Intrapulmonary autologous transplant of bone marrow-derived mesenchymal stromal cells improves lipopolysaccharide-induced acute respiratory distress syndrome in rabbit

Background Lung diseases such as acute respiratory distress syndrome (ARDS) have a high incidence worldwide. The current drug therapies for ARDS have supportive effects, making them inefficient. New methods such as stromal cell therapy are needed for this problem. Methods This research was performed with ten New Zealand rabbits in two groups. Bone marrow aspiration was performed on the treated group, and mesenchymal stem cells were isolated and cultured. The experimental model of ARDS was induced using LPS from Escherichia coli strain O55:B5. Then, 1010 bone marrow mesenchymal stem cells (BM-MSCs) were autologously transplanted intrapulmonary in the treatment group, and 1–2 ml of PBS in the control group. The clinical signs, computed tomographic (CT) scans, echocardiography, blood gas analysis, complete blood count, and cytokine levels were measured before and at 3, 6, 12, 24, 48, 72, and 168 h after BM-MSC transplant. Finally, the rabbits were killed, and histopathological examination was performed. Results The results showed that BM-MSCs decreased the severity of clinical symptoms, the number of white blood cells and heterophils in the blood, the total cell count, and number of heterophils and macrophages in bronchoalveolar lavage, and balanced the values of arterial blood gases (increase in partial pressure of oxygen and O2 saturation and decrease in the partial pressure of carbon dioxide). They also downregulated the tumor necrosis factor (TNF)-α and interleukin (IL)-6 concentrations and increased the IL-10 concentrations at different times compared with time 0 and in the control group, significantly. In the CT scan, a significant decrease in the Hounsfield units and total lung volume was found by echocardiography, and in comparing the two groups, a significant difference in the parameters was noticed. The histopathology demonstrated that the BM-MSCs were able to reduce the infiltration of inflammatory cells and pulmonary hemorrhage and edema. Conclusions This study indicated that BM-MSCs play a significant role in the repair of lung injury. Electronic supplementary material The online version of this article (10.1186/s13054-018-2272-x) contains supplementary material, which is available to authorized users.


Background
Lung disorders have significant morbidity and mortality rates worldwide, both in humans and in animals. Acute respiratory distress syndrome (ARDS) is one of the leading causes of respiratory failure around the world. Although early diagnosis, timely medical care, and treatment may lead to improvement of symptoms, the signs return after a period of time. Causes of ARDS are different. It can be caused by direct or indirect damage to the lung epithelium. ARDS is described by severe hypoxemia, decreased pulmonary compliance, diffuse alveolar damage, and bilateral pulmonary infiltrates after cardiac edema [1,2] and confirmed by a combination of clinical, physiological, and chest imaging parameters. Pulmonary inflammation with disruption of the mechanism of the alveolar-capillary barrier is an important direct cause of ARDS [3,4]. Therapeutic approaches include mechanical ventilation, neuromuscular blocking agents, fluid management, drug and antimicrobial therapy, and prone positioning [3,5,6]. These therapeutic strategies have a supportive role and cannot prevent the progression of the disease [7][8][9][10]. The ultimate approach is lung transplant, but it has many problems for recipient patients such as lack of suitable donors and the use of immunosuppressive drugs over a lifetime to prevent rejection of the transplant [11]. Therefore, the recognition of new therapeutic approaches such as stromal cell therapy is essential [12]. MSCs confer immunomodulatory and anti-inflammatory effects, enhance bacterial clearance, reduce cell injury and death, and are angiogenic [5,13]. The mechanism of the MSCs' effects includes several pathways mediated through differentiation, proliferation, soluble intermediate release, extracellular vesicles, transfer of organelles, and direct cell-to-cell contact, which decrease activation of inflammatory cell secretion of paracrine mediators [14,15]. Recent studies have shown positive effects of MSC-based therapy for ARDS. Induction of inflammation by the LPS of Escherichia coli O55:B5 is one of the best and simplest methods for making an experimental model of ARDS. Although the ARDS animal models cannot reflect human ARDS accurately, the rabbit model is similar and hence suitable for translating the results from pilot to clinical conditions [16]. Anatomical, physiological, genetic, and biochemical similarity to humans simulates human lung disease, and as the rabbit is easy to handle, it is considered as a suitable model for pulmonary research [17,18]. Moreover, the rabbit serves as an excellent platform for treatment based on stromal cells [19,20]. Thus, in this study, the rabbit was used as a model for causing ARDS, and then it was treated with stromal cells. The aim of this study was evaluation of therapeutic potential intrapulmonary administration of BM-MSCs in an experimental model of E. coli LPS-induced ARDS in the rabbit.

Isolation, primary culture, and expansion of BM-MSCs
Bone marrow (BM) samples were obtained from the humerus of rabbits in aseptic surgical conditions. After 30 min of centrifugation (400 relative centrifugal force), mononuclear cells were collected from the interphase, and eventually the cell pellets were seeded into 25-cm 2 flasks (SPL Life Sciences, Pocheon, South Korea) with DMEM-high glucose, 20% FBS (Life Technologies, Carlsbad, CA, USA), and 100 U/ml penicillin/streptomycin (Biowest, Nuaillé, France) and incubated at 37°C in humid air with 5% CO 2 (Memmert, Eagle, WI, USA). When the adhesion of the cells was near confluence (more than 70%), the cells were trypsinized by trypsinethylenediaminetetraacetic acid of 0.25% (Life Technologies) and replated at dilutions of 1:2 under conditions of the same cultivation. The characteristics of the BM-MSCs were labeled with phycoerythrin-conjugated antibodies against CD45 (BioLegend, San Diego, CA, USA), CD90 (eBioscience, San Diego, CA, USA) and CD34 and CD29 (Abcam, Cambridge, UK), and the multilineage differentiation ability of BM-MSCs to engage in osteogenic and adipogenic differentiation was checked in vitro. This is described in more detail in the additional files.
Experimental design ARDS experimental model Ten healthy adult male New Zealand white rabbits were chosen, and an ARDS experimental model was induced with LPS from E. coli O55:B5 [21] (Sigma-Aldrich, St. Louis, MO, USA) at 400 μg/kg dissolved in 0.1 ml of PBS intrapulmonary the under bronchoscopic guidance. After the ARDS confirmation, rabbits were randomly distributed into two groups: (1) the control group (ARDS + PBS) and (2) the treatment group (ARDS + BM-MSC). Protocol details are available in the additional files.

BM-MSC autologous transplant
A total of 10 10 BM-MSCs suspended in 0.1 ml of PBS [5] were autologously transplanted intrapulmonary under bronchoscopic guidance 24 h after induction of ARDS. Details of the method are provided in the additional files.

Clinical assessment
During the study, the clinical signs of rabbits were calculated and recorded on the basis of clinical scores for each rabbit. Heart rate (HR), respiratory rate (RR), body temperature, twitch, abnormal breathing, nasal discharge, cough, appetite, and physical condition were measured using a clinical score. The scoring is based on clinically evaluated criteria that were individually defined and measured for each rabbit (Additional file 1: Table S1).

Computed tomography and echocardiography
Computed tomographic (CT) scans of the lung of rabbits were taken with the SOMATOM Spirit Class II (Siemens, Erlangen, Germany), and echocardiographic examinations were performed using a Vivid 7 ultrasound system (GE Healthcare, Milwaukee, WI, USA) with a 4.4-10.0-MHz phased-array transducer (10S) during experimental modeling of ARDS before and 12, 24, 48, 72, and 168 h after transplant in each animal under the same circumstances. More details are provided in the additional files.

Blood and bronchoalveolar lavage samples
Blood samples were collected from the central ear artery for blood gas analyses using blood gas analyzers (OPTI CCA-TS; OPTI Medical Systems, Roswell, GA, USA) and from the ear vein for hematologic parameter analysis and measurement of cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-6, and IL-10) with a commercially available enzyme-linked immunosorbent assay kit (EASTBIOPHARM, Hangzhou, China) before transplant of BM-MSCs and then for 3, 6, 12, 24, 48, 72, and 168 h after transplant. Also, bronchoalveolar lavage (BAL) samples were collected by fiberoptic bronchoscope (11262 BC; Karl Storz, Tuttlingen, Germany) before and 24, 48, 72, and 168 h after transplant. Then, the centrifuged BALs were stored at − 80°C for measurement of cytokines. Protocol details are available in the additional files.

Histopathology
The rabbits were killed 7 days after BM-MSC transplant. First, the lungs and hearts were macroscopically examined, and then sections of them were routinely prepared, stained with H&E, and observed by use of an E600 Eclipse optical microscope (Nikon Instrument, Tokyo, Japan). More details are provided in the additional files.

Statistical analysis
The results were analyzed statistically using IBM SPSS Statistics version 24 software (IBM, Armonk, NY, USA). For variables in this study, data were analyzed with the repeated measures independent samples t test, Friedman test, and Mann-Whitney U test, and p < 0.05 was considered statistically significant.

Culture of BM-MSCs
The plastic adherent BM-MSCs proliferated 5-7 days after seeding and reached 80% confluence about 2 weeks later. After three passages, the adherent cells were observed by microscopy to display homogeneous spindle fibroblast-like morphology (Additional file 1: Figure S1).

Flow cytometric analysis
Flow cytometric analysis demonstrated that cultured BM-MSCs expressed a particular pattern of cell surface markers of CD29 and CD90, 92% and 89%, respectively, but were uniformly negative for CD34 and CD45 (Additional file 1: Figure S2), which indicates cultured adherent cells were MSCs with high purity. Thus, the pure MSCs whose immunophenotype was confirmed were used in this study.

Differentiation
Multilineage differentiation ability of BM-MSCs to engage in osteogenic and adipogenic differentiation in vitro confirmed potential pluripotent MSCs, and their ability to form osteoblasts and adipocytes when incubated in differentiation medium was retained (Additional file 1: Figure S3).

Confirmation of ARDS experimental model
Twenty-four hours after the intrapulmonary administration of LPS, inflammation and edema were stabilized in the lung. Two different evaluations proved ARDS occurrence: (1) Clinical examination showed changes in respiratory sounds during auscultation, such as crackle and wheeze, increased respiratory rate/hyperpnea (p = 0.004), heart rate/ tachycardia (p = 0.008) and body temperature/hyperthermia (p = 0.011), cough, mucus hyperemia, abnormal discharge, and reduced appetite; and (2) plain chest radiograph showing significant bilateral radiologic density (air bronchogram and air alveologram patterns and lung edema and bronchial and bronchiolar septum thickness) were also confirmed (Additional file 1: Figure S4). These results confirmed the experimental model of ARDS compared with the baseline 24 h after injection of LPS. In both groups, after inflammation, unilateral or bilateral mucosal secretions from the nose were produced that were occasionally accompanied by color changes. But after transplant of BM-MSCs, statistical comparison showed a significant decrease in nasal discharge compared with the control group at 24 h (p = 0.011), 48 h (p = 0.007), 72 h (p = 0.007), and 168 h (p = 0.008). The regular rhythm of nasal twitching in the rabbit is a reason for the rabbit's health and alertness. Nasal twitching was reduced at the time of inflammation. But there was a significant difference between results at 12 h (p = 0.005), 24 h (p = 0.005), 72 h (p = 0.014), and 168 h (p = 0.014) after transplant.
Also, there was a significant decrease in cough count between the two groups at 12 h (p = 0.

MSCs cause blood cells and BAL cells to balance Blood cells
The measured blood parameters at different times are shown in the Table 1. Rabbits in the two groups had significant leukocytosis 1 day after inflammation (p < 0.005). But the BM-MSC transplant reduced the number of white blood cells. The changes were significant at 12 h (p = 0.046), 24 h (p = 0.019), 48 h (p = 0.022), 72 h (p = 0.044), and 168 h (p = 0.043) compared with time 0. Also, comparison of the two groups showed that cell transplant was effective at 6 h (p = 0.043), 12 h (p = 0.000), and 24 h (p = 0.047) ( Table 1).
Comparison of heterophil band numbers between the two groups showed that cell transplant was affected at 48 h (p = 0.046), 72 h (p = 0.021), and 168 h (p = 0.038).
Statistical analysis showed that there was no significant difference in the lymphocyte count, monocytes, platelets, hematocrit in or between the treatment and control groups during the study. There was a significant difference in the number of red blood cells (p = 0.007) and concentration of hemoglobin (p = 0.027) in the treatment group compared with the control group only at 48 h.

Cells from BAL
In BAL, the total nucleated cell count included ciliated epithelial cells, squamous epithelial cells, alveolar macrophages, leukocytes, heterophils, eosinophils, and plasma cells. Typically, total leukocyte cells in treatment group included macrophages and lymphocytes, and in the control group, they included macrophages and heterophils (Fig. 2).
The results demonstrated that the BAL cell count was significantly increased in inflammatory conditions, but    Table S3).

Regulation of arterial blood gases with MSCs
The partial pressure of oxygen (PO 2 ) and O 2 saturation (SatO 2 ) levels were decreased, and partial pressure of carbon dioxide (PCO 2 ) levels were increased, in ARDS (time 0) compared with baseline (− 24 h), significantly.  Table S4).
Additionally, PCO 2 was significantly decreased in the treatment group at 24 h (p = 0.036), 48 h (p = 0.034), and 72 h (p = 0.01) after transplant, and comparison between the two groups displayed a significant decrease of PCO 2 at 48 h and 72 h (p = 0.016).
Also, statistical analysis for pH value indicated significant differences between the two groups at 24 h (p = 0.019). Respiratory acidosis occurred in both groups at 3 and 6 h via pH decrease and increase in PCO 2 . Analysis of bicarbonate, base excess, and anion gap data showed no significant difference in and between the two groups (Fig. 4).

Effect of MSCs on arterial blood electrolytes
The results showed nonsignificant reduction in the value of Na + , K + , and Cl − in and between the treatment and control groups at the different times and alone. There was a significant difference in the Cl − value in the treatment group compared with the control group at 24 h (p = 0.029) (Additional file 1: Table S5).  In contrast, when MSCs were administered, IL-10 was significantly increased in the BAL and plasma. Concentration of IL-10 in BAL was significant at 12 h (p = 0.047), 24 h (p = 0.011), 48 h (p = 0.001), and 168 h (p = 0.041) compared with time 0, and comparison of the two groups showed significant differences at 12 h (p = 0.032), 24 h (p = 0.018), 72 h (p = 0.041), and 168 h (p = 0.008) (Fig. 5). Also, increase of IL-10 concentration in plasma was significant at 48 h (p = 0.047), 72 h (p = 0.044), and 168 h (p = 0.022) against time 0. MSCs also increased plasma IL-10 concentrations compared with control group at 48 h (p = 0.043) and 72 h (p = 0.029), significantly (Fig. 5), so that BMSCs reduced lung injury and inflammation via significant immunomodulatory properties and attenuated the severity of ARDS.

Imaging findings Tomodensitometric and volumetric findings of lung CT scans
Hounsfield units and volumes of the aerated and nonaerated areas of the right and left lungs were measured on CT scans. Quantitative estimation (the Hounsfield unit measurement) was done for different adjacent CT sections with the Leonardo workstation and software tools (Siemens). Lung parenchymal margins were manually demarcated, and then average Hounsfield units were obtained for each section. Also, the 3D pattern was observed for a better evaluation of the lung parenchyma (data not shown). These measurements demonstrated that Hounsfield units had increased 1 day after the experimental inflammation (before stromal cells therapy), which represents replacement of alveolar air with mucous and inflammatory cells (Fig. 6). A significant decrease in the Hounsfield units was seen at 48 h (p = 0.032), 72 h (p = 0.036), and 168 h (p = 0.025) post-transplant, which indicates an increase in aerated volume of the lung in the treatment group. Also, variation volumes were compared and showed that total lung volume (aerated + nonaerated + tissue + edematous fluids) in both groups increased after ARDS, but transplant of BM-MSCs had decreased the process at 72 h (p = 0.047) and 168 h (p = 0.027). On CT scans, most nonaerated areas were observed in lower lobes in the caudoventral area ( Fig. 7 and Additional file 1: Table S7).

Echocardiography findings
The images and amounts of Doppler and M-mode echocardiographic parameters are shown in Additional file 1: Figure S6. The amounts of BAL and plasma cytokines of

Findings of gross pathology and histopathology
The macroscopic examination of the lungs showed hyperemia, hemorrhage, emphysema, edema, and hepatization in the control group (Fig. 8a), but brief hyperemia and edema were observed in the treatment   (Fig. 8f). Sections of the lung demonstrated different histopathological patterns between the control and treatment groups. Microscopically, lungs showed more severe damage in the control group than the treatment group as hemorrhage in parenchyma and alveoli, moderate to severe vascular hyperemia, moderate to severe interstitial pneumonia, severe alveolar injuries and edema, neutrophilic margination in the capillary vessels, abundant presence of inflammatory cells, epithelial cells and other cell debris (cellularity) in interstitial spaces and alveoli, and thickness of the alveolar septum (Fig. 8b-d). But treatment with BM-MSCs reduced the infiltration rate of inflammatory cells in the alveolar septum, hyperemia, hemorrhage, and edema, and lung structure was approximately normal and only slightly increased the thickness of the alveolar septum. Also, in most sections of the treatment group, injury in the bronchus, bronchioles, and vessels was not observed (Fig. 8g-i).
In macroscopic observations of the heart, no lesions were found in both treatment and control groups, but in the microscopic examination of the heart sections in the control group, necroses were observed in a small number of myofibers (Fig. 8e), whereas in treatment group, no injury was observed (Fig. 8k).

Discussion
BM-MSCs are an ideal choice for cell therapy because there are fewer complications for cell isolation and also BM autologous cells that are capable of eliminating immune response and transplant rejection [22]. MSCs have positive effects in the repair of ARDS [23].
Although fibroblasts play a role in normal and pathological repair, and an accumulation of fibrocytes, fibroblasts, and myofibroblasts in the alveolar compartment, leading to excessive deposition components of the extracellular matrix but also according to prior findings the effect of them remains controversial [23][24][25] and unknown, so that reduction of its deposition or enhancement of its degradation could be treatment strategies. [24] The results of this study showed that the BM-MSCs significantly decreased the severity of clinical symptoms induced by LPS, the number of inflammatory cells in blood and BAL, and balanced the values of arterial blood gases and cytokines. On the CT scans, a significant decrease in the Hounsfield units was observed, which is indicative of an increase in aerated volume of the lung in the treatment group. The echocardiographic parameters did not reveal a significant difference in the treatment group, and compared with the two groups together it was significant. Also, the histopathology demonstrated reduction in the infiltration of inflammatory cells and pulmonary hemorrhage and edema in the recipients of BM-MSCs.
In natural conditions, cells from bone marrow migrated to chemotactic gradients, but the amount of engraftment was low, and use of exogenous stromal cells can be helpful to this mechanism. The exact mechanisms of MSCs' actions are not precise [25], and sometimes the results of stromal cell research using animal models are incompatible with each other [23]. However, three mechanisms have been defined for MSCs' actions consisting of differentiation, cell-cell contact and paracrine function via the soluble factors [25,26]. Researchers are likely to focus on manipulation of inflammatory pathways and optimizing lung repair while preventing ARDS progression [6]; whereas management of inflammatory pathways forbids the development of ARDS, some researchers believe in the immunomodulatory effects of MSCs [6]. Most trials have used local delivery of autologous BM-MSCs with the aim increasing the concentration of growth factors and cytokines in damaged tissue to improve possible engraftment and repair [23]. For the first time, Gupta et al. reported effects of the local delivery of MSCs in ARDS that are consistent with this study [26]. The anti-inflammatory role of MSCs may vary in humans and animals, but the useful effects of MSCs have been shown in several animal models [27] and indicate the significant role of identification feature of MSCs [22]. LPS of gram-negative bacterial wall binds to the CD14/TLR4/MD2 receptor complex, activating pathway and transcription of some inflammation-and apoptosis-related genes and activating innate immune response [18,25] that produces the acute phase of ARDS. Therefore, the LPS-induced animal models could be suitable for cell therapy. In prior findings, only a few animal models have been used to investigate the mechanism of MSC therapy in ARDS, most of which used rat and mouse [25,[28][29][30][31]. Although  [32]. The reduction of heterophils, macrophages, and the number of total cells were observed in the treated group, unlike the results of other research [26]. The complete blood count findings indicated that despite intrapulmonary administration of LPS and MSCs, there is a relationship between the numbers of intravascular and intra-alveolar heterophils and lymphocytes. Direct administration of LPS effects on gas exchange process and induces acute hypoxemia [33]. The results of studies suggest that MSC transplant can improve hypoxemia via reducing alveolar-arterial oxygen gradient [29,30] which is in agreement with the present study. But the research results of Moodley et al., (2016) conflicted with this study [27]. The difference in injury model and type of cell therapy may be the cause of the variable results. There are not any studies that have reported clinical signs, including HR, RR, and RT, after stromal cell therapy; this study is the first report in which clinical signs were evaluated using a previously described scoring system.
The protective effects of BM-MSCs on ARDS are a result of the immune regulation function and inhibitory T-cell function [31]. Monocytes and macrophages can release inflammatory factors such as TNF-α that play a role in phagocytes of the necrotic and apoptotic cells [34]. Because MSC soluble factors may be the therapeutic basis of MSCs, this result is confirmed by recent reports that demonstrated immunomodulatory properties of MSCs [26]. MSCs' effects are explained by a shift from a proinflammatory to an anti-inflammatory response [26]. Reduced cytokines of proinflammatory (TNF-α and IL-6) and increased cytokines of anti-inflammatory (IL-10) in BAL and plasma samples play principal roles in the treatment mechanism of ARDS [26,29,30]. In the present study, the TNF-α concentration decreased following MSC therapy, which is similar to many studies [31]. The IL-10 level increased after the use of MSCs and inhibited antigen-presenting cell function and inflammation and enhanced tolerance, but our findings are not similar to some other studies [26,30]. Beneficial effects of MSCs may be mediated by a decrease of TNF that indicated the attenuation of the inflammation [26]. Prostaglandin E2 (PGE2) has a critical role in IL-10 and IL-6 secretion from macrophages [31]. It is possible that TNF-and IL-6-dependent PGE2 production plays a major role as a clinical sign and in particular hyperthermia in the acute phase of inflammation [34]. TNF has roles in local inflammation complications such as leukocyte stimulation (neutrophils and macrophage) in BAL and lung endothelium that was shown in the pathology. Also, TNF has roles in systemic complications such as hyperthermia, C-reactive protein, leukocytosis, necrosis, and apoptosis, decreasing appetite, and even decreases of cardiac output and vascular permeability and increases in edema and hypotension.
In this study, CT scans and echocardiography were used to follow improvement and management of ARDS during BM-MSC therapy. One particular feature of ARDS is lack of aerated lung volume caused by inflammation and edema, and total lung volume reduction (air Fig. 8 Necropsy and histopathological findings in the rabbit. a-e Control group. a Macroscopic examination of the lung shows hyperemia, hemorrhage, and edema. b Interstitial edema and pneumonia (arrow). b and c Inflammatory cell infiltration (arrowhead). d Hyperemia and severe hemorrhage in alveoli and parenchyma (arrows). e Myofibrils necrosis of heart (arrow). f-k Treatment group. f Macroscopic examination of the lung shows brief hyperemia and edema. g-i Histopathological examination reveals a decrease in damage in the alveoli and parenchyma of the lung. k Lack of damage to the heart. Note: In all of the tables, data were presented as mean ± SD (n = 5 rabbit per group). *p < 0.05 significant compared with inflammation time in the same group. #p < 0.05 significant compared with the control group at the same time + parenchyma) mostly in the lower lobes can explain hypoxemia, low respiratory compliance, and alveolar dead space and increased pulmonary vascular permeability [35,36]. CT scanning is a common clinical diagnostic tool, a specific, repeatable, and noninvasive technique for diagnosis that until now has rarely been used as a research tool, and there is not any valid data of the distribution region of the existence or absence of air in alveolus during ARDS and especially after cell-based therapy. Our findings based on CT scanning are matched with the diagnosis of pathology and all the other outcomes. Therefore, it can be said that CT scanning is a suitable method for evaluation of the stromal cell therapy effects in pulmonary inflammation and edema, but further studies are needed in this field. Interactions between lung, right ventricle, and pulmonary circulation are critical in ARDS. Serial echocardiographic measurements have a potential clinical diagnosis in the early stage ARDS as a prognostic and therapeutic method. We showed that ARDS causes changes in echocardiographic parameters and reduces cardiac function, whereas transplant of BM-MSCs was able to prevent these changes.
We demonstrated that MSCs could attenuate the inflammation by reducing pathological lung changes [31], but in some research, no significant data were reported regarding pathology. This variation may be caused by short study duration so that further studies are needed. Our histopathological examination, like other findings from imaging and all of the laboratory results, showed that BM-MSC transplant could improve ARDS.

Conclusions
Many studies on cell-based therapies in ARDS have been done, but most of them focused on molecular and signal tests. Although these studies could clear pathways, they are still far from tissue function. In this study, we tried to explain the MSCs' effects on organ function. We investigated effects of BM-MSCs in an experimental model of ARDS and confirmed that MSCs decrease inflammation and improve alveolar fluid clearance and have a protective role in ARDS. Improvement in clinical signs, the decrease of inflammatory cells in blood and BAL, the balance in blood gases and cytokines, the decrease in the Hounsfield units, no changes in echocardiographic parameters. and the reduction of pulmonary hemorrhage and edema in pathology were observed. Despite these results, subsequent studies are required to confirm the decrease in inflammation, and physiological parameters over the long term and many experiments should be performed until stromal cell therapy is validated as a method of routine clinical treatment.