Characterization of intercellular communication and mitochondrial donation by mesenchymal stromal cells derived from the human lung
© The Author(s). 2016
Received: 29 April 2016
Accepted: 23 June 2016
Published: 12 July 2016
Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are capable of repairing wounded lung epithelial cells by donating cytoplasmic material and mitochondria. Recently, we characterized two populations of human lung-derived mesenchymal stromal cells isolated from digested parenchymal lung tissue (LT-MSCs) from healthy individuals or from lung transplant recipients’ bronchoalveolar lavage fluid (BAL-MSCs). The aim of this study was to determine whether LT-MSCs and BAL-MSCs are also capable of donating cytoplasmic content and mitochondria to lung epithelial cells.
Cytoplasmic and mitochondrial transfer was assessed by co-culturing BEAS2B epithelial cells with Calcein AM or Mitotracker Green FM-labelled MSCs. Transfer was then measured by flow cytometry and validated by fluorescent microscopy. Molecular inhibitors were used to determine the contribution of microtubules/tunnelling nanotubes (TNTs, cytochalasin D), gap junctions (carbenoxolone), connexin-43 (gap26) and microvesicles (dynasore).
F-actin microtubules/TNTs extending from BM-MSCs, LT-MSCs and BAL-MSCs to bronchial epithelial cells formed within 45 minutes of co-culturing cells. Each MSC population transferred a similar volume of cytoplasmic content to epithelial cells. Inhibiting microtubule/TNTs, gap junction formation and microvesicle endocytosis abrogated the transfer of cytoplasmic material from BM-MSCs, LT-MSCs and BAL-MSCs to epithelial cells. In contrast, blocking connexin-43 gap junction formation had no effect on cytoplasmic transfer. All MSC populations donated mitochondria to bronchial epithelial cells with similar efficiency. Mitochondrial transfer was reduced in all co-cultures after microtubule/TNT or endocytosis inhibition. Gap junction formation inhibition reduced mitochondrial transfer in BM-MSC and BAL-MSC co-cultures but had no effect on transfer in LT-MSC co-cultures. Connexin-43 inhibition did not impact mitochondrial transfer. Finally, bronchial epithelial cells were incapable of donating cytoplasmic content or mitochondria to any MSC population.
Similar to their bone marrow counterparts, LT-MSCs and BAL-MSCs can donate cytoplasmic content and mitochondria to bronchial epithelial cells via multiple mechanisms. Given that BM-MSCs utilize these mechanisms to mediate the repair of damaged bronchial epithelial cells, both LT-MSCs and BAL-MSCs will probably function similarly.
KeywordsMesenchymal stromal cells Mesenchymal stem cells Mitochondrial donation Intercellular communication Bronchoalveolar lavage
Recent studies suggest that adult stem cells reside within niches where they coordinate organ homeostasis and repair after injury. In the bone marrow, mesenchymal stromal cells (MSCs) and haematopoietic stem cells (HSCs) reside proximal to adrenergic nerve fibres to form the HSC niche . Bone marrow-derived MSCs (BM-MSCs) are an essential component of this structure, forming gap junctions with HSCs and expressing HSC maintenance genes which directly contribute to the HSC volume and capacity to mobilize to extramedullary sites during haematopoiesis . Emerging evidence suggests that the function of MSCs within stem cell niches, irrespective of organ, is to provide support for and coordinate the behaviour of parenchymal progenitor cells [1–4]. In order to accomplish this, BM-MSCs must be able to effectively communicate and interact with their target cells. Known mechanisms already characterized for BM-MSCs include microtubules , tunnelling nanotubes (TNTs) [3, 6–8], gap junctions , microvesicles [9–12] and exosomes [13, 14]. While the transferred material is yet to be fully defined, it is known to include growth factors [1, 7], microRNA , immuno-modulatory molecules [15–17] and mitochondria [5, 6, 8, 9]. Notably, mitochondrial transfer is emerging as an important mechanism by which MSCs can stabilize or enhance the bioenergetic potential of target cells, which includes alveolar epithelial cells  and alveolar macrophages . These mechanisms are thought to contribute to the therapeutic benefit of MSCs by enhancing epithelial proliferation [5, 9], modulating T-lymphocyte proliferation [19, 20] and inducing the polarization of macrophages towards the M2 phenotype [18, 21, 22]. As such, MSCs have been demonstrated to promote tissue homeostasis and tissue repair in multiple models of lung injury including heat shock [5, 23], cigarette smoke-mediated wounding , acute lung injury [8, 9], experimental asthma [8, 24, 25] and bleomycin-induced pulmonary fibrosis [26, 27]. Despite this knowledge, whether these modes of behaviour exist in MSC populations which reside endogenously within the human lung remains to be elucidated.
The relationship between MSCs and parenchymal progenitor cells is yet to be fully described in the human lung, but is probably analogous to that of the bone marrow. We suspected that human lung MSCs promote tissue homeostasis and repair by donating cytoplasmic material and mitochondria to parenchymal cells such as bronchial epithelium. Evidence to support this hypothesis comes from murine studies which demonstrate that lung-derived MSCs produce growth factors such as fibroblast growth factor-10 and hepatocyte growth factor, which promote the proliferation and propagation of lung epithelial progenitor cells into complex airway and alveolar structures [2, 28]. Recently, we described two populations of human lung MSCs isolated from digested parenchymal lung tissue (LT-MSCs) from healthy individuals or from lung transplant recipients’ bronchoalveolar lavage fluid (BAL-MSCs) . The aim of this study was to test the hypothesis that, like their bone marrow counterparts, human lung-derived MSCs donate cytoplasmic content and mitochondria to human bronchial epithelial cells, in vitro, and to characterize the underlying mechanisms.
Cell lines and ethics
Human BM-MSCs were kindly donated by Cell and Tissue Therapies, WA, Australia. BEAS2B epithelial cell lines were purchased from American Type Tissue Culture Collection.
Parenchymal lung tissue was obtained from patients undergoing a blebectomy to treat recurrent pneumothorax. Samples of macroscopically normal lung were mechanically digested into 1 mm3 pieces and seeded onto plastic culture dishes at a density of 1 g per dish (10 cm diameter). Cells were maintained in Dulbecco’s modified Eagle medium (DMEM; Gibco), supplemented with 10 % fetal calf serum and penicillin/streptomycin/glutamine (Gibco). Between 7 and 14 days after seeding, colony-forming units comprised of LT-MSCs were observable. MSC colonies were isolated and transferred to individual flasks where they were allowed to clonally expand up to 60–80 % confluence, after which they were cryopreserved in liquid nitrogen. All cells used were between passages 2 and 4 and were preserved in liquid nitrogen prior to use.
We and others have reported previously that BAL-MSCs are rarely isolated from healthy individuals [29, 30]. Bronchoalveolar lavage (BAL) fluid was therefore collected from lung transplant recipients undergoing a post-transplant bronchoscopy. BAL was performed by wedging the bronchoscope in the middle lobe or lingula, infusing 100 ml of 0.9 % saline via the working channel and aspirating the effluent. BAL samples were immediately centrifuged and seeded into six-well plates at a density of (0.3-0.4) × 106 cells/well. Colony-forming units comprised of BAL-MSCs were observable within 14–21 days after seeding. Cells were expanded and maintained as already detailed.
As described previously [29, 31], BAL-MSCs and LT-MSCs are classified as MSCs based on the diagnostic criteria outlined by The Internationally Society for Cellular Therapy  and transcriptional profiling . Briefly, both populations are plastic adherent, express the cell surface proteins CD105, CD73 and CD90, and lack CD45 expression. Cells also have a demonstrable capacity for osteoblast, chondroblast and adipocyte differentiation [29, 30, 31]. Finally, both populations qualify as MSCs according to transcriptional profiling [30, 33]. Additionally, BAL-MSCs lack characteristic fibrocyte markers and express a HLA serotype consistent with that of the lung donor.
Cytoplasmic exchange and mitochondrial donation assays
Inhibitory compounds (or vehicle controls; Additional file 3: Table S1) used to determine the mechanisms underlying cytoplasmic/mitochondrial transfer were added to culture simultaneously with MSCs and remained in culture for the duration of the assay.
Co-cultures were fixed with 4 % paraformaldehyde (BD), permeabilized with 1 % Triton X (Scharlau) in PBS and blocked for 1 hour in 1 % bovine serum albumin (Bovogen). Co-cultured cells were stained with Phalloidin (AbCam CytoPainter Kit) for 90 minutes for microtubules/TNTs, as per the manufacturer’s instructions, and α-cytokeratin-FITC (15 μG; Milltenyi) for epithelial cells, washed with PBS and mounted in ProLong Gold (containing DAPI; Life Technologies). Fluorescent labelling was visualized using an Axiolmager Z1 microscope (Zeiss) and Axiovision image acquisition software. For live cell confocal microscopy, MSCs were labelled with Mitotracker Green FM and BEAS2B cells with DiD, as already described. Cells were seeded onto 10 mm diameter glass-bottom culture dishes (Ibidi). Dishes were imaged at 37 °C at a rate of 1 image per 10 seconds using a 63× oil immersion lens, coupled with a 10× eyepiece lens and a differential interference contrast filter using a Leica TCS SP2 microscope.
Statistical analysis was performed using Stata v11 (StataCorp) with the Mann–Whitney U test or Wilcoxon signed-rank test, as appropriate. All data are expressed as median and interquartile range unless otherwise stated. Statistical significance was defined as p < 0.05.
Patient demographics for bronchoalveolar lavage-derived mesenchymal stromal cells
Lung transplant patients (n = 14)
Age at transplantation (years)
Donor age (years)
Male donor sex
Time post transplant (years)
Chronic obstructive pulmonary disease
Idiopathic pulmonary fibrosis
BM-MSCs, LT-MSCs and BAL-MSCs connect with BEAS2B epithelial cells via microtubules and TNTs
Microtubules and TNTs were first observed connecting MSCs and BEAS2B epithelial cells after 45 minutes of co-culturing cells. These projections stained strongly with phalloidin indicating that they are comprised of F-actin, consistent with previous reports for BM-MSCs  (Fig. 1).
BM-MSCs, LT-MSCs and BAL-MSCs have a similar efficiency for uni-directional cytoplasmic transfer to BEAS2B epithelial cells
We aimed to determine whether the observed interaction between MSCs and epithelial populations mediated the transfer of cytoplasmic material. Calcein AM transfer from MSCs to epithelial cells was rapid and first observed after 4 hours of co-culture, as shown by an increase in BEAS2B cell Calcein AM fluorescence (Fig. 2) and a corresponding decrease in MSC Calcein AM fluorescence (Additional file 4: Figure S3A).
Cytoplasmic transfer occurred at a similar level for all MSC populations (Fig. 2d). After co-culture with BM-MSCs, the Calcein AM MFI in BEAS2B cells was increased 223.5-fold (172.2–565.9, n = 14). Similarly, BEAS2B Calcein AM MFI was increased 252.1-fold (150.9–252.1, n = 9) after co-culture with LT-MSCs and was increased 192.3-fold (156.9–513.4, n = 12) after co-culture with BAL-MSCs. We did not observe Calcein AM transfer when the cells were separated by transwell culture (Fig. 2c). Finally, cytoplasmic transfer was uni-directional and could not be observed to occur from BEAS2B cells to any MSC population (Fig. 2b).
Cytoplasmic transfer from BM-MSCs, LT-MSCs and BAL-MSCs to BEAS2B epithelial cells is mediated by contact-dependent and contact-independent mechanisms
We next sought to characterize the mechanisms of cytoplasmic transfer between MSCs and epithelial cells (Fig. 3). In line with a previous report , we used the dynamin inhibitor, Dynasore, to inhibit BEAS2B cells endocytosing MSC-produced microvesicles. Endocytosis inhibition reduced cytoplasmic transfer by 61.3 % (58.7–64.1), 47.7 % (47.1–48.7) and 57.7 % (45.68–57.7) in BM-MSC, LT-MSC and BAL-MSC co-cultures, respectively (n = 6, p < 0.05 for all; Fig. 3). Blocking microtubule/TNT formation using the actin polymerase inhibitor cytochalasin D  reduced cytoplasmic transfer by 43.0 % (31.7–44.3), 40.5 % (19.3–41.2) and 36.0 % (34.6–39.4) from BM-MSCs, LT-MSCs and BAL-MSCs, respectively (n = 6, p < 0.05 for all). The greatest reduction in cytoplasmic transfer was observed using the non-specific gap junction blocker carbenoxolone [35, 36] (Fig. 3). Cytoplasmic transfer from BM-MSCs to BEAS2B cells was reduced by 90.8 % (88.9–93.2, p < 0.05, n = 6) after carbenoxolone treatment. Similarly, transfer was reduced by 85.0 % (79.4–87.5, p < 0.05, n = 6) and 88.5 % (85.6–90.9, p < 0.05, n = 6) in LT-MSC and BAL-MSC co-cultures respectively. To determine the contribution of connexin-43-dependent gap junctions, which have previously been demonstrated to mediate cytoplasmic transfer in murine studies , we employed the inhibitor gap26 [37, 38]. Blocking connexin-43 did not prevent cytoplasmic transfer with any MSC cell type.
BM-MSCs, LT-MSCs and BAL-MSCs share similar efficiency for uni-directional mitochondrial transfer to BEAS2B epithelial cells
BM-MSCs can promote the regeneration of damaged lung epithelial cells through the donation of mitochondria using similar mechanisms to those described for cytoplasmic transfer [5, 9]. We found that all MSC populations can donate mitochondria to BEAS2B epithelial cells, as indicated by increased BEAS2B Mitotracker Green fluorescence (Fig. 4) and decreased MSC Mitotracker Green fluorescence (Additional file 4: Figure S3B) after co-culture. When visualized using live cell confocal microscopy, mitochondria localized within MSC microtubules or TNTs, tracked towards BEAS2B cells and crossed the cell membrane border. In addition, individual mitochondrion could be seen within endosomes (Fig. 4e, white arrowhead) and microvesicles (Fig. 4e, f, white arrows). Following co-culture with BM-MSCs, BEAS2B Mitotracker Green MFI increased 15.9-fold (10.7–22.4, n = 10). Similarly, BEAS2B Mitotracker Green MFI increased 26.7-fold (12.1–44.6, n = 10) after co-culture with LT-MSCs and increased 15.9-fold (12.7–25.2, n = 9) after co-culture with BAL-MSCs (Fig. 4d). Mitochondrial transfer was not observed when BEAS2B cells were co-cultured with any MSC populations using a transwell set-up (Fig. 4c). Additionally, we did not observe any mitochondrial transfer from BEAS2B cells to any MSC populations (Fig. 4b).
Mitochondrial transfer from BM-MSCs, LT-MSCs and BAL-MSCs to BEAS2B epithelial cells is mediated by contact-dependent and non-dependent mechanisms
Inhibiting endocytosis in BEAS2B cells resulted in a 15.8 % (14.6–31.2, p < 0.05, n = 7) decrease in transfer in BM-MSC co-cultures (Fig. 5). Mitochondrial donation was also decreased after endocytosis inhibition by 34.7 % (32.3–37.4, p < 0.05, n = 7) in LT-MSCs and by 25.2 % (20.0–30.3, p < 0.05, n = 7) in BAL-MSC co-cultures (Fig. 5). Inhibiting microtubule/TNT formation reduced mitochondrial transfer by 25.5 % (23.6–29.7, p < 0.05, n = 7) in BM-MSC co-cultures. Transfer was reduced by 13.8 % (12.4–16.2, p < 0.05, n = 7) in LT-MSC co-cultures and by 11.0 % (10.0–30.3, p < 0.05, n = 7) in BAL-MSC co-cultures. The non-specific inhibition of gap junction formation reduced mitochondrial donation by 26.8 % (23.8–32.5, p < 0.05, n = 7) in BM-MSC co-cultures and by 10.9 % (7.5–15.6, p < 0.05, n = 7) in BAL-MSC co-cultures. Gap junction inhibition did not reduce mitochondrial donation in LT-MSC co-cultures. Finally, connexin-43 inhibition did not reduce mitochondrial transfer in any MSC-BEAS2B cell co-culture (Fig. 5). When all inhibitory compounds were added together we observed greater attenuation of mitochondrial transfer (Fig. 5), suggesting synergy between microtubule/TNT, endocytosis and gap junction mediated mechanisms.
MSCs exhibit therapeutic effects in multiple models of acute and chronic lung epithelial injury and represent a promising therapeutic approach for the treatment of end-stage lung diseases including idiopathic pulmonary fibrosis  and acute lung injury  in humans. These studies have utilized MSCs isolated from tissue sources such as the bone marrow, adipose and placenta. Comparative studies have demonstrated that, irrespective of source, MSCs from any two tissues are strikingly similar [41, 42]. Despite this, each population possesses subtle nuances, specific to their source [30, 43], which may confer additional benefit or efficiency in a therapeutic context. One of the mechanisms by which MSCs support epithelial cell repair is thought to be via direct transfer of cytoplasmic contents [9, 35], but the nature of the transferred material, mechanisms of transfer, effects on target cells and effects of the tissue microenvironment have yet to be fully defined. In this study, we demonstrate that human MSCs isolated from bone marrow, parenchymal lung tissue and BAL fluid donate cytoplasmic contents and mitochondria to bronchial epithelial cells. Transfer was rapid, uni-directional and mediated by multiple mechanisms including microvesicles, microtubules/TNTs and non-connexin-43 gap junctions. Similar mechanisms contributed to mitochondrial donation in BM-MSCs and BAL-MSCs, whereas microvesicles and microtubules/TNTs, but not gap junctions, contributed to mitochondrial transfer by LT-MSCs. Inhibiting these pathways did not completely prevent mitochondrial donation, however, suggesting the existence of additional mechanisms. These findings add to a growing body of literature which demonstrates MSCs isolated from different sources are similar but exhibit subtle differences which may be reflective of adaption to a specific tissue. Our data provide further support to the idea that human lung-derived mesenchymal populations contribute to tissue homeostasis and repair through direct interaction with epithelial cell populations.
BAL-MSCs are a unique population of transplant donor-derived cells that are generally only recoverable from lung allografts [29, 30]. Their absence from healthy individuals and correlation with disease has led to the suggestion that BAL-MSCs are either a causal factor for lung disease [44, 45] or an epiphenomenon. We have previously postulated that BAL-MSCs are derived from an in-tissue source which has mobilized into the alveolar space to provide support for injured epithelial cells . This is supported by previous studies demonstrating that BAL-MSCs administered to mice preferentially engraft in close proximity to alveolar progenitor cells and donate keratinocyte growth factor via connexin-43 gap junctions . Our findings expand on this by demonstrating that cytoplasmic content is transferred from human LT-MSCs and BAL-MSCs to human bronchial epithelial cells and by identifying a capacity for mitochondrial donation. Unlike previous studies, we were unable to identify a role of connexin-43 in the transfer of cytoplasmic content or mitochondria. These findings provide a foundation for future investigations into the reparative capacity of BAL-MSCs and LT-MSCs.
Although it is now clear that LT-MSCs and BAL-MSCs transfer cytoplasmic material to the lung epithelium, the composition of transferred material remains to be characterized. We hypothesize that LT-MSCs and BAL-MSCs transfer material analogous to that produced by BM-MSCs. While this material remains incompletely defined, it is known to include growth factors [26, 46], immuno-modulatory molecules [16, 47], transcription factors  and micro-RNAs [11, 14]. Human BAL-MSCs are thought to transfer keratinocyte growth factor to murine type 2 alveolar epithelial cells, in vivo . Additionally, murine LT-MSCs have been demonstrated to produce fibroblast growth factor-10 and hepatocyte growth factor which drives the differentiation of epithelial progenitor cells into complex airway and alveolar structures, in vitro . Together, these findings support the notion that the role of MSCs within tissue is to provide support, be it by the production of paracrine factors or mitochondrial donation to the surrounding parenchymal populations.
Our data suggest that human BM-MSCs and BAL-MSCs employ a non-connexin-43 gap junction in the transfer of cytoplasmic content and mitochondria to bronchial epithelial cells. This finding was surprising as connexin-43 has previously been demonstrated to mediate both cytoplasmic and mitochondrial transfer from BM-MSCs to alveolar epithelial cells . Together, these findings suggest MSCs utilize gap junctions in a region-specific manner. To date, no other connexins have been implicated in the transfer of cytoplasmic content or mitochondria. The most logical alternative candidate is connexin-26 which is expressed by basal cells . Notably, basal cells serve as a progenitor cell for the bronchial epithelium  and interaction between basal cells and local MSCs would fit with the developing narrative of the pulmonary stem cell niche.
There are several limitations to this study. Firstly, our study used a human bronchial epithelial cell line which may limit the generalisability of our findings to primary cells. This may explain why we were unable to demonstrate a role for connexin-43 in cytoplasmic or mitochondrial donation. However, healthy human lung epithelium is difficult to obtain and requires a heavily supplemented growth medium, which in itself can lead to artefacts. Secondly, the isolation and ex-vivo expansion of MSCs can lead to functional alterations, including their interaction with lung epithelial cells . Therefore, we used MSCs at the lowest passage feasibly possible and passage-matched all samples.
LT-MSCs and BAL-MSCs share a similar capacity for cytoplasmic and mitochondrial transfer to bronchial epithelial cells. Transfer is rapid, ubiquitous and unidirectional from MSCs to the epithelium. These findings offer novel insight into the potential role of lung MSCs in organ homeostasis and repair.
BAL, bronchoalveolar lavage; BAL-MSC, bronchoalveolar lavage-derived mesenchymal stromal cell; BEGM, basal epithelial growth medium; BM-MSC, bone marrow-derived mesenchymal stromal cell; DMEM, Dulbecco’s modified Eagle medium; HSC, haematopoietic stem cell; LT-MSC, lung tissue-derived mesenchymal stromal cell; MFI, mean fluorescent intensity; MSC, mesenchymal stromal cell; PBS, phosphate-buffered saline; TNT, tunnelling nanotube
The authors would like to acknowledge the generous support of The Prince Charles Hospital Foundation. Additionally, they wish to acknowledge the assistance of Katharine Irvine in the preparation of this manuscript.
This body of work was funded by The Prince Charles Hospital Foundation from grant NR2013-06.
Availability of data and materials
Additional supplementary data and information are available on request. This manuscript does not refer to a dataset such as those used in microarray data analysis.
KAS was responsible for concept and design (85 %), provision of study material or patients (70 %), collection and/or assembly of data (100 %), data analysis and interpretation (90 %), manuscript writing (55 %) and final approval of the manuscript (25 %). DCC was responsible for concept and design (5 %), financial support (33 %), provision of study material or patients (20 %), data analysis and interpretation (5 %), manuscript writing (10 %) and final approval of manuscript (25 %). STY was responsible for concept and design (5 %), financial support (33 %), data analysis and interpretation (5 %), manuscript writing (10 %) and final approval of the manuscript (25 %). PM-AH was responsible for concept and design (5 %), manuscript writing (5 %), financial support (33 %), provision of study material or patients (10 %) and final approval of manuscript (25 %). Additionally, we wish to recognize KI, whom is not listed as an author but did contribute to ~20 % of the manuscript writing.
The authors of this publication are a part of the Queensland Lung Transplant Service. KAS is a PhD candidate whose thesis focuses on the role of lung-derived stromal populations and their contribution to tissue homeostasis and disease. STY is the chief scientist and statistician responsible for integrating clinical and scientific aspects of our work. DCC and PM-AH are both thoracic physicians interested in the potential of MSCs as a cellular therapy for advanced lung diseases.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study was approved by The Human Research Ethics Committee (HREC/13/QPCH/96), The Prince Charles Hospital. All patients provided written informed consent before enrolling in the study.
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