Comparison of the therapeutic effects of human and mouse adipose-derived stem cells in a murine model of lipopolysaccharide-induced acute lung injury
© Zhang et al.; licensee BioMed Central Ltd. 2013
Received: 8 November 2012
Accepted: 23 January 2013
Published: 29 January 2013
Adipose-derived stem cells (ASCs) have emerged as important regulators of inflammatory/immune responses in vitro and in vivo and represent attractive candidates for cell-based therapies for diseases that involve excessive inflammation. Acute lung injury (ALI) is an inflammatory condition for which treatment is mainly supportive due to lack of effective therapies. In this study, the therapeutic effects of ASC-based therapy were assessed in vivo by comparison of the anti-inflammatory properties of both human and murine ASCs in a mouse model of lipopolysaccharide (LPS)-induced ALI.
Human ASCs (hASCs) or mouse ASCs (mASCs) were delivered to C57Bl/6 mice (7.5 × 105 total cells/mouse) by oropharyngeal aspiration (OA) four hours after the animals were challenged with lipopolysaccharide (15 mg/kg). Mice were sacrificed 24 and 72 hours after LPS exposure, and lung histology examined for evaluation of inflammation and injury. Bronchoalveolar lavage fluid (BALF) was analyzed to determine total and differential cell counts, total protein and albumin concentrations, and myeloperoxidase (MPO) activity. Cytokine expression in the injured lungs was measured at the steady-state mRNA levels and protein levels for assessment of the degree of lung inflammation.
Both human and mouse ASC treatments provided protective anti-inflammatory responses. There were decreased levels of leukocyte (for example neutrophil) migration into the alveoli, total protein and albumin concentrations in BALF, and MPO activity after the induction of ALI following both therapies. Additionally, cell therapy with both cell types effectively suppressed the expression of proinflammatory cytokines and increased the anti-inflammatory cytokine interleukin 10 (IL-10). Overall, the syngeneic mASC therapy had a more potent therapeutic effect than the xenogeneic hASC therapy in this model.
Treatment with hASCs or mASCs significantly attenuated LPS-induced acute lung injury in mice. These results suggest a potential benefit for using an ASC-based therapy to treat clinical ALI and may possibly prevent the development of acute respiratory distress syndrome (ARDS).
Acute lung injury (ALI) is a common clinical occurrence that results from a number of localized and systemic pathological conditions including sepsis, trauma, shock, pneumonia, gastric aspiration, toxic ingestion, and pancreatitis. ALI can progress into a life-threatening condition known as acute respiratory distress syndrome (ARDS) particularly in critically ill patients. ALI/ARDS is characterized by acute onset of overwhelming pulmonary inflammation, bilateral infiltrates, and diffuse alveolar damage. Inflammation may progress to the point of widespread pulmonary edema and poor lung compliance that ultimately result in severe hypoxemia and devastating respiratory failure [1–3]. Studies indicate that the age-adjusted incidence of ALI/ARDS in the United States is 86.2 per 100,000 person-years, and the mortality rate is 36 to 44% due to lack of an effective therapy [4, 5]. Current treatment for ALI/ARDS is mainly limited to supportive care including mechanical ventilation with concomitant treatment of underlying diseases or initiating factors . Given the severe complications and high mortality rate of ALI/ARDS, a novel and more effective therapy is needed.
Multipotent stromal cells (or mesenchymal stem cells, MSCs) are a heterogeneous subset of progenitor cells that have self-renewal and multilineage differentiation capabilities [6–8]. In recent years, bone marrow-derived MSCs (BMSCs) have emerged as promising cell-based therapeutic agents for various inflammatory diseases given their more recently recognized property of potent immunomodulation and immunosuppression [8–12].
Encouraging results have been obtained using BMSC therapy in both rodent and human tissue models of ALI/ARDS [13–15]. Gupta et al. reported that intrabronchial delivery of murine BMSCs improved survival and attenuated lipopolysaccharide (LPS)-induced ALI in mice. This study showed that bronchoalveolar lavage fluid (BALF) and plasma from endotoxin-challenged mice treated with BMSCs had lower concentrations of tumor necrosis factor alpha (TNF-α) and macrophage inflammatory protein 2 (MIP-2) and higher concentrations of interleukin 10 (IL-10) compared with untreated mice . Recently, our group reported that, in a mouse model of LPS-induced acute lung injury, administration of human BMSCs by oropharyngeal aspiration (OA) significantly reduced the expression of proinflammatory cytokines, neutrophil counts, total BALF protein and pulmonary edema. Our study demonstrated that xenogeneic BMSCs recapitulated the observed immunomodulatory effects of syngeneic BMSCs . However, a critical hindrance in clinical translation of BMSCs is that bone marrow has a high degree of viral exposure [16, 17], and it may not be practical to obtain BMSCs from some morbid donors [18, 19].
In contrast, adipose tissue is abundant, expendable, and easily accessible, and adipose-derived MSCs (that is adipose-derived stem cells (ASCs) or adipose stem cells) can be isolated from adipose tissue using a simple protocol that results in high yields, due in part to the higher frequency of mesenchymal stem cells in the adipose tissue compared to bone marrow. Because they are easy to acquire and exhibit some of the same immunosuppressive and immunomodulatory characteristics as BMSCs , ASCs are an attractive alternative source of readily available adult stem cells. However, very little is known regarding the effect of adipose-derived stem cells in experimental models of ALI/ARDS. The current study tests the ability of ASCs isolated from either syngeneic mice or humans to ameliorate lung injury and suppress inflammation after administration of LPS via an OA route. This may be the first report describing the therapeutic role of mouse syngeneic ASC or human xenogeneic ASC in attenuating ALI in a murine model of LPS-induced ALI.
Materials and methods
Preparations of human and mouse ASCs
Human and mouse ASCs were isolated and characterized using standard procedures developed at the Pennington Biomedical Research Center [21–23]. All protocols were reviewed and approved by the Pennington Biomedical Research Center Institutional Review Board and all human participants provided written informed consent (PBRC number 23040). The mASCs were isolated from inbred transgenic C57Bl/6-Tg(UBC-GFP)30Scha/J mice (Jackson Laboratories, Bar Harbor, ME, USA), which ubiquitously express enhanced green fluorescent protein (eGFP) in all tissues. The cells were negative for CD11b, CD31, and CD45, and they were positive for CD29 and CD106 via flow cytometry analysis (data not shown). The cells were able to generate colony-forming units-fibroblasts (CFU-Fs, data not shown) and were able to differentiate along adipogenic and osteogenic lineages (data not shown). Frozen vials of 1 × 106 human or mouse ASCs (passage 3) were thawed and plated onto a 145 cm2 culture dish (Nunc, Rochester, NY, USA) in 20 ml complete culture medium (CCM) that consisted of DMEM/F-12 (GIBCO, Grand Island, NY, USA), 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA), and 1% Anti-Anti (antibiotic-antimycotic, GIBCO). The cells were incubated at 37°C with humidified air containing 5% CO2. After 18 to 20 hours, the medium was removed and adherent cells were washed once with 1 × phosphate-buffered saline (PBS, GIBCO), harvested with 0.25% trypsin/1mM EDTA (GIBCO), and replated at 200 cells/cm2 in CCM for expansion. Media were changed every 3 to 4 days until the cells reached approximately 70% confluence. The culture medium was changed to antibiotic-antimycotic-free CCM one day before the cells were to be delivered to mice. ASCs were harvested with 0.25% trypsin/1 mM EDTA, washed with PBS, resuspended in Hank's balanced salt solution (HBSS, GIBCO) and administered to mice via OA as described below. HBSS alone was used as a vehicle-only control.
ALI induction and treatment protocols
All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Tulane University and conformed to the requirements of the Animal Welfare Act.
To induce acute lung injury, female C57Bl/6 mice (National Cancer Institute-Frederick, Frederick, MD, USA) aged 8 to 10 weeks were first anesthetized by inhalation of 2% isoflurane (VetOne, Meridian, ID, USA) vapor mixed with oxygen as previously described [14, 24]. Anesthetized mice were suspended by their cranial incisors, and the tongue was extracted to full extension to prevent the swallowing reflex. LPS from Escherichia coli 055:B5 (15 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) or PBS vehicle alone (75 µl each) was pipetted into the back of the throat, and the nares were pinched shut to force breathing through its mouth and liquid aspiration. Four hours after LPS challenge, two doses of human or mouse ASCs (3.75 × 105, suspended in 75 µl HBSS) or HBSS vehicle-only control (75 µl each) were delivered by OA 30 minutes apart, for a total dose of 7.5 × 105 cells (150 µl total volume) per mouse for each test group. The animals were sacrificed 24 or 72 hours after LPS exposure by exsanguination under anesthesia with 80 mg/kg ketamine plus 8 mg/kg xylazine. The lungs were processed for bronchoalveolar lavage (BAL) collection, RNA or protein isolation, or histology as described below.
Immediately after exsanguination, the lungs were cannulated with a 20-gauge intravenous (IV) catheter (Exel International Medical Products, St. Petersburg, FL, USA) and gently washed five times with 530 μl (right lung) or 1 ml (whole lung) PBS supplemented with 0.4 mM EDTA (GIBCO) and protease inhibitor cocktail (Roche, Indianapolis, IN, USA). The lavage fluid was spun at 1,500 × g for 5 minutes at 4°C to pellet the cells. Cells from all five lavage collections were pooled for total cell counting while the supernatant from the first lavage was stored at-80°C for biochemical analysis. The protein concentration in the bronchoalveolar lavage fluid was measured using the micro bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL, USA). For differential cell counts, the cells were spun onto glass slides by cytospin (Thermo-Shandon, Wilmington, DE, USA) and stained with a modified Wright-Giemsa stain (Diff-Quik, Fisher Scientific, Pittsburgh, PA, USA). The numbers of neutrophils, macrophages, eosinophils, basophils, and lymphocytes, up to a total of 100 cells, were determined in three random viewing fields per sample.
Hematoxylin and eosin (H&E) staining
Lungs were inflation-fixed with 10% neutral buffered formalin (Sigma-Aldrich) through the trachea at 25 cm H2O pressure for 20 minutes, excised from the mice, and placed in fresh 10% neutral buffered formalin at 4°C. The fixed lungs were then embedded in paraffin, and 6 μm sections were stained with H&E to reveal anatomic details. Whole section images were captured on an Aperio ScanScope (Aperio Technologies, Vista, CA, USA) and viewed using Aperio ImageScope (Aperio Technologies).
Lung homogenization, RNA isolation, and cDNA synthesis
Excised lung tissue was weighed and homogenized using a Bio-Plex cell lysis kit (Bio-Rad, Hercules, CA, USA) as per the manufacturer's instruction. The homogenates were centrifuged at 10,000 × g for 15 minutes, and the supernatant was aliquoted and stored at-80°C until analyzed. The protein concentration in the supernatant was measured by BCA assay. Total RNA was isolated from lung tissue homogenized in TriPure Isolation Reagent (Roche) and was purified with the RNeasy mini kit (Qiagen, Valencia, CA, USA). The RNA was first treated with DNase I (Amplification grade, Invitrogen) and then converted into cDNA using iScript cDNA Synthesis Kit (Bio-Rad) following the manufacturer's instructions in a PTC-200 Peltier Thermal Cycler (MJ Research, Ramsey, MN, USA).
The persistence of viable hASCs or mASCs in the lung was determined at 20 hours after the cells were delivered using real-time RT-PCR for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or eGFP mRNA, respectively, as previously described [14, 25]. TaqMan Gene Expression Assay (Hs00266705_g1, Applied Biosystems, Foster City, CA, USA) was used to specifically quantify human GAPDH. The following primer/probe set was used to quantify eGFP: forward 5'-CCACATGAAGCAGCAGGACTT-3', reverse 5'-GGTGCGCTCCTGGACGTA-3', and probe 5'-6FAM-TTCAAGTCCGCCATGCCCGAA-TAMRA-3'. Briefly, 7.5 × 105 hASCs or mASCs were added to a whole lung tissue sample just prior to homogenization and RNA isolation. Standard curves were generated by adding serial dilutions of mRNA isolated from lung tissue plus ASCs to mRNA isolated only from lung tissue. Real-time RT-PCR was performed, and Ct values were used to determine the persistence of hASCs or mASCs according to the standard curves. The final value for total cDNA in the sample was corrected by parallel real-time RT-PCR assays with primers that amplified both the human and the mouse genes for GAPDH (forward 5'-CAGCGACACCCACTCCTCCACCTT-3', reverse 5'-CATGAGGTCCACCACCCTGTTGCT-3') and mouse ribosomal protein, large, P0 (Rplp0, forward 5'-CGACCTGGAAGTCCAACTAC-3', reverse 5'-ATCTGCTGCATCTGCTTG-3'). Results were presented as the percentage of the calculated cell number relative to the delivered cell number (7.5 × 105). For cytokine/chemokine expression analysis, the real-time PCR reactions were performed as previously described by Ripoll et al. with modifications . Briefly, each reaction mixture contained 1 µl of commercially available TaqMan GeneExpression Assay primer/probe set (Applied Biosystems), 10 µl TaqMan Gene Expression Master Mix (Applied Biosystems), 50 ng cDNA template and RNase-free water with a total volume of 20 µl. Beta-actin (β-actin) was used as an endogenous control. The reaction was performed at 50°C for 2 minutes, 95°C for 10 minutes followed by a 40-cycle two-step PCR (95°C for 15 seconds and 60°C for 1 minute) using the CFX96 Real-Time System (Bio-Rad). TaqMan GeneExpression Assays (Applied Biosystems) used for murine genes were: β-actin (Mm00607939_s1), macrophage inflammatory protein 2 (MIP-2, Mm00436450_m1), interleukin 1 alpha (IL-1α, Mm00439621_m1), IL-1β (Mm01336189_m1), MIP-1α (Mm00441258_m1), and tumor necrosis factor alpha (TNF-α, Mm00443258_m1), and IL-10 (Mm01288386_m1).
Myeloperoxidase activity assay, ELISA, and multiplex immunoassay
BALF myeloperoxidase (MPO) activity assay was performed as previously described . Briefly, BALF was added to a reaction buffer containing 50 mM potassium phosphate (pH 6.0), 0.0005% (v/v) H2O2, and 0.167 mg/ml o-dianisidine dihydrochloride (Sigma-Aldrich); the absorbance at 460 nm was monitored for 30 minutes using the Synergy HT multi-detection microplate reader (Bio-Tek Instruments, Winooski, VT, USA). The albumin levels in BALF were measured using a mouse albumin ELISA kit purchased from Bethyl Laboratories (Montgomery, TX, USA). Measurement of cytokines and chemokines in lung homogenates was performed by multiplex immunoassay using a Millipore mouse cytokine/chemokine 32-plex kit (Millipore, Billerica, MA, USA).
Data are summarized as mean ± SEM. One-way ANOVA was performed for statistical analysis, followed by Bonferroni multiple comparison tests. The statistical significance value was set at P <0.05.
Engraftment of hASCs and mASCs in the lung
Change in body weight following treatment
The body weight of each mouse was determined immediately prior to LPS (or PBS control) challenge and again at the time of sacrifice (24 hours after LPS exposure). The change in body weight was calculated as percentage relative to the basal body weight. While intact, untreated mice (N = 7) gained about 2% body weight during this 24-hour period, mice in the PBS + hASC (N = 6) and PBS + mASC (N = 6) control groups lost 2 to 3% body weight during the 24-hour period. For the diseased mice induced by LPS, the percentage of body weight lost was reduced following hASC (10.19 ± 0.85%, N = 10, P >0.05) and mASC (7.14 ± 0.52%, N = 10, P <0.001) treatment, as compared to the HBSS vehicle-only controls (11.82 ± 0.66%, N = 16) (Figure 1b).
Assessment of lung histology and microvascular permeability
Massive inflammation causes vascular leakage in the lung in this LPS-induced ALI mouse model [13, 14], as indicated by the elevated levels of protein and albumin in BALF (Figure 2d, e). To assess the effect of hASCs or mASCs on the integrity of the alveolar-capillary membrane barrier and pulmonary vascular leakage, the levels of total protein and albumin in the bronchoalveolar lavage fluid were examined 24 hours after PBS or LPS challenge. Results demonstrated that total protein concentration in BALF was reduced in both the hASC (N = 9) and mASC (N = 10) treatment groups compared to the HBSS-only control group (N = 10), however, statistical significance (P <0.001) was reached only in the mASC treatment group. Administration of hASCs (N = 9) and mASCs (N = 10) resulted in significantly lower concentrations of BALF albumin, about 41% and about 56% lower than those of HBSS (N = 10) vehicle-treated mice (P <0.01 and P <0.001, respectively). Interestingly, these parameters showed a trend that treatment with mASCs decreased BALF total protein and albumin even more than hASC treatment, although this effect was not statistically significant.
Effect of hASCs and mASCs on the infiltration of inflammatory cells
Regulation of cytokine expression by hASCs and mASCs
According to the Berlin definition of ARDS, the term acute lung injury (ALI) has been eliminated since 2011 , however, the majority of the field still uses ALI as a valid denomination. For the purpose of this article, we still use the term ALI. Given that excessive inflammatory responses play a crucial role in the pathophysiology of ALI/ARDS , immunomodulatory therapy is an attractive new approach in the treatment of such afflictions [28, 29]. Multipotent stromal cells provide unique opportunities for developing novel treatments for a large array of inflammatory diseases, because MSCs possess potent immunomodulatory properties and are often considered to be hypoimmunogenic or 'immunoprivileged' [10, 30]. An emerging body of data indicates that either human or mouse bone marrow-derived MSCs (BMSCs) alleviate tissue damage and suppress inflammatory reactions in different models of ALI [13–15, 31]. As an alternative source of mesenchymal stem cells, adipose tissue is abundant and easily accessible, and high yields of adipose-derived MSCs (ASCs) are easier to isolate with less invasive procedures. ASCs have been reported to have similar immune regulatory capabilities as BMSCs [20, 32]. While the use of BMSCs to treat ALI/ARDS is promising, there is little known about the potential therapeutic effects of ASCs in this disease setting. In this study, the therapeutic effect of human-and mouse-derived ASCs was investigated in a mouse model of LPS-induced ALI.
Endotoxin (bacterial LPS)-induced ALI in mice is a widely used experimental model to investigate the pathogenesis and treatment of ALI/ARDS. However, it should be noted that no single ALI animal model can completely reproduce all the pathophysiological features of ALI/ARDS in humans. The airway LPS administration model employed in these studies results in massive alveolar neutrophil infiltration, which is also seen in human ALI/ARDS [33, 34].
In this study the relatively noninvasive OA method  was used to administer both LPS and ASCs. A previous study from our group showed a comparable beneficial effect using the IV, intraperitoneal (IP), and OA methods to deliver human BMSCs to LPS-challenged mice .
Taking advantage of the cross-species nature of hASCs and the eGFP marker of mASCs in our experiments, the persistence of the cells in the lungs after delivery through OA was confirmed by real-time RT-PCR for human GAPDH and eGFP. It is expected that the cells detected reflect persistence of live cells since mRNA for human GAPDH and eGFP was detected as markers for human and mouse ASCs, respectively.
These results demonstrated that treatment of ALI with either hASCs or mASCs decreased the body weight loss following ALI induction, which suggests lower disease severity following treatment. Both treatments decreased LPS-induced lung damage and neutrophil infiltration substantially, as shown by H&E staining. This was further corroborated by decreased levels of total protein and albumin in BALF, indicating reduced endothelial and epithelial permeability in the lung following either hASCs or mASCs treatment.
Because neutrophil activation and transmigration into lung interstitium and broncheoalveolar space plays a crucial role in the development of ALI , it can be inferred that decreasing infiltration of neutrophils using ASCs administration may have clinically relevant therapeutic potential. Activated neutrophils secrete proteinases and potent enzymes such as MPO that generate oxidants, all of which can damage lung tissue leading to edema and loss of lung function . Decreased MPO activity after cell treatment indicates that both hASCs and mASCs are capable of decreasing the lung inflammatory responses that ultimately cause the neutrophil-associated tissue damage common in ALI.
Nemeth et al. and Anderson et al. showed that mouse BMSCs and ASCs were able to reprogram macrophages to increase their IL-10 production, and the administration of these cells protected mice from sepsis [12, 32]. In this study, both hASC and mASC treatments led to a reduction of the expression of various proinflammatory cytokines/chemokines, while increasing the production of the anti-inflammatory cytokine IL-10. These data suggest that the anti-inflammatory effects of hASC and mASC in this model were mediated, at least in part, by the increased production of IL-10 in the lung. The decrease of the production of proinflammatory cytokines/chemokines at the protein level was found to be not as pronounced as that at the mRNA level 24 hours after LPS exposure. This may be explained by the time lag between translation and transcription.
It is important to note that while cytokine and chemokine levels were quantified in the lungs of mASC-treated mice, the cellular source of the cytokines or chemokines (that is, lung epithelial cells, alveolar macrophages, inflammatory cells or administered ASCs) was not able to be determined. Our previous study showed that, when stimulated with LPS or BALF, mASCs did secrete some cytokines and chemokines (for example IL-1α and MIP-1α) in vitro (data not shown). These data suggest that decreased cytokine levels in animals treated with mASCs may actually be lower than recorded since the mASC may act as an exogenous source of these cytokines. However, the expression of cytokines and chemokines from human ASCs could not be detected since human cytokines and chemokines would not be detected by mouse specific primers. Therefore, using human ASCs offers the possibility of determining how the ASCs are responding to the injured lung, because human specific primers or antibodies can be used.
To the best of our knowledge, this is the first report to demonstrate the beneficial effects of ASCs for the treatment of LPS-induced acute lung injury in vivo. Moreover, the therapeutic effects of human and mouse ASCs have been compared side by side. While both hASCs and mASCs significantly attenuated the lung injury and inflammation, some notable differences between the two cell types were observed. For most of the parameters examined, mASCs had a more beneficial effect than xenogeneic hASCs. A possible explanation for this observation is that the cross-talk between mASCs and the injured mouse lung is more effective than in the xenotransplantation model using hASCs. In addition, studies have demonstrated species variation in the mechanisms of bone marrow-derived MSC-mediated immunosuppression. Ren et al. showed that under the same culture conditions, immunosuppression by human or monkey BMSCs was mediated by indoleamine 2,3-dioxygenase, while mouse BMSCs utilized nitric oxide . The species variation may also be an explanation to the different therapeutic effects of hASCs and mASCs in this ALI model.
To advance the use of stem cell therapy in the treatment of ALI/ARDS it is essential that the molecular mechanisms behind any observed beneficial effects be understood. As discussed above, there is increasing evidence that both mouse and human BMSCs have similar anti-inflammatory properties, however the mechanisms mediating these outcomes may be unique to each cell type. Similarly, our unpublished data suggest that human and mouse ASCs suppress inflammation in the lung by different mechanisms. Therefore, it is essential that human cells be used in preclinical studies employing animal models. However concerns that the protective factors produced by human MSCs may not be effective in other species have limited such studies. Results from this study demonstrate potent anti-inflammatory effects of both human and mouse ASCs in wild-type mice exposed to LPS, a well-characterized model of ALI. While syngeneic mouse ASCs were expectedly more effective at suppressing inflammation, our results clearly demonstrate the feasibility of using immunocompetent mice to interrogate the molecular mechanisms by which human ASCs suppress inflammation.
There are currently more than 60 clinical trials registered with clinicaltrial.gov testing ASCs in a variety of disorders, including Crohn's fistula, type 2 diabetes, and chronic obstructive pulmonary disease. As discussed above, ASCs are easy to isolate and expand in vitro, and they are also 'immunoprivileged' , which makes allogeneic transplantation to humans feasible. Despite the promising results we obtained in the mouse model, clinicians should use caution when considering the use of ASCs as a therapy for ALI/ARDS in human subjects. A recent study showed that in both humans and mice, intravenous infusion of ASCs did not induce any serious adverse events related to ASC transplantation or any tumor development (even in nude mice) . However, other studies have indicated that ASCs enhance tumorigenesis and metastasis of breast cancer cell lines and primary breast cancer samples [39, 40]. Our recently published data showed that ASCs isolated from the subcutaneous abdominal adipose tissue of obese subjects have enhanced invasion toward breast cancer cells . Prantl et al. demonstrated that ASCs promote prostate tumor growth . Therefore, before use in human subjects, the safety of ASCs should be fully investigated and evaluated, particularly in patients with breast cancer and prostate cancer.
This study suggests that the transplantation of human or mouse ASCs by oropharyngeal aspiration effectively attenuates lung injury and inflammation in a mouse model of LPS-induced ALI. These therapeutic effects may be partially due to the increased production of IL-10 in the lung following the cell-based treatment. A side-by-side comparison indicated that mASCs are more potent than hASCs in mediating the therapeutic effects following LPS-induced ALI in this mouse model. Therefore, adipose-derived stem cells may serve as a novel treatment modality for ALI, a medical challenge that currently lacks efficient therapies beyond supportive care.
acute lung injury
acute respiratory distress syndrome
adipose-derived stem cells
bronchoalveolar lavage fluid
bone marrow-derived mesenchymal stem cells
complete culture medium
Dulbecco's modified Eagle's medium
enhanced green fluorescent protein
enzyme-linked immunosorbent assay
granulocyte macrophage colony-stimulating factor
hematoxylin and eosin
human adipose-derived stem cells
Hank's balanced salt solution
macrophage inflammatory protein
mesenchymal stem cells
mouse adipose-derived stem cells
reverse transcription-polymerase chain reaction
tumor necrosis factor alpha
We thank Margie McCants for her technical assistance with cell culture and Dina Gaupp for her tremendous help with H&E staining. The research was supported by funds from Tulane University.
- Ware LB, Matthay MA: The acute respiratory distress syndrome. N Engl J Med. 2000, 342: 1334-1349. 10.1056/NEJM200005043421806.View ArticlePubMedGoogle Scholar
- Dushianthan A, Grocott MP, Postle AD, Cusack R: Acute respiratory distress syndrome and acute lung injury. Postgrad Med J. 2011, 87: 612-622. 10.1136/pgmj.2011.118398.View ArticlePubMedGoogle Scholar
- Bonvillain RW, Zhang S, Eagle ME, Danchuk SD, Bunnell BA, Sullivan DE: Battling inflammation in acute lung injury and acute respiratory distress syndrome: stem cell-based therapy targeting the root cause of acute lung injury. J Pulmonar Respirat Med.
- Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, Stern EJ, Hudson LD: Incidence and outcomes of acute lung injury. N Engl J Med. 2005, 353: 1685-1693. 10.1056/NEJMoa050333.View ArticlePubMedGoogle Scholar
- Phua J, Badia JR, Adhikari NK, Friedrich JO, Fowler RA, Singh JM, Scales DC, Stather DR, Li A, Jones A, Gattas DJ, Hallett D, Tomlinson G, Stewart TE, Ferguson ND: Has mortality from acute respiratory distress syndrome decreased over time?: A systematic review. Am J Resp Crit Care Med. 2009, 179: 220-227. 10.1164/rccm.200805-722OC.View ArticlePubMedGoogle Scholar
- Prockop DJ: Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997, 276: 71-74. 10.1126/science.276.5309.71.View ArticlePubMedGoogle Scholar
- Pittenger MF: Multilineage potential of adult human mesenchymal stem cells. Science. 1999, 284: 143-147. 10.1126/science.284.5411.143.View ArticlePubMedGoogle Scholar
- Uccelli A, Moretta L, Pistoia V: Mesenchymal stem cells in health and disease. Nat Rev Immunol. 2008, 8: 726-736. 10.1038/nri2395.View ArticlePubMedGoogle Scholar
- Ripoll CB, Flaat M, Klopf-Eiermann J, Fisher-Perkins JM, Trygg CB, Scruggs BA, McCants ML, Leonard HP, Lin AF, Zhang S, Eagle ME, Alvarez X, Li YT, Li SC, Gimble JM, Bunnell BA: Mesenchymal-lineage stem cells have pronounced anti-inflammatory effects in the twitcher mouse model of Krabbe's disease. Stem Cells. 2010, 29: 67-77.View ArticleGoogle Scholar
- Nauta AJ, Fibbe WE: Immunomodulatory properties of mesenchymal stromal cells. Blood. 2007, 110: 3499-3506. 10.1182/blood-2007-02-069716.View ArticlePubMedGoogle Scholar
- Singer NG, Caplan AI: Mesenchymal stem cells: mechanisms of inflammation. Annual Review of Pathology: Mechanisms of Disease. 2011, 6: 457-478. 10.1146/annurev-pathol-011110-130230.View ArticleGoogle Scholar
- Nemeth K, Leelahavanichkul A, Yuen PS, Mayer B, Parmelee A, Doi K, Robey PG, Leelahavanichkul K, Koller BH, Brown JM, Hu X, Jelinek I, Star RA, Mezey E: Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med. 2009, 15: 42-49. 10.1038/nm.1905.PubMed CentralView ArticlePubMedGoogle Scholar
- Gupta N, Su X, Popov B, Lee JW, Serikov V, Matthay MA: Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice. J Immunol. 2007, 179: 1855-1863.View ArticlePubMedGoogle Scholar
- Danchuk S, Ylostalo JH, Hossain F, Sorge R, Ramsey A, Bonvillain RW, Lasky JA, Bunnell BA, Welsh DA, Prockop DJ, Sullivan DE: Human multipotent stromal cells attenuate lipopolysaccharide-induced acute lung injury in mice via secretion of tumor necrosis factor-alpha-induced protein 6. Stem Cell Res Ther. 2011, 2: 27-10.1186/scrt68.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee JW, Fang X, Gupta N, Serikov V, Matthay MA: Allogeneic human mesenchymal stem cells for treatment of E. coli endotoxin-induced acute lung injury in the ex vivo perfused human lung. Proc Natl Acad Sci USA. 2009, 106: 16357-16362. 10.1073/pnas.0907996106.PubMed CentralView ArticlePubMedGoogle Scholar
- Yoo KH, Jang IK, Lee MW, Kim HE, Yang MS, Eom Y, Lee JE, Kim YJ, Yang SK, Jung HL, Sung KW, Kim CW, Koo HH: Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues. Cell Immunol. 2009, 259: 150-156. 10.1016/j.cellimm.2009.06.010.View ArticlePubMedGoogle Scholar
- Rubinstein P, Rosenfield RE, Adamson JW, Stevens CE: Stored placental blood for unrelated bone marrow reconstitution. Blood. 1993, 81: 1679-1690.PubMedGoogle Scholar
- Zheng B, Cao B, Li G, Huard J: Mouse adipose-derived stem cells undergo multilineage differentiation in vitro but primarily osteogenic and chondrogenic differentiation in vivo. Tissue Eng. 2006, 12: 1891-1901. 10.1089/ten.2006.12.1891.View ArticlePubMedGoogle Scholar
- Jurgens WJ, Oedayrajsingh-Varma MJ, Helder MN, Zandiehdoulabi B, Schouten TE, Kuik DJ, Ritt MJ, van Milligen FJ: Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies. Cell Tissue Res. 2008, 332: 415-426. 10.1007/s00441-007-0555-7.PubMed CentralView ArticlePubMedGoogle Scholar
- Gonzalez-Rey E, Anderson P, Gonzalez MA, Rico L, Buscher D, Delgado M: Human adult stem cells derived from adipose tissue protect against experimental colitis and sepsis. Gut. 2009, 58: 929-939. 10.1136/gut.2008.168534.View ArticlePubMedGoogle Scholar
- Yu G, Floyd ZE, Wu X, Halvorsen YD, Gimble JM: Isolation of human adipose-derived stem cells from lipoaspirates. Methods Mol Biol. 2011, 702: 17-27. 10.1007/978-1-61737-960-4_2.View ArticlePubMedGoogle Scholar
- Yu G, Wu X, Kilroy G, Halvorsen YD, Gimble JM, Floyd ZE: Isolation of murine adipose-derived stem cells. Methods Mol Biol. 2011, 702: 29-36. 10.1007/978-1-61737-960-4_3.View ArticlePubMedGoogle Scholar
- Strong AL, Semon JA, Strong TA, Santoke TT, Zhang S, McFerrin HE, Gimble JM, Bunnell BA: Obesity-associated dysregulation of calpastatin and MMP-15 in adipose-derived stromal cells results in their enhanced invasion R1. Stem Cells. 2012, 30: 2774-2783. 10.1002/stem.1229.View ArticlePubMedGoogle Scholar
- Lakatos HF, Burgess HA, Thatcher TH, Redonnet MR, Hernady E, Williams JP, Sime PJ: Oropharyngeal aspiration of a silica suspension produces a superior model of silicosis in the mouse when compared to intratracheal instillation. Experimental lung research. 2006, 32: 181-199. 10.1080/01902140600817465.View ArticlePubMedGoogle Scholar
- Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL, Semprun-Prieto L, Delafontaine P, Prockop DJ: Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell. 2009, 5: 54-63. 10.1016/j.stem.2009.05.003.PubMed CentralView ArticlePubMedGoogle Scholar
- Yan C, Wu M, Cao J, Tang H, Zhu M, Johnson PF, Gao H: Critical role for CCAAT/enhancer-binding protein in immune complex-induced acute lung injury. J Immunol. 2012, 189: 1480-1490. 10.4049/jimmunol.1200877.PubMed CentralView ArticlePubMedGoogle Scholar
- Force ADT, Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, Fan E, Camporota L, Slutsky AS: Acute respiratory distress syndrome: the Berlin Definition. JAMA. 2012, 307: 2526-2533.Google Scholar
- Yeh CC, Kao SJ, Lin CC, Wang SD, Liu CJ, Kao ST: The immunomodulation of endotoxin-induced acute lung injury by hesperidin in vivo and in vitro. Life Sci. 2007, 80: 1821-1831. 10.1016/j.lfs.2007.01.052.View ArticlePubMedGoogle Scholar
- Schaefer MB, Ott J, Mohr A, Bi MH, Grosz A, Weissmann N, Ishii S, Grimminger F, Seeger W, Mayer K: Immunomodulation by n-3-versus n-6-rich lipid emulsions in murine acute lung injury--role of platelet-activating factor receptor. Crit Care Med. 2007, 35: 544-554. 10.1097/01.CCM.0000253811.74112.B6.View ArticlePubMedGoogle Scholar
- Patel SA, Sherman L, Munoz J, Rameshwar P: Immunological properties of mesenchymal stem cells and clinical implications. Archivum immunologiae et therapiae experimentalis. 2008, 56: 1-8.View ArticlePubMedGoogle Scholar
- Ortiz LA, Dutreil M, Fattman C, Pandey AC, Torres G, Go K, Phinney DG: Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury. Proc Natl Acad Sci USA. 2007, 104: 11002-11007. 10.1073/pnas.0704421104.PubMed CentralView ArticlePubMedGoogle Scholar
- Anderson P, Souza-Moreira L, Morell M, Caro M, O'Valle F, Gonzalez-Rey E, Delgado M: Adipose-derived mesenchymal stromal cells induce immunomodulatory macrophages which protect from experimental colitis and sepsis. Gut. 2012,Google Scholar
- Rittirsch D, Flierl MA, Day DE, Nadeau BA, McGuire SR, Hoesel LM, Ipaktchi K, Zetoune FS, Sarma JV, Leng L, Huber-Lang MS, Neff TA, Bucala R, Ward PA: Acute lung injury induced by lipopolysaccharide is independent of complement activation. J Immunol. 2008, 180: 7664-7672.PubMed CentralView ArticlePubMedGoogle Scholar
- Matute-Bello G, Matthay MA: Animal models of acute lung injury. [http://www.thoracic.org/clinical/critical-care/critical-care-research/animal-models-of-acute-lung-injury.php]
- Grommes J, Soehnlein O: Contribution of neutrophils to acute lung injury. Mol Med. 2011, 17: 293-307.PubMed CentralView ArticlePubMedGoogle Scholar
- Ren G, Su J, Zhang L, Zhao X, Ling W, L'Huillie A, Zhang J, Lu Y, Roberts AI, Ji W, Zhang H, Rabson AB, Shi Y: Species variation in the mechanisms of mesenchymal stem cell-mediated immunosuppression. Stem Cells. 2009, 27: 1954-1962. 10.1002/stem.118.View ArticlePubMedGoogle Scholar
- Lindroos B, Suuronen R, Miettinen S: The potential of adipose stem cells in regenerative medicine. Stem cell Rev. 2011, 7: 269-291. 10.1007/s12015-010-9193-7.View ArticlePubMedGoogle Scholar
- Ra JC, Shin IS, Kim SH, Kang SK, Kang BC, Lee HY, Kim YJ, Jo JY, Yoon EJ, Choi HJ, Kwon E: Safety of intravenous infusion of human adipose tissue-derived mesenchymal stem cells in animals and humans. Stem Cells Dev. 2011, 20: 1297-1308. 10.1089/scd.2010.0466.View ArticlePubMedGoogle Scholar
- Zimmerlin L, Donnenberg AD, Rubin JP, Basse P, Landreneau RJ, Donnenberg VS: Regenerative therapy and cancer: in vitro and in vivo studies of the interaction between adipose-derived stem cells and breast cancer cells from clinical isolates. Tissue Eng Part A. 2011, 17: 93-106.PubMed CentralView ArticlePubMedGoogle Scholar
- Jotzu C, Alt E, Welte G, Li J, Hennessy BT, Devarajan E, Krishnappa S, Pinilla S, Droll L, Song YH: Adipose tissue derived stem cells differentiate into carcinoma-associated fibroblast-like cells under the influence of tumor derived factors. Cellular oncology. 2011, 34: 55-67.View ArticleGoogle Scholar
- Strong AL, Semon JA, Strong TA, Santoke TT, Zhang S, McFerrin HE, Gimble JM, Bunnell BA: Obesity-associated dysregulation of calpastatin and MMP-15 in adipose-derived stromal cells results in their enhanced invasion. Stem Cells. 2012, 30: 2774-2783. 10.1002/stem.1229.View ArticlePubMedGoogle Scholar
- Prantl L, Muehlberg F, Navone NM, Song YH, Vykoukal J, Logothetis CJ, Alt EU: Adipose tissue-derived stem cells promote prostate tumor growth. Prostate. 2010, 70: 1709-1715. 10.1002/pros.21206.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.