Anjan Kumar Das

The current landscape of the mesenchymal stromal cell secretome: A new paradigm for cell-free regeneration.

The unique properties of mesenchymal stromal/stem cells (MSCs) to self-renew and their multipotentiality have rendered them attractive to researchers and clinicians. In addition to the differentiation potential, the broad repertoire of secreted trophic factors (cytokines) exhibiting diverse functions such as immunomodulation, anti-inflammatory activity, angiogenesis and anti-apoptotic, commonly referred to as the MSC secretome, has gained immense attention in the past few years. There is enough evidence to show that the one important pathway by which MSCs participate in tissue repair and regeneration is through its secretome. Concurrently, a large body of MSC research has focused on characterization of the MSC secretome; this includes both soluble factors and factors released in extracellular vesicles, for example, exosomes and microvesicles. This review provides an overview of our current understanding of the MSC secretome with respect to their potential clinical applications.

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)

Diverse effects of dimethyl sulfoxide (DMSO) on the differentiation potential of human embryonic stem cells

In vitro disease modeling using pluripotent stem cells can be a fast track screening tool for toxicological testing of candidate drug molecules. Dimethyl sulfoxide (DMSO) is one of the most commonly used solvents in drug screening. In the present investigation, we exposed 14- to 21-day-old embryoid bodies (EBs) to three different concentrations of DMSO [0.01% (low dose), 0.1% (medium dose) and 1.0% (high dose)] to identify the safest dose that could effectively be used as solvent. We found that DMSO treatment substantially altered the morphology and attachment of cells in concurrence with a significant reduction in cell viability in a dose-dependent manner. Gene expression studies revealed a selective downregulation of key markers associated with stemness (Oct-4, Sox-2, Nanog and Rex-1); ectoderm (Nestin, TuJ1, NEFH and Keratin-15); mesoderm (HAND-1, MEF-2C, GATA-4 and cardiac-actin); and endoderm (SOX-17, HNF-3β, GATA-6 and albumin), indicating an aberrant and untimely differentiation trajectory. Furthermore, immunocytochemistry, flow cytometry and histological analyses demonstrated substantial decrease in the levels of albumin and CK-18 proteins coupled with a massive reduction in the number of cells positive for PAS staining, implicating reduced deposits of glycogen. Our study advocates for the first time that DMSO exposure not only affects the phenotypic characteristics but also induces significant alteration in gene expression, protein content and functionality of the differentiated hepatic cells. Overall, our experiments warrant that hESC-based assays can provide timely alerts about the outcome of widespread applications of DMSO as drug solvent, cryoprotectant and differentiating agent.

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.

In vitro and in vivo neurogenic potential of mesenchymal stem cells isolated from different sources

Regenerative medicine is an evolving interdisciplinary topic of research involving numerous technological methods that utilize stem cells to repair damaged tissues. Particularly, mesenchymal stem cells (MSCs) are a great tool in regenerative medicine because of their lack of tumorogenicity, immunogenicity and ability to perform immunomodulatory as well as anti-inflammatory functions. Numerous studies have investigated the role of MSCs in tissue repair and modulation of allogeneic immune responses. MSCs derived from different sources hold unique regenerative potential as they are self-renewing and can differentiate into chondrocytes, osteoblasts, adipocytes, cardiomyocytes, hepatocytes, endothelial and neuronal cells, among which neuronal-like cells have gained special interest. MSCs also have the ability to secrete multiple bioactive molecules capable of stimulating recovery of injured cells and inhibiting inflammation. In this review we focus on neural differentiation potential of MSCs isolated from different sources and how certain growth factors/small molecules can be used to derive neuronal phenotypes from MSCs. We also discuss the efficacy of MSCs when transplanted in vivo and how they can generate certain neurons and lead to relief or recovery of the diseased condition. Furthermore, we have tried to evaluate the appropriate merits of different sources of MSCs with respect to their propensity towards neurological differentiation as well as their effectiveness in preclinical studies.

Co-culture of mesenchymal-like stromal cells derived from human foreskin permits long term propagation and differentiation of human embryonic stem cells.

Among the different parameters governing the successful derivation and expansion of human embryonic stem cells (hESC), feeder layers play the most important role. Human feeders in form of human mesenchymal stromal cells (hMSCs) and human foreskin fibroblasts (HFFs) lay the foundation for eradication of animal‐derived hESC culture system. In this study we explored the potential of human foreskin derived mesenchymal like stromal cells (HF‐MSCs) to support self renewal and pluripotency of hESC. The MSCs isolated from human foreskin were found to be resistant to standard concentrations and duration of mitomycin‐C treatment. Growth pattern, gene profiling (Oct‐4, Nanog, Sox‐2, Rex‐1), cytoskeletal protein expression (vimentin, nestin) and tri‐lineage differentiation potential into adipocytes, chondrocytes and osteocytes confirmed their mesenchymal stromal cell status. Further, the HF‐MSCs were positive for CD105, CD166, CD73, CD44, CD90, SSEA‐4, and negative for CD34, CD45, HLA‐DR cell‐surface markers and were found to exhibit BM‐MSC‐like characteristics. hESC lines co‐cultured with HF‐MSC feeders showed expression of expected pluripotent transcription factors Oct‐4, Nanog, Sox‐2, GDF‐3, Rex‐1, STELLAR, ABCG2, Dppa5, hTERT; surface markers SSEA‐4, TRA‐1‐81 and maintained their cytogenetic stability during long term passaging. These novel feeders also improved the formation of embryoid bodies (EBs) from hESC which produced cell types representing three germ layers. This culture system has the potential to aid the development of clinical‐grade hESCs for regenerative medicine and drug screening. Further, we envisage foreskin can serve as a valuable source of alternative MSCs for specific therapeutic applications. J. Cell. Biochem. 112: 1353–1363, 2011.

Induced pluripotent stem cells (iPSCs): the emergence of a new champion in stem cell technology‐driven biomedical applications

Pluripotent stem cells possess the unique property of differentiating into all other cell types of the human body. Further, the discovery of induced pluripotent stem cells (iPSCs) in 2006 has opened up new avenues in clinical medicine. In simple language, iPSCs are nothing but somatic cells reprogrammed genetically to exhibit pluripotent characteristics. This process utilizes retroviruses/lentiviruses/adenovirus/plasmids to incorporate candidate genes into somatic cells isolated from any part of the human body. It is also possible to develop disease‐specific iPSCs which are most likely to revolutionize research in respect to the pathophysiology of most debilitating diseases, as these can be mimicked ex vivo in the laboratory. These models can also be used to study the safety and efficacy of known drugs or potential drug candidates for a particular diseased condition, limiting the need for animal studies and considerably reducing the time and money required to develop new drugs. Recently, functional neurons, cardiomyocytes, pancreatic islet cells, hepatocytes and retinal cells have been derived from human iPSCs, thus re‐confirming the pluripotency and differentiation capacity of these cells. These findings further open up the possibility of using iPSCs in cell replacement therapy for various degenerative disorders. In this review we highlight the development of iPSCs by different methods, their biological characteristics and their prospective applications in regenerative medicine and drug screening. We further discuss some practical limitations pertaining to this technology and how they can be averted for the betterment of human life. Copyright

Stem Cells in Stroke Repair: Current Success & Future Prospects

Stroke causes a devastating insult to the brain resulting in severe neurological deficits because of a massive loss of different neurons and glia. In the United States, stroke is the third leading cause of death. Stroke remains a significant clinical unmet condition, with only 3% of the ischemic patient population benefiting from current treatment modalities, such as the use of thrombolytic agents, which are often limited by a narrow therapeutic time window. However, regeneration of the brain after ischemic damage is still active days and even weeks after stroke occurs, which might provide a second window for treatment. Neurorestorative processes like neurogenesis, angiogenesis and synaptic plasticity lead to functional improvement after stroke. Stem cells derived from various tissues have the potential to perform all of the aforementioned processes, thus facilitating functional recovery. Indeed, transplantation of stem cells or their derivatives in animal models of cerebral ischemia can improve function by replacing the lost neurons and glial cells and by mediating remyelination, and modulation of inflammation as confirmed by various studies worldwide. While initially stem cells seemed to work by a ‘cell replacement’ mechanism, recent research suggests that cell therapy works mostly by providing trophic support to the injured tissue and brain, fostering both neurogenesis and angiogenesis. Moreover, ongoing human trials have encouraged hopes for this new method of restorative therapy after stroke. This review describes up-to-date progress in cell-based therapy for the treatment of stroke. Further, as we discuss here, significant hurdles remain to be addressed before these findings can be responsibly translated to novel therapies. In particular, we need a better understanding of the mechanisms of action of stem cells after transplantation, the therapeutic time window for cell transplantation, the optimal route of cell delivery to the ischemic brain, the most suitable cell types and sources and learn how to control stem cell proliferation, survival, migration, and differentiation in the pathological environment. An integrated approach of cell-based therapy with early-phase clinical trials and continued preclinical work with focus on mechanisms of action is needed.

Comparative analysis of cardiomyocyte differentiation from human embryonic stem cells under 3-D and 2-D culture conditions

Post-myocardial infarction cardiomyocytes are the most important target cell types for cardiac repair. Many of the applications envisaged for human embryonic stem cells (hESC)-derived cardiomyocytes demand that the differentiation procedure be robust, cost effective and high yielding. Various lines of evidence including our earlier study suggest that hESCs have distinct preferences to become heart cells. However, a direct comparison between different protocols has not yet been reported to date. Here, we performed a logical and systematic comparison of cardiomyocytes obtained from hESCs via embryoid bodies (EBs) in suspension versus adherent static cultures of feeder-free hES colonies representing three-dimensional (3-D) and two-dimensional (2-D) culture systems, respectively. An in-depth characterization of the beating cells revealed appropriate cardiac marker expression both at gene and protein levels. Despite using similar media, 3-D and 2-D cultures showed significant variation in growth and ability to form beating areas. While the expression of pre-cardiac mesoderm markers like GATA-4, HAND1, Myf5, Msx1, and BMP-IIR remained unaltered; levels of functional heart-specific markers such as MLC-2A/2V, cTnT, ANP, Phospholamban, α-MHC and KV4.3 were substantially up-regulated in 3-D compared to 2-D cultures. Concurrently we observed a sharp decline in the expression of ESC, ectoderm and endoderm markers including Oct-4, Sox-2, NFH, Sox-1, Sox-17 and AFP. Further immunocytochemistry and flow cytometry demonstrated a higher percentage of cells positive for Brachyury, desmin and cardiac troponin in 3-D cultures. Our results underscore the higher efficiency of cardiomyocytes derived via 3-D cultures. This finding enriches our basic understanding of the differentiation pattern in hESC-derived cardiomyocytes.

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.