Lung-derived mesenchymal stromal cell post-transplantation survival, persistence, paracrine expression, and repair of elastase-injured lung

Abstract

While multipotent mesenchymal stromal cells have been recently isolated from adult lung (L-MSCs), there is very limited data on their biological properties and therapeutic potential in vivo. How L-MSCs compare with bone marrow-derived MSCs (BM-MSCs) is also unclear. In this study, we characterized L-MSC phenotype, clonogenicity, and differentiation potential, and compared L-MSCs to BM-MSCs in vivo survival, retention, paracrine gene expression, and repair or elastase injury after transplantation. L-MSCs were highly clonogenic, frequently expressed aldehyde dehydrogenase activity, and differentiated into osteocytes, chondrocytes, adipocytes, myofibroblasts, and smooth muscle cells. After intravenous injection (2 h), L-MSCs showed greater survival than BM-MSCs; similarly, L-MSCs were significantly more resistant than BM-MSCs to anchorage independent culture (4 h) in vitro. Long after transplantation (4 or 32 days), a significantly higher number of CD45(neg) L-MSCs were retained than BM-MSCs. By flow cytometry, L-MSCs expressed more intercellular adhesion molecule-1 (ICAM-1), platelet derived growth factor receptor alpha (PDGFRα), and integrin α2 than BM-MSCs; these proteins were found to modulate endothelial adherence, directional migration, and migration across Matrigel in L-MSCs. Further, L-MSCs with low ICAM-1 showed poorer lung retention and higher phagocytosis in vivo. Compared with BM-MSCs, L-MSCs expressed higher levels of several transcripts (e.g., Ccl2, Cxcl2, Cxcl10, IL-6, IL-11, Hgf, and Igf2) in vitro, although gene expression in vivo was increased by L-MSCs and BM-MSCs equivalently. Accordingly, both L-MSCs and BM-MSCs reduced elastase injury to the same extent. This study demonstrates that tissue-specific L-MSCs possess mechanisms that enhance their lung retention after intravenous transplantation, and produce substantial healing of elastase injury comparable to BM-MSCs.

Figures

FIG. 1.

The culture phenotype and differentiation potential of L-MSCs. Lung-derived MSCs (L-MSCs) were cultured in basal media (alpha MEM, 15% FBS) by the outgrowth method on plastic. (A) L-MSCs grown on plastic at low density, passage 7, phase contrast, 200× and (B) confluent for 7 days, 100×; (C) colony formation of passage 7 L-MSCs, seeded at 2,000 cells/78 cm2, crystal violet stain and (D) appearance of single colony at 10 days of growth, 50×; immunohistochemistry of subconfluent cultures of L-MSCs show strong expression of collagen IV (E), laminin (F), and vimentin (G), weak expression of fibronectin (H) and negligible immunoreactivity for collagen 1 (I), (200×); (J) isotype control: rabbit IgG; L-MSCs were differentiated to osteocytes as evidenced by calcium phosphate staining with Alizarin Red, 50× (K); chondrocyte mucopolysaccharides stained with Alcian Blue, 50× (L); adipocytes, neutral lipid droplet formation from 21 to 28 days, phase contrast (M), and specific neutral lipid staining using LipidTox (Invitrogen), 200× (N); tube formation after 12 h culture in endothelial growth media (O); endothelial growth media L-MSCs grown on plastic (shown) or Matrigel display uptake of Dil-Ac-LDL (P), 100×; shift in phenotype from undifferentiated L-MSCs (Q), to myofibroblastic cells (R) after exposure to TGFβ1 (10 ng/mL, 2 days) as evidenced by increase in frequency and intensity of alpha smooth muscle actin expression and shift in morphology to large, stellate, filamentous cells, 200×; L-MSCs differentiated into smooth muscle cells when placed in low serum (2.5%) (S, control L-MSCs), exposed to 5 Aza-cytidine (24 h), and supplemented with ascorbic acid (50 μg/mL) for 10 days (T, smooth muscle cells): cells exhibited a marked increase in surface area and appearance of stress fibers (100×). L-MSCs, lung-derived mesenchymal stromal cells; MEM, minimum essential media; FBS, fetal bovine serum; TGFβ1, transforming growth factor beta 1. Color images available online at www.liebertonline.com/scd

FIG. 2.

Retention of L-MSCs versus BM-MSCs was evaluated 32 days after syngeneic transplantation (1×106 cells IV) in a murine model of emphysema. (A) Representative flow cytometry plots showing higher frequency of PKH-positive (FL2) cells after L-MSCs transplantation. (B) Based on flow cytometry retention of L-MSCs was significantly greater than BM-MSCs (*P<0.001, n=11/group). (C) Phagocytosis evidenced by CD45 expression was significantly lower in L-MSCs than BM-MSCs. (D) Appearance of PKH-positive (red) clusters of L-MSCs around both injured and noninjured zones on day 32 after transplantation (top panels), and example of CD45pos cells adjacent to CD45neg PKHpos L-MSCs (lower left panel); BM-MSCs (lower right panel) were rarer and appeared as single cells rather than clusters (100–400×). (E) Flow cytometry analysis of PKH-positive cells in mice injected with L-MSCs showing low expression of hematopoietic markers (CD45 and CD11b) and endothelial markers (CD31 and Sca-1) but enrichment for markers found in L-MSCs in vitro (CD73 and CD105). (F) Cryosections containing PKH-labeled L-MSCs were co-stained for a variety of phenotypic markers: PKH stained in close proximity to vimentin, CD31, laminin, col IV, and aqp5, but pixel-by-pixel analysis showed high correlation coefficients (>0.5) only for vimentin with PKH (Pearson r=0.82) or beta-actin with PKH (r=0.71). BM-MSCs, bone marrow-derived mesenchymal stromal cells. Color images available online at www.liebertonline.com/scd

FIG. 3.

Transplantation assays comparing L-MSCS and BM-MSCs retention and evidence for phagocytosis (day 4), early survival (2 h), and in vitro assay to explore potential mechanism for survival (anoikis resistance). (A) Retention of PKH26-labeled lung (L-MSCs) versus bone marrow (BM-MSCs)-derived multipotential stromal cells (1×106 cells in 200 μL by tail vein, n=11 mice/group; *P<0.05 vs. L-MSCs or ‘L’). L-MSCs were retained in the lung at a significantly higher rate than BM-MSCs (P<0.001), whether cultured L-MSCs were harvested at log or stationary phase and independent of whether recipient mice were elastase injured or not; phagocytosis as evidenced by CD45 or CD11b (Mac-1) was lower for L-MSCs at 4 days after injection. (B) Two hours after injection, the total percentage of L-MSCs that were viable was greater than the percentage of viable BM-MSCs (*P<0.05) and there was a trend (†P=0.07) toward a lower proportion of L-MSCs that were apoptotic (Annexin V positive); in vitro assay of anoikis resistance: cells were cultured for up to 4 h in suspension (37°C, 5% CO2); samples (4/time point) were analyzed throughout this period. (C) Viability was significantly (*P<0.05) higher for L-MSCs than for BM-MSCs 2 and 4 h after start of incubation; L-MSCs showed no decrement in viability during this period. (D) Apoptosis was significantly greater (*P<0.05) in BM-MSCs than in L-MSCs; both cell types showed a significant increase in apoptosis over time (†P<0.05 vs. 1–2 h).

FIG. 4.

Comparison of surface proteins expressed on L-MSCs versus BM-MSCs used for transplantation assays by flow cytometry. (A) Antigens shown are organized into phenotypic categories, including endothelial markers (from CD31 through CD34), stem cell markers (CD133 through Sca-1), endothelial cell receptor ligands (CD62E through CD24), integrins (Itg α2 to Itg β6), mesenchymal stem cell markers (CD44 through CD106), inter-cellular adhesion proteins (CD54 through CD166), fibroblasts (PDGFR-α and -β), pericytes (CD271/NGFR), and hematopoietic cell markers (CD45, CD11b, CD14, and CD11c) or other leukocyte ligands (CD11a). L-MSCs lacked any markers specific for hematopoietic cells, endothelial cells, or pericytes; high level of expression for mesenchymal stem cells was noted including CD44, CD73, CD105, CD106, and Sca-1. L-MSCs differed from BM-MSCs by >70% incidence (*) for ICAM-1 (CD54), Itg α2, and PDGFRα, which was supported by western blots (B–E). (F) Effect of 24 h culture with TNFα (rhTNFα, Sigma,10 ng/mL); TNFα increased ICAM-1 but difference between L-MSCs and BM-MSCs remained >70% (n=3 experiments/group). PDGFR-α and -β, platelet derived growth factor receptor alpha and beta; NGFR, nerve growth factor receptor; ICAM-1, intercellular adhesion molecule 1; PDGFRα, platelet derived growth factor receptor alpha; rhTNFα, recombinant human tumor necrosis factor; TNFα, tumor necrosis factor alpha.

FIG. 5.

Endothelial attachment was compared at 1 h between L-MSCs and BM-MSCs. (A) Significantly more L-MSCs than BM-MSCs attached to human umbilical vein endothelial cells (HUVECs) (*P<0.05). Anti-ICAM-1 blocking antibody (clone YN1/1.7.4, 10 μg/mL) significantly reduced the adherence of L-MSCs to HUVEC relative to treatment with isotype Ab. Phase-contrast images (100×) of study showing adherence of L-MSCs or BM-MSCs against darker background of HUVEC monolayer (lower panel). Higher magnification (400×) images showing spreading of adherent L-MSCs (inset). (B) Endothelial adherence study employing PKH-labeled BM-MSCs or L-MSCs and analyzed on the basis of total fluorescence. Group data showing statistically significant increased total pixel density of adherent L-MSCs incubated with isotype or no antibody versus BM-MSCs incubated with isotype antibody (rat IgG2b) and a significant reduction in attachment and spreading of L-MSCs when incubated (1 h) with anti-ICAM-1 functional blocking Ab (†P<0.05 vs. isotype Ab). (C) Blocking Ab effect on attachment of L-MSCs to endothelial monolayers was also concentration dependent (ICAM-1 group, *P<0.05, 10 vs. 1 μg/mL). (D) Association between ICAM-1 expression and lung retention of L-MSCs in elastase-injured mice. Explant-derived L-MSCs with high (87.8%) versus low (23.0%) incidence of ICAM-1 expression were generated by repeated passage using trypsin-free reagent (TrypLE; Invitrogen) versus trypsin/ethylenediaminetetraacetic acid, respectively. Passage 7 high versus low ICAM-1 expressing L-MSCs (0.5×106/mouse) were injected intravenously and retention quantified 4 days after treatment (n=5 mice/group) using flow cytometry. Representative flow cytometry plots from mice injected with high, low, or no L-MSCs injected showed significantly higher retention of high ICAM-1 versus low ICAM-1 expressing L-MSCs. (E) High ICAM-1 expression resulted in significantly (*P=0.003) greater retention efficiency (% total cells/injected cells in millions) and (F) Lower phagocytosis rates by total leukocytes or macrophages. Color images available online at www.liebertonline.com/scd

FIG. 6.

Migration and invasion assays in vitro. (A) L-MSCs and BM-MSCs were compared for their capacity to migrate across porous membranes with and without Matrigel (growth factor reduced) interposed (6 wells/condition). Both baseline migration in the absence of a chemo-attractant (“random migration”) and migration in response to serum (15% FBS) was significantly greater in L-MSCs (P<0.05 vs. respective BM-MSCs) in both uncoated (*) and Matrigel-coated † wells. (B) Specific chemo-attractants (PDGFAA, PDGFBB, and FGF-2, 25 ng/mL, 24 h) were effective to increase migration of L-MSCs (*P<0.05 vs. negative control), whereas only PDGFBB improved migration of BM-MSCs (†P<0.05 vs. respective L-MSCs groups, or PDGFBB-stimulated BM-MSCs). (C) Migration of L-MSCs across Matrigel-coated wells was significantly greater than BM-MSCs (*P<0.01); migration across Matrigel was impaired by prior incubation of cells with functional anti-Itg α2 antibody († vs. isotype); blocking with this antibody was ineffective in BM-MSCs.

FIG. 7.

Effect of L-MSCs versus BM-MSCs on gene expression in vivo and repair of elastase injury. (A) Quantitative real-time polymerase chain reaction measurements of mRNA expression in lung tissues after administration of L-MSCs or BM-MSCs (0.5×106 IV) versus saline treatment to mice with prior elastase injury (n=4/group). Primers are listed in the on-line Supplementary Table 4. Results are expressed as fold changes (*P<0.05 L-MSCs/PBS or BM-MSC/PBS; †P<0.05 L-MSCs/BM-MSCs). (B–C) Administration of either L-MSCs or BM-MSCs (0.5×106 IV) significantly reduced MLI (shown is mean±standard deviation) relative to control treatment on day 22 (n=7/group); (D) Repeated administration of L-MSCs (0.33×106 IV every 2 weeks for a total or 3 treatments) significantly reduced MLI 28 days after last treatment (n=10/group; *P<0.05 vs. PBS treatment). (E) Picrosirius staining was evaluated in the latter experiment by color-intensity thresholding of digital images (10 images/section, 2 sections/animal) and quantification of mean total pixel intensity of these 20 images using SigmaScanPro5. There was no significance difference in picrosirius staining between PBS and L-MSCs-treated mice. PBS, phosphate-buffered saline; MLI, mean linear intercept.