Amniotic mesenchymal stem cells: a new source for hepatocyte-like cells and induction of CFTR expression by coculture with cystic fibrosis airway epithelial cells

Abstract

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with lung and liver manifestations. Because of pitfalls of gene therapy, novel approaches for reconstitution of the airway epithelium and CFTR expression should be explored. In the present study, human amniotic mesenchymal stem cells (hAMSCs) were isolated from term placentas and characterized for expression of phenotypic and pluripotency markers, and for differentiation potential towards mesoderm (osteogenic and adipogenic) lineages. Moreover, hAMSCs were induced to differentiate into hepatocyte-like cells, as demonstrated by mixed function oxidase activity and expression of albumin, alpha1-antitrypsin, and CK19. We also investigated the CFTR expression in hAMSCs upon isolation and in coculture with CF airway epithelial cells. Freshly isolated hAMSCs displayed low levels of CFTR mRNA, which even decreased with culture passages. Following staining with the vital dye CM-DiI, hAMSCs were mixed with CFBE41o- respiratory epithelial cells and seeded onto permeable filters. Flow cytometry demonstrated that 33-50% of hAMSCs acquired a detectable CFTR expression on the apical membrane, a result confirmed by confocal microscopy. Our data show that amniotic MSCs have the potential to differentiate into epithelial cells of organs relevant in CF pathogenesis and may contribute to partial correction of the CF phenotype.

Figure 1

hAMSCs morphology and growth. Cell morphology at passage one (a) and passage three (b), original magnification 10x. Growth kinetics of hAMSCs in culture (c).

Figure 2

hAMSCs pluripotent stem cells and ESC marker expression. Flow cytometry representative expression of the pluripotent and embryonic stem cells markers in freshly isolated hAMSCs. (a) For each staining, a gate on viable cells (red) was drawn; (b) hAMSCs were gated on the basis of morphological features; (c) cells incubated with isotypic control were used as negative controls; (d) Nanog, (e) Oct-4, and (f) SSEA4 expression in hAMSCs.

Figure 3

Immunofluorescence characterization of hAMSCs. Representative images of fluorescence microscopy staining. The upper left panel (denoted as “negative”) shows cells incubated with the secondary antibody only. hAMSCs were positive for CD29, CD90, fibronectin, vimentin, and CK 18 and negative for ZO-1 and CK 7. Nuclei were counterstained with DAPI. Original magnification 20x.

Figure 4

In vitro differentiation capability. Representative images of in vitro osteocyte (b), adipocyte (d), and hepatocyte (f) differentiation of hAMSCs. (a), (c), and (e) represent respective negative controls (i.e., uninduced cells). Original magnification 20x.

Figure 5

Immunophenotype of mesenchymal stem cells prior (left) and after (right) hepatocyte differentiation for 21 days. (a–f) negative controls, (b–g) CK7, (c–h) albumin, (d–i) alpha1-antitrypsin, and (e–j) CK19. Original magnification 20x. The insert h1 represents an enlargement of the cell highlighted by the white square.

Figure 6

CFTR mRNA expression in hAMSCs and hAECs. CFTR on hAMSCs and hAECs upon isolation (T0) and on hAMSCs at passages one (P1) and three (P3). Upper panels: CFTR; lower panels: β-actin. M: molecular weight markers; Ctr+: positive control (nasal brushing); Ctr−: negative control (no RT). On the right, arrows indicate the specific band along with PCR-product length.

Figure 7

CFTR immunodetection by confocal analysis. Confocal scans are shown in the horizontal cross-section (xy) plane and vertical cross-section (xz) plane. (a) CFBE41o- cells; (b) hAMSCs- CFBE 1 : 5 ratio; (c) hAMSCs- CFBE 1 : 10 ratio; (d) hAMSCs- CFBE 1 : 15 ratio; (e) hAMSCs alone. The white arrows point to CM-DiI-labelled hAMSCs expressing CFTR on their membrane (green signal). Note in (d) that hAMSCs harbour some CFTR-specific signal in discrete regions under the apical plasma membrane.