Functional characterisation of bone marrow-derived mesenchymal stromal cells from COPD patients

Abstract

Autologous bone marrow-derived mesenchymal stromal cells (BM-MSCs) are evaluated for clinical use in chronic obstructive pulmonary disease (COPD) patients, but it is unclear whether COPD affects BM-MSCs. To investigate this, BM-MSCs from nine COPD patients and nine non-COPD age-matched controls were compared with regard to immunophenotype, growth and differentiation potential, and migration capacity. Other functional assays included the response to pro-inflammatory stimuli and inducers of the nuclear factor (erythroid derived 2)-like 2 antioxidant response element (Nrf2-ARE) pathway, and effects on NCI-H292 airway epithelial cells. No significant differences were observed in terms of morphology, proliferation and migration, except for increased adipocyte differentiation potential in the COPD group. Both groups were comparable regarding mRNA expression of growth factors and inflammatory mediators, and in their potential to induce mRNA expression of epidermal growth factor receptor ligands in NCI-H292 airway epithelial cells. MSCs from COPD patients secreted more interleukin-6 in response to pro-inflammatory stimuli. Activation of the Nrf2-ARE pathway resulted in a comparable induction of mRNA expression of four target genes, but the expression of the NAD(P)H:quinone oxidoreductase 1 gene NQO1 was lower in MSCs from COPD patients. The observation that MSCs from COPD patients are phenotypically and functionally comparable to those from non-COPD controls implies that autologous MSCs can be considered for use in the setting of clinical trials as a treatment for COPD.

Conflict of interest statement

can be found alongside this article at openres.ersjournals.com

Figures

FIGURE 1

Mesenchymal stromal cell (MSC) phenotype from chronic obstructive pulmonary disease (COPD) and non-COPD patients. Bone marrow-derived MSCs (BM-MSCs) from COPD and non-COPD patients cultured in vitro were characterised by fluorescence-activated cell sorter (FACS) analysis and by their potential to differentiate into mesenchymal lineages. a) Morphology of MSCs from COPD and non-COPD donors: example of MSC culture. Scale bar s=100 μm. b) FACS data. Per antibody, data are shown as mean±sem. n=9 per group. c) Differentiation into adipocytes and osteoblasts, and mineralisation potential. Staining intensity of Oil red O, alkaline phosphatase and Alizarin red (calcium) was quantified on a microplate reader, and per donor optical density (OD) values were calculated as fold change compared with control (co-cultured undifferentiated MSCs). Plots represent median, interquartile range, and minimum and maximum values. *: p<0.05, n=9 per group (except osteoblast/mineralisation: n=6–8 per group).

FIGURE 2

Mesenchymal stromal cell (MSC) proliferation and migration capacity. In vitro proliferation of MSCs from chronic obstructive pulmonary disease (COPD) and non-COPD patients was followed over time. a) Time needed for cultures to become near-confluent and the number of MSCs that were obtained per passage were assessed. b) Population doubling time (PDT) was calculated by dividing the natural logarithm of 2 by the exponent of growth. Plots represent median, interquartile range, and minimum and maximum values. n=9 per group. c) Migration as assessed using electric cell-substrate impedance sensing (ECIS). MSCs were cultured in ECIS arrays in the presence of an electrical fence (EF), which prevented cell adherence across the electrode area. Resistance (at 500 Hz) and capacitance (at 40 kHz) were measured continuously and followed up to 15 h after removal of the electrical fence (t=0 h). Restoration of the resistance and capacitance to control values (corresponding to full coverage of the electrode) was used as a measure for migration capacity of MSCs. Normalised data were obtained by correcting for the resistance/capacitance values obtained in control wells without an electric fence, at the time the electric fence was removed (t=0 h). Data are presented as mean±sem. n=5–8 per group.

FIGURE 3

Induction of immune mediators and growth factors upon stimulation with pro-inflammatory cytokines. Mesenchymal stromal cells (MSCs) were stimulated with tumour necrosis factor (TNF)-α and interleukin (IL)-1β (20 ng·mL−1 each) or plain culture medium as a negative control (NC) during 6 h. a) mRNA expression of immune mediators and growth factors, normalised for housekeeping genes, shown for chronic obstructive pulmonary disease (COPD) (circles) and non-COPD donors (squares). AREG: amphiregulin; HBEGF: heparin-binding epidermal growth factor-like growth factor; FGF2: fibroblast growth factor 2; ADM1: adrenomedullin; CCL20: chemokine (C-C motif) ligand 20; IL6: IL-6; CXCL8: chemokine (C-X-C motif) ligand 8; TSG6: TNF-stimulated gene 6. Individual data are shown in graphs, horizontal bars represent mean. *: p<0.05, n=9 per group. b) IL-6 and IL-8 protein secretion by MSCs from COPD and non-COPD donors, assessed 24 h after addition of TNF-α and IL-1β. Individual data points are shown; horizontal bars represent mean. *: p<0.05, n=9 per group.

FIGURE 4

Induction of the nuclear factor (erythroid derived 2)-like 2 antioxidant response element (Nrf2-ARE) target genes in mesenchymal stromal cells (MSCs) from chronic obstructive pulmonary disease (COPD) and non-COPD donors. Gene expression of glutathione peroxidase 2 (GPX2), haem oxygenase 1 (HMOX1), NAD(P)H:quinone oxidoreductase 1 (NQO1) and smoke and cancer-associated long noncoding RNA-1 (SCAL1) was assessed in MSCs from COPD and non-COPD donors 6 h after addition of cigarette smoke extract (CSE) or sulforaphane (SFN), which are both inducers of the Nrf2-ARE pathway, or plain culture medium as a negative control (NC). a) Normalised mRNA expression in response to increasing concentrations of CSE. Data are presented as mean±sem. *: p<0.05, n=9 per group (except CSE 1.0 AU·mL−1: n=7 in COPD group). b) Normalised mRNA expression in response to 25 μM SFN, compared with housekeeping genes. Individual data points are shown (circles: COPD; squares: non-COPD); horizontal bars represent mean. *: p<0.05, n=9 per group.

FIGURE 5

Paracrine effects of mesenchymal stromal cells (MSCs) on H292 airway epithelial cells. NCI-H292 airway epithelial cells were incubated with conditioned medium obtained from MSCs (MSC-CM). mRNA expression of epidermal growth factor receptor ligands after 9 h incubation with conditioned medium from MSCs cultured in serum-free low-glucose Dulbecco's modified Eagle medium (LG-DMEM) (Ctrl, from MSC-CMCTRL) or in serum-free LG-DMEM supplemented with TNF-α and IL-1β (Stim, from MSC-CMSTIM). AREG: amphiregulin; HBEGF: heparin-binding epidermal growth factor-like growth factor; TGFA: transforming growth factor-α; CCDN1: cyclin D1. Normalised values compared with housekeeping genes are shown. Individual data points are shown (circles: chronic obstructive pulmonary disease (COPD) donor-derived MSC-CM; squares: non-COPD donor-derived MSC-CM); horizontal bars represent mean. *: p<0.05, n=9 per group. For all genes investigated, mRNA expression was lower in cells cultured in plain culture medium (LG-DMEM without additional stimulation) compared with mRNA expression in cells cultured in MSC-CM (data not shown).