H&E (B) and immunofluorescence (B & C) of ALI culture sections derived from esophageal basal cells (P12) and of mouse esophageal. Scale bar: 20 µm. D. Schematic and example of organoid differentiation of expanded mouse epididymis basal cells isolated from KRT5-tdTomato-B1EGFP mouse. E. Phase contrast images and immunofluorescence of mouse epididymis basal cells at indicated passages expanded with TGFβ/BMP4 inhibitors. Scale bar: 100 µm. F. Fluorescence images of tdTomato and EGFP in differentiated organoids from D (upper panels are whole mount 3D organoids (Scale bar: 100 µm) and bottom panels are sections of the organoids (Scale bar: 20 µm). G. Co-immunofluorescence of indicated markers with tdTomato on epididymis organoid sections. Scale bar: 20 µm. (See also Figure S6 and S7)

The expanded cells also retain the capacity to differentiate efficiently. We demonstrate that expanded esophageal basal cells generate a stratified epithelium on transwell membranes efficiently (Figure 7B–C). The differentiated stratified esophageal epithelium has appropriate cellular morphology and markers, consistent with those of esophageal tissue (Figure 7B–C). Similarly, the expanded laryngeal basal cells (Figure S6C) and forestomach basal cells (Figure S6F) produce stratified epithelia when differentiated on ALI, and they possess the same cellular architecture and marker expression as their corresponding tissues (Figure S6C and S6F).

Expanded epididymis basal cells and mammary basal cells generate organoids efficiently in Matrigel. Epididymis basal cells from a KRT5-tdTomato-B1EGFP mouse (V-ATPase B1 is a differentiated clear cell marker, V-ATPase B1-GFP is referred to as “B1EGFP”) (Miller et al., 2005) generate organoids contain GFP+ cells, indicating the presence of clear cells (Figure 7F). The epithelia are also positive for typical markers found in the epididymis tissue (Figure G and Figure S7G). Furthermore, the expanded epididymis basal cells form spheres after subcutaneous injection in vivo (Figure S7E–F). Tubular organoids contain a pseudostratified epididymal epithelium characterized by KRT5+ basal cells and KRT8+ luminal cells, as well as principal cells marked by AQP9 (Figure S7F). Similarly, derived mammospheres from expanded mammary basal cells possess typical cellular architecture and marker expression characteristic of mammary tissue (S7C–D). Taken together, we have identified a method to expand epithelial basal cells from a diverse collection of murine tissues and demonstrated preserved differentiation potential of the expanded basal cells.

DISCUSSION

Dual SMAD signaling inhibition permits the expansion of adult epithelial basal cells from a wide array of organs and highlights a commonality of the role of the TGFβ/BMP/SMAD signaling pathway in regulating epithelial basal cell behavior. Prior cultures that were considered to have arrested because of senescence likely failed due to unwanted differentiation. Interestingly, dedifferentiation can be enhanced using TGFβ/BMP/SMAD signaling inhibitors demonstrating that the pathway has consistent effects on differentiation and dedifferentiation, implying that differentiation and dedifferentiation are mechanistically two sides of the same coin. Strictly speaking, TGFβ/BMP/SMAD signaling inhibition compromises terminal differentiation. A nearly identical phenotype has been reported following YAP overexpression in airway basal stem cells (Zhao et al., 2014). These converging phenotypes are no doubt related to the mechanisms governing specific steps of the differentiation cascade. We speculate that the effect of dual SMAD signaling inhibition reflects the presence of a highly evolutionarily conserved signaling network that regulates p63 and its associated pathways to maintain epithelial basal identity and prevent differentiation.

A single bronchoscopic lavage reproducibly generates the substrate for large quantities of airway epithelium. Once bovine pituitary extract and laminin-enriched 804G coating is eliminated, our method is completely defined with no limiting reagents or possibility to introduce xeno-contaminants. Remarkably, in 1 out of 5 cases, even sputum from CF patients was sufficient to generate diseased airway epithelium. If methods for sputum induction could be improved, this would represent the least invasive way to generate human stem cells in any existing organ. It is possible that organs that are damaged in a disease process would be more likely to yield stem cells since more epithelial cell are being shed. Similar non-invasive methodologies might be used to generate epithelia from the secretions of many other epithelial organs, so that the collection of human stem cells is less invasive than a routine blood test. For now, a bronchoscopic lavage or nasal brushing is required for perfect yield but again sinus lavage is likely sufficient to reproducibly yield large quantities of respiratory epithelium for study. Furthermore, we hope our newfound ability to clone human epithelial stem cells will enable the creation of reproducible human model systems that can be genetically modified.

Despite these promising results, it is necessary to recognize that in vitro culture still has limitations. For example, we observed that expanded human airway stem cell-derived epithelia gradually lose CFTR-mediated chloride and ENaC-mediated sodium conductance, but interestingly they do maintain transepithelial resistance and differentiation potential. In fact, several studies (Pavlov et al., 2014; Staruschenko et al., 2004) have demonstrated the negative effect of ROCK inhibitor Y27632 and EGF on ENaC activity, which may explain the gradual decrease in ENaC-mediated sodium conductance in cells expanded in the presence of Y27632 and EGF. Partial loss of cellular physiological function in in vitro culture systems may be more common than previously recognized and may be tissue-specific and function-specific. For example, airway epithelium appears to preserve club cell differentiation potential longer than ciliated cell differentiation potential, and CFTR-mediated function appears to be lost even faster than ciliated cell differentiation. We speculate that the mechanisms involved in the differential deterioration of different physiologic cellular functions at distinct passage numbers are likely epigenetic. If so, epigenetic modulation is likely to be of value.

EXPERIMENTAL PROCEDURES

Human and mouse airway stem cell isolation and serial expansion

Human and mouse airway stem cells are isolated from fresh human tissues (trachea and mainstem bronchi) or mouse trachea (Zhao et al., 2014). The cells are cultured in the medium either alone or with 5–10 µM ROCKi, 0.5–1 µM A-83-01, 0.5–1 µM DMH-1 and 1 µM CHIR99021 separately or in various combinations on plates pre-coated with laminin-enriched 804G–conditioned medium. To split the cell cultures, the cells are dissociated with trypsin and re-seeded at 1:10 ratio (See supplemental information for detailed isolation and culture method).

Mucociliary differentiation on air-liquid interface

Human or mouse airway stem cell cultures are seeded onto 0.4 µm transwell membranes pre-coated with 804G-conditioned medium with a density of >6000 cells/mm2. After cell attachment for >12 hours, excess cells are removed and the medium is replaced with complete Pneumacult-ALI medium (StemCell Technology, Cat. 05001) or vertex ALI medium (Neuberger et al., 2011) filling both the upper and lower chambers. The next day, ALI medium is added only in the lower chamber to initiate airway-liquid interface. Then the medium is changed daily until differentiation is well established. Ciliogenesis is monitored by inverted-phase microscopy for beating cilia. We culture ALI for 14–16 days as a standard protocol. For some experiments, we extend the ALI culture for 4–5 weeks.

TSA-based immunofluorescence

All Phospho-SMAD staining is performed using Tyramide Signal Amplification (TSA) method. After antigen retrieval, the slides are incubated with diluted primary antibody at 4°C overnight. Of note, the primary antibodies used in TSA staining are usually diluted at 1:5000–1:10,000. The next day, the slides are incubated with HRP-conjugated antibody at 1:2000 for 1–2 hours and then with TSA working solution (1:100 dilution) for 15 min and followed by incubating with Streptavidin-594 diluted at 1: 200 for 10–30 min (see supplemental information for detailed staining method).

Supplementary Material

Highlights.

  • SMAD activity is active in suprabasal cells but weaker in basal epithelial cells

  • SMAD signaling activity correlates with mucociliary differentiation in the airway

  • Dual TGFβ/BMP inhibition prevents spontaneous differentiation in culture

  • Dual TGFβ/BMP inhibition allows prolonged culture of diverse epithelial basal cells

Acknowledgments

J.R. is a New York Stem Cell Foundation Robertson Investigator and a Maroni Research Scholar at MGH. We thank Marina Mazur (the University of Alabama at Birmingham) for assistance with cell culture and the entire Rajagopal Laboratory for constructive comments. This research was supported by grants from the NIH (R01HL116756, R01HL118185 to J.R.; DK047967, HL051670 to J.F.E; K08HL124298 to V.V.; T32HL087735 to H.M.; R00 AR063127 to Y-C. H.), the Cystic Fibrosis Foundation (RAJAGO12G0 and RAJAGO12I0 to J.R.), and the Smith Family Awards and March of Dimes Basil O’Connor Starter Scholar Awards (to Y-C.H.)

Footnotes

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SUPPLEMENTAL INFORMATION

Full Experimental Procedures and 7 Supplemental Figures.

AUTHOR CONTRIBUTIONS

H.M. designed and conducted experiments and wrote manuscript. V.V. implemented IRB protocols. V.V., P.R.T, and K.B. assisted in mouse work. S.H.C., A.K.C., and J.F.E. performed ferret and pig tracheal stem cells culture and analysis. B.Z. and Y.H. performed human keratinocyte culture and analysis. G.M.S., B.T., J.H, H.B., M.M and S.R. conducted CFTR physiological function analysis. A.L., C.C and C. K. collected sputum and BAL. A.F and S.A performed the TRAP and TRF. V.V., P.R.T and J.R edited manuscript. J.R. oversaw the work.

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