An insight into non-integrative gene delivery approaches to generate transgene-free induced pluripotent stem cells.

Over a decade ago, a landmark study that reported derivation of induced Pluripotent Stem Cells (iPSCs) by reprogramming fibroblasts has transformed stem cell research attracting the interest of the scientific community worldwide. These cells circumvent the ethical and immunological concerns associated with embryonic stem cells, and the limited self-renewal ability and restricted differentiation potential linked to adult stem cells. iPSCs hold great potential for understanding basic human biology, in vitro disease modeling, high-throughput drug testing and discovery, and personalized regenerative medicine. The conventional reprogramming methods involving retro- and lenti-viral vectors to deliver reprogramming factors in somatic cells to generate iPSCs nullify the clinical applicability of these cells. Although these gene delivery systems are efficient and robust, they carry an enormous risk of permanent genetic modifications and are potentially tumorigenic. To evade these safety concerns and derive iPSCs for human therapy, tremendous technological advancements have resulted in the development of non-integrating viral- and non-viral approaches. These gene delivery techniques curtail or eliminate the risk of any genomic alteration and enhance the prospects of iPSCs from bench-to-bedside. The present review provides a comprehensive overview of non-integrating viral (adenoviral vectors, adeno-associated viral vectors, and Sendai virus vectors) and DNA-based, non-viral (plasmid transfection, minicircle vectors, transposon vectors, episomal vectors, and liposomal magnetofection) approaches that have the potential to generate transgene-free iPSCs. The understanding of these techniques could pave the way for the use of iPSCs for various biomedical applications.

An Insight into DNA-free Reprogramming Approaches to Generate Integration-free Induced Pluripotent Stem Cells for Prospective Biomedical Applications.

More than a decade ago, a pioneering study reported generation of induced Pluripotent Stem Cells (iPSCs) by ectopic expression of a cocktail of reprogramming factors in fibroblasts. This study has revolutionized stem cell research and has garnered immense interest from the scientific community globally. iPSCs hold tremendous potential for understanding human developmental biology, disease modeling, drug screening and discovery, and personalized cell-based therapeutic applications. The seminal study identified Oct4, Sox2, Klf4 and c-Myc as a potent combination of genes to induce reprogramming. Subsequently, various reprogramming factors were identified by numerous groups. Most of these studies have used integrating viral vectors to overexpress reprogramming factors in somatic cells to derive iPSCs. However, these techniques restrict the clinical applicability of these cells as they may alter the genome due to random viral integration resulting in insertional mutagenesis and tumorigenicity. To circumvent this issue, alternative integration-free reprogramming approaches are continuously developed that eliminate the risk of genomic modifications and improve the prospects of iPSCs from lab to clinic. These methods establish that integration of transgenes into the genome is not essential to induce pluripotency in somatic cells. This review provides a comprehensive overview of the most promising DNA-free reprogramming techniques that have the potential to derive integration-free iPSCs without genomic manipulation, such as sendai virus, recombinant proteins, microRNAs, synthetic messenger RNA and small molecules. The understanding of these approaches shall pave a way for the generation of clinical-grade iPSCs. Subsequently, these iPSCs can be differentiated into desired cell type(s) for various biomedical applications.

Prospective applications of induced pluripotent stem cells in military medicine.

Soldiers involved in combat operations worldwide may be subjected to a wide array of tissue-specific injuries of varying degrees, thereby undergoing complicated medical treatments and prolonged rehabilitations. In many cases involving inadequate recovery, soldiers are further mentally traumatized as they can no longer serve their beloved country. In addition, many severe injuries can lead to soldiers being incapacitated for life and unable to perform even the most basic day-to-day activities. Present therapy for combat injuries is majorly aimed at alleviating pain and limiting further tissue damage from secondary infections. Cell-based therapy using stem cells is a promising tissue regenerative source, which will help our soldiers to recuperate from the severe injuries, and in some cases, even continue their service for the country after complete recovery. In this context, we would like to discuss the yet fully untapped potential of induced pluripotent stem cells (iPSCs) in regenerative medicine on the battlefield. In this review, we shall try to explore the rationale behind the use of these cells for military medicine, as well as the conventional and novel approaches to produce them for therapeutic applications. We shall also attempt to elucidate the evolving trends of battlefield injuries throughout history and the ongoing research on regeneration of tissues of specific interest using iPSCs and their potential role in combat medicine in the future. Additionally, we shall also discuss the concept of stem cell bio-banking for military personnel as a personalized safeguard against crippling and traumatic combat injuries.

Small Molecule Inhibiting Nuclear Factor-kB Ameliorates Oxidative Stress and Suppresses Renal Inflammation in Early Stage of Alloxan-Induced Diabetic Nephropathy in Rat.

Diabetic nephropathy is one of the major microvascular complications of diabetes mellitus which ultimately gives rise to cardiovascular diseases. Prolonged hyperglycaemia and chronic renal inflammation are the two key players in the development and progression of diabetic nephropathy. Nuclear factor kB (NF-kB)-mediated inflammatory cascade is a strong contributor to the renovascular inflammation in diabetic nephropathy. Here, we studied the effects of piceatannol, a potent NF-kB inhibitor, on various oxidative stress markers and NF-kB dependent diabetic renoinflammatory cascades in rat induced by alloxan (ALX). Experimental diabetes was induced in male Wistar rats by a single intraperitoneal dose, 150 mg/kg body-weight (b.w.) of ALX. Diabetic rats were treated with Piceatannol (PCTNL) at a dose of 30 and 50 mg/kg b.w. After 14 days of oral treatment, PCTNL significantly restored blood sugar level, glomerular filtration rate, serum markers and plasma lipids. PCTNL administration also reversed the declined activity of cellular antioxidant machineries namely superoxide dismutase and glutathione and the elevated levels of malondialdehyde and nitric oxide. Moreover, piceatannol-treated groups showed marked inhibition of renal pro-inflammatory cytokines and NF-kB p65/p50 binding to DNA. Renal histopathological investigations also supported its ameliorative effects against diabetic kidney damage. Importantly, effects were more prominent at a dose of 50 mg/kg, and in terms of body-weight gain, PCTNL failed to effect significantly. However, overall findings clearly demonstrated that PCTNL provides remarkable renoprotection in diabetes by abrogating oxidative stress and NF-kB activation - and might be helpful in early stage of diabetic nephropathy.

Diosmin Modulates the NF-kB Signal Transduction Pathways and Downregulation of Various Oxidative Stress Markers in Alloxan-Induced Diabetic Nephropathy.

Hyperglycaemia-mediated oxidative stress plays an imperative role in the progression of diabetic nephropathy. NF-kB is an important transcription factor in eukaryotes which regulates a diverse array of cellular process, including inflammation, immunological response, apoptosis, growth and development. Increased expression of NF-kB plays a vital role in the pathogenesis of many inflammatory diseases including diabetic nephropathy. Hence, the present study was designed to explore the nephroprotective nature of diosmin by assessing the various biochemical parameters, markers of oxidative stress and proinflammatory cytokine levels in alloxan-induced diabetic Wistar rats. Type 2 diabetes was induced in Wistar rats by single intraperitoneal injection of alloxan (120 mg/kg body weight). Seventy-two hours after the conformation of diabetes (blood glucose level ≥ 250 mg/dl), the rats were segregated into four groups, each group having six animals. Diabetic rats were treated with diosmin at a dose of 50 mg and 100 mg/kg body weight respectively. After the 28th day of treatment, rats were sacrificed, blood serum, plasma and kidney tissue were collected for various biochemical analysis. Inflammatory cytokine levels were measured through ELISA kit. Diosmin treatment produces significant reduction in the blood glucose and plasma insulin level and increases the body weight when compared with diabetic rats. Elevated level of malondialdehyde (MDA) and decrease levels of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and nitric oxide (NO) were significantly restored after 28 days of diosmin treatment. Diosmin treatment group also restores the normal architecture of the kidney tissue which was confirmed by histopathological examination. Moreover, oral administration of diosmin shows a significant normalization in the level of NF-kB, proving its pivotal role in maintaining renal function. The above ameliorative effects were more pronounced with diosmin at a dose of 100 mg/kg body weight. The above results permit us to conclude that treatment with diosmin halts hyperglycaemia-mediated oxidative stress and decline in pro-inflammatory cytokines and thus has beneficial anti-diabetic activity.