Airway epithelial stem cell renewal and differentiation: overcoming challenging steps towards clinical-grade tissue engineering

Abstract

Background: Despite their life-saving potential, tissue engineering approaches for the treatment of extensive tracheal and bronchial defects still face significant limitations. A major challenge is the inability to regenerate a functional airway epithelium containing the appropriate amount of stem cells required for long-term tissue renewal following transplantation of the bioengineered graft. In this scenario, extensive cell culture characterization, validation assays and quality controls are needed to guide each step of the regeneration process.

Methods: Stem cell depletion is often due to suboptimal culture conditions, therefore we tested the ability of a clinical-grade culture system to support the safe and efficient in vitro expansion and differentiation of primary human tracheal and bronchial epithelial cells. Single-cell clonal analysis was used to unravel the heterogeneity of airway basal cells and to understand tissue-specific regeneration and differentiation mechanisms. Functional assays were used to investigate the wound healing ability and tightness of the regenerated epithelium under the selected culture conditions.

Results: Primary tracheobronchial epithelial cells showed an impressive proliferative potential, allowing the regeneration of a mature and functional epithelium without immortalisation events. Analysis at the single cell level allowed the identification of the subpopulation of basal cells endowed with in vitro self-renewal, distinguishing them from transient amplifying cells. This approach has further defined the hierarchy of cellular differentiation and its correlation with regenerative and differentiation potential.

Conclusions: Our results show that primary airway epithelial cell cultures can maintain stem cells together with their differentiation lineages in vitro. Airway cells can be safely and effectively used in autologous tissue engineering approaches when cultured under appropriate and well-standardised conditions. In addition to the validation assays proposed for the development of new advanced therapy products, this study outlines possible quality controls to enhance therapeutic success and maximise patient safety in future clinical applications.

Keywords: Airway differentiation; Airway holoclones; Airway reconstruction; Clinical-grade expansion; Epithelial stem cells; Regenerative medicine; Respiratory mucosa; Stem cell markers; Tissue engineering; Tracheobronchial epithelium.

Fig. 1

Cell extraction and long-term proliferative potential of primary human airway epithelial cells. (A) Comparison of the clonogenicity of cells directly extracted from AT (n = 4) and AB (n = 5) biopsies to that after the first expansion passage. Each independent strain is indicated by a different shape. Unpaired, biparametric, two-tailed t test. (B) Representative images of the CFE assay performed at cell extraction (biopsy, 1000 cells seeded) and after one cell passage (primary culture, 400 cells seeded). (C) Graphs showing quantification of the CFE assay during the AT (left, n = 3) and AB (right, n = 3) lifespans. The blue lines indicate the percentage of clonogenic cells (number of grown colonies/total seeded cell ratio); the red lines indicate the percentage of aborted colonies (aborted colonies/total grown colonies ratio). Independent human strains are indicated by different shapes. (D) Histograms showing the cell doubling (left) and doubling time (right) of AT (n = 3, green bar) and AB (n = 3, orange bar) independent strains, indicated by different shapes. Unpaired, biparametric, two-tailed t test. The values are presented as mean ± SD. *** p < 0.001, ** p < 0.01. (E) Representative images of CFE indicator dishes showing the trend of clonogenic and aborted colonies in AT3 and AB2 lifespans. The number of cells seeded in AT3 serial passages was 300 (p2), 500 (p12), 2000 (p20), and 67,000 (p27), and the number of cells seeded in AB2 serial passages was 400 (p2), 600 (p10), 700 (p22), and 250,000 (p28). p: passages

Fig. 2

Characterization of airway epithelial cell cultures. (A) IF staining of AT (left) and AB (right) cultures at four time points during the lifespan of airway epithelial cells (representative images of three AT and three AB primary independent human strains). Scale bar, 50 μm. (B) WB analysis of total cell extracts from six expansion passages (AT2 strain) that represent five consecutive lifespan intervals (I-V), immunostained with the indicated antibodies. The experiment was conducted with n = 3 AT strains (n = 5 technical replicates) and n = 3 AB strains (n = 4 technical replicates). (C) Histograms showing the quantification of the expression levels of CK14, involucrin, p63α, BMI1 (all normalized per GAPDH), and SOX2 (normalized per Vinculin) from the top left to the bottom right. Average and SD of n = 3 AT and n = 3 AB are displayed per time range (see Methods). Independent strains are indicated by different shapes. For multiple pellets analysed within the same interval, the mean expression value was considered. Unpaired, biparametric, two-tailed t test * p < 0.05; ** p < 0.01; and *** p < 0.001

Fig. 3

Regenerative and differentiation properties of human airway cultured cells. (A) IF images showing goblet (MUC5AC-positive) and club (uteroglobin-positive) cells in progressive passages of the AT1 (upper panel) and AB2 (lower panel) lifespans. The same analysis was repeated for the 3 AT and three AB primary strains. Scale bar, 50 μm. (B) Graphical representation of the abundance of goblet and club cells during AT (upper panel) and AB (lower panel) lifespans. Data collected from n = 3 AT and n = 3 AB independent strains are indicated by different shapes. Grading: -, none; +, low; ++, moderate; +++, high; and ++++, very high (see Methods). (C) IF images of MUC5AC (red) and uteroglobin (green) staining in AT and AB cultures. The merged image highlights the presence of double-positive cells coexpressing MUC5AC and Uteroglobin. The white triangles highlight MUC5AC/Uteroglobin double-positive cells. These hybrid cells were observed in n = 3 AT and n = 3 AB-independent strains. Scale bar, 50 μm. (D) Cartoon showing air-liquid interface (ALI) culture protocol. From left to right: cells are seeded onto a de-epithelialized human matrix, cultured for 7 days under submerged conditions, and exposed to ALI for 28 days. (E) Representative images of the epithelium regenerated by AT (n = 3) and AB (n = 3) independent human strains (n = 9 technical replicates). The dotted line indicates the epithelial basal layer; the asterisk indicates the mucous released by goblet cells. Scale bar, 50 μm

Fig. 4

Basal cell heterogeneity and hierarchy. (A) Representative images of clones positive for CK14, Uteroglobin, and MUC5AC. The brightfield image shows the clone morphology. Clones are indicated by white dotted lines. Scale bar, 50 μm. (B) Graph showing the percentage of clones expressing specific markers quantified in: CK14, n = 63 AT and n = 91 AB clones; Uteroglobin, n = 24 AT and n = 29 AB clones; and MUC5AC, n = 48 AT and n = 29 AB clones. (C) Scheme of the clonal analysis procedure. (D) CFE dishes used to classify the different types of clones. Analysis was conducted in n = 3 independent AT strains (384 total AT clones analysed via n = 9 clonal analyses) and in n = 2 AB independent strains (168 total AB clones analysed via n = 4 clonal analyses). (E) Clonogenicity after self-renewal assay. For each clone, the total percentage of grown colonies after stratification is presented as the sum of the percentage of aborted colonies, (light brown) and the percentage of growing colonies, (light yellow). AT2 analysed clones: H, n = 7; EM, n = 4; IM, n = 12; LM, n = 5; P, n = 1.(F) Graphs showing average AT and AB residual clonogenic potential values after stratification of H, EM, IM, LM, and P progeny. The high standard deviation reflects the heterogeneity of H (0–6% of aborted colonies). Analysis was conducted using n = 3 AT strains (H, n = 14; EM, n = 14; IM, n = 29; LM, n = 13 and P, n = 20) and n = 1 AB strain (H, n = 15; EM, n = 4; IM, n = 7; and LM, n = 2). Unpaired, biparametric, two-tailed Welch’s t test was used. The values indicate means ± SD. ***p < 0.001. ****p < 0.0001

Fig. 5

Morphological and molecular characterization of airway clones. (A) Violin plot showing the size measured in mm² of AT (left) and AB (right) clones. AT clones: H, n = 36; EM, n = 86; IM, n = 112; LM, n = 56; P, n = 45 belonging to n = 3 independent AT strains; AB clones: H, n = 35; EM, n = 44; IM, n = 52; LM, n = 13; P, n = 9 belonging to n = 2 independent AB strains. Dots represent single clones. Median, first and third quartiles are displayed. (B) WB analysis of total cell extracts from the progeny of AT2 clones (H, n = 3; EM, n = 2; IM, n = 1; LM, n = 2) immunostained with the indicated antibodies (images representative of n = 2 analyses conducted with independent clones). (C) Bar graphs showing the quantification of the expression levels of p63α, BMI1 (normalized to GAPDH), and SOX2 (normalized to Vinculin in AT and to Actin in AB) from left to right. Averages and SD are displayed per clonal category (see Methods). AT2 clones: H, n = 3; EM, n = 5; IM, n = 5; LM, n = 3. AB1 clones: H, n = 3; EM, n = 2; IM, n = 3; LM, n = 3. Due to their very limited residual proliferative potential, the amount of material collected from the progenies of the AT2 and AB1 paraclones was insufficient to carry out a reliable WB analysis. Unpaired, biparametric, two-tailed t test * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001

Fig. 6

Differentiation potential of the clones. (A) Representative IF images of the different types of progenies of AT clones cultured under standard submerged conditions. Analysis was conducted using n = 3 AT (H, n = 11; EM, n = 10; IM, n = 25; LM, n = 13; P, n = 3) and n = 1 AB strains (H, n = 7; EM, n = 6; IM, n = 4; LM, n = 4; P, n = 1). The dotted line indicates the epithelial basal layer. (B) Double IF staining of AT and AB epithelial cells revealing coexpression of MUC5AC (red) and Uteroglobin (green) in the progeny of airway H, EM, IM, and LM. White triangles highlight MUC5AC/Uteroglobin-positive cells. (C) Representative IF images of the different types of progenies of AT clones cultured under ALI conditions. Analysis was conducted on n = 3 AT (H, n = 11; EM, n = 19; IM, n = 17; LM, n = 14; P, n = 13) and n = 1 AB strains (H, n = 2; EM, n = 1). Dotted line indicates the epithelial basal layer

Fig. 7

Assays to evaluate the functionality of the regenerated airway epithelium. (A) Representative images of airway epithelia grown in CG (left) or BEGM (right) culture systems at five consecutive time points after the scratch (0 h); images were acquired via live imaging (Cell Observer^®, Zeiss). The assay was conducted with n = 1 AT and n = 1 AB primary cultures from independent donors. Scale bar, 50 μm. (B) Quantification of the percentage of in vitro wound closure in AT and AB epithelia under CG or BEGM culture conditions (see Methods). The wounded area was quantified via AxioVision version 4.8. (C) Graph showing the TEER measurements obtained at six consecutive time points during AT (green bars) and AB (orange bars) ALI cultures. Average. The average and standard deviation are displayed per time point based on three technical replicates of n = 1 AT and n = 1 AB strain. TEER measurements were conducted with an ERS-2 voltmeter (see Methods). (D) Representative SEM images of AT and AB regenerated epithelia showing apical cilia (c) and microvilli (m). The dotted square highlights the area magnified in the corresponding inferior image

Fig. 8

Airway clone renewal and differentiation potential. Cartoon showing a model of in vitro human airway epithelium renewal and differentiation potential of clones. H, EM, IM, and LM basal clones display multipotency in differentiating into club, goblet, and ciliated cells, whereas paraclones can only differentiate into goblet cells. Among specialized cell types, club cells may differentiate into goblet cells