Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 7 (5), e35639

Comparison of Epithelial Differentiation and Immune Regulatory Properties of Mesenchymal Stromal Cells Derived From Human Lung and Bone Marrow

Affiliations

Comparison of Epithelial Differentiation and Immune Regulatory Properties of Mesenchymal Stromal Cells Derived From Human Lung and Bone Marrow

Mario Ricciardi et al. PLoS One.

Abstract

Mesenchymal stromal cells (MSCs) reside in many organs including lung, as shown by their isolation from fetal lung tissues, bronchial stromal compartment, bronchial-alveolar lavage and transplanted lung tissues. It is still controversial whether lung MSCs can undergo mesenchymal-to-epithelial-transition (MET) and possess immune regulatory properties. To this aim, we isolated, expanded and characterized MSCs from normal adult human lung (lung-hMSCs) and compared with human bone marrow-derived MSCs (BM-hMSCs). Our results show that lung-MSCs reside at the perivascular level and do not significantly differ from BM-hMSCs in terms of immunophenotype, stemness gene profile, mesodermal differentiation potential and modulation of T, B and NK cells. However, lung-hMSCs express higher basal level of the stemness-related marker nestin and show, following in vitro treatment with retinoic acid, higher epithelial cell polarization, which is anyway partial when compared to a control epithelial bronchial cell line. Although these results question the real capability of acquiring epithelial functions by MSCs and the feasibility of MSC-based therapeutic approaches to regenerate damaged lung tissues, the characterization of this lung-hMSC population may be useful to study the involvement of stromal cell compartment in lung diseases in which MET plays a role, such as in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Characterization of lung-hMSCs.
(A) Morphology of lung-hMSCs. Inverted phase-contrast microscopy of primary colonies of lung-hMSCs (left panel) and expanded colonies at confluence (right panel). (B) (left panel) Characterization by qRT-PCR of stemness profile of cells extracted from lung tissues (dark grey columns) in comparison with BM-hMSCs (ligh grey columns). Relative gene expression fold induction of nestin (nes), smad4, nanog, pou5f1(or Oct4), sox2 and tdgf1 (or Cripto). Gene expression is normalized on actb gene (beta actin) and related to BM-hMSCs as basal expression. Mean ± SD; * = p<0.01; (B) (right panel) Analysis of nestin expression detected by flow cytometry in lung-hMSCs (dark grey column) and BM-hMSCs (light grey column). Columns show Nestin fluorescence ratio normalized on the fluorescence of the relative isotype. Mean ± SD. * p<0.01. (C) Schematic representation of clonogenic and self-renewal assays with a representative case of a single colony obtained from CFU-F assay, consisting of more than 50 cells. Scale bar: 1 mm. (D) Multilineage differentiation potential. Lung-hMSCs, under appropriate stimuli, can generate adipocytes (left panel), osteocytes (middle panel) and chondrocytes (right panel), as confirmed by the staining with Oil-red-O, alkaline phosphatase (ALP) and Tolouidin Blue dyes, respectively.
Figure 2
Figure 2. Immune regulatory properties.
(A) Effect of the coculture of MSCs derived from human BM (light grey) and human lung (dark grey) on the proliferation of B, NK and T cells. Different MSC:Effector ratios were assessed as shown in the legend. Means±SD. * p<0.01, ** p<0.03. (B) Molecular mechanisms underlying the immune regulatory properties of BM-hMSCs (light grey) and lung-hMSCs (dark grey) towards T cells. Proliferation of T cells was evaluated without MSCs or at 10∶1 ratio with hMSCs from BM and lung, in presence of specific inhibitors of known pathways: IDO-inhibitor L-1-methyl-tryptophan (L-1MT), inhibitor of heme-oxigenase 1 (snPP), COX2 -inhibitor (NS398), iNos-inhibitor (L-NMMA) and IFN-gamma blocking antibody.
Figure 3
Figure 3. In vivo localization of lung-hMSCs at perivascular level.
Immunofluorescence on human lung tissue sample with anti-NG2 and anti-CD31 (left panel), anti-NG2 and anti-CD146 (middle panel), and anti-NG2 and anti-Nestin (right panel) mAbs. White arrows: cells of interest. Nuclei are stained with Topro3 in blue.
Figure 4
Figure 4. Epithelial differentiation.
(A) Morphology changes after epithelial differentiation. Inverted phase-contrast microscopy (Zeiss Observer.Z1; 100x) of BM-hMSCs and lung-hMSCs without and with RA treatment (Ctrl; +RA). (B) Evaluation of epithelial differentiation by qRT-PCR on BM-hMSCs (light grey) and lung-hMSCs (dark grey), before (basal) and after RA treatment for the induction of epithelial differentiation. Panel shows the expression of the epithelial genes cytokeratin 18 (krt18), tight junction protein (tjp1), also named zona occludens 1, occludin (ocln) and mesenchymal genes vimentin (vim) and e-cadherin repressor snai1. Data are normalized on actb (beta actin) expression and related to basal BM-hMSCs. The error bars represent standard deviation; * =  p<0.01; ** = p<0.03; *** = p<0.05. (C) Evaluation of epithelial differentiation by Western Blot with anti-vimentin (left panel) and anti-cytokeratin 18 (right panel) antibodies on protein extract of BM-hMSCs (lane 1-2) and lung-hMSCs (lane 3–4), either untreated (Ctrl) or treated with RA (+RA). (D) Evaluation by immunofluorescence of epithelial differentiation of lung-hMSCs after RA treatment. Staining with anti-cytokeratin 18 (CK18) and Occludin (OCLN) (left panel), and with E-cadherin (Ecad) and anti-smooth muscle actin (SMA) (right panel) mAbs. Nuclei are stained with Topro3 in blue. (E) Functional evaluation of epithelial differentiation with Trans-Epithelial Electric Resistance (TEER). Histograms show the value of TEER (Ω*cm2) on BM-hMSCs (light grey) and lung-hMSCs (dark grey), before and after RA treatment. The error bars represent standard deviation; ** = p<0.03; *** = p<0.05.

Similar articles

See all similar articles

Cited by 20 articles

See all "Cited by" articles

References

    1. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol. 1976;4:267–274. - PubMed
    1. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997;276:71–74. - PubMed
    1. Krampera M, Glennie S, Dyson J, Scott D, Laylor R, et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood. 2003;101:3722–3729. - PubMed
    1. Krampera M, Pasini A, Pizzolo G, Cosmi L, Romagnani S, et al. Regenerative and immunomodulatory potential of mesenchymal stem cells. Curr Opin Pharmacol. 2006;6:435–441. - PubMed
    1. Conese M, Rejman J. Stem cells and cystic fibrosis. Journal of Cystic Fibrosis. 2006;5:141–143. - PubMed

Publication types

MeSH terms

Feedback