Charan Thej

Are serum-free and xeno-free culture conditions ideal for large scale clinical grade expansion of Wharton’s jelly derived mesenchymal stem cells? A comparative study

IntroductionMesenchymal stromal/stem cells (MSCs) for clinical use have largely been isolated from the bone marrow, although isolation of these cells from many different adult and fetal tissues has been reported as well. One such source of MSCs is the Whartons Jelly (WJ) of the umbilical cord, as it provides an inexhaustible source of stem cells for potential therapeutic use. Isolation of MSCs from the umbilical cord also presents little, if any, ethical concerns, and the process of obtaining the cord tissue is relatively simple with appropriate consent from the donor. However, a great majority of studies rely on the use of bovine serum containing medium for isolation and expansion of these cells, and porcine derived trypsin for dissociating the cells during passages, which may pose potential risks for using these cells in clinical applications. It is therefore of high priority to develop a robust production process by optimizing culture variables to efficiently and consistently generate MSCs that retain desired regenerative and differentiation properties while minimizing risk of disease transmission.MethodsWe have established a complete xeno-free, serum-free culture condition for isolation, expansion and characterization of WJ-MSCs, to eliminate the use of animal components right from initiation of explant culture to clinical scale expansion and cryopreservation. Growth kinetics, in vitro differentiation capacities, immunosuppressive potential and immunophenotypic characterization of the cells expanded in serum-free media have been compared against those cultured under standard fetal bovine serum (FBS) containing medium. We have also compared the colony-forming frequency and genomic stability of the large scale expanded cells. Secretome analysis was performed to compare the angiogenic cytokines and functional angiogenic potency was proved by Matrigel assays.ResultsResults presented in this report identify one such serum-free, xeno-free medium for WJ expansion. Cells cultured in serum-free, xeno-free medium exhibit superior growth kinetics and functional angiogenesis, alongside other MSC characteristics.ConclusionsWe report here that WJ-MSCs cultured and expanded in Mesencult XF, SF Medium retain all necessary characteristics attributed to MSC for potential therapeutic use.

Higher propensity of Wharton's jelly derived mesenchymal stromal cells towards neuronal lineage in comparison to those derived from adipose and bone marrow.

Mesenchymal stromal cells (MSCs) derived from different tissue sources are capable of differentiating into neural and glial cell types. However, the efficiency of differentiation varies between MSCs derived from different tissues. We compared the efficiency of neural progenitor population generation between adipose (AD), bone marrow (BM) and Whartons jelly (WJ) derived MSCs. MSCs isolated from the three sources were induced to form primary neurospheres using epidermal growth factor (20 ng/mL) and bFGF (20 ng/mL). The self‐renewal potential of the primary neurospheres was assessed by secondary neurosphere assay. Primary neurospheres were differentiated to neuronal lineage on fibronectin‐coated dishes. The neurospheres and the resulting differentiated cells were characterized by immunocytochemistry and the RT‐PCR analyses. We have also investigated the secretome profile of neuronal‐related growth factors using Ray biotech cytokine array. The results show that MSCs from the three sources can be induced to generate neurospheres and they expressed neural progenitor markers nestin, Sox2 and Pax6 transcription factors. When differentiated on fibronectin coated dishes in mitogen free culture conditions, the primary spheres from all three sources were able to generate neuron/glial ‐ like cells which expressed Nfl, Map2 and GFAP with varied efficiency. Self‐renewal potential of these progenitors was determined by secondary sphere formation. WJ‐ and BM‐derived neurospheres were able to self‐renew, while AD derived progenitors failed to do so. Comparison of the secretome profile suggested that WJ derived MSCs secrete more neurotrophic factors. The data suggest that human WJ derived MSCs can be induced to make neural progenitors with higher efficiency compared to BM and AD derived MSCs.

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

Mesenchymal Stromal Cells: Emerging Treatment Option for Diabetic Wounds

The overwhelming rise in the incidence of diabetes worldwide has become a huge concern for both healthcare professionals of different specialties as well as for the patients. Long-term diabetic patients may develop diabetic foot ulcers which are often difficult to heal and can lead to limb amputation. Mesenchymal stromal cell (MSC) derived from various tissue sources has shown significant promise in cutaneous wound healing including diabetic foot ulcer (DFU). The mechanism of wound healing by MSC has largely been attributed to various cytokines and growth factors secreted by these cells delivered in the vicinity of the wounds. We have recently reported on the development and characterization of an allogeneic, pooled bone marrow-derived MSC product, Stempeucel®, which is safe and efficacious to administer in humans. Here, we present evidence that these cells produce several cytokines/growth factors that are important in healing diabetic wounds. More importantly, we demonstrate that administration of the pooled cells significantly enhances wound closure in diabetic mice and may be a prospective cell therapy product to initiate clinical trial in humans.