Adult stem cells from bone marrow stroma differentiate into airway epithelial cells: potential therapy for cystic fibrosis

Abstract

Cystic fibrosis (CF), the most prevalent, fatal genetic disorder in the Caucasian population, is caused by mutations of CF transmembrane conductance regulator (CFTR). The mutations of this chloride channel alter the transport of chloride and associated liquid and thereby impair lung defenses. Patients typically succumb to chronic bacterial infections and respiratory failure. Restoration of the abnormal CFTR function to CF airway epithelium is considered the most direct way to treat the disease. In this report, we explore the potential of adult stem cells from bone marrow, referred to as mesenchymal or marrow stromal stem cells (MSCs), to provide a therapy for CF. We found that MSCs possess the capacity of differentiating into airway epithelia. MSCs from CF patients are amenable to CFTR gene correction, and expression of CFTR does not influence the pluripotency of MSCs. Moreover, the CFTR-corrected MSCs from CF patients are able to contribute to apical Cl(-) secretion in response to cAMP agonist stimulation, suggesting the possibility of developing cell-based therapy for CF. The ex vivo coculture system established in this report offers an invaluable approach for selection of stem-cell populations that may have greater potency in lung differentiation.

Figures

Fig. 1.

Air–liquid-interface culture of human MSCs with human AECs. (a) Diagram of the air–liquid-interface culture model. (b) Primary human AECs grown on a semipermeable filter. Paraffin section and hematoxylin/eosin staining shows typical well differentiated and pseudostratified airway epithelia. (c) An electron micrograph of this culture demonstrates three types of AECs: ciliated cells, nonciliated cells, and basal cells. (d and e) Fluorescent micrographs demonstrate the primitive morphology of GFP-expressing MSCs in a submerged culture on plastic surfaces. (f) Morphology of RFP-tagged CF AECs in a submerged culture on a plastic surface. (g) Coculture of GFP-MSCs with RFP-tagged CF AECs. A confocal microscopic x-y section demonstrates that GFP-MSCs assumed epithelia-like cuboidal or columnar morphology. (h) A confocal microscopic x-z image shows the vertical section view of the GFP-MSC.

Fig. 2.

Coculture of GFP-MSCs with human AECs induces the MSCs to express epithelia-specific genes. The cocultured cells were stained with CK-18 antibody (a–d) or isotype mouse IgG1 as a control (e–h). By confocal microscopy, x-y sections demonstrated that the airway cells express abundant CK-18 proteins (a), and in the same view GFP-MSCs are interspersed among the airway cells (c). (d) The x-z vertical sections demonstrated that the applied MSCs had changed morphologically from the spindle fibroblast-like shape to the columnar epithelial shape with a positive CK-18 staining. Control staining with the isotype IgG1 antibody (e and f) confirmed the staining specificity. The enlarged (i and j) and the merged (k) images display details of the double-positive cells. (l–n) Flow-cytometric analysis of occludin gene expression. The x axis indicates GFP green fluorescence intensity, and the y axis indicates occludin fluorescence staining intensity. (l) Flow cytometry of unstained human AECs. (m) Coculture of human MSCs with human AECs stained with an isotype mouse IgG1 as a negative control. (n) Coculture of human MSCs with human AECs stained with the antibody against human occludin. Approximately 1% of total cells (i.e., ≈10% of the applied MSCs) were occludin- and GFP-double-positive. To confirm the FACS data, the occludin antibody-stained cells for the FACS analysis were cytospun onto a microscopic slide, and examined by fluorescent microscopy. (o and r) 4′,6-Diamidino-2-phenylindole (DAPI) fluorescence. (p and s) GFP fluorescence. (q and t) Occludin-phycoerythrin fluorescence. (o–q) A representative AEC was positive for occludin and negative for GFP. (r–t) A cluster of GFP-positive stem cells with occludin-positive stainings. Large arrows point to a GFP- and occludin-double-positive cell. Small arrows point to a cell with positive GFP fluorescence but negative occludin staining, serving as an internal control. In contrast, arrowheads point to a cell with negative GFP but positive occludin staining.

Fig. 3.

Expression of CFTR in GFP-MSCs after coculture. (a and b) FACS sorting of the GFP-MSCs population. AECs alone (a) or cells from a coculture of MSCs with AECs (1:10) (b) were subjected to FACS sorting. The R2 frame was used for gating and purification of the GFP-expressing cells. RT-PCR was performed on the cells from the R2 gate frame to detect the CFTR mRNA transcriptions by using the primer set: human (hu)CFTRWT(+) and huCFTRWT(-) as shown (e). (c) CFTR mRNA transcripts were detected in the cocultured MSCs and AECs but not in MSCs alone. (d) In a separate RT-PCR control, for which a primer set of the TBP gene was used, the three cultures are all expressing TBP. To confirm the RT-PCR results discussed above, another coculture was performed in which MSCs from normal individuals were mixed with ΔF508 CF AECs in a ratio of 1:20. RT-PCR using the identical CFTR primer set (e) demonstrated the WT CFTR expression in the coculture sample but not the culture with MSCs alone or ΔF508 CF AECs alone (f, lanes 1–3). In Calu-3 cells, derived from airway submucosal gland epithelium, CFTR gene expression was detected, serving as a positive control (f, lane 4). The ubiquitously expressed TBP gene was transcribed in all four cultures, serving as an RNA quality and loading-quantity control.

Fig. 4.

CFTR-corrected CF-patient MSCs retained their multipotency and responded to cAMP stimulation by secreting chloride to the apical side. (a) Schematic for CF-patient MSC isolation, expansion, gene correction, and positive drug selection. (b) RT-PCR to verify the successful CFTR gene transfer. RT-PCR was performed to amplify WT CFTR transcripts but not ΔF508 mutant transcripts. The gene-corrected CF MSCs and positive control Calu-3 cells have WT CFTR transcription, whereas non-gene-corrected CF MSCs and the no-RT control show negative amplification. In the TBP RT-PCR control, all of the samples except the no-RT control show positive PCR products. (c) Phase-contrast microscopic view of the CFTR gene-corrected CF-patient MSCs. (d) Photomicrograph of a representative stem cell colony plate. Purple-stained foci are the MSC colonies. (e) Osteogenesis of the CFTR gene-corrected CF-patient MSCs. After differentiation in an osteogenic medium, cells had mineral deposits visualized in red by Alizarin red staining. (f) Adipogenesis of the CFTR gene-corrected CF-patient MSCs. After differentiation in an adipogenic medium, cells had lipid droplet accumulation stained in red with oil red O. (g and h) CFTR gene-corrected MSCs from CF patients contributed to the apical cAMP-stimulated Cl- secretion. CFTR gene-corrected CF-patient MSCs or non-gene-corrected CF-patient MSCs were mixed with ΔF508 CF AECs at different ratios as indicated. After 1 month in culture at the air–liquid interface, chloride efflux assays were performed as described in Materials and Methods. A two-way ANOVA test revealed that cocultures with the CFTR gene-corrected CF-patient MSCs (•) had a greater chloride secretion in response to the IBMX and forskolin stimulation than the cocultures with non-gene-corrected CF-patient MSCs (▴) (n = 4; P ≤ 0.05).