Anish Sen Majumdar

A double blind randomized placebo controlled phase I/II study assessing the safety and efficacy of allogeneic bone marrow derived mesenchymal stem cell in critical limb ischemia

BackgroundPeripheral vascular disease of the lower extremities comprises a clinical spectrum that extends from no symptoms to presentation with critical limb ischemia (CLI). Bone marrow derived Mesenchymal Stem Cells (BM- MSCs) may ameliorate the consequences of CLI due to their combinatorial potential for inducing angiogenesis and immunomodulatory environment in situ. The primary objective was to determine the safety of BM- MSCs in patients with CLI.MethodsProspective, double blind randomized placebo controlled multi-center study was conducted in patients with established CLI as per Rutherford classification in category II-4, III-5, or III-6 with infra-inguinal arterial occlusive disease and were not suitable for or had failed revascularization treatment. The primary end point was incidence of treatment – related adverse events (AE). Exploratory efficacy end points were improvement in rest pain, increase in Ankle Brachial Pressure Index (ABPI), ankle pressure, healing of ulcers, and amputation rates. Twenty patients (BM-MSC: Placebo = 1:1) were administered with allogeneic BM-MSCs at a dose of 2 million cells/kg or placebo (PlasmaLyte A) at the gastrocnemius muscle of the ischemic limb.ResultsImprovement was observed in the rest pain scores in both the arms. Significant increase in ABPI and ankle pressure was seen in BM-MSC arm compared to the placebo group. Incidence of AEs in the BM-MSC arm was 13 vs. 45 in the placebo arm where as serious adverse events (SAE) were similar in both the arms (5 in BM-MSC and 4 in the placebo group). SAEs resulted in death, infected gangrene, amputations in these patients. It was observed that the SAEs were related to disease progression and not related to stem cells.ConclusionBM-MSCs are safe when injected IM at a dose of 2 million cells/kg body weight. Few efficacy parameters such as ABPI and ankle pressure showed positive trend warranting further studies.Trial registrationNIH website (http://www.clinicaltrials.gov/ct2/show/NCT00883870)

Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation

The clinical application of human bone marrow derived multipotent mesenchymal stromal cells (MSC) requires expansion, cryopreservation, and transportation from the laboratory to the site of cell implantation. The cryopreservation and thawing process of MSCs may have important effects on the viability, growth characteristics and functionality of these cells both in vitro and in vivo. More importantly, MSCs after two rounds of cryopreservation have not been as well characterized as fresh MSCs from the transplantation perspective. The objective of this study was to determine if the effect of successive cryopreservation of pooled MSCs during the exponential growth phase could impair their morphology, phenotype, gene expression, and differentiation capabilities. MSCs cryopreserved at passage 3 (cell bank) were thawed and expanded up to passage 4 and cryopreserved for the second time. These cells (passive) were then thawed and cultured up to passage 6, and, at each passage MSCs were characterized. As control, pooled passage 3 cells (active) after one round of cryopreservation were taken all the way to passage 6 without cryopreservation. We determined the growth rate of MSCs for both culture conditions in terms of population doubling number (PDN) and population doubling time (PDT). Gene expression profiles for pluripotency markers and tissue specific markers corresponding to neuroectoderm, mesoderm and endoderm lineages were also analyzed for active and passive cultures of MSC. The results show that in both culture conditions, MSCs exhibited similar growth properties, phenotypes and gene expression patterns as well as similar differentiation potential to osteo‐, chondro‐, and adipo‐lineages in vitro. To conclude, it appears that successive or multiple rounds of cryopreservation of MSCs did not alter the fundamental characteristics of these cells and may be used for clinical therapy. J. Cell. Biochem. 113: 3153–3164, 2012.

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.

Explant culture: a simple, reproducible, efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate.

Adipose tissue has emerged as a preferred source of mesenchymal stem/stromal cells (MSC), due to its easy accessibility and high MSC content. The conventional method of isolation of adipose tissue‐derived stromal cells (ASC) involves enzymatic digestion and centrifugation, which is a costly and time‐consuming process. Mechanical stress during isolation, use of bacterial‐derived products and potential contamination with endotoxins and xenoantigens are other disadvantages of this method. In this study, we propose explant culture as a simple and efficient process to isolate ASC from human adipose tissue. This technique can be used to reproducibly isolate ASC from fat tissue obtained by liposuction as well as surgical resection, and yields an enriched ASC population free from contaminating haematopoietic cells. We show that explanting adipose tissue results in a substantially higher yield of ASC at P0 per gram of initial fat tissue processed, as compared to that obtained by enzymatic digestion. We demonstrate that ASC isolated by explant culture are phenotypically and functionally equivalent to those obtained by enzymatic digestion. Further, the explant‐derived ASC share the immune privileged status and immunosuppressive properties implicit to MSC, suggesting that they are competent to be tested and applied in allogeneic clinical settings. As explant culture is a simple, inexpensive and gentle method, it may be preferred over the enzymatic technique for obtaining adipose tissue‐derived stem/stromal cells for tissue engineering and regenerative medicine, especially in cases of limited starting material. Copyright

Comparison of chemokine and receptor gene expression between Wharton ' s jelly and bone marrow-derived mesenchymal stromal cells

BACKGROUND AIMS Because of their multilineage differentiation capacity, immunomodulatory role and homing ability, mesenchymal stromal cells (MSC) are emerging as a new therapeutic strategy for treating a variety of disorders. Although bone marrow (BM) is the best characterized source of MSC, Whartons jelly (WJ) of the umbilical cord holds great promise as an alternative. As delivery direct to the site of injury is not always feasible, efficient homing of MSC to the site of injury is critical for inducing tissue repair and regeneration. MSC express a wide variety of growth factors, chemokines and receptors that are important for cell migration, homing and re-establishment of blood supply for recovery of damaged tissues. METHODS Detailed chemokine and receptor gene expression profiles of WJ MSC were established, and subsequently compared with those of BM-derived MSC using a polymerase chain reaction (PCR) array. Secretion of growth factors was analyzed and evaluated using culture supernatant from WJ and BM MSC. RESULTS Our results revealed a differential expression pattern of the chemokines and their receptors between WJ- and BM-derived MSC. Several Glutamic acid-Leucine-Arginine; ELR-positive CXC chemokine genes and secretion of growth factors, which promote angiogenesis, were found to be up-regulated in WJ MSC. CONCLUSIONS To understand better the localization and mechanism of tissue repair by transplanted WJ MSC, we attempted chemokine and their receptor transcription profiling, followed by analysis of growth factors secreted by WJ MSC, and compared them against those of BM MSC. The data suggest that MSC from different sources can be explored for distinct therapeutic roles.

Impact of passing mesenchymal stem cells through smaller bore size needles for subsequent use in patients for clinical or cosmetic indications.

BackgroundNumerous preclinical and clinical studies have investigated the regenerative potential and the trophic support of mesenchymal stem cells (MSCs) following their injection into a target organ. Clinicians favor the use of smallest bore needles possible for delivering MSCs into vascular organs like heart, liver and spleen. There has been a concern that small needle bore sizes may be detrimental to the health of these cells and reduce the survival and plasticity of MSCs.MethodsIn this report, we aimed to investigate the smallest possible bore size needle which would support the safe delivery of MSCs into various tissues for different clinical or cosmetic applications. To accomplish this we injected cells via needle sizes 24, 25 and 26 G attached to 1 ml syringe in the laboratory and collected the cells aseptically. Control cells were ejected via 1 ml syringe without any needle. Thereafter, the needle ejected cells were cultured and characterized for their morphology, attachment, viability, phenotypic expression, differentiation potential, cryopreservation and in vivo migration abilities. In the second phase of the study, cells were injected via 26 G needle attached to 1 ml syringe for 10 times.ResultsSimilar phenotypic and functional characteristics were observed between ejected and control group of cells. MSCs maintained their cellular and functional properties after single and multiple injections.ConclusionsThis study proves that 26 G bore size needles can be safely used to inject MSCs for clinical/therapeutics purposes.

Isolation, characterization, and gene expression analysis of Wharton's jelly-derived mesenchymal stem cells under xeno-free culture conditions.

Mesenchymal stem cells (MSCs) have become an attractive tool for tissue engineering and targets in clinical transplantation due to their regeneration potential and immuno-suppressive capacity. Although MSCs derived from bone marrow are the most widely used, their harvest requires an invasive procedure. The umbilical cord, which is discarded at birth, can provide an inexhaustible source of stem cells for therapy. The Wharton’s jelly-derived MSCs (WJ-MSCs), from the umbilical cord, have been shown to have faster proliferation rates and greater expansion capability compared with adult MSCs. The standard isolation and in vitro culture protocol for WJ-MSCs utilizes fetal bovine serum (FBS) or calf serum as a nutrient supplement. However, FBS raises potential safety concerns such as transmission of viral/prion disease and may initiate xenogeneic immune reactions against bovine antigens. Therefore, for therapeutic applications, there is an urgent requirement to establish an alternative nutrient supplement which would favor cell proliferation, retain MSC characteristics, and prove safe in human subjects. In the present study, we isolated and expanded WJ-MSCs in 5% pooled, allogeneic human serum (HS) supplemented with 2 ng/mL of basic fibroblast growth factor. For cell dissociation, porcine trypsin was replaced with TrypLE, a recombinant enzyme, and a protease-free protocol was adapted for isolation of MSCs from WJ. We determined their growth kinetics, in vitro differentiation potential, surface marker expression, and colony-forming unit potential and compared them against standard WJ-MSC cultures expanded in 10% FBS. All these parameters matched quite well between the two MSC populations. To test whether there is any alteration in gene expression on switching from FBS to HS, we analyzed a panel of stem cell and early lineage markers using Taqman® low density array. No significant deviation in gene expression was observed between the two populations. Thus we established an efficient, complete xeno-free protocol for propagation of human WJ-MSCs.

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. Copyright

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