Integrin α6β4 Identifies Human Distal Lung Epithelial Progenitor Cells with Potential as a Cell-Based Therapy for Cystic Fibrosis Lung Disease

<div><p>To develop stem/progenitor cell-based therapy for cystic fibrosis (CF) lung disease, it is first necessary to identify markers of human lung epithelial progenitor/stem cells and to better understand the potential for differentiation into distinct lineages. Here we investigated integrin α6β4 as an epithelial progenitor cell marker in the human distal lung. We identified a subpopulation of α6β4⁺ cells that localized in distal small airways and alveolar walls and were devoid of pro-surfactant protein C expression. The α6β4⁺ epithelial cells demonstrated key properties of stem cells <i>ex vivo</i> as compared to α6β4^- epithelial cells, including higher colony forming efficiency, expression of stem cell-specific transcription factor Nanog, and the potential to differentiate into multiple distinct lineages including basal and Clara cells. Co-culture of α6β4⁺ epithelial cells with endothelial cells enhanced proliferation. We identified a subset of adeno-associated virus (AAVs) serotypes, AAV2 and AAV8, capable of transducing α6β4⁺ cells. In addition, reconstitution of bronchi epithelial cells from CF patients with only 5% normal α6β4⁺ epithelial cells significantly rescued defects in Cl^- transport. Therefore, targeting the α6β4⁺ epithelial population via either gene delivery or progenitor cell-based reconstitution represents a potential new strategy to treat CF lung disease. </p> </div

Human α6β4⁺ cells differentiate into both basal and Clara cells <i>ex</i><i>vivo</i>.

<p>α6⁺ cells were isolated from normal human distal lung and cultured in Matrigel for 33 days. (A, B, C) Colonies were stained with (A) basal cell marker K-5 (red) and (B) Clara cell marker CC10 (green). (C) Merged image of K-5 and CC10. (D, E, F) Immunostaining of colonies with (D) type II AEC marker SPC (red) and (E) β4 (green). (F) Merged image of SPC and β4 staining. Nuclei were stained with DAPI (blue). Scale bar=100 µm.</p

Identification and localization of α6β4⁺ cells in the human distal lung.

<p>(A) Epithelial cells were isolated from a normal human lung, and surface markers α6 and E-cadherin were labelled with Alexa Fluor-568 and Alexa Fluor-647, respectively, and analyzed by FACS. Left panel: staining with primary antibody isotype control IgG; right panel: staining with antibodies against α6 and E-cadherin. The P4 gate indicates cells that were positive for both α6 and E-cadherin, whereas the P5 gate indicates E-cadherin⁺ but α6^- cells. (B) Populations of cells from the P4 or P5 gate in (A) were isolated, cytospinned on slides, and immediately immunostained with antibodies against SPC, CC10 or K-5. Data are the quantitation of expression of SPC, CC10 and K-5 on either α6⁺ (left pie chart) or α6^- (right pie chart) cells. (C, D) Localization of α6β4⁺ cells was determined by co-immunostaining the alveolar (C) region or distal airway (D) in normal human lungs with β4 (green) and SPC (red) or K-5 (red). Nuclei were stained with DAPI (blue). (C) Upper panel, single channel images of β4. Lower panel, merged image of cells co-immunostained with β4 and SPC. Arrow indicates a β4⁺/SPC^- cell in the alveolar region. (D) Upper panel, merged image of distal airway at low magnification. Lower panels show enlarged view of dotted box. Lower left panel, single channel images of β4. Lower right panel, merged image of cells co-immunostained with β4 and K-5. Arrow indicates a β4⁺/K-5^- cell in the distal airway; arrowhead indicates a β4⁺/K-5⁺ cell in the distal airway. Scale bar= 50 µm.</p

Co-culture of α6β4⁺ cells with HUVECs <i>ex</i><i>vivo</i> enhances proliferation but does not alter differentiation.

<p>(A, B) α6⁺ cells were isolated from normal human distal lung, mixed with HUVECs, and cultured in Matrigel for two weeks. (A) Phase contrast images of α6⁺ cells cultured without HUVECs (left panel) or with HUVECs (right panel). Arrow indicates colonies arising from α6β4⁺ cells; arrowhead indicates single HUVEC cells. Scale bar= 200 µm. (B) Colony forming efficiency was analyzed by counting the number of colonies present 14 days after seeding 5000 α6⁺ cells in the presence or absence of HUVECs. * <i>P</i><0.05 compared to α6β4⁺ cells alone. Data are representative of 4 experiments from 4 independent donors. Lines denote paired samples from the same donor. (C) Merged image of colonies arisen from co-culture of α6β4⁺ cells and HUVEC stained with K-5 (red) and CC10 (green). Scale bar=100 µm. (D) Merged image of immunostaining with SPC (red) and α6 (green) in colonies arisen from co-culture of α6β4⁺ cells and HUVEC. Scale bar=100 µm. (E) Merged image of immunostaining with goblet cell marker Muc5AC (green) and CC10 (red) in colonies arisen from co-culture of α6β4⁺ cells and HUVEC. Nuclei were stained with DAPI (blue). Scale bar=50 µm.</p

Reconstitution with only 8% of normal human α6β4⁺ cells rescues CFTR function in epithelia from patients with CF.

<p>(A) Wild type α6β4⁺ epithelial progenitor cells at various percentages (1%-8%) were mixed with bronchial epithelial cells from patients with CF (CF) and cultured for two weeks in transwells at the air-liquid interface. Responses to cAMP agonists (forskolin + IBMX) were determined by Ussing chamber. Non-CF: bronchial epithelial cells from non-CF lung. Data are shown as mean ± SEM; n=4-5 experiments from 4-5 different donors. *<i>P</i><0.05 compared to CF 0%. (B-D) Normal human GFP⁺ cells derived from α6β4⁺ epithelial cells were mixed with CF bronchial epithelial cells and cultured for two weeks in transwells at the air-liquid interface. Scale bar=100 µm. (B) Representative enface confocal images of GFP⁺ cells in co-culture with CF bronchial epithelial cells. Scale bar=100 μm. (C) Percent (%) of GFP-positive cells was quantified by flow cytometry. (D) Quantitation of the surface area containing GFP⁺ cells. For (C and D), data are shown as mean ± SEM; n=3 random fields per sample in three independent experiments in D (single donor). </p

Human α6β4+ cells express stem cell-specific transcription factor Nanog.

<p>α6⁺ (A) and α6^- (B) cells were isolated from normal human distal lung and cultured in Matrigel for one week, and then colonies were stained with Nanog (red) and α6 (green). Nuclei were stained with DAPI (blue) days. Scale bar=20 µm.</p

Specific adeno-associated viruses (AAVs) transduce α6β4⁺ cells.

<p>A series of AAVs encoding GFP were used to infect α6β4⁺ cells. (A) GFP expression in cells transduced with either 10⁷ AAV2 viral genomes/cell (Vg/cell, left panel) or Ad-5 at an MOI=100 (right panel). (B) GFP⁺ cells/colonies were quantified two weeks after infection with 10⁶ (white bars) or 10⁷ (gray bars) AAV Vg/cell or Ad-5 at MOI=10 (white bar) or MOI=100 (gray bar). CTL: no virus control infection. Data are shown as mean ± SEM; n=3 experiments from 3 independent donors. * <i>P</i><0.05 vs. AAV9 at the same MOI by ANOVA. </p