Label extension, single-arm, phase III study shows efficacy and safety of stempeucel® in patients with critical limb ischemia due to atherosclerotic peripheral arterial disease.

BACKGROUND: Peripheral arterial disease (PAD) of lower extremities comprises a clinical spectrum that extends from asymptomatic patients to critical limb ischemia (CLI) patients. 10% to 40% of the patients are at the risk of primary amputation. This study was planned in "no-option" patients of CLI due to atherosclerotic PAD to assess the efficacy and safety of pooled, allogeneic, adult human bone marrow-derived mesenchymal stromal cells which is already approved for marketing in India for CLI due to Buerger's disease. METHODS: This was a single-arm, multi-centric, phase III study where mesenchymal stromal cells was injected as 2 million cells/kg body weight in the calf muscle and around the ulcer. Twenty-four patients of lower extremity CLI due to PAD with Rutherford III-5 or III-6 and ankle-brachial pressure index ≤ 0.6 and having have at least one ulcer with area between 0.5 and 10 cm2 were included in the study. These patients were evaluated over 12 months from drug administration. RESULTS: Over a period of 12 months, statistical significant reduction of rest pain and ulcer size along with improvement in ankle-brachial pressure index and ankle systolic was observed. The quality of life of patients improved together with increase in total walking distance and major amputation-free survival time. CONCLUSION: Mesenchymal stromal cells may be a feasible option to treat "no-option" patients with atherosclerotic PAD. Trial registration This study is registered prospectively in National Institutes of Health and Clinical Trials Registry-India (CTRI) website: CTRI/2018/06/014436. Registered 6th June 2018. http://ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=24050&EncHid=&userName=stempeutics .

Characteristics of Pooled Wharton's Jelly Mesenchymal Stromal Cells (WJ-MSCs) and their Potential Role in Rheumatoid Arthritis Treatment.

INTRODUCTION: Mesenchymal stromal cells (MSC) from Wharton's jelly of umbilical cord is primitive and serve as an inexhaustible source of stem cells with greater potential in clinics. The existence of heterogeneity among the donor MSCs makes it difficult to predict the properties and clinical outcome of WJ-MSCs. We developed a strategy to minimize the donor to donor heterogeneity and produce consistency in biological properties by pooling three individual donors WJ-MSCs. Further, evaluated the effectiveness of the pooled MSCs in regulating the disease severity of Rheumatoid arthritis (RA) in animal models. METHODS: WJ-MSCs were isolated from umbilical cord obtained from different donors, characterised and pooled based on the gender of baby. The biological properties of the pooled WJ-MSCs were compared to the individual WJ-MSCs. Further, the pooled WJ-MSCs were analysed for their safety profile in both in vitro and in vivo settings. The efficiency of pooled WJ-MSCs in regulating RA pathogenesis was also analysed in mice models of Collagen induced arthritis (CIA). RESULTS: We identified differences in proliferation capacity, pro inflammatory gene expression levels among individual WJ-MSCs isolated from different donors and the variation is also attributed to gender difference. WJ-MSCs pooled and cultured from different donor's exhibit all the MSC characteristics and exhibited superior immunosuppressive capabilities. In the in vivo toxicity study, pooled MSCs are found to be safe, and further in the RA preclinical studies, they were found to decrease the disease severity in these animals. CONCLUSIONS: Pooled WJ-MSCs reduces heterogeneity of individual donors and have superior immunosuppressive property. It is also effective in reducing the disease severity in the experimental animal models of RA.

Phase IV postmarketing surveillance study shows continued efficacy and safety of Stempeucel in patients with critical limb ischemia due to Buerger's disease.

Buerger's disease or thromboangiitis obliterans is a type of obstructive vascular diseases categorized as vasculitis and usually present in 95% of young smoker men. The main pathogenetic mechanism is interplay between immune system and inflammation. Earlier our phase II study has shown that Stempeucel is safe when injected at 2 million cells/kg body weight by virtue of its anti-inflammatory, immunomodulatory, and angiogenetic properties. The present study was conducted to further assess the safety and efficacy of Stempeucel in critical limb ischemia due to Buerger's disease after obtaining approval from Indian FDA based on the data generated in the phase II study. This is an open label, multicenteric phase IV PMS study conducted across India with experienced vascular surgeons. Fifty patients of critical limb ischemia due to Buerger's disease with Rutherford III-5 or III-6 were included in the study and each individual received a dose of 2 million cells/kg body weight of Stempeucel in the calf muscles and around the ulcer. These patients were evaluated over 12 months from drug administration. The present study showed the continued long term efficacy over a period of 12 months follow up in these patients corroborating the result obtained in the previous phase II studies. There was significant improvement in rest pain, ankle systolic pressure, and ankle brachial pressure index with accelerated ulcer healing. In conclusion, the present study shows that the intramuscular administration of Stempeucel continues to be safe, tolerable, and effective alternative treatment in patients with Buerger's disease.

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.

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.

Efficacy and safety of adult human bone marrow-derived, cultured, pooled, allogeneic mesenchymal stromal cells (Stempeucel®): preclinical and clinical trial in osteoarthritis of the knee joint.

BACKGROUND: Osteoarthritis (OA) is a common and debilitating chronic degenerative disease of the joints. Currently, cell-based therapy is being explored to address the repair of damaged articular cartilage in the knee joint. METHODS: The in vitro differentiation potential of adult human bone marrow-derived, cultured, pooled, allogeneic mesenchymal stromal cells (Stempeucel®) was determined by differentiating the cells toward the chondrogenic lineage and quantifying sulfated glycosaminoglycan (sGAG). The mono-iodoacetate (MIA)-induced preclinical model of OA has been used to demonstrate pain reduction and cartilage formation. In the clinical study, 60 OA patients were randomized to receive different doses of cells (25, 50, 75, or 150 million cells) or placebo. Stempeucel® was administered by intra-articular (IA) injection into the knee joint, followed by 2 ml hyaluronic acid (20 mg). Subjective evaluations-visual analog scale (VAS) for pain, intermittent and constant osteoarthritis pain (ICOAP), and Western Ontario and McMaster Universities Osteoarthritis (WOMAC-OA) index-were performed at baseline and at 1, 3, 6, and 12 months of follow-up. Magnetic resonance imaging of the knee was performed at baseline, and at 6 and 12 months follow-up for cartilage evaluation. RESULTS: Stempeucel® differentiated into the chondrogenic lineage in vitro with downregulation of Sox9 and upregulation of Col2A genes. Furthermore, Stempeucel® differentiated into chondrocytes and synthesized a significant amount of sGAG (30 ± 1.8 µg/µg GAG/DNA). In the preclinical model of OA, Stempeucel® reduced pain significantly and also repaired damaged articular cartilage in rats. In the clinical study, IA administration of Stempeucel® was safe, and a trend towards improvement was seen in the 25-million-cell dose group in all subjective parameters (VAS, ICOAP, andWOMAC-OA scores), although this was not statistically significant when compared to placebo. Adverse events were predominant in the higher dose groups (50, 75, and 150 million cells). Knee pain and swelling were the most common adverse events. The whole-organ magnetic resonance imaging score of the knee did not reveal any difference from baseline and the placebo group. CONCLUSION: Intra-articular administration of Stempeucel® is safe. A twenty-five-million-cell dose may be the most effective among the doses tested for pain reduction. Clinical studies with a larger patient population are required to demonstrate a robust therapeutic efficacy of Stempeucel® in OA. TRIAL REGISTRATION: Clinicaltrials.gov NCT01453738 . Registered 13 October 2011.

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.