Bone marrow aspiration, preparation of master cell banks, working cell banks, and Stempeucel® from two different aspirations from the same donors at different times
The methodology of isolating BM, preparation of master cell banks (MCB), working cell banks (WCB), and preparation Stempeucel® has been published previously [13, 15] and has also been patented (US8956862). Briefly, the MCB comprises of BMMSCs derived from individual donors and the WCB comprises a pooled population of BMMSCs. The working cell bank-1 (WCB-1) was prepared from the first BM aspirates from three donors, and subsequently, the WCB-1A was prepared from the BM aspirated obtained from the same three donors 2 years apart. Stempeucel®-1 is manufactured from WCB-1, and similarly, Stempeucel®-1A is manufactured from WCB-1A batch. Stempeucel®-1 is formulated and cryopreserved in 85% Plasmalyte A (Baxter, Illinois, USA), 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Missouri, USA), and 5% human serum albumin (HSA) (Sigma-Aldrich, Missouri, USA), and Stempeucel®-1A is formulated in CS5 (CryoStor 5, Biolife Solutions, Washington, USA). In this study, we evaluated the comparability of the Stempeucel®-1 and Stempeucel®-1A manufactured from two different WCB batches 1 and 1A and cryopreserved in plasmalyte A and CS5 formulations respectively. The basic criteria for the characterization of Stempeucel®-1 and Stempeucel®-1A are given in supplementary Table 1. The techniques used to characterize Stempeucel® have been published previously [14, 15].
Gene expression profiling by microarray and analysis of Stempeucel®-1, Stempeucel®-1A, and human foreskin fibroblasts (HFF)
Total RNA was isolated using the RNeasy Micro kit (Qiagen, Hilden, Germany) as per the manufacturer’s protocol. The quality of the extracted RNA was checked using Nanodrop Spectrophotometer (Thermo Fisher Scientific, Waltham, USA). The OD 260/280 ratio was > 1.9 and OD 260/230 was > 1.8 for all samples. Further, RNA integrity was assessed using 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, USA) and RNA 6000 Nano kit. The RIN number of all RNA samples was > 8.0. For each sample, 250 ng of total RNA was amplified and labeled using the Ambion WT Expression Kit (Thermo Fisher Scientific, Waltham, USA) and Affymetrix GeneChip WT terminal labelling kit (Affymetrix, Santa Clara, USA), respectively, according to the protocol provided by the supplier. The labeled second cycle cDNA was processed for further analysis using a previously published method [19]. Unsupervised hierarchical clustering of differentially expressed genes was done using a Euclidian algorithm with Centroid linkage rule to identify gene clusters whose expression levels are significantly reproduced across the replicates.
Immunophenotypic characterization by flow cytometry
The cells were incubated with the below-mentioned antibodies for half an hour at room temperature, following which the samples were analyzed using the FACS DIVA and WinMDI 2.9 software. The expression levels of CD73, CD105, CD44, CD166, CD90, HLA-ABC, CD34, CD45, HLA-DR, CD40, CD80, and CD86 were analyzed. The details and concentrations of the antibodies used are provided in supplementary Table 1.
Assessment of in vitro immunomodulatory properties of Stempeucel®-1 and Stempeucel®-1A
For immunosuppression assays, a one-way mixed lymphocyte reaction (MLR) assay was performed at a ratio of 1:2.5, 1:5, and 1:10 (peripheral blood mononuclear cells (PBMSC): MSC) as described before [20]. For inflammatory cytokine priming, the MSC growth medium was replaced with 10 ml/flask of complete medium supplemented with interferon-γ (IFN-γ) (10 ng/ml) (Thermo Fisher Scientific, Waltham, USA) and tumor necrosis factor α (TNFα) (15 ng/ml) (Thermo Fisher Scientific, Waltham, USA). After 40 h of priming, cells were used for the MLR assay. Cell proliferation was measured using a fluorimetric immunoassay kit (Millipore-Sigma, Burlington, USA) to quantify Bromodeoxyuridine (BrdU) incorporation, according to the manufacturer’s instructions. A one-way MLR cultured in the absence of MSCs would be considered as the 100% proliferation control. All treatments were performed in triplicates.
Enzyme-linked immunosorbent assay
Human angiogenic cytokines, VEGF, Ang-1, SDF-1α, IL-6, IL-8, HGF, and TGFβ1 in the conditioned media (CM), which was collected at the 72-h time point as described before [17], were estimated using Enzyme-Linked Immunosorbent Assay (ELISA) kits (R&D Systems, Minneapolis, USA) according to the manufacturer’s directions. The samples were assayed in duplicates. Error bars are expressed as mean value ± SEM.
In vitro angiogenic activity assessment of Stempeucel®-1 and Stempeucel®-1A
The CM collected at the 72-h time point from Stempeucel®-1 and Stempeucel®-1A was used to evaluate three in vitro angiogenic functional assays using human umbilical vein endothelial cells (HUVECs). The HUVEC migration, proliferation, and tube formation assays were performed as described in our previous publication [17].
Animal experiments
Animal model and cell injection procedure
Unilateral hind limb ischemia was established in 10–12-week-old BALB/c nude (OlaHsd-Fox1nu) mice as described before [13]. Stempeucel®-1 and Stempeucel®-1A were administered at a dose of 5 × 106 cells in 50 μl of Plasmalyte A as determined to be the maximum effective dose for Stempeucel®-1 and published previously [7], or vehicle (Plasmalyte A) was administered by i.m injection using a 26G needle, around the ligation site, at five different places (approximately 10 μl at each site), following 2-5 h post-induction of limb ischemia to enable animals to recover from the trauma of surgery and anesthesia. All the animals were observed for 28 days at regular intervals.
Limb necrosis and functional scoring
The therapeutic effect of Stempeucel® in ameliorating the progression of tissue necrosis was assessed by examining the number of toes necrosed following ligation. Necrotic scoring was performed on days 0, 7, 14 & 28. Similarly, clinical and functional outcome following Stempeucel® treatment was assessed by Tarlov score, ischemic score, and ambulatory scores as described before [21].
Blood flow measurement by laser Doppler imaging
The rate of blood perfusion was measured for both ischemic and normal limb for both cell and vehicle-treated groups using laser Doppler imaging system (LDI2, Moor Instruments, UK) at the Department of Pharmacology, PSG College of Pharmacy, Coimbatore, India. The blood flow measurements were expressed as a ratio of the flow in the ischemic limb versus the normal limb.
DiI Labelling of Stempeucel®-1 followed by intramuscular administration for biodistribution analysis
To examine the persistence of bone marrow-derived MSC in the hindlimb ischemia model in BALB/c nude mice, the cells were stained with Vybrant™ CM-DiI Cell-Labeling Solution (Thermo Fisher Scientific, Waltham, USA) as described before [22], prior to injecting the cells into the animals. Five million viable cells were resuspended in 50 μl PlasmaLyte A and injected i.m in both ischemic and normal limbs. Evaluation of signal intensity from CM-DiI-labeled cells was performed after cell injection on days 1, 3, 6, 11, 14, 21, and 28 using the In Vivo Imaging System (IVIS) Xtreme Imaging System (Bruker, Massachusetts, USA). The images were quantified using Carestream molecular imaging software. As per the IVIS Xtreme Imaging System guidelines, all the animals were imaged before the cell injection to normalize autofluorescence. Average pixel intensity from each group was calculated from the individual animal’s pixel area intensity.
Histopathological evaluation
The therapeutic effect of Stempeucel® was also evaluated based on histological analysis of the various muscle sections (adductor, soleus, gastrocnemius, and semimembranosus/gracilus), stained with H & E. The stained muscle sections were scored for muscle degeneration, inflammation, and muscle necrosis from five separate fields in four distinct areas. Total numbers of each incident of degeneration, inflammation, and necrosis were calculated from each group. In animals that presented auto-amputation, the muscles were not collected, and in such cases, the maximum histological scoring of 5 (severe) was given for the degeneration, inflammation, and muscle necrosis. Muscle fiber area was quantified using QWin Software (Leica Biosystems, Wetzlar, Germany). Immunohistochemical analysis was performed on paraffin-embedded muscle tissue sections (5 μm thickness) of mice using anti-mouse CD31 antibody (Cat no. SAB4502167; Sigma-Aldrich, Missouri, USA), anti-human nuclear antigen (HNA) (Cat no. ab191181; Abcam, Cambridge, UK), and anti-human VEGF antibody (Cat no. ab46154; Abcam, Cambridge, UK). HRP-conjugated secondary antibodies, anti-rabbit IgG (EnVision+, Agilent technologies, CA, USA), goat anti-mouse IgG (Cat no.205719; Abcam, Cambridge, UK), and goat anti-rabbit IgG (Cat no. 205718; Abcam, Cambridge, UK) were used for CD31, HNA, and VEGF staining respectively. The immunoreactive products were visualized as described before [23]. The evaluation was carried out by blinded pathologists who were not aware to which group the animals were allocated.
Statistical analysis
The results were represented as mean ± SD or SEM. All the groups were compared by one-way ANOVA followed by Dunnett’s multiple comparisons using GraphPad Prism 5 software. P < 0.05 was considered a significant change.
