A comprehensive analysis of mRNA expression profiles of Esophageal Squamous Cell Carcinoma reveals downregulation of Desmoglein 1 and crucial genomic targets.

AIM: Esophageal Squamous Cell Carcinoma (ESCC) is a histological subtype of esophageal cancer that begins in the squamous cells in the esophagus. In only 19% of the ESCC-diagnosed patients, a five-year survival rate has been seen. This necessitates the identification of high-confidence biomarkers for early diagnosis, prognosis, and potential therapeutic targets for the mitigation of ESCC. METHOD: We performed a meta-analysis of 10 mRNA datasets and identified consistently perturbed genes across the studies. Then, integrated with ESCC ATLAS to segregate 'core' genes to identify consequences of primary gene perturbation events leading to gene-gene interactions and dysregulated molecular signaling pathways. Further, by integrating with toxicogenomics data, inferences were drawn for gene interaction with environmental exposures, trace elements, chemical carcinogens, and drug chemicals. We also deduce the clinical outcomes of candidate genes based on survival analysis using the ESCC related dataset in The Cancer Genome Atlas. RESULT: We identified 237 known and 18 novel perturbed candidate genes. Desmoglein 1 (DSG1) is one such gene that we found significantly downregulated (Fold Change =-1.89, p-value = 8.2e-06) in ESCC across six different datasets. Further, we identified 31 'core' genes (that either harbor genetic variants or are regulated by epigenetic modifications) and found regulating key biological pathways via adjoining genes in gene-gene interaction networks. Functional enrichment analysis showed dysregulated biological processes and pathways including "Extracellular matrix", "Collagen trimmer" and "HPV infection" are significantly overrepresented in our candidate genes. Based on the toxicogenomic inferences from Comparative Toxicogenomics Database we report the key genes that interacted with risk factors such as tobacco smoking, zinc, nitroso benzylmethylamine, and drug chemicals such as cisplatin, Fluorouracil, and Mitomycin in relation to ESCC. We also point to the STC2 gene that shows a high risk for mortality in ESCC patients. CONCLUSION: We identified novel perturbed genes in relation to ESCC and explored their interaction network. DSG1 is one such gene, its association with microbiota and a clinical presentation seen commonly with ESCC hints that it is a good candidate for early diagnostic marker. Besides, in this study we highlight candidate genes and their molecular connections to risk factors, biological pathways, drug chemicals, and the survival probability of ESCC patients.

Small extracellular vesicle-loaded bevacizumab reduces the frequency of intravitreal injection required for diabetic retinopathy.

Diabetic retinopathy (DR) is associated with retinal neovascularization, hard exudates, inflammation, oxidative stress and cell death, leading to vision loss. Anti-vascular endothelial growth factor (Anti-VEGF) therapy through repeated intravitreal injections is an established treatment for reducing VEGF levels in the retina for inhibiting neovascularization and leakage of hard exudates to prevent vision loss. Although anti-VEGF therapy has several clinical benefits, its monthly injection potentially causes devastating ocular complications, including trauma, intraocular hemorrhage, retinal detachment, endophthalmitis, etc. Methods: As mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (MSC-EVs) demonstrated safety in clinical studies, we have tested the efficacy of MSC-derived small EVs (MSC-sEVs) loaded anti-VEGF drug bevacizumab in a rat model of DR. Results: The study identified a clinically significant finding that sEV loaded with bevacizumab reduces the frequency of intravitreal injection required for treating diabetic retinopathy. The sustained effect is observed from the reduced levels of VEGF, exudates and leukostasis for more than two months following intravitreal injection of sEV loaded with bevacizumab, while bevacizumab alone could maintain reduced levels for about one month. Furthermore, retinal cell death was consistently lower in this period than only bevacizumab. Conclusion: This study provided significant evidence for the prolonged benefits of sEVs as a drug delivery system. Also, EV-mediated drug delivery systems could be considered for clinical application of retinal diseases as they maintain vitreous clarity in the light path due to their composition being similar to cells.

Polyphenol-based targeted therapy for oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a chronic, progressive, and precancerous condition mainly caused by chewing areca nut. Currently, OSF therapy includes intralesional injection of corticosteroids with limited therapeutic success in disease management. Therefore, a combined approach of in silico, in vitro and in vivo drug development can be helpful. Polyphenols are relatively safer than other synthetic counterparts. We used selected polyphenols to shortlist the most suitable compound by in silico tools. Based on the in silico results, epigallocatechin-3-gallate (EGCG), quercetin (QUR), resveratrol, and curcumin had higher affinity and stability with the selected protein targets, transforming growth factor beta-1 (TGF-ß1), and lysyl oxidase (LOX). The efficacy of selected polyphenols was studied in primary buccal mucosal fibroblasts followed by in vivo areca nut extract induced rat OSF model. In in vitro studies, the induced fibroblast cells were treated with EGCG and QUR. EGCG was safer at higher concentrations and more efficient in reducing TGF-ß1, collagen type-1A2 and type-3A1 mRNA expression than QUR. In vivo studies confirmed that the EGCG hydrogel was efficient in improving the disease conditions compared to the standard treatment betamethasone injection with significant reduction in TGF-ß1 and collagen concentrations with increase in mouth opening. EGCG can be considered as a potential, safer and efficient phytomolecule for OSF therapy and its mucoadhesive topical formulation help in the improvement of patient compliance without any side effects. Highlights Potential polyphenols were shortlisted to treat oral submucous fibrosis (OSF) using in silico tools Epigallocatechin 3-gallate (EGCG) significantly reduced TGF-ß1 and collagen both in vitro and in vivo EGCG hydrogel enhanced antioxidant defense, modulated inflammation by reducing TGF-ß1 and improved mouth opening in OSF rat model.

An overview of the current advances in the treatment of inflammatory diseases using mesenchymal stromal cell secretome.

The growing interest in mesenchymal stromal cell (MSC) therapy has been leading to the utilization of its therapeutic properties in a variety of inflammatory diseases. The clinical translation of the related research from bench to bedside is cumbersome due to some obvious limitations of cell therapy. It is evident from the literature that the MSC secretome components mediate their wide range of functions. Cell-free therapy using MSC secretome is being considered as an emerging and promising area of biotherapeutics. The secretome mainly consists of bioactive factors, free nucleic acids, and extracellular vesicles. Constituents of the secretome are greatly influenced by the cell's microenvironment. The broad array of immunomodulatory properties of MSCs are now being employed to target inflammatory diseases. This review focuses on the emerging MSC secretome therapies for various inflammatory diseases. The mechanism of action of the various anti-inflammatory factors is discussed. The potential of MSC secretome as a viable anti-inflammatory therapy is deliberated.

Mannosylated-Chitosan-Coated Andrographolide Nanoliposomes for the Treatment of Hepatitis: In Vitro and In Vivo Evaluations.

A key diterpene lactone of Andrographis paniculata, i.e., andrographolide (AG), exhibits a variety of physiological properties, including hepatoprotection. The limited solubility, short half-life, and poor bioavailability limits the pharmacotherapeutic potential of AG. Therefore, in this study we aimed to formulate and optimize AG-loaded nanoliposomes (AGL) using the Design of Experiment (DOE) approach and further modify the surface of the liposomes with mannosylated chitosan to enhance its oral bioavailability. Physical, morphological, and solid-state characterization was performed to confirm the formation of AGL and Mannosylated chitosan-coated AGL (MCS-AGL). Molecular docking studies were conducted to understand the ligand (MCS) protein (1EGG) type of interaction. Further, in vitro release, ex vivo drug permeation, and in vivo pharmacokinetics studies were conducted. The morphological studies confirmed that AGL was spherical and a layer of MCS coating was observed on their surface, forming the MCS-AGL. Further increase in the particle size and change in the zeta potential of MCS-AGL confirms the coating on the surface of AGL (375.3 nm, 29.80 mV). The in vitro drug release data reflected a sustained drug release profile from MCS-AGL in the phosphate buffer (pH 7.4) with 89.9 ± 2.13% drug release in 8 h. Ex vivo permeation studies showed higher permeation of AG from MCS-AGL (1.78-fold) compared to plain AG and AGL (1.37-fold), indicating improved permeability profiles of MCS-AGL. In vivo pharmacokinetic studies inferred that MCS-AGL had a 1.56-fold enhancement in AUC values compared to plain AG, confirming that MCS-AGL improved the bioavailability of AG. Additionally, the 2.25-fold enhancement in the MRT proves that MCS coating also enhances the in vivo stability and retention of AG (stealth effect). MCS as a polymer therefore has a considerable potential for improving the intestinal permeability and bioavailability of poorly soluble and permeable drugs or phytoconstituents when coated over nanocarriers.

Advancements in cell membrane camouflaged nanoparticles: A bioinspired platform for cancer therapy.

The idea of employing natural cell membranes as a coating medium for nanoparticles (NPs) endows man-made vectors with natural capabilities and benefits. In addition to retaining the physicochemical characteristics of the NPs, the biomimetic NPs also have the functionality of source cell membranes. It has emerged as a promising approach to enhancing the properties of NPs for drug delivery, immune evasion, imaging, cancer-targeting, and phototherapy sensitivity. Several studies have been reported with a multitude of approaches to reengineering the surface of NPs using biological membranes. Owing to their low immunogenicity and intriguing biomimetic properties, cell-membrane-based biohybrid delivery systems have recently gained a lot of interest as therapeutic delivery systems. This review summarises different kinds of biomimetic NPs reported so far, their fabrication aspects, and their application in the biomedical field. Finally, it briefs on the latest advances available in this biohybrid concept.

Novel bioactive formulation derived from the conditioned medium of mesenchymal stromal cells reduces under-eye dark circles in human volunteers.

BACKGROUND: Under-eye dark circles are a common condition observed in dermatology practice. Mesenchymal stromal cell-derived conditioned medium (MSC-CM) contains an array of growth factors and cytokines reported to promote periorbital rejuvenation and may be useful in removing the dark circle around the eyes. AIMS: The aim of the present study was to evaluate the safety and efficacy of developed bioactive formulation containing mesenchymal stromal cell-derived conditioned medium in reducing the under-eye dark circles. PATIENTS/METHODS: We tested the safety profile of MSC-CM along with antioxidants, in vitro using human melanocytes cultures. The bioactive formulation containing MSC-CM was developed and tested for physicochemical parameters. The dermatological safety was evaluated by primary irritant patch-test under complete occlusion on healthy human subjects. To elucidate its safety and efficacy, monocentric, open-label, single-arm study was carried out in 20 Indian female subjects for the duration of 12 weeks. Parameters such as eye puffiness, radiance, skin smoothness, even skin tone, periorbital fine lines and wrinkles, crow's feet, whitening, pigmentation, skin tightening, and refreshing/soothing effect were used to investigate the rejuvenating property of the bioactive formulation. RESULTS: Mesenchymal stromal cell-derived conditioned medium along with antioxidants decreased the melanin content compared to the CM alone in the melanocyte cultures. Besides, the bioactive formulation was safe and emerged as a non-irritant product. Improvement in the majority of the clinical parameters assessed through efficacy study was observed within 4 weeks of topical application of the formulation twice daily, and showed continued improvement for 12 weeks as evaluated by the dermatologists as well as self-assessment by the subjects. CONCLUSION: The bioactive formulation containing MSC-CM was safe and effective in reducing the under-eye dark circles and was beneficial in improving the overall appearance of the eye area.

Safety and therapeutic potential of human bone marrow-derived mesenchymal stromal cells in regenerative medicine.

Regenerative medicine is considered as an alternative approach to healthcare. Owing to their pluripotent abilities and their relative lack of ethical and legal issues, adult stem cells are considered as optimal candidates for use in the treatment of various diseases. Bone marrow-derived mesenchymal stem cells are among the most promising candidates for clinical applications as they have expressed a higher degree of plasticity in vitro. Many investigators have begun to examine how bone marrow stem cells might be used to rebuild damaged tissues. The systemic administration of cells for therapeutic applications requires efficient migration and homing of cells to the target site. Cell adhesion molecules and their ligands, chemokines, extracellular matrix components and specialized bone marrow niches all participate in the proper regulation of this process. MSCs suppress the pathophysiological process that is mediated by chronic inflammation and contributes to a modification of the microenvironment and tissue regeneration. Due to the intricacy of the mesenchymal stem cell, there is ever-increasing amount of data emerging about their migration and regenerative mechanisms. Many factors influence MSC mobilization and their homing to injured tissues. This review summarizes the current clinical and pre-clinical data available in literature regarding the use of MSC in tissue repair and their prospective therapeutic role in various diseases and the underlying repair mechanisms will be discussed.

Human bone marrow-derived, pooled, allogeneic mesenchymal stromal cells manufactured from multiple donors at different times show comparable biological functions in vitro, and in vivo to repair limb ischemia.

BACKGROUND: We have previously demonstrated that a pooled population of bone marrow-derived, allogeneic mesenchymal stromal cells (BMMSC), Stempeucel®-1, produced under good manufacturing practices (GMP) conditions, showed clinical efficacy and safety in patients suffering from critical limb ischemia (CLI) due to Buerger's disease. While Stempeucel®-1 is currently used for CLI and other clinical indications, we wanted to ensure that the product's continuity is addressed by developing and characterizing a second generation of pooled product (Stempeucel®-1A), manufactured identically from second BM aspirates of the same three donors after a 2-year interval. METHODS: The two versions of Stempeucel® were manufactured and subjected to gene and protein expression analysis. The nature of various growth factors/cytokines secreted and immunomodulatory activity of these two cell populations were compared directly by various in vitro assays. The preclinical efficacy of these two cell types was compared in an experimental model of hind limb ischemia (HLI) in BALB/c nude mice. The reversal of ischemia, blood flow, and muscle regeneration were determined by functional scoring, laser Doppler imaging, and immunohistochemical analyses. RESULTS: Qualitative and quantitative analyses of genes and proteins involved in promoting angiogenic activity and immune regulatory functions revealed high levels of correlation between Stempeucel®-1 and Stempeucel®-1A cell populations. Moreover, intramuscular (i.m) administration of these two cell products in the ischemic limbs of BALB/c nude mice showed significant repair (≥ 70%) of toe and foot necrosis, leading to improved ambulatory function and limb salvage. Furthermore, a biodistribution kinetics study showed that Stempeucel®-1 was mostly localized in the ischemic muscles of mice for a significantly longer time compared to normal muscles, thus playing an essential role in modulating and reversing HLI damage. CONCLUSIONS: This study shows that with a reproducible manufacturing procedure, it is possible to generate large numbers of pooled mesenchymal stromal cells from human bone marrow samples to establish product equivalence. We conclude from these results that, for the first time, two pooled, allogeneic BMMSC products can be repeatedly manufactured at different time intervals using a two-tier cell banking process with robust and comparable angiogenic properties to treat ischemic diseases.

Optimization of culture conditions for human bone marrow-derived mesenchymal stromal cell expansion in macrocarrier-based Tide Motion system.

BACKGROUND: With high cell doses required for mesenchymal stromal cell (MSC) clinical trials, there is a need to upgrade technologies that facilitate efficient scale up of MSCs for cell therapy. Conventional expansion with 2D culture vessels becomes the bottleneck when large cell dosages are required. Tide Motion bioreactors offer a robust, scalable platform using BioNOC II macrocarriers developed for the production of adherent cells. METHODS: We evaluated the growth and expansion of bone marrow-derived MSCs (BM-MSCs) on the macrocarrier-based culture system by optimizing key parameters such as cell seeding densities, culturing conditions, and harvesting procedures to achieve optimal cell growth. BM-MSCs expanded in conventional 2D adherent cultures were seeded into BioNOC II macrocarriers and grown in serum-containing or serum-free medium. RESULTS: BM-MSCs attained a maximum cell density of 0.49 ± 0.07 × 106 cells/carrier after 12 days of culture in BioNOC II macrocarriers with cell viability > 86% while retaining MSC specific characteristics such as surface marker expression, tri-lineage differentiation potential, immunosuppressive properties, and potency. CONCLUSION: These results reveal the feasibility of BM-MSC expansion in the scalable macrocarrier-based Tide Motion system both under serum and serum-free conditions and represent an important step for the large-scale production system of BM-MSC based cellular therapies.

Expansion and characterization of bone marrow derived human mesenchymal stromal cells in serum-free conditions.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are gaining increasing importance in the field of regenerative medicine. Although therapeutic value of MSCs is now being established through many clinical trials, issues have been raised regarding their expansion as per regulatory guidelines. Fetal bovine serum usage in cell therapy poses difficulties due to its less-defined, highly variable composition and safety issues. Hence, there is a need for transition from serum-based to serum-free media (SFM). Since SFM are cell type-specific, a precise analysis of the properties of MSCs cultured in SFM is required to determine the most suitable one. Six different commercially available low serum/SFM with two different seeding densities were evaluated to explore their ability to support the growth and expansion of BM-MSCs and assess the characteristics of BM-MSCs cultured in these media. Except for one of the SFM, all other media tested supported the growth of BM-MSCs at a low seeding density. No significant differences were observed in the expression of MSC specific markers among the various media tested. In contrary, the population doubling time, cell yield, potency, colony-forming ability, differentiation potential, and immunosuppressive properties of MSCs varied with one another. We show that SFM tested supports the growth and expansion of BM-MSCs even at low seeding density and may serve as possible replacement for animal-derived serum.

Hepatoprotective effect of bone marrow-derived mesenchymal stromal cells in CCl4-induced liver cirrhosis.

Bone marrow mesenchymal stromal cells (BM-MSCs) are multipotent stem cells which are ideal candidates for use in regenerative medicine. The objectives of this study were to evaluate the hepatoprotective effect of BM-MSC and its combination treatment with silymarin in carbon tetrachloride (CCl4)-induced liver cirrhosis animal model and to investigate whether tail vein or portal vein infusion was the ideal route for BM-MSC transplantation. 36 female Wistar rats were randomly divided into six groups (n = 6): Group 1 (normal control), Group 2 (received only CCl4, disease model), Group 3 (CCl4 + BM-MSCs through tail vein), Group 4 (CCl4 + BM-MSCs through portal vein), Group 5 (CCl4 + silymarin), Group 6 (CCl4 + BM-MSCs + silymarin). On the 21st day after treatment, blood samples were collected for biochemical estimations. After the experiment, the rats were sacrificed. Liver was dissected out and processed for histopathology and scanning electron microscopy studies. Liver enzyme and marker analysis, histopathological studies indicated that the combination of BM-MSCs and silymarin was effective in treating liver cirrhosis. Transplanted BM-MSCs in combination with silymarin ameliorated the liver tissue damage through their immunoregulatory activities. Among the two routes, the intravenous administration of cells through the tail vein was found to be more effective and safe.

A nanocomposite hydrogel delivery system for mesenchymal stromal cell secretome.

BACKGROUND: Mesenchymal stromal cell conditioned medium (MSC-CM) contains a cocktail of bioactive factors that act synergistically to induce therapeutic effects. This has been clearly demonstrated by in vivo applications of MSC-CM, but the establishment of controlled delivery systems is an unmet requirement for clinical translation. METHODS: We developed a nanocomposite-hydrogel (NP-H) comprised of poly-L-lactide nanoparticles (NPs) embedded in gelatin/hyaluronic acid (Gel/HA) hydrogel as a delivery vehicle for MSC-CM. First, we optimized the culture conditions for bone marrow-derived MSCs using serum-containing medium (SCM) and serum-free medium (SFM) and characterized the corresponding CM (serum-containing conditioned medium (ScCM) and serum-free conditioned medium (SfCM), respectively) for its potency and xeno markers. Then we prepared a composite matrix followed by physiochemical characterization and functional assays were performed. RESULTS: Nanocomposite hydrogel displayed an even distribution of NPs along with high porosity (> 60%) and swelling ratios > 1500%, while its protein release pattern corresponded to a mix of degradation and diffusion kinetics. Functional evaluation of the composites was determined using MSCs and human fibroblasts (HFFs). The cells seeded directly onto the composites displayed increasing metabolic activities over time, with ScCM-NP-H groups having maximum activity. The cells treated in vitro with 5% and 10% extracts of ScCM-NP-H and SfCM-NP-H exhibited a dose- and duration-dependent response. Cell activities reduced considerably for all groups, except 10% ScCM-NP-H, which displayed a significant increase over time. CONCLUSION: We observed that sustained release of MSC-CM is required to prevent dose-dependent cytotoxicity. The proposed nanocomposite hydrogel for MSC-CM delivery can open up a new array for its clinical application.

Human bone marrow-derived mesenchymal stromal cells in combination with silymarin regulate hepatocyte growth factor expression and genotoxicity in carbon tetrachloride induced hepatotoxicity in Wistar rats.

BACKGROUND: To evaluate the antimutagenic potential of combination treatment of human bone marrow-derived mesenchymal stromal cells (BM-MSCs) and silymarin and its effect on hepatocyte growth factor levels in CCl4 induced hepatotoxicity in Wistar rats. METHODS: Hepatotoxicity was induced in adult female Wistar rats using carbon tetrachloride (CCl4 ). Thirty-six rats were randomly divided into six groups with six rats in each group: Group 1 (normal control group), Group 2 (received only CCl 4 ), Group 3 (CCl 4 +low dose BM-MSCs), Group 4 (CCl 4 +high dose BM-MSCs), Group 5 (CCl 4 + silymarin), Group 6 (CCl 4 +silymarin+high dose BM-MSCs). Thirty days after the treatment, blood samples were collected for hepatocyte growth factor estimation. The rats were then killed, bone marrow was extracted for chromosomal aberration assay. Liver tissue was processed for evaluating the DNA fragmentation assay, histopathology, and scanning electron microscopy study. RESULTS: Combination treatment of silymarin and high dose BM-MSCs significantly (P < 0.05) restored the plasma hepatocyte growth factor levels which were comparable with normal levels and exhibited significant antimutagenic and antiapoptotic activity by decreasing the frequency of structural chromosomal aberrations and suppressing the DNA fragmentation in liver tissue samples. The combination treatment produced significant hepatoprotective effect which was supported by histopathology and scanning electron microscopy study. CONCLUSION: Results indicate that the treatment of BM-MSCs in combination with silymarin had a better hepatoprotective and antimutagenic effect and represents a novel strategy for the treatment of hepatotoxicity.

Haemostatic potential of human bone marrow-derived mesenchymal stromal cells in Wistar rats with carbon tetrachloride induced liver cirrhosis.

BACKGROUND: To evaluate the haemostatic potential of human bone marrow-derived mesenchymal stromal cells (BM-MSCs) in carbon tetrachloride (CCl4) induced liver cirrhosis in Wistar rats. METHODS: This was an experimental study. Liver cirrhosis was induced in adult female Wistar rats using CCl4. Rats were randomly divided into 6 groups with ten rats in each group: group 1 (normal control group), group 2 (received only CCl4), group 3 (CCl4 + low dose BM-MSCs), group 4 (CCl4 + high dose BM-MSCs), group 5 (CCl4 + silymarin), group 6 (CCl4 + high dose BM-MSCs + silymarin). Thirty days after the treatment, blood samples were collected for liver enzyme level analysis, prothrombin time test and plasma fibrinogen estimations. The rats were then sacrificed, livers were excised and used for histopathological and scanning electron microscopy (SEM) study. RESULTS: BM-MSCs and the combination treatment of high dose BM-MSCs and silymarin effectively decreased the prothrombin time and increased plasma fibrinogen concentration in rats with CCl4 induced liver cirrhosis. BM-MSCs treatment produces significant anti-fibrotic effect which was supported by the liver enzyme level analysis, histopathology and SEM study. CONCLUSIONS: Results indicate that treatment of BM-MSCs in combination with silymarin had a better haemostatic effect when compared to the administration of BM-MSCs alone.

Safety Assessment of Human Bone Marrow-derived Mesenchymal Stromal Cells Transplantation in Wistar Rats.

INTRODUCTION: Bone Marrow-derived Mesenchymal Stromal Cells (BM-MSCs) are multipotent stem cells isolated from adult human bone marrow. Properties of MSCs make them potentially ideal candidates for regenerative medicine. The preclinical data available in the literature regarding the safety assessment of MSCs at different dosage group is scanty. AIM: To evaluate the safety of BM-MSCs transplantation in Wistar rats. MATERIALS AND METHODS: Eighteen adult female Wistar rats were used in the study. They were randomly divided into normal control, low dose MSCs and high dose MSCs groups. Low dose group received 3.25 million BM-MSCs/kg body weight; high dose group received 9.75 million BM-MSCs/kg body weight intravenously. Body weight, food and water intake of each rat were measured statistically using SPSS version 16.0; animals were observed for changes in behaviour, general clinical signs, presence of any abnormal response, mortality for thirty days. RESULTS: Repeated measures ANOVA indicated a significant increase in body weight, food, and water intake of all animals at all weeks of the study period compared to week zero (p<0.05). Between the low dose and high dose MSCs group, increase in absolute body weight was seen at the end of the 30th day which was statistically significant (p=0.01). There was no significant difference in body weights, food and water intake in MSCs group when compared to normal control. All the animals survived for the entire duration of the study. Further, there was no change in the behaviour of the animals, no adverse clinical signs or complications following the MSCs treatment. CONCLUSION: Results indicate that administration of BM-MSCs is safe when given by a slow intravenous infusion as it did not alter the food and water intake behaviour of the animals and did not have any negative effect on its body weight.

Administration of Adult Human Bone Marrow-Derived, Cultured, Pooled, Allogeneic Mesenchymal Stromal Cells in Critical Limb Ischemia Due to Buerger's Disease: Phase II Study Report Suggests Clinical Efficacy.

Critical limb ischemia (CLI) due to Buerger's disease is a major unmet medical need with a high incidence of morbidity. This phase II, prospective, nonrandomized, open-label, multicentric, dose-ranging study was conducted to assess the efficacy and safety of i.m. injection of adult human bone marrow-derived, cultured, pooled, allogeneic mesenchymal stromal cells (BMMSC) in CLI due to Buerger's disease. Patients were allocated to three groups: 1 and 2 million cells/kg body weight (36 patients each) and standard of care (SOC) (18 patients). BMMSCs were administered as 40-60 injections in the calf muscle and locally, around the ulcer. Most patients were young (age range, 38-42 years) and ex-smokers, and all patients had at least one ulcer. Both the primary endpoints-reduction in rest pain (0.3 units per month [SE, 0.13]) and healing of ulcers (11% decrease in size per month [SE, 0.05])-were significantly better in the group receiving 2 million cells/kg body weight than in the SOC arm. Improvement in secondary endpoints, such as ankle brachial pressure index (0.03 [SE, 0.01] unit increase per month) and total walking distance (1.03 [SE, 0.02] times higher per month), were also significant in the group receiving 2 million cells/kg as compared with the SOC arm. Adverse events reported were remotely related or unrelated to BMMSCs. In conclusion, i.m. administration of BMMSC at a dose of 2 million cells/kg showed clinical benefit and may be the best regimen in patients with CLI due to Buerger's disease. However, further randomized controlled trials are required to confirm the most appropriate dose. Stem Cells Translational Medicine 2017;6:689-699.

Large-scale expansion of pre-isolated bone marrow mesenchymal stromal cells in serum-free conditions.

The regenerative potential of mesenchymal stromal or stem cells (MSCs) has generated tremendous interest for treating various degenerative diseases. Regulatory preference is to use a culture medium that is devoid of bovine components for stem cell expansion intended for therapeutic applications. However, a clear choice an alternative to fetal bovine serum (FBS) has not yet emerged. We have screened five different commercially available serum-free media (SFM) for their ability to support the growth and expansion of pre-isolated undifferentiated bone marrow-derived MSCs (BM-MSCs) and compared the results with cells grown in standard FBS-containing medium as control. In addition, based on initial screening results, BD Mosaic™ Mesenchymal Stem Cell Serum-free (BD-SFM) medium was evaluated in large-scale cultures for the performance and culture characteristics of BM-MSCs. Of the five different serum-free media, BD-SFM enhanced BM-MSCs growth and expansion in Cell STACK (CS), but the cell yield per CS-10 was less when compared to the control medium. The characteristics of MSCs were measured in terms of population doubling time (PDT), cell yield and expression of MSC-specific markers. Significant differences were observed between BD-SFM and control medium in terms of population doublings (PDs), cell yield, CFU-F and morphological features, whereas surface phenotype and differentiation potentials were comparable. The BD-SFM-cultured MSCs were also found to retain the differentiation potential, immune-privileged status and immunosuppressive properties inherent to MSCs. Our results suggest that BD-SFM supports large-scale expansion of BM-MSCs for therapeutic use.

Isolation, expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) heralded a new beginning for regenerative medicine and generated tremendous interest as the most promising source for therapeutic application. Most cell therapies require stringent regulatory compliance and prefer the use of serum-free media (SFM) or xeno-free media (XFM) for the MSC production process, starting from the isolation onwards. Here, we report on serum-free isolation and expansion of MSCs and compare them with cells grown in conventional fetal bovine serum (FBS)-containing media as a control. The isolation, proliferation and morphology analysis demonstrated significant differences between MSCs cultured in various SFM/XFM in addition to their difference with FBS controls. BD Mosaic™ Mesenchymal Stem Cell Serum-Free media (BD-SFM) and Mesencult-XF (MSX) supported the isolation, sequential passaging, tri-lineage differentiation potential and acceptable surface marker expression profile of BM-MSCs. Further, MSCs cultured in SFM showed higher immune suppression and hypo-immunogenicity properties, making them an ideal candidate for allogeneic cell therapy. Although cells cultured in control media have a significantly higher proliferation rate, BM-MSCs cultured in BD-SFM or MSX media are the preferred choice to meet regulatory requirements as they do not contain bovine serum. While BM-MSCs cultured in BD-SFM and MSX media adhered to all MSC characteristics, in the case of few parameters, the performance of cells cultured in BD-SFM was superior to that of MSX media. Pre-clinical safety and efficiency studies are required before qualifying SFM or XFM media-derived MSCs for therapeutic applications.

Effect of bFGF on HLA-DR expression of human bone marrow-derived mesenchymal stem cells.

There has been a steady rise in the therapeutic applications of bone marrow mesenchymal stem cells (BM-MSCs) because of their unique properties of multilineage differentiation and immune modulation as well as the ease in isolation. However, up-regulation of surface HLA-DR levels when maintaining MSCs in culture under the influence of mitotic factors such as Basic fibroblast growth factor (bFGF) is an area of concern when considering them for the purpose of clinical applications. Thus, we investigated the association of bFGF supplemented to the culture media and the surface expression levels of HLA-DR in BM-MSCs in order to optimize the yield, while keeping HLA-DR levels under permissible levels. Human BM-MSCs were culture expanded in the absence of bFGF and in the presence of 1 ng/ml or 2 ng/ml bFGF. The HLA-DR profile of the cultures was analyzed at the end of each passage. On comparing the percent HLA-DR+ cell population at different concentrations as well as absence of bFGF, significant differences were not observed in the HLA-DR expression levels of the MSC cultures which had reached complete confluence. However, variations in HLA-DR expressions levels were seen which could be traced to the age of cells in culture with values drastically reduced to below 4% on maintaining MSCs typically two to three days beyond achieving full confluence. On the basis of the findings from this study, no significant correlation could be established on the effect of bFGF in modulating HLA-DR surface expression of BM-MSCs. Instead, the data are suggestive of the reasoning that the duration for which BM-MSCs are maintained in culture directly influences their phenotypic characteristics in terms of HLA-DR expression levels, with lowest levels achieved on their prolonged maintenance in culture.