Treatment of acute respiratory distress syndrome with allogeneic adipose-derived mesenchymal stem cells: a randomized, placebo-controlled pilot study

This was a single-center, randomized, double-blind, and placebo-controlled study. The study protocol complied with the declaration of Helsinki and was approved by the Research Ethics Committee at Shaoxing Second Hospital (Clinical Trials.gov Identifier: NCT01902082). Written informed consent was obtained from the patient or legally authorized representative before enrolling each patient. Study enrollment occurred between January and April 2013. ARDS was defined and classified according to the Berlin definition [19]. In the new Berlin definition, diagnostic criteria for ARDS rely on 4 categories: (1) timing: within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) radiography: bilateral opacities – not fully explained by effusions, lobar/lung collapse or nodule; (3) origin of lung edema: respiratory failure not fully explained by cardiac failure or fluid overload, and (4) oxygenation impairment: subdivided into 3 categories according to the degree of hypoxemia severity (mild, moderate and severe). The Berlin definition eliminated the concept of acute lung injury, which now falls in the category of mild ARDS. Eligible patients were at least 18 years of age and diagnosed within 48 hours with a PaO₂/FiO₂ ratio of < 200. Exclusion criteria included pre-existing severe disease of any major organs, pregnancy, pulmonary hypertension, malignant disease, human immunodeficiency virus (HIV) infection or if informed consent could not be obtained.

Patients were randomized upon study enrollment. For all patients, a negative fluid balance was maintained by diuretics and fluid restriction. ARDS Network low tidal volume protocol was adopted for standardized ventilator management, targeting a tidal volume of 8 ml/kg of the patient’s ideal body weight and a plateau pressure less than 30 mmHg [20]. Per the requirement of Research Ethics Committee at Shaoxing Second Hospital, frozen MSCs with DMSO and fetal bovine serum were not allowed to be infused to patients directly. For the MSCs group, frozen cells were immediately thawed, cultured with patient’s own serum and harvested in 24-48 hours. Freshly harvested MSCs, at a dose of 1 × 10⁶ cells/kg body weight, were suspended in 100 ml normal saline for peripheral intravenous infusion and administered over 1 hour within 48 hours of enrollment. For the placebo group, a bag of 100 ml normal saline was infused at similar time point. After administration of the MSCs or placebo at day 0, patients were assessed daily at days 1, 3, 5, 7, 14, and 28 (or until hospital discharge or death, whichever occurred first). Patients who were discharged from the hospital before day 28 were asked to return to the study site for assessment. All other aspects of the therapeutic management of the patients were left to the discretion of the clinical team. The primary endpoint was the occurrence of adverse events. Secondary efficacy endpoints included the following: PaO₂/FiO₂ ratio, hospital indices (length of hospital stay, ventilator-free days and ICU-free days at day 28), and serum biomarkers of ARDS including IL-6, IL-8 and SP-D.

Normal human adipose-derived MSCs were purchased from ATCC (Cat # PCS-500-011, LOT 59753760, passage 2, Manassas, VA). The donor of the MSCs was a 23 year-old female of Hispanic origin. MSCs were certified to be negative for HIV, HBV, HCV, bacteria, yeast and mycoplasma. After purchase, sterility, viral, and endotoxin tests of the MSCs were performed at the pathology lab of Shaoxing Second Hospital to confirm the certificate of analysis. Cells were then resuspended in expansion media containing Dulbecco’s Modified Eagle’s Medium (DMEM) -low glucose supplemented with penicillin and streptomycin and 2% fetal bovine serum (FBS) (life technologies, Grand Island, NY) plus EGF and FGF (R&D Systems, Minneapolis, MN) at a density of 4000 cells/cm². Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂ in 150 mm dishes (life technologies, Grand Island, NY). When the cultures reached near confluence (>80%), the cells were detached by treatment with trypsin/EDTA and replated at a density of 4000 cells/cm². MSCs were passaged up to a maximum of four times. After sufficient MSCs were expanded, cells were harvested and cryopreserved in 70% culture media, 20% fetal bovine serum and 10% DMSO. Sterility, viral, and endotoxin tests were carried out again after the expansion. Right after each enrollment, 50 ml of peripheral blood was collected and serum harvested from the patients. If a patient was randomized to MSCs treatment, cryopreserved MSCs were immediately thawed, washed with phosphate-buffered saline (PBS), and cultured with the same expansion media above except supplemented with 2% of the patient’s own serum at a density of 15000 cells/cm² for 24-48 hours. Cells were harvested with trypsin/EDTA and quantitated with a hemocytometer. Viral and endotoxin tests were performed prior to the infusion. All cell culture procedures were carried out in good manufacturing practice (GMP) conditions by personnel who had received formal training in GMP within a facility with highly controlled temperature, room air, pressure, etc.

Morphology was monitored twice a week throughout the culture period by light microscopy. Immunophenotyping of cultured MSCs was performed using flow cytometry. The following markers were analyzed: CD73, CD90, CD105, CD34, CD45, and human leukocyte antigen (HLA)-DR (BD Biosciences, Franklin Lakes, New Jersey). The samples were analyzed on a FACSCalibur using CellQuest Pro software (BD Biosciences).

For osteogenic differentiation of the expanded MSCs, cells were further cultured with osteogenic medium containing 10% FBS, 0.2 mM L-ascorbic acid 2-phosphate and 0.01 M β-glycerophosphate in DMEM. After 2-3 weeks, the cultures were stained for alkaline phosphatase (ALP) activity. For adipogenic differentiation, MSCs were cultured in adipogenic medium consisting of DMEM and 10% FBS supplemented with 10 μg/ml Insulin, 100 nM dexamethasone, 250 μM isobutylmethylxanthine and 200 μM indomethacin. After 2-3 weeks of differentiation, the cultures were stained with Oil Red O.

Sixteen C57BL/6 male mice aged 6-8 week-old were randomized into 4 study groups: short term MSCs (2 days), short term placebo, long term MSCs (28 days), and long term placebo. Animal studies were approved by the Institutional Animal Care and Use Committee at Zhejiang University. Mice received one high dose of intravenous infusion of 2 × 10⁸ expanded cells/kg of body weight or normal saline at day 0. Mice were sacrificed at day 2 or day 28. At the sacrifice, serum was harvested for monitoring renal function, liver function, cardiac enzymes, and pancreatic enzymes. Kidney, liver and lung samples were paraffin-fixed for histopathological analysis.

Five milliliters (ml) of peripheral blood were collected from patients immediately before MSCs or saline treatment (day 0) and day 5 after treatment. Serum samples were collected by centrifugation at 1,500 g for 10 minutes and stored at -70°C until assay at the end of the trial. IL-6, IL-8 and SP-D levels were determined by commercial enzyme-linked immunosorbent assays (ELISA) (R&D Systems, Minneapolis, MN).

Continuous variables were expressed as mean ± standard deviation (SD). Comparisons of continuous variables between two groups were performed by using unpaired Student’s t-test. Comparisons within groups were performed by using paired t-test. Differences were deemed statistically significant at p < 0.05.

Adipose-derived MSCs were spindle-shaped with a fibroblast-like morphology and were attached to the plate during cell culture. These characteristics were well preserved during subculture for a total of 4 passages before harvest. For phenotypic characterization of MSCs, surface protein expression at the end of expansion was examined by flow cytometry. The MSCs were positive for CD73 (96.7%), CD90 (97.1%), and CD105 (98.3%), but were negative for CD34 (0.75%), CD45 (1.28%), and HLA-DR (0.73%). The expanded MSCs preserved the abilities of osteogenesis as determined by alkaline phosphatase staining (Figure 1A) and adipogenesis as assayed by Oil Red O staining (Figure 1B).

After administering one high dose of 2 × 10⁸ MSCs/kg or normal saline at day 0 via intravenous infusion, no mouse death was observed during the 28-day study period. There were no significant differences in liver (alanine aminotransferase and total bilirubin) and kidney (creatinine and blood urea nitrogen) function between the two groups on both day 2 and day 28 (Table 1). MSCs treatment did not alter cardiac enzymes, pancreatic enzymes and body weight (data not shown). Mice treated with MSCs did not show any histopathological changes in the liver, lungs, or kidneys at both day 2 (Figure 2) and day 28 (data not shown).

A total of 25 ARDS patients were screened for enrollment in the study. Of this number, 13 patients were not enrolled because of the exclusion criteria or refusal to participate the study. The study population is comprised of 6 patients randomized to the MSCs group and 6 patients to the placebo group. Baseline demographics with no statistically significant differences between the study groups are summarized in Table 2.

Within 48 hours of randomization, patients received one dose of 1 × 10⁶ cells/kg body weight or saline as a single intravenous infusion over 60 minutes. Study drugs were well tolerated. No adverse events were recorded during infusions. One patient from each group presented with diarrhea one day after study drug treatment and resolved within 48 hours. One patient in the MSCs group developed rash in the chest area after the infusion and resolved spontaneously over 24 hours. During the study period, one patient in the MSCs group died of multiple organ failure. Deaths occurred in two patients in the placebo group with one multiple organ failure and the other sepsis. None of the deaths were considered to be related to the study drugs by the clinical investigators and were consistent with the patients’ existing disease processes. All the remaining patients completed the 28-day follow-up period. There were no other adverse events or serious adverse events.

As part of the safety and efficacy assessment, an evaluation of the oxygenation index and patient outcomes was conducted. Significant improvements in oxygenation index (PaO2/FiO2) from baseline were observed in all data points in the MSCs group. In the placebo group, there were no significant improvements at days 5 (p = 0.05) and 7 (p = 0.05) as compared to baseline. The PaO₂/FiO₂ did not differ significantly between MSCs and placebo groups at all time points (Figure 3). Assessment of hospital indices did not reveal significant differences in length of hospital stay, ventilator-free days and ICU-free days at day 28 between the two study groups (Table 3).

There were no statistically significant differences in serum SP-D, IL-6 or IL-8 levels between the MSCs and placebo groups at both day 0 and day 5 (Table 4). In the placebo group, SP-D, IL-6 or IL-8 levels were similar between day 0 and day 5 (Figure 4B, D, F). These findings are in agreement with those reported in other ARDS studies which showed no changes in biomarkers during the first week of ARDS development [21, 22]. In the MSCs group, serum SP-D levels at day 5 were significantly lower than those at day 0 (p = 0.027) (Figure 4A). The IL-6 levels at day 5 showed a trend towards lower levels as compared with day 0, but this trend was not statistically significant (p = 0.06) (Figure 4E). Although the mean value for IL-8 at day 5 was much lower than that of day 0 (Table 4), the difference was not statistically significant due to the variation of the data (p = 0.19) (Figure 4C).