Approximately, 100 ml of human lipoaspirate (n=10) were obtained by vaser assisted liposuction from abdominal subcutaneous fat from voluntary healthy human donors. All the donors were pre-screened for infectious diseases and comprehensive health conditions before included in the study. Written informed consent was obtained before lipoaspiration and project was duly approved by Institutional Committee for Stem Cell Research and Therapy (ICSCRT) and Institutional Ethics Committee (IEC). Sample processing was done inside a class 100 bio-safety hood in a class B cGMP: current good manufacturing practices, facility. Adipose tissues were washed thoroughly with Dulbecco’s phosphate buffered saline (DPBS) (Invitrogen, Bangalore, India) and subjected to digestion with freshly prepared 2 mg/ml collagenase Type-I (MP Biomedical, Santa Ana, CA) for 60 minutes at 37°C under gentle agitation. To the digested adipose tissue, an equal volume of growth medium, consisting of Dulbecco’s modified Eagle’s medium-knockout (DMEM-KO) (Invitrogen) supplemented with 10% fetal bovine serum (FBS), (Hyclone, South Logan UT), 2 ηg/ml basic fibroblast growth factor (bFGF) and 1% GlutaMAX (Invitrogen) was added to neutralize the enzyme. The suspension was then centrifuged at 2500 rpm for 10 minutes. The resulting cell pellet was then re-suspended in growth medium. The resulting cell suspension obtained was the stromal vascular fraction (SVF). The SVF was first filtered through a 100-μm filter and then through a 40-μm filter (Millipore India, Bangalore, India).

Clinical grade MCB, WCB and IP was prepared as per the method described earlier (21). Briefly, SVF from each donor was seeded separately on to 2-chamber cell stack (corning plastic wares supplied by Sigma Aldrich, Bangalore, India) in growth medium at a density of 85,000~100,000 cells/cm² and incubated at 37°C in 5% humidified CO₂. The medium was changed every third day, and cells were harvested once they became confluent ~70% with 0.05% trypsin plus 0.2% Ethylenediaminetetraacetic acid (EDTA), washed with DPBS and counted with haemocytometer. The cells from the individual donors were tested for quality parameters. The cells were aliquoted and frozen at the concentration of 1 million cells/ml and stored in vapour phased liquid nitrogen at passage-0.

For WCB, frozen vials of MCB from five different donors were thawed and cells were pooled together. Mixed donor cells were then seeded onto 5-chamber cells stack (Corning) in growth medium at the seeding density of 3000 cells/cm² and cultured as described above. Cells were harvested once they reached ~70% confluence. Cells of the WCB were tested for quality parameters and were frozen at the concentration of 3 million cells/vial and stored in vapour phase liquid nitrogen at passage-1.

The IP was prepared by seeding frozen-thawed WCB at a seeding density of 3000 cells/cm² in a 10-chamber cells stack (Corning) containing growth medium and cultured as described above. The cells were harvested once they reached ~70% confluent. IP was then tested for quality parameters and used for pre-clinical study. Remaining batch was frozen at the concentration of 100 million cells/cryocyte bag and stored in vapour phased liquid nitrogen at passage-2.

The MCB, WCB and IP were tested for quality parameters as per the guidelines of Central Drug Standard Control Organization (CDSCO), India. Details are given in Table 1. Briefly, quality control was carried out broadly under following categories. Purity was tested by analysing cell morphology and immunophenotypic analysis for hematopoietic and mesenchymal stem cell markers using flow cytometry for CD34, CD45, CD73, CD90, CD105, CD166 and HLA-DR as per the method described earlier (15). Potency of the cells was carried out by testing cell viability by flow cytometry using 7-aminoactinomycin D (7AAD), tri-lineage differentiation potential for osteoblasts, adipocytes, chondrocytes as per the method described earlier (22), secretome analysis using multiplex Elisa (Invitrogen), immunomodulation by testing suppression of T-cell proliferation by AD-MSC and pluripotent markers like OCT-4, Nanog, SOX-2, REX-1 and ABCG2 by real time PCR (15, 23). Safety of the cells was tested by bacterial endotoxin using Lal Test (Charles River, Wilmington, MA (specification <0.5 EU/ml), mycoplasma testing using PCR (Venor Gem, Minerva Biolab, Berlin, Germany), sterility by BactAlert 3D automated system (Biomeurix, France) for 7 days, karyotyping by G banding, viral screening for human immunodeficiency virus (HIV) 1,2, hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), and venereal disease research laboratory (VDRL) tests by polymerase chain reaction (PCR) and transformation markers like MYC, ERCC3, XRCC4 and RAD51 (15). Toxicity studies was carried out at third party accredited good laboratory practice (GLP) animal facility for acute toxicity in mice and rat using two infusion routes like intravenous (IV) and intramuscular (IM) and four doses like 5, 10, 15 and 20×10⁶ cells/kg body weight. Twenty one days repeated dose toxicity study in rat using IV route was also carried out. Pharmacodynamics was carried out using bio-distribution study and pharmacokinetics study was carried out as efficacy model study as stated in this paper and finally stability testing was carried out for MCB, WCB and IP and determined by viability, MSC cell surface markers analysis, tri-lineage differentiation capacity and colony forming unit (CFU) assays. Final product was tested for trace elements of trypsin and fetal bovine serum (Table 1). Batches were released for pre-clinical and clinical applications once they satisfied quality parameters set by the CDSCO and drug controller general of India (DCGI) (21).

All the animals were scanned for HRCT at Advanced Centre for Treatment, Research, Education in Cancer (ACTREC), Mumbai, India, as per the method described earlier (25). Briefly, experimental animals were scanned on day 21 post-administration of bleomycin. Mice were anesthetized intraperitoneally with ketamine hydrochloride (Sun Pharma, Mumbai, India; 100 mg/kg) and xylazine (Indian Immunological, Hyderabad, India; 10 mg/kg) in order to maintain hypnosis during HRCT. Scanning was performed with a FLEX-Triumph™ CT-scanner imaging platform (Gamma Medica). Acquisition parameters were 50 kw, 512 projections at 360°C (1 projection= 280 ms), average field of view 5 cm, thin-section slices, each of 0.625 mm in thickness, and spaced 1 mm apart covered the complete mouse lung from the apex to the hemi-diaphragm. Images were acquired in axial plane and the bone algorithm was applied to better visualize the lung. Around 20 to 22 slices covered the entire animal lungs. Following the scan, the mice were placed in their cages, recovering from the anaesthesia. Post-scan care was taken and animals were supplied with standard laboratory diet and water ad libitum.

The hydroxyproline content was assessed to indicate the extent of collagen deposition in the lungs. To estimate the hydroxyproline content, lung tissue homogenates were hydrolysed in 6N hydrochloric acid (Sigma Aldrich) for 16 h at 120°C. An aliquot of these hydrolysates was then assayed using a hydroxyproline estimation kit (BioVision Research Products, Milpitas, CA, USA). Briefly, hydrolysates were incubated with Chloramine-T, followed by colour development with Ehrlich’s reagent at 65°C for 90 min. The absorbance at 550 nm was measured by iMark Microplate Reader (Bio-Rad, Hercules, CA, USA). Hydroxyproline levels were determined by plotting standard curve using serial dilutions of hydroxyproline standard. Results were quantified as μg/lung.

Lung biopsies were fixed in 10% neutral buffered formalin. Paraffin embedded sections (5 μm thickness) of the lung specimens were stained with haematoxylin (Sigma Aldrich) and eosin (Loba Chemie, Mumbai, MH, India) and Masson’s trichrome (Sigma Aldrich) stain for collagen deposition. Sections were graded by an investigator blinded to the treatment groups. Modified Ashcroft’s scoring was performed on a scale of 0 to 8 to determine the extent of fibrosis as described elsewhere (26).

To qualitatively assess the homing potential of systemically administered AD-MSCs, cells were stained with PKH-67 fluorescent cytosolic dye (Sigma Aldrich) prior to injection into mice. Post sacrifice, fluorescence imaging of cryotome sections of the lung tissues was carried out using the Nikon Eclipse Ti inverted fluorescence microscope (Nikon Instruments Inc., Melville, NY, USA). PKH-67 tagged cells were visualized using the green fluorescent filter (excitation - 490 nm, emission - 502 nm). To observe their distribution in the lung tissues, the sections were then stained with 10 EM Hoechst 33342 (AnaSpec Inc.; Fremont, CA, USA) and visualized under the blue fluorescent filter (excitation - 358 nm, emission - 461 nm). The images were then superimposed using the NIS elements imaging software to create a composite image.

Total cellular RNA was extracted from lung tissues of mice and purified using the PureLink RNA Mini Kit (Invitrogen). RTQ-PCR analysis was carried out using the express SYBR GreenER qPCR Supermix kit (Invitrogen). The primers used were designed using primer 3 software and custom synthesized (Bioserve, Hyderabad, India). Melting curve analysis was performed at the end of PCR and changes in gene expression were analysed using the ‘StepOne Plus’ software (version 1.1). The relative expression of each gene was calculated using the comparative Ct method. Relative quantification of target mRNA expression was calculated and normalized to GAPDH expression. The results are presented as the log2 fold change of mRNA expression as compared to the amount present in vehicle control samples.

Data was pooled from both the experiments and expressed as mean±SEM unless otherwise stated. Comparisons between two groups were determined using a two-tailed Student’s t-test. For multiple comparisons, one-factor analysis of variance was used, followed by the appropriate ad-hoc test as dictated by the normality and distribution of the data. The survival curves were calculated by the Kaplan-Meier method, and differences between survival curves were compared by the log-rank test. All statistical analysis was performed using SPSS software (version 13 for Windows; SPSS, Inc., Chicago, IL, USA). p values less than or equal to 0.05 were considered significant and expressed as p<0.01; p<0.001.

Purity of the MSCs derived from adipose tissue as MCB, WCB and IP maintained normal phenotypes which were typically small, spindle-shaped appearance in all the passages (Fig. 1A). Immunophenotypic analysis by flow cytometry showed less than 0.2% hematopoietic cell surface markers like CD34 and CD 45 (Fig. 1E, F) and more than 99% MSC stem cell population characterized by CD73, CD90, CD105, CD166 (Fig. 1H–K) that represent homogeneous cell population. Expression of HLA-DR was also found to be less than 0.2% (Fig. 1G).

Potency of the MSC was tested by cell viability, tri-lineage differentiation, secretome analysis and immunomodulation assays. Cell viability by 7-AAD staining for exclusion of non-viable cells, followed by flow cytometric analysis was found to be >97% viable population (Fig. 1D). Potential of tri-lineage differentiation into adipocyte showed uniform Oil Red O staining for neutral lipid vacuoles (Fig. 2A). Similarly, Von-Kossa assay showed mineralized deposits in osteo-induced culture of AD-MSC (Fig. 2B) and also retained the ability to differentiate into chondrocytes as shown by Alcian blue staining (Fig. 2C). We did not find any visible detectable differences in the potential of tri-lineage differentiation in MCB, WCB and IP. Potency of AD-MSCs was also tested by secretome analysis by multiplex Elisa and showed high amount of secretary growth factors, cytokines and chemokines. Level of secretome was at increasing trend with highest at IP level (Fig. 2E).

Safety of the cells was tested and found negative for mycoplasma, bacterial endotoxin was less than 0.06 EU/ml, and infectious diseases like HIV 1,2, HCV, HBV, CMV and VDRL testing was found negative. Safety was also proven by acute toxicity and 21 days repeated dose toxicity studies. All the AD-MSC batches were passed through sterility test, had normal karyotyping (Fig. 2D) and high expression of pluripotent markers.

Stability data for MCB, WCB and IP was also shown very robust MSC batch with no significant detectable differences in viability, tri-lineage differentiation capacity, cell surface marker analysis and colony forming unit potential even after 3 years of MCB batch study. Detail quality analysis and its specification and results obtained are given in Table 1 and 3.

Dose of 0.004 U/g of bleomycin induced severe pulmonary fibrosis in Swiss–albino mice. Survival analysis was carried out by Kaplan-Meier survival analysis and compared with vehicle control group. Results showed that, over the first 15 days, half of the vehicle control animals failed to survive the single bleomycin infusion. On the contrary, survivability was significantly prolonged in the mice treated with AD-MSCs and 66.66+3.3 percent animals were survived at the end of the study compared to vehicle control group (p<0.001). Survivability was also significantly improved in pirfenidone treated mice compared to vehicle control group and showed 53.33+3.3 percent survivability at the end of the study (p<0.01) However, survivability was significantly better in AD-MSCs treated mice than pirfenidone (p<0.05) (Fig. 3). Results therefore concluded that, AD-MSCs treatment significantly improve survivability better than pirfenidone in bleomycin induced IPF.

The observations that uncontrolled inflammatory responses in the lungs and aberrant repair mechanisms have been implicated in the pathogenesis of IPF, combined with the findings that therapies targeting inflammation and fibrosis have proved quite successful in reducing the severity of experimental pulmonary fibrosis, led us to hypothesize that human AD-MSCs (which exhibit immunomodulatory properties) could be efficacious in a murine model of pulmonary fibrosis.

Bleomycin-induction of pulmonary fibrosis was manifested with a significant increase in collagen deposition in lungs of mice due to excessive scar tissue formation (Fig. 4B) and led to a marked increase in the overall weight of the lungs (Fig. 4E). Consistent with these observations, bleomycin treatment significantly increases the collagen deposition in the lungs as evidenced by increased lung weights and a corresponding increase in levels of hydroxyproline (Fig. 4F). More importantly, intravenous administration of AD-MSCs showed significant inhibition of bleomycin-induced lung fibrosis (Fig. 4C) and significant reduction in collagen deposition as evidenced by significant reduction of lung weights (p<0.001) and decrease in levels of hydroxyproline (p<0.001) (Fig. 4E, F). These results were significantly better than pirfenidone treated group.

Histological analysis confirmed the bleomycin-induction of fibrosis. Lung tissue sections from IPF induced mice, but not from normal mice, revealed severe fibrosis, characterized by the presence of fibrotic masses, collapsed alveoli with severely thickened alveolar septa along with extensive tissue scarring (Fig. 5B). In contrast, tissue sections from AD-MSC (Fig. 5C), pirfenidone-treated bleomycin mice (Fig. 5D) revealed attenuation in fibrosis with mild thickening of alveolar septa, protection against bleomycin-induced lung fibrosis and maintenance of alveolar architecture. Modified Aschcroft’s scores were performed in a blinded fashion, to quantify lung damage. This analysis revealed significant difference between bleomycin groups demonstrating that AD-MSC and pirfenidone treatment improved the lung architecture (p<0.01) (Fig. 5E).

Intra-tracheal administration of bleomycin in mice induces localized inflammation that can be evidenced by the disruption of normal alveolar architecture and lung parenchyma. This chemokine rich environment at the site of injury acts as a potent inducer of MSC migration that results in the homing of systemically administered AD-MSCs into the lung after bleomycin induced tissue damage. Prior studies have also demonstrated that AD-MSCs maximally retain into the lung when administered intravenously through the tail vein. Therefore, in order to investigate the homing and engraftment potential of AD-MSCs, cells were tagged with PKH-67 cytosolic stain, immediately before intravenous administration to mice. Fluorescence photomicrographs of lung sections of AD-MSC treated animals revealed that PKH-67 labelled cells demonstrated homing and engraftment potential towards the damaged lung tissue and were detected even at day 24 after administration (Fig. 6).

To demonstrate the reduction in inflammation and fibrosis of bleomycin-induced lung injury by AD-MSCs, we studied mechanisms that mediate this effect. We observed that AD-MSCs down-regulated the expression of pro-inflammatory cytokines IL2, IL1b, tumour necrosis factor (TNF) and TGF β which led to a reduction in inflammation (Fig. 7A) and the expression of pro-fibrotic mediators, bFGF, connective tissue growth factor (CTGF), collagen (COL)3a1 and CoL1a1 which led to reduction of pulmonary fibrosis (Fig. 7B). Furthermore, we observed that AD-MSCs downregulated the elevated expression of matrix metalloproteinases (MMPs) (Fig. 7B) which in turn downregulated the expression of tissue inhibitor of metalloproteinases (TIMPs) (Fig. 7B), thus maintaining the MMP-TIMP balance and preventing the restructuring of the matrix caused due to bleomycin-induced lung injury. This reduction in cellular restructuring led to a reduction of collagen deposition and subsequent attenuation of fibrosis.

The HRCT features of IPF are more representative of the pathologic process. Selected parameters, such as distribution of fibrotic and honeycombing, consolidation of lung, ground glass opacities were used to diagnose the severity of the disease. Honeycombing like structure was not observed in any of the animal suggested that honeycombing structure may not be a characteristic of experimentally induced fibrosis in mice. After 3 intravenous infusion of AD-MSC, disease characteristics like fibrosis observed by HRCT scan in bleomycin induced animals had significantly disappeared on day 21 with only few foci of ground glass opacities. This suggested that AD-MSC treatment protected lung against bleomycin induced fibrosis and improved lung architecture (Fig. 8).