A comparative study of mesenchymal stem cell transplantation and NTG-101 molecular therapy to treat degenerative disc disease

Cellular replacement therapy using mesenchymal stem cells (MSCs) and/or the delivery of growth factors are at the forefront of minimally invasive biological treatment options for Degenerative Disc Disease (DDD). In this study, we compared the therapeutic potential of a novel drug candidate, NTG-101 to MSCs, including rat cartilage derived stem cells (rCDSCs), bone marrow stem cells (rBMSCs) and human Umbilical Cord Derived Mesenchymal Stem Cells (hUCMSCs) for the treatment of DDD. We induced DDD using a validated image-guided needle puncture injury in rat-tail IVDs. Ten weeks post-injury, animals were randomized and injected with MSCs, NTG-101 or vehicle. At the end of the study, histological analysis of the IVD-Nucleus Pulposus (NPs) injected with NTG-101 or rCDSCs showed a healthy or mild degenerative phenotype in comparison to vehicle controls. Immunohistochemical analysis revealed strong expression of aggrecan, collagen 2, brachyury and Oct4 in IVD-NPs injected with NTG-101. Our results also demonstrated suppression of inflammation induced p38 and NFκB resulting in inhibition of catabolic genes, but activation of Smad-2/3, Erk-1/2 and Akt-dependent signaling inducing anabolic genes in IVD-NP on treatment with NTG-101. In conclusion, a single injection of NTG-101 into the degenerative disc demonstrated superior benefits compared to stem cell transplantation.

In humans, a healthy IVD-NP is rich in proteoglycans such as aggrecan as well as collagen type 2 that resist loading forces placed upon the spine. Changes in lifestyle, aging and gene expression lead to chronic inflammation resulting in the loss of NP cells, triggering the development and progression of DDD 19,20 . In humans, the IVD-NP is rich in notochordal cells (NCs) and Nucleus Pulposus Progenitor Cells (NPPCs) from birth until early adolescence and are gradually replaced by chondrocyte like cells (CLCs). Sakai et al. 21 , demonstrated that IVD-specific progenitors are exhausted with ageing and degeneration of the IVD. The loss of IVD stem cells  Confocal Laser Scan Microscopy (CLSM) of MSC surface markers and cell type specific protein markers in rat CDSCs and BMSCs. Rat cartilage and bone marrow derived mesenchymal stem cells were plated on matrigel coated glass coverslips and MSC surface markers (CD44, CD133 and CD166) and cell type specific protein markers (Sox2, Oct4, Sox9 and Brachyury) were analyzed using specific antibodies. The protein expression was detected using Alexa 568 fluorophore and DAPI was used as nuclear stain. As shown, panel shows cytoplasmic and membranous staining of CD44 and CD133 in (a) BMSCs and (b) CDSCs; CD166 expression was not observed in CDSCs, but membranous and diffused, faint cytoplasmic staining was observed BMSCs as shown in panel (a) and (b). As shown in (a) rBMSCs showed diffused cytoplasmic Sox2 staining, nuclear Oct4 and Sox9 (b) rCDSCs demonstrated nuclear staining of both pluripotency markers (Sox2, Oct4) and chondrocyte transcription factor (Sox9). Notochordal cell marker, Brachyury was not detected in both rBMSCs and rCDSCs as shown in the panel (Scale bar: 10 µm).  2). Both rCDSCs and rBMSCs showed nuclear expression of the chondrogenic transcription factor Sox9 with no detectable expression of brachyury ( Fig. 2a,b).
To confirm the stemness characteristics of the cartilage and bone marrow derived rat MSCs, we differentiated these MSCs into osteogenic, chondrogenic and adipogenic lineages as defined in "Materials and methods". Both rCDSCs and rBMSCs differentiated into osteogenic, chondrogenic and adipogenic lineages as shown by Alizarin red, Safranin O and Oil red O staining respectively confirming their stemness ( Supplementary Fig. S1a,b).
As described in "Materials and methods", we used GFP-expressing rat MSCs (rCDSCs/rBMSCs) to track and detect these cells in rat tail IVDs post-injection. Our immunohistochemical analysis using GFP-specific mouse monoclonal antibodies demonstrated no detectable expression of GFP-/GFP-expressing cells in IVD-NPs injected with rCDSCs or rBMSCs 10 weeks post-injury ( Supplementary Fig. S2). Green fluorescent protein (GFP) expressing Wistar rat tissue sections used as positive control (spine/caudal IVDs) showed strong membranous staining of GFP on NP-cells ( Supplementary Fig. S2). of time to elucidate the mechanism of action of NTG-101 in vitro. Inflammation is considered as the most important player in the development of a degenerative IVD. Treatment with the pro-inflammatory cytokine, IL-1β (10 ng/ml) for 24 h induced significant expression of ECM degrading enzymes (MMP-3, MMP-13) and the pain associated enzyme, cyclooxygenase-2 (Cox-2) in rat IVD-NP cells as compared to no treatment control (NTC) cells ( Fig. 6a-c). However, the presence of NTG-101 significantly suppressed IL-1β induced expression of MMP-3, MMP-13 and Cox-2 within 24 h of treatment as revealed by qPCR analysis (Fig. 6a-c). Further, our results showed that treatment with IL-1β (10 ng/ml) resulted in phosphorylation of the p38 and p65-subunits of the NFκB complex ( Fig. 6d), key proteins regulating expression of several inflammation associated genes such as MMPs, Cox-2 and several other pro-inflammatory cytokines. Of note, western blots further showed that treatment with NTG-101 suppressed IL-1β induced phosphorylation of both the p38 and p65-subunits of NFκB in rat IVD-NP cells within 30 min of treatment ( Fig. 6d, Supplementary Fig. S4). Furthermore, with the reduction in inflammation in IVD-NP, treatment with NTG-101 induced NP-cell proliferation in vitro (> 50%) for up to 96 h as compared to no treatment control cells (NTC) cells (Fig. 6e). Treatment with NTG-101 resulted in phosphorylation of extra-cellular signal regulated kinase-1/2 (Erk-1/2, also known as p42/44, Thr202/Tyr204), Akt (Ser473 and Thr308) and receptor Smads including Smad-2 (Ser465/467) and Smad-3(423/425) proteins in rat IVD-NP cells in comparison to untreated controls (NTC, Fig. 6f, Supplementary Fig. S5).

Discussion
Degenerative disc disease (DDD) significantly impacts the quality of life in patients suffering from this condition globally 45,46 . However, only a few biological therapeutics have made their way from pre-clinical models into human clinical trials for treatment of DDD. Among these, an intra-discal injection of MSCs is currently in clinical trial studies (Phase 1/2/3) focusing on safety and efficacy of BMSCs and IVD-NP derived cells [35][36][37][38] . In an effort to better understand the mechanisms involved with cell-based therapy, we used a rat-tail disc injury model of DDD to compare the efficacy of mesenchymal stem cells (MSCs) derived from three different sources including rat cartilage (rCDSCs), bone marrow (rBMSCs) and human umbilical cord (hUCMSCs). We compared the results of stem cell transplants with the non-cellular therapeutic, NTG-101 containing a combination of rhCTGF and rhTGF-β1 within an excipient solution. The results of this study demonstrated differences in the regenerative effects of growth factor-based therapeutics (NTG-101) and MSC-based therapies in a pre-clinical rodent model of DDD. Injured rat tail IVD-NPs injected with NTG-101 showed the presence of notochordal and stem cells as well as increased expression of healthy ECM proteins (aggrecan, Col2A1). However, IVDs injected with rCDSCs    www.nature.com/scientificreports/ showed limited regeneration potential with moderate expression of aggrecan and ColA1 but failed to restore notochordal and stem cells in injured IVDs. Additionally, the loss of NP cells and lower expression of aggrecan was observed in IVD-NPs injected with rBMSCs and hUCMSCs, in a manner akin to IVDs injected with vehicle. These findings clearly demonstrated the differences in the regenerative capabilities of MSCs depending on the source from which these cells are derived. These results also indicate the need to understand the complexities involved with transplantation of MSCs into a degenerative IVD that is typified by a pro-inflammatory, hypoxic, acidic, and reduced nutrient milieu. Although rCDSCs, rBMSCs and hUCMSCs show mesenchymal characteristics, only IVDs injected with rCD-SCs showed features comparable to a healthy, uninjured age-matched IVD-NPs. These CDSCs were derived from rat healthy articular cartilage from the knee, the milieu of which shares similarities with the IVD-NP niche (low nutrient supply, avascular and hypoxia). Since CDSCs have a similar origin and resemblance to chondrocyte-like cells (CLCs) within the IVD-NP, rCDSCs have a higher propensity to differentiate into NP-like cells and regenerate a healthy ECM in degenerate IVDs. In contrast, our study showed injured and degenerative IVDs injected with rBMSCs or hUCMSCs showed features of degenerative IVD-NPs such as reduced cellularity and an excess of fibrocartilaginous matrix. Among degenerative IVDs receiving an intra-discal injection of rBMSCs or human UCMSCs, 70% of the IVD-NPs showed a fibrocartilaginous, metaplastic IVD phenotype like the vehicle control. These results clearly demonstrated the differential ability of MSCs of variable origin to repair and regenerate a degenerating IVD. These considerations are particularly relevant given the oxygen rich, nutrient and growth factor enriched micro-environments from which rBMSCs or hUCMSCs are derived. In contrast, several studies have suggested that BMSC transplantation reverses IVD degeneration [33][34][35][36][37][38] . The characteristics of BMSCs including low immunogenicity, ability to differentiate into NP-like cells, and possible capacity to preserve the structure and function of IVD may characterize them as a preferred choice for transplantation. It has been reported that an intra-discal injection of BMSCs induced an increase in the endogenous cell population in vivo, suggesting BMSCs promoted cell survival and proliferation as well as prevented apoptosis in a degenerative IVD-NP [32][33][34] . By tracking the fate of GFP-tagged BMSCs, Yang et al. 47 showed that a proportion of BMSCs could survive up to 24 weeks in the degenerated murine intervertebral discs and arrest the progressive degeneration of the IVD-NP www.nature.com/scientificreports/ through self-differentiation and stimulatory effect on endogenous NP-cells. However, the decreased number of BMSCs observed at 24 weeks post-injection suggested that the degenerative environment may be detrimental to the survival of the BMSCs in IVD-NP. Several studies have investigated and proposed a bidirectional transfer of proteins between MSCs and NP cells that may provide a new mechanism for the interaction between MSCs and NP cells 39,40,[48][49][50][51][52] . To understand the impact of the IVD-NP microenvironment, in vitro co-culture experiments have been used to study the possible influence of the respective cells upon each other. Cytokines and/or growth factors secreted from MSCs can stimulate and regulate the viability and function of NP cells in their proximity and vice versa. Granulocyte colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), leukemia inhibitory factor (LIF) and interleukins (IL-6, IL-10 and IL-11) are constitutively expressed by MSCs 52 . In addition, MSCs have also been reported to secrete insulin growth factor-1 (IGF-1), epithelial growth factor (EGF) and several members of transforming growth factor beta (TGF-β) superfamily including BMP-2, BMP-4, BMP-6, BMP-7, and TGF-β1 [48][49][50] . Richardson et al. 51 showed a sharp increase in the expression levels of Sox9, aggrecan, collagen I, collagen II, and collagen VI genes in BMSCs after a 7-day cell-cell contact with NP cells. In addition, direct cell-cell contact with autologous human BMSCs enhanced the cell proliferation and proteoglycan synthesis in IVD-NP cells 51 . These results suggested that paracrine signaling and interaction between MSCs and NP cells benefit both the biological activities of NP cells and the differentiation of MSCs. However, the engraftment and possible change in stem cell phenotype post-transplantation, as well as the integration of these transplanted cells within the IVD-NP remains controversial, with little evidence of cellular survival. Therefore, it raises the question that if the restorative effects of stem cells are dependent on secreted factors, would it not be preferable to deliver the requisite proteins more directly and avoid the risks of cellular transplant? The present study demonstrated that a single injection of NTG-101, containing a combination of rhCTGF and rhTGF-β1, provided superior regenerative potential in a pre-clinical model of DDD as compared to MSCs derived from three different sources (cartilage, bone marrow and umbilical cord). Our data showed that transplanted MSCs do not survive and integrate within the harsh, inflammatory microenvironment of the degenerative IVD. These results suggest that a minimally invasive intervention capable of inducing a regenerative effect upon the IVD without the associated risks of cell-based therapy may prove to be the most effective therapy for DDD. In our earlier reports, we showed that NTG-101 plays an important role in the suppression of inflammation and the restoration of ECM proteins both in vitro and in in vivo 27,28 . Among the components of NTG-101, both rhCTGF and rhTGF-β1 are known for their anti-inflammatory and cell growth promoting functions. Our results demonstrated the anti-inflammatory and pro-anabolic effects of the combination of rhCTGF and rhTGF-β1 in rat, canine and human NP cells 27,28 . Herein, we showed activation of multiple signaling cascades including suppression of IL-1β induced p38 and NFκB signaling and activation of pro-anabolic pathways such as Smad-2/3, Erk-1/2 and PI-3K/Akt leading to the development of an anabolic environment that catalyzes regeneration in IVD-NPs (Fig. 7). Transforming growth factor-β1 (TGF-β1), an important component of NTG-101 initiates multiple cellular signaling pathways by binding to and activating its specific cell surface receptors, TGF-β receptors-R2/R1, that possess an intrinsic serine/-threonine kinase activity [53][54][55] . These activated receptors stimulate the phosphorylation of receptor Smad proteins, Smad-2 and Smad-3 which form complexes with other co-Smads like Smad-4 [53][54][55] . The hetero-trimeric complex of Smad-2/3/4 accumulates in the nucleus and regulate the transcription of several target genes involved in the development of a healthy extra-cellular matrix (aggrecan, Collagen 2A) and other proteoglycans 56,57 . In addition, TGF-β1 is also known to induce cell proliferation and activate pro-survival signaling by activating non-Smad pathways involving phosphorylation of Erk-1/2 and Akt in a Smad-dependent or independent manner via TGF-β activated kinase 1 (TAK1) in cell type specific manner 58,59 . Both the canonical and non-canonical TGF-β signaling pathways have been reported to promote glycosaminoglycans (GAG) and proteoglycan (PG) biosynthesis in NP cells of the disc 59 . Further, TGF-β growth factor signaling and tonicity-responsive enhancer binding protein (TonEBP), a transcription factor that regulates cellular osmolarity in NP cells work synergistically to maintain GAG and PG biosynthesis in NP cells 59 . In addition, rhCTGF also acts synergistically with rhTGF-β1 and hypoxia in regulating proteoglycan synthesis in IVD-NPs, supporting our in vivo results that treatment with the combination of these growth factor enhanced viability and ECM protein synthesis, while suppressing inflammation in vitro and in pre-clinical animal models of DDD 27, 28 . Our results strongly suggest that a single injection of NTG-101 into the degenerative disc can overcome the injurious and pro-inflammatory effects conferred by needle puncture injury and induce repair.
In conclusion, our results demonstrated that an intra-discal injection of NTG-101 into the degenerative IVD-NP conferred superior results as compared to an injection of MSCs (rCDSCs/rBMSCs/hUCMSCs). The NTG-101 injection promoted the maintenance of a healthy IVD-NP in terms of its morphology and cellular phenotype promoting the preservation of notochordal and stem cells in comparison to IVDs injected with MSCs alone that showed limited regenerative potential. (rCDSCs) and bone marrow stem cells (rBMSCs) were derived from 12-week-old GFP-expressing Wistar rats (n = 8), following humane euthanasia using CO 2 asphyxiation. Rat CDSCs were cultured in serum free media in low adherent cell culture flasks under hypoxia (3.5% O 2 ) at 37 °C as suspension culture (Fig. 1a). While rBMSCs were cultured in adherent cell culture flasks in complete growth medium containing Advanced Dulbecco's Modified Eagles Medium (AMDEM), fetal bovine serum (FBS, 10%), penicillin-streptomycin (1×) and glutamax (1×) under normoxia i.e., 5% CO 2 at 37 °C (Fig. 1b). See Supplementary data for detailed protocol. These cells were characterized for MSC cell surface markers, pluripotency and lineage differentiation as described below.

Cell culture of human umbilical cord derived stem cells (hUCMSCs). Human Umbilical Cord
Derived Stem cells (hUCMSCs) were purchased from American Type Cell Culture (ATCC, PCS 500 010). Cells were cultured in adherent flasks containing low serum MSC-growth media (ATCC PCS-500-030) and supplement (ATCC PCS-500-040) obtained from ATCC, under normoxia i.e., 5% CO 2 at 37 °C as per manufacturer's instructions. Human UCMSCs were characterized by ATCC for MSC markers and their differentiation into osteogenic, chondrogenic and adipogenic lineages 60 .

Isolation, cell culture and treatment of rat caudal IVD-NP cells.
Healthy rat caudal IVDs were removed aseptically from 32-week-old Wistar rats. The nucleus pulposus (NP) was separated and enzymatically digested according to our established methods 27,28 . The next day, the cells were filtered with a 70 μm cell strainer and cultured within a hypoxic incubator (NuAire, MN, USA) in 3.5% O 2 , 5% CO 2 , 37 °C in Advanced Dulbecco's modified Eagle's medium (ADMEM) supplemented with 8% fetal bovine serum (FBS), penicillin and streptomycin (100 U/mL) until passage (P2) as described earlier 27,28 . The NP cells obtained from rat IVDs were pooled together for treatment with respective agents as follows. The cells were either cultured in serum free DMEM used as no treatment controls (NTC) or treated with NTG-101, IL-1β (10 ng/mL) or IL-1β + NTG-101 for various time points under hypoxic conditions. The effect of NTG-101 on cell viability, expression of catabolic genes and associated cell signaling proteins regulating ECM synthesis or degradation, inflammation and pain in presence or absence of pro-inflammatory cytokine, IL-1β in NP cells was determined using qPCR and/or Western blotting as described below (also see "Supplementary Data S1" for details). www.nature.com/scientificreports/ RNA isolation, reverse transcription and quantitative real time PCR (qPCR). Total RNA was isolated from spheroids of rCDSCs and adherent rBMSCs and hUCMSCs (passage, P2/3) using Trizol following manufacturer's instructions. In addition, total RNA was also isolated from rat caudal IVD-NP cells treated with NTG-101, IL-1β (10 ng/ml), IL-1β (10 ng/ml) + NTG-101 and no treatment controls (NTC). See Supplementary data for detailed protocol. Total RNA was quantified using a NanoVue Plus (Biochrom, MA). cDNA was prepared from total RNA (~ 1 µg) using iScript Reverse Transcriptase (Biorad, CA). We determined the expression of mesenchymal stem cell surface markers (CD29, CD44, CD90, CD105, CD133, CD166), pluripotency markers (Oct4, Sox2), chondrogenic marker (Sox9), notochordal cell marker (Brachyury) and the hematopoietic progenitor cell markers (CD34, CD45) in MSCs using species and gene specific primers with the SYBR Green reagent (Thermo Fisher Scientific, ON) using quantitative real time-Polymerase Chain Reaction (qPCR) on the ABI 7900HT 96-well Fast block machine. For control and treated rat IVD-NP cells, expression of catabolic genes including MMP-3, MMP-13 and Cox-2 was also determined using qPCR. The sequence of the primers used is given in Supplementary data (Supplementary Table S1a,b). To check the specificity of the amplification products, melt curve analysis was carried out after each reaction. Species specific (rat/human) hypoxanthine phosphoribosyl-transferase (HPRT) gene expression was used as an internal control for normalization. No template controls, i.e., master mix without cDNA, was used as negative controls for the qPCR experiments.

Intra-discal injection of vehicle, NTG-101 and MSCs (rCDSCs/rBMSCs/hUCMSCs) in pre-clinical rodent model of DDD.
We used our established image-guided rat-tail needle puncture injury model of DDD using 12-week old female Wistar rats (n = 30), 5-caudal discs/animal using fluoroscopic image guidance as described earlier 27,28 . We have previously shown that 10-week post needle puncture injury, the IVD assumes an 'end-stage' fibrocartilaginous degenerative phenotype 27 . In this study, we injured the rat-tail IVDs and waited 10 weeks before the PBS or therapeutic injections. For injuries, anesthesia was achieved using isofluorane (5 L/ min plus 1 L/min O 2 ) and maintained at 3 L/min. Once deeply anaesthetized, the animal was affixed on a stereotactic procedure apparatus (Model 900, Kopf Instruments, CA, USA) with nose cone inhalation. For disc injury, we used a 26-gauge (G) needle (Hamilton Company, USA) mounted on a Hamilton syringe. The needle was advanced completely through the selected tail IVD under fluoroscopic guidance. The animals were then removed from the stereotactic apparatus and allowed to recover in a warmed cage. Ten weeks post injury, animals were randomized, and the injured discs were injected with 8.0 µL of vehicle (i.e., phosphate buffer saline, PBS, 1×, pH = 7.2), NTG-101 or 150,000 cells (rCDSCs/rBMSCs/hUCMSCs) suspended in PBS (1×, pH = 7.2) using a 32G needle under fluoroscopic guidance. At the end of the experiment (i.e., 20 weeks post-injury), animals were humanely euthanized using CO 2 asphyxiation and each vertebral lumbar/caudal motion segment was dissected aseptically. The IVDs were harvested and fixed in formalin for histological and immunohistochemical analysis. Age-matched (32-week-old) healthy IVDs were obtained from rat tails that served as uninjured, healthy controls.
Histological analysis. For histology, rat caudal IVDs (healthy or treated with potential therapeutic agents) were removed, decalcified using CalEx (Fisher Scientific, ON) and paraffinized using histological cassettes as described earlier 27,28 . The tissue block was trimmed with a microtome (Leica Biosystems, CA) until tissue was exposed, followed by cutting 5 µ thick ribbons containing 10-12 paraffin-embedded tissue sections. Each section was collected on a single slide and checked for integrity of the tissue on the slide using bright field microscope. www.nature.com/scientificreports/ to ensure quality of the tissue sections, at least three sections (1st, middle and the last) were used for Safranin O staining for assessment of histology and proteoglycan content, followed by immunohistochemical staining of proteins using specific antibodies on concurrent sections. Histological grading of the IVD-NPs (injury followed by treatment) was carried out based on morphology, cellularity and Safranin O staining intensity in these paraffin embedded, tissue sections representing the histology of respective IVDs from each treatment group as described earlier 27,28 . See Supplementary Table S3 for histological scoring criteria in rat IVDs. The histological scoring was done by three observers (ME, AM and HG), and scores were recorded independently. In the case of inter-observer variability of scores, all three observers reviewed scores and arrived at a final consensus score.
Immunohistochemistry (IHC). Immunohistochemical analysis was performed in serial sections for ECM proteins (Aggrecan, Collagen 2), the notochordal cell marker (Brachyury), stem cell marker (Oct4), chondrocyte marker (Sox 9) and Green Fluorescent Protein (GFP) using the Vectastain tm rabbit or mouse kit (Vector Labs, ON). Briefly, after Safranin-O staining, subsequent serial tissue sections were deparaffinized in xylene followed by hydration in gradient alcohol. Antigen retrieval was performed using microwave-based heat retrieval method. Thereafter, slides were washed three times with Tris-buffered saline (TBS, 1X, pH = 7.4) containing 0.025% Triton-X-100 (TBS-T), followed by blocking with appropriate serum, provided in the kit. The slides were then incubated with rabbit polyclonal or mouse monoclonal primary antibodies at appropriate dilutions for 1 h at room temperature followed by washing with TBS-T, three times). The sections were incubated with hydrogen peroxide (0.3% v/v) for 15 min to block endogenous peroxidase activity, followed by three washes with TBS-T. Tissue sections were incubated with the goat anti-rabbit or mouse secondary antibody at appropriate dilution for 30 min. Protein expression was detected using diaminobenzidine (DAB) as chromogen. The sections were counterstained with Meyer's hematoxylin and mounted with DPX mountant. For scoring, ECM and cytoplasmic staining for Aggrecan, Col2A1 and nuclear expression of Brachyury, Oct4 and Sox-9 was considered as positive staining for IHC analysis (Supplementary Table S4). The bright field sections were evaluated semi-quantitatively for % positivity and staining intensity in IVD-NP tissue by light microscopic examination using a Nikon bright field microscope (Nikon Eclipse TE2000-U).
Cell viability assay. Rat IVD-NP (4000 cells per well) were plated in 96-well flat bottom plates to evaluate the effect of treatment with NTG-101 on cell viability in time dependent manner. Rat NP cells (passage, P2) were treated with NTG-101 in serum free media for 48-96 h and cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, USA) as described earlier 27, 28 . Western blotting. For Western blotting, protein estimation was performed using Bradford assay (BioRad, CA). Briefly, equal amounts of rat IVD-NP cell lysates prepared using NP-40 lysis buffer containing protease and phosphatase inhibitors (Millipore-Sigma, ON), were subjected to Western blotting as described earlier 27 . Whole cell lysates (30 μg) were resolved on 10% or 4-15% gradient sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE, Biorad, CA) under reducing conditions and proteins were electro-transferred onto polyvinyledendifluoride (PVDF) membranes using Trans-Blot Turbo System (BioRad, CA). After blocking with 5% non-fat powdered milk (for non-phospho-proteins) or 1% bovine serum albumin (BSA, for phosphorylated signaling proteins) in Tris-buffered saline (TBS, 0.1 M, pH = 7.4), blots were incubated with rabbit polyclonal or mouse monoclonal primary antibodies at 4 °C overnight. Membranes were washed three times with Tween (0.1%)-Tris-buffer saline (TTBS) and then incubated for 2 h at room temperature with the respective horse radish peroxidases (HRP)-conjugated anti-IgG secondary antibodies (BioRad, CA), diluted as per the manufacturer's instructions in 2% non-fat milk in TBS (pH = 7.2, 1×). Blots were washed three times with TTBS for 15 min, protein bands were detected by the enhanced chemiluminescence reagents (BioRad, CA) and images captured using GE AI600RGB Imager (Cytiva, MA). β-actin was used as a loading control for each experiment.
Statistical analysis. For qPCR analysis, gene expression was evaluated based on Ct-values. Each sample was run in duplicate, and the fold change was calculated using 2 −(ddCt) method. For MSCs, histograms represent fold change of gene expression in rCDSCs and hUCMSCs with respect to rBMSCs. For viability assays and expression of catabolic genes (MMP-3, MMP-13 and Cox-2), histograms represent viability and expression in treated cells with respect to no treatment control (NTC) cells. For histological analysis, histograms showing average scores for morphology (M), cellularity (C), Safranin O staining intensity (I) and total score (M + C + I) were plotted for all variables considered in this study. For immunohistochemical analysis, total score was evaluated as the sum of score on % positivity and intensity in each tissue section as described earlier 27,28 . All data are expressed as average score ± standard deviation (S.D.). Statistical analysis was performed using MS-Excel using 2-tailed Student's t-test. P-value ≤ 0.05 was defined as statistically significant for all tests.