Search

ObjectiveAssess the impact of introducing a consensus guideline incorporating an adapted Sepsis Risk Calculator (SRC) algorithm, in the management of early onset neonatal sepsis (EONS), on antibiotic usage and patient safety.DesignMulticentre prospective studySettingTen perinatal hospitals in Wales, UK.PatientsAll live births ≥34 weeks’ gestation over a 12-month period (April 2019–March 2020) compared with infants in the preceding 15-month period (January 2018–March 2019) as a baseline.MethodsThe consensus guideline was introduced in clinical practice on 1 April 2019. It incorporated a modified SRC algorithm, enhanced in-hospital surveillance, ongoing quality assurance, standardised staff training and parent education. The main outcome measure was antibiotic usage/1000 live births, balancing this with analysis of harm from delayed diagnosis and treatment, disease severity and readmissions from true sepsis. Outcome measures were analysed using statistical process control charts.Main outcome measuresProportion of antibiotic use in infants ≥34 weeks’ gestation.Results4304 (14.3%) of the 30 105 live-born infants received antibiotics in the baseline period compared with 1917 (7.7%) of 24 749 infants in the intervention period (45.5% mean reduction). All 19 infants with culture-positive sepsis in the postimplementation phase were identified and treated appropriately. There were no increases in sepsis-related neonatal unit admissions, disease morbidity and late readmissions.ConclusionsThis multicentre study provides evidence that a judicious adaptation of the SRC incorporating enhanced surveillance can be safely introduced in the National Health Service and is effective in reducing antibiotic use for EONS without increasing morbidity and mortality.

Background Age-related macular degeneration (AMD) is a result of degeneration/damage of the retinal pigment epithelium (RPE) while retinitis pigmentosa (RP), an inherited early-onset disease, results from premature loss of photoreceptors. A promising therapeutic approach for both is the replacement of lost/damaged cells with human induced pluripotent stem cell (hiPSC)-derived retinal cells. Methods The aim of this study was to investigate the in vivo functionality of RPE and photoreceptor progenitor (PRP) cells derived from a clinical-grade hiPSC line through a unified protocol. De novo-generated RPE and PRP were characterized extensively to validate their identity, purity, and potency. Results RPE expressed tight junction proteins, showed pigmentation and ciliation, and secreted polarization-related factors vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). PRP expressed neural retina proteins and cone and rod markers, and responded to KCl-induced polarization. Transcriptomic analysis demonstrated an increase in the expression of mature retinal tissue-specific genes coupled with concomitant downregulation of genes from undesired lineages. RPE transplantation rescued visual function in RCS rats shown via optokinetic tracking and photoreceptor rescue. PRP transplantation improved light perception in NOD.SCID-rd1 mice, and positive electroretinography signals indicated functional photoreceptor activity in the host’s outer nuclear layer. Graft survival and integration were confirmed using immunohistochemistry, and no animals showed teratoma formation or any kind of ectopic growth in the eye. Conclusions To our knowledge, this is the first demonstration of a unified, scalable, and GMP-adaptable protocol indicating strong animal efficacy and safety data with hiPSC-derived RPE and PRP cells. These findings provide robust proof-of-principle results for IND-enabling studies to test these potential regenerative cell therapies in patients.

Inherited retinal diseases (IRDs) are a diverse group of rare eye disorders, resulting in vision loss or blindness. The underlying reason is mutation in one or more than 250 different genes associated with the development and normal physiology of retina largely comprising of rod/cone photoreceptors and retinal pigment epithelium. Interestingly, the sub retinal region of an eye has been shown to be immune privileged, broadening the scope of cell-replacement therapies for patients suffering from retinal degeneration. Several groups around the globe, including ours, have demonstrated safety and efficacy in preclinical studies by employing various approaches of retinal cell therapy. This had largely been possible with the advent of induced pluripotent stem cells (iPSC)-reprogrammed from adult somatic cells, that serves as a starting material for generating retinal cells de novo. Here, we describe a detailed procedure for reprogramming peripheral blood mononuclear cells (PBMC) into iPSC using episomal vectors without any physical disruption in the host genome. The lines thus created were tested for sterility, cytogenetic stability, identity, absence of episomal plasmids and further authenticated for pluripotency and tri-lineage differentiation capacity by embryoid body formation and immunocytochemistry. We believe that this feeder-cell free, animal-product free and gene-insertion free protocol would help people to develop and bank patient-specific cell lines for autologous cell therapies for incurable rare diseases.

The lung is the most vulnerable target for the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, and respiratory failure causing acute respiratory distress syndrome is its foremost outcome. However, the current primary in vitro models in use for SARS-CoV-2 display apparent limitations for modeling such complex human respiratory disease. Although patient cells can directly model the effects of a drug, their availability and capacity for expansion are limited compared with transformed/immortalized cells or tumor-derived cell lines. An additional caveat is that the latter may harbor genetic and metabolic abnormalities making them unsuitable for drug screening. Therefore, it is important to create physiologically relevant human-cell models that can replicate the pathophysiology of SARS-CoV-2, thus facilitating drug testing. In this study, we show preliminary data on how human induced pluripotent stem cells-derived lung epithelial cell system could emerge as a relevant and sensitive platform for modeling SARS-CoV-2 infection and drug screening.

Human mesenchymal stromal cells (MSCs) are currently the leading candidate for cell-based therapeutics. While the use of MSCs in transplantation therapies is widely expanding, still, there is a lot of scope for better understanding of the mechanisms underlying their effects. We have generated MSCs from pre- and post-natal human tissue sources such as Wharton's jelly (WJ), stem cells from human exfoliated deciduous teeth (SHED), and bone marrow (BM). We then expanded, banked, and characterized them based on morphology, growth kinetics, senescence, immunophenotype, gene expression, and secretion of growth factors. Although the immunophenotype was very similar across MSCs from the three types of donor tissues, they showed minor variations in their growth kinetics. Further, a higher percentage of senescent cells were observed in BM-MSCs than in WJ-MSCs and SHED. Gene expression analysis showed the increased expression of INF-γ, PDGFA, VEGF, IL10, and SDF in SHED over WJ-MSC and BM-MSC. Comparative secretome profiling by ELISA demonstrated the presence of FGF-2, IL-10, PDGF, SDF-1, Ang-1, TGF-β3, HGF, INF-γ, VEGF, and IL-6 in cell culture supernatants. Based on our findings, WJ-MSC and SHED appear more potent than BM-MSC for managing inflammation, immunomodulation, angiogenesis, fibrosis, and scarring. Due to widespread application of MSCs in cell replacement therapy, these subtle differences need to be taken into consideration while designing stem cell-based clinical trials.

SUMMARY Primary cilia are sensory organelles that protrude from the cell membrane. Defects in the primary cilium cause ciliopathy disorders, with retinal degeneration as a prominent phenotype. Here, we demonstrate that the retinal pigment epithelium (RPE), essential for photoreceptor development and function, requires a functional primary cilium for complete maturation and that RPE maturation defects in ciliopathies precede photoreceptor degeneration. Pharmacologically enhanced ciliogenesis in wild-type induced pluripotent stem cells (iPSC)-RPE leads to fully mature and functional cells. In contrast, ciliopathy patient-derived iPSC-RPE and iPSC-RPE with a knockdown of ciliary-trafficking protein remain immature, with defective apical processes, reduced functionality, and reduced adult-specific gene expression. Proteins of the primary cilium regulate RPE maturation by simultaneously suppressing canonical WNT and activating PKCδ pathways. A similar cilium-dependent maturation pathway exists in lung epithelium. Our results provide insights into ciliopathy-induced retinal degeneration, demonstrate a developmental role for primary cilia in epithelial maturation, and provide a method to mature iPSC epithelial cells for clinical applications., Graphical Abstract, In Brief May-Simera et al. show that primary cilia regulate the maturation and polarization of human iPSC-RPE, mouse RPE, and human iPSC-lung epithelium through canonical WNT suppression and PKCδ activation. RPE cells derived from ciliopathy patients exhibit defective structure and function. These results provide insights into ciliopathy-induced retinal degeneration.

Human induced pluripotent stem cells (hiPSCs) not only offer great opportunities for the study of human development but also have tremendous potential for future clinical cell-based therapies. The protocol outlined here is used to differentiate hiPSCs into lung epithelial cell types through a process that faithfully recapitulates the stepwise events observed in vivo. From pluripotency, cells are differentiated to a definitive endoderm fate, followed by progression into anteriorized foregut endoderm that has the ability to give rise to both proximal and distal epithelial cells. Furthermore, this methodology allows for the study of lung dysfunction and disease modeling using patient-derived cells, as well as high-throughput pharmacological screening and eventually personalized therapies. Recently we were able to reproduce this protocol using the working cell bank of an hiPSC line made under current Good Manufacturing Practice (cGMP) conditions, a necessary step for the future clinical application of these cells. © 2019 by John Wiley & Sons, Inc.

Inherited retinal diseases (IRDs) are clinically and genetically heterogenous diseases affecting the neural retina and retinal pigment epithelium resulting in irreversible blindness. Owing to advantages like ease of access for treatment, one eye being a perfect natural control for the other, and immune privileged environment, research exploring treatment for these retinal diseases has advanced remarkably. We describe the generation of induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMC) of three patients with IRDs. These well-characterized iPSC lines provide an ideal platform to investigate normal and pathological retinogenesis for drug screening and personalized cell therapy.

Neural crest cells (NCC) are a population of epithelial cells that arise from the dorsal tube and undergo epithelial-mesenchymal transition (EMT) eventually generating tissues from peripheral nervous system, melanocytes, craniofacial cartilage, and bone. The antidiabetic drug metformin reportedly inhibits EMT in physiological conditions like cancer and fibrosis. We hypothesize that perturbation of EMT may also contribute to developmental disabilities associated with neural crest (NC) development. To understand the molecular network underlying metformin action during NC formation, we first differentiated murine embryonic stem (ES) cells into NCC and characterized them by demonstrating spatiotemporal regulation of key markers. Metformin treatment prompted a delay in delamination of NCC by inhibiting key markers like Sox-1, Sox-9, HNK-1, and p-75. We then revealed that metformin impedes Wnt axis, a major signaling pathway active during NC formation via DVL-3 inhibition and impairment in nuclear translocation of β-catenin. Concomitantly we identified and tested a candidate set of miRNAs that play a crucial role in NC cell fate determination. Further studies involving loss and gain of function confirmed that NCC specifiers like Sox-1 and Sox-9 are direct targets of miR-200 and miR-145, respectively and that they are essentially modulated by metformin. Our in vitro findings were strongly supported by in vivo studies in zebrafish. Given that metformin is a widely used drug, for the first time we demonstrate that it can induce a delayed onset of developmental EMT during NC formation by interfering with canonical Wnt signaling and mysregulation of miR-145 and miR-200. Video Highlight: https://youtu.be/TZWqwMMIQ6k

Primary cilia are sensory organelles that protrude from the cell membrane. Cilia defects cause ciliopathy disorders with retinal degeneration as a prominent phenotype. Here, we demonstrate that the retinal pigment epithelium (RPE), essential for photoreceptor development and function, requires a functional primary cilium for complete maturation, and RPE maturation defects in ciliopathies precede photoreceptor development. Pharmacologically enhanced ciliogenesis in wildtype induced pluripotent stem cells (iPSCs)-RPE leads to fully-mature and functional cells. Whereas, ciliopathy patient-derived iPSCs-RPE and wildtype iPSC-RPE with a knockdown of ciliary-trafficking protein remain immature, with defective apical processes, reduced functionality, and reduced adult-specific gene expression. Primary cilia proteins regulate RPE maturation by simultaneously suppressing canonical-WNT and activating PKC-δ pathways. A similar cilia-dependent maturation pathway exists in lung epithelium. Our results provide novel insights into ciliopathy-induced retinal degeneration, demonstrate a developmental role for primary cilia in epithelial maturation, and provide a method to mature iPSC-epithelial cells for clinical applications.

Age-related macular degeneration (AMD) is the foremost cause of blindness in people over the age of 60 worldwide. Clinically, this disease starts with distortion in central vision eventually leading to legal blindness. Vision loss has a significant impact on quality of life and incurs a substantial cost to the economy. Furthermore, AMD is a complex and progressive neurodegenerative disorder that triggers visual impairment due to the loss of retinal pigmented epithelium (RPE) and the light-sensitive photoreceptors that they support, protect and provide nutrition. Currently, there is no curative treatment for the most common form of this disease, i.e., dry AMD. A novel approach to treat AMD involves the transplantation of RPE cells derived from human induced pluripotent stem cells (iPSCs) in the outer retina. These iPSC-derived RPE cells not only show characteristics similar to native RPE but also could replace as well as regenerate damaged pathologic RPE and produce supportive growth factors and cytokines. Several clinical trials are being conducted taking advantage of a variety of cell- and tissue engineering-based approaches. Here, we present a simple, cost effective, and scalable cell-culture model for generation of purified RPE thus providing the foundation for developing an allogeneic cell therapy for AMD.

Retinal degenerative disorders are a leading cause of the inherited, irreversible and incurable vision loss. While various rodent model systems have provided crucial information in this direction, lack of disease-relevant tissue availability and species-specific differences have proven to be a major roadblock. Human induced pluripotent stem cells (iPSC) have opened up a whole new avenue of possibilities not just in understanding the disease mechanism but also potential therapeutic approaches towards a cure. In this review, we have summarized recent advances in the methods of deriving retinal cell types from iPSCs which can serve as a renewable source of disease-relevant cell population for basic as well as translational studies. We also provide an overview of the ongoing efforts towards developing a suitable in vitro model for modeling retinal degenerative diseases. This basic understanding in turn has contributed to advances in translational goals such as drug screening and cell-replacement therapies. Furthermore we discuss gene editing approaches for autologous repair of genetic disorders and allogeneic transplantation of stem cell-based retinal derivatives for degenerative disorders with an ultimate goal to restore vision. It is pertinent to note however, that these exciting new developments throw up several challenges that need to be overcome before their full clinical potential can be realized.

Stem cell transplantation is a generic term covering different techniques. However there is argument over the pros and cons of autologous and allogeneic transplants of mesenchymal stem cells (MSCs) for regenerative therapy. Given that the MSCs have already been proven to be safe in patients, we hypothesize that allogeneic transplantation could be more effective and cost-effective as compared to autologous transplantation specifically in older subjects who are the likely victims of degenerative diseases. This analysis is based on the scientific logic that allogeneic stem cells extracted in large numbers from young and healthy donors could be physiologically, metabolically and genetically more stable. Therefore stem cells from young donors may be expected to exhibit higher vigor in secreting trophic factors leading to activation of host tissue-specific stem cells and also be more efficient in remodeling the micro-environmental niche of damaged tissue.

Advances in dental pulp stem cell (DPSC) biology and behaviour have promised much in the field of regenerative medicine. Their recent use in clinical trials for bone repair enforces the notion that DPSCs can be used successfully in patients; however they display diverse characteristics under different culture conditions. Since the success of any stem cell culture is regulated by its own micro-environment, it is imperative to optimise the growth conditions and establish a generic protocol for maintenance and scale-up. This study focused on optimisation of long-term culture conditions of human exfoliated deciduous teeth (SHED) in comparison with DPSCs, employing three commonly used basal media - knockout Dulbecco's modified Eagle's medium (KO-DMEM), α-MEM and DMEM/F12. Based on their characterisation with respect to morphology, growth kinetics, cell surface marker expression, differentiation capacity and plating density, our findings suggest that cells can be expanded with the highest efficiency in KO-DMEM medium supplemented with 10% FBS. Additionally, under our standardised xeno-free (10% human plasma) growth conditions, DPSCs displayed and retained their multipotent attributes until late passages. The differences in the growth and differentiation characteristics between SHED and DPSCs are shown, and certify SHED can be a key element in tissue engineering.

Neurodegenerative diseases are devastating because they cause increasing loss of cognitive and physical functions and affect an estimated 1 billion individuals worldwide. Unfortunately, no drugs are currently available to halt their progression, except a few that are largely inadequate. This mandates the search of new treatments for these progressively degenerative diseases. Neural stem cells (NSCs) have been successfully isolated, propagated, and characterized from the adult brains of mammals, including humans. The confirmation that neurogenesis occurs in the adult brain via NSCs opens up fresh avenues for treating neurological problems. The proof‐of‐concept studies demonstrating the neural differentiation capacity of stem cells both in vitro and in vivo have raised widespread enthusiasm toward cell‐based interventions. It is anticipated that cell‐based neurogenic drugs may reverse or compensate for deficits associated with neurological diseases. The increasing interest of the private sector in using human stem cells in therapeutics is evidenced by launching of several collaborative clinical research activities between Pharma giants and research institutions or small start‐up companies. In this review, we discuss the major developments that have taken place in this field to position stem cells as a prospective candidate drug for the treatment of neurological disorders.

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.

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. © 2011 Wiley-Liss, Inc.

Techniques to evaluate gene expression profiling, including real-time quantitative PCR, TaqMan low-density arrays, and sufficiently sensitive cDNA microarrays, are efficient methods for monitoring human embryonic stem cell (hESC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turnaround time, and the involvement of highly specialized technical expertise. Hence, there is a paucity of rapid, cost-effective, robust, yet sensitive methods for routine screening of hESCs. A critical requirement in hESC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all germ layers, including ecto-, meso-, and endoderm. To quantify the modulation of gene expression in hESCs during their propagation, expansion, and differentiation via embryoid body (EB) formation, the authors developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR (mxPCR) platform technology. Among the 15 gene primers tested, 4 were pluripotent markers comprising set 1, and 3 lineage-specific markers from each ecto-, meso-, and endoderm layers were combined as sets 2 to 4, respectively. The authors found that these 4 sets were not only effective in determining the relative differentiation in hESCs, but were easily reproducible. In this study, they used the HUES-7 cell line to standardize the technique, which was subsequently validated with HUES-9, NTERA-2, and mouse embryonic fibroblast cells. This single-reaction mxPCR assay was flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation.

Human embryonic stem cells (hESC) differentiate spontaneously in culture and develop a complex microenvironment comprising of autologously derived niche that in turn supports their pluripotency. The basic hypothesis that we deal with is that hESCs undergoing differentiation, sequentially generate trophectoderm and endoderm lineages and thereafter influence further events through the production of growth factors. These factors control the fate of hESCs either by promoting or retarding the recruitment of new cells in the differentiation program. This scenario therefore represents an analog of the in vivo situation in which extra-embryonic tissues influence the behavior of the inner cell mass (ICM). The premise of the paper is the Rho kinase inhibitor Y27632 that can spatiotemporally alter this balance between pluripotency and differentiation. To evaluate the composition and inclination of lineage specification during spontaneous differentiation, we have studied the hESC colonies and their surrounding niche as interdependent entities. We show that the population of fibroblastic niche that surrounds hESC colonies co-expresses trophectoderm and niche cell markers including SSEA1, hCG, progesterone, HAND1, pSmad1 and FGFR1 as early as day 4. A sudden increase in the expression of GATA4 and AFP secretion indicated putative endoderm formation on day 6 in both control and Y27632 treated cultures. On day 6, 20 microM of Y27632 supplementation significantly reduced the trophectoderm-like niche population without affecting endoderm formation, enhanced the average size and number of hESC colonies, decreased IGF1 secretion thereby improving the pluripotency. Overall our findings support the afore mentioned hypothesis and demonstrate that closely packed epithelial trophectoderm-like cells bordering the hESC colonies present an initial and imminent localized niche which is spatiotemporally regulated. Such advances in understanding the behavior and modulation of hESC and its surrounding niche would facilitate better differentiation protocols for applications in regenerative medicine and drug screening.

During the last decade, embryonic stem cells (ESC) have unleashed new avenues in the field of developmental biology and emerged as a potential tool to understand the molecular mechanisms taking place during the process of differentiation from the embryonic stage to adult phenotype. Their uniqueness lies in retaining the capacity of unlimited proliferation and to differentiate into all somatic cells. Together with promising results from rodent models, ESC has raised great hope among for human ESC-based cell replacement therapy. ESC could potentially revolutionize medicine by providing a powerful and renewable cell source capable of replacing or repairing tissues that have been damaged in almost all degenerative diseases such as Parkinson's disease, myocardial infarction (MI) and diabetes. Somatic stem cells are an attractive option to explore for transplantation because they are autologous, but their differentiation potential is very limited. Currently, the major sources of somatic cells used for basic research and clinical trials come from bone marrow. But their widespread acceptability has not been gained because many of the results are confusing and inconsistent. The focus here is on human embryonic stem cells (hESCs), using methods to induce their differentiation to cardiomyocytes in vitro. Their properties in relation to primary human cardiomyocytes and their ability to integrate into host myocardium have been investigated into how they can enhance cardiac function. However, important aspects of stem cell biology and the transplantation process remain unresolved. In summary, this review updates the recent progress of ES cell research in cell therapy, discusses the problems in the practical utility of ESC, and evaluates how far this adjunctive experimental approach can be successful.

Epithelial-mesenchymal transition (EMT) is one of the key biological phenomena behind cancer and metastasis. Clinical studies suggest that patients undergoing metformin therapy are less predisposed to cancer but the underlying mechanism is far from clear. Given that metformin also acts as TGF-β inhibitor, we sought to explore whether and how metformin could modulate EMT in a cancer like microenvironment. Our data using human cell lines revealed that metformin induced a distinct change from stromal-shaped mesenchymal cells to cuboidal-shaped epithelial cells with upregulation of epithelial markers and mitigation of their invasive property. One of the key regulatory pathways, which intersect tumorigenesis and metformin activity, is AMPK. We demonstrated that metformin attenuates ERK signaling by activating AMPK pathway leading to suppression of Snail and Slug resulting in upregulation of crucial tumor suppressor gene E-cadherin. ChIP assay confirmed insufficient binding of repressors like Slug to the E-cadherin promoter. Further, our data revealed reduction in HDAC activity prompting hypomethylation of E-cadherin promoter thus reflecting an epigenetic modification. To expand the translational significance of the study we verified these findings in diabetic patients undergoing metformin treatment. To our knowledge this is the first report representing an inverse relationship of AMPK and ERK signaling axis in promoting mesenchymal to epithelial transition (MET) via re-expression of E-cadherin upon metformin treatment thus rationalizing lower incidence of cancer in metformin-administered patients.Metformin promotes reversal of the epithelial-mesenchymal transition. Metformin attenuates ERK signaling by activating AMP kinase. Metformin induces hypomethylation of the E-cadherin gene promoter. Epigenetic modification of the E-cadherin promoter was observed in leukocytes from diabetic subjects. These findings provide a potential basis for decreased cancer incidence in metformin-treated subjects.

Cell replacement therapy, while being a promising approach, is challenged by the limited supply of appropriate cells and incomplete understanding of the cardiac differentiation process. In this manuscript, we show the generation of spontaneously beating cardiomyocytes without using dimethylsulfoxide (DMSO), the most well-known cardiotrophic factor. Here, we employ basic fibroblast growth factor (FGF), a commonly used growth factor for embryonic stem (ES) cell differentiation, to initiate and maintain in vitro cardiac differentiation. Upon differentiation, beating or pulsating cardiac cells grown on tissue culture plates that interspersed with oval-shaped cells appeared after 1 week in culture. The number of beating colonies increased with time, and cells remained contractile for at least 45 days. Within 12 days of differentiation, these cells expressed markers characteristic of cardiomyocytes and hepatocytes such as GATA4, Nkx2.5, beta-myosin heavy chain, myosin light chain 2V, cardiac troponin T, sodium calcium exchanger and HNF-4alpha, alpha-fetoprotein, albumin, transthyretin, and CK-18, respectively. Thus, molecular and cellular characterization of these oval-shaped cells identified them as hepatocyte-like cells. Furthermore, we have identified a candidate set of signaling molecules like bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) and demonstrated their interactive role in in vitro cardiogenesis. To our knowledge, this is the first report elucidating the intrinsic signaling pathway of hepatocyte-like cells in the differentiation of cardiomyocytes from mouse ES cells without employing co-culture techniques. Hence, the study provides a significant insight into the mechanism of in vitro derivation of cardiomyocytes, mediated through interactive signaling with adjoining endodermal derivatives.

Vitiligo is an autoimmune disorder that leads to depigmentation of skin via melanocyte dysfunction. Keratinocyte-induced toxicity is one among the several etiological factors implicated for vitiligo, and hence, autologous keratinocyte grafting is projected as one of the primary mode of treatment for vitiligo. However, reports indicate that perilesional keratinocytes not only display signatures of apoptosis but also could secrete cytokines and mediators which have antagonistic effect on proliferation or survival. Therefore, we investigated how vitiligo patients' derived keratinocytes respond to surplus amounts of inflammatory cytokines and whether they recapitulate events that take place during conventional wound healing. The primary objective of our study was to determine whether keratinocytes isolated from a vitiligo patient would undergo epithelial-mesenchymal transition similar to their normal counterparts upon induction with inflammatory cytokines such as TGF-b1 and EGF. We found that these keratinocytes undergo EMT during wound repair accompanied with increase in the levels of mesenchymal markers and ECM proteins; decrease in the levels of epithelial markers and enhanced migratory ability. Besides, we also demonstrated that EMT induction leads to activation of SMAD and MAPK pathways via Ras, Raf, PAI 1, Snail, Slug and ZO1. To our knowledge, this is the first report on the characterization of primary keratinocytes isolated from vitiligo patients with respect to their wound healing capacity.

The staggering number of publications featuring the use of stem cells has revolutionized regenerative medicine research. Preclinical studies indicate that allogeneic human mesenchymal stem cells (MSCs) may be useful for the treatment of several clinical disorders, including sepsis, acute renal failure, acute myocardial infarction, and more recently, acute lung injury (ALI). However, considerable success would not be obtained in clinical trials due to poor survival of transplanted cells under the influence of inflammatory conditions. Despite robust approaches like cellular reprogramming, scaffolds and conditioned media have been tested to overcome this problem; however the success rate of these approaches remain questionable. Recently, pretreatment of bioactive compounds in vitro have been shown to suppress cell apoptosis and promote cell survival. Quite likely a similar phenomenon can take place in vivo. Based on such studies, we hypothesize that MSCs derived from human post-natal tissues could be conditioned and prepared for targeted disease therapy. Depending on the disease condition, the MSCs could be treated prior to delivery with appropriate bioactive compounds to allow them survive longer and perform a better role as biocatalyst. The advantage of this approach could be the tailor made availability of MSCs preconditioned with appropriate bioactive compounds for disease specific therapy. Therefore, the choice of suitable bioactive molecule is likely to enhance the efficacy of targeted stem cell therapy and preconditioning may provide a novel strategy in maximizing biological and functional properties of MSCs.

Metformin is not only a widely used oral antidiabetic drug, which acts as an insulin sensitizer and suppressor of hepatic gluconeogenesis, but it also exhibits antitumor properties. Besides, it has been utilized in the treatment of polycystic ovary syndrome (PCOS) for infertile women with glucose intolerance and as a component of combination therapy to reduce early (first trimester) pregnancy loss or spontaneous abortion (SAB). Based on recent studies demonstrating its beneficial effects on mothers and the fetus, metformin is even recommended for later stages of pregnancy. Probing into the mechanism of action revealed that it can activate a stress modulatory pathway, none other than the AMP-activated protein kinase (AMPK) via LKB 1. It is well accepted that AMPK signaling plays a crucial role during implantation by combating stress in multiple ways. Stress factors commonly encountered during pregnancy are malnutrition, diabetes, and hypoxia, which may result in SABs or other complications. For instance, the elevated levels of insulin, which are a typical characteristic of hyperinsulinemic, obese, or PCOS patients, can impair the development of the blastocyst and the preimplantation embryo. Further, a severe hypoxic environment prompts early and untimely differentiation of the embryonic cells leading to abnormal growth and development. Therefore, the modulation of stress-related pathways could be pivotal in ameliorating such stress responses during implantation. Here we hypothesize a putative noncanonical pathway underpinning the role of metformin in high-risk pregnancies to counteract stress by recreating an in vitro replica of human implantation, engaging embryonic stem cells, trophoblast stem cells, and endometrial stromal cells in a three-dimensional scaffold.

It is well established that fibroblasts and mesenchymal stem cells (MSC) share several characteristics with subtle differences. However, no study highlighting the versatility of fibroblasts beyond their multipotentiality has been reported so far. Mouse embryonic fibroblasts (MEFs) are widely used as feeder layers to support the growth of embryonic stem cells (ESC). We hypothesized that MEF may retain ES-like features in concurrence to their developmental hierarchy in addition to their multipotent nature. Hence, we performed a comparative assessment of MEF and ESC to determine their ability to differentiate into cell types other than mesoderm as well as capacity to form teratoma using routine in vitro and in vivo techniques. MEF were derived by trypsin/ EDTA (ethylenediaminetetraacetic acid) digestion from E13.5 embryos after removing heads and viscera following plastic adherence. MEFs robustly proliferated in culture until passage 15 and formed aggregates by hanging drop method. Flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry revealed the presence of key MSC markers such as CD90, CD73, Sca-1, CD44, CD29, Vimentin and absence of CD45. Additionally, they expressed SSEA-1, Oct-4, Nanog, Sox-2 and ABCG2 as pluripotency markers; Nestin, β-III tubulin, Otx-2 (ectoderm); MEF-2, Mesp2, GATA-2 (mesoderm) and GATA-4, α-amylase, PDX-1 (endoderm) as tri-lineage markers. Furthermore, MEFs formed representative tissues from all three germ layers upon transplantation into Balb/c mice. These unique abilities of MEF to exhibit pluripotency, in addition to fibroblast characteristics and their ready availability with less ethical concerns and low maintenance requirements make them an attractive model for further exploration as a possible tool for regenerative medicine.

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.

The in vitro derived hepatocytes from human embryonic stem cells (hESC) is a promising tool to acquire improved knowledge of the cellular and molecular events underlying early human liver development under physiological and pathological conditions. Here we report a simple two-step protocol employing conditioned medium (CM) from human hepatocellular carcinoma cell line, HepG2 to generate functional hepatocyte-like cells from hESC. Immunocytochemistry, flow cytometry, quantitative RT-PCR, and biochemical analyses revealed that the endodermal progenitors appeared as pockets in culture, and the cascade of genes associated with the formation of definitive endoderm (HNF-3b, SOX-17, DLX-5, CXCR4) was consistent and in concurrence with the up-regulation of the markers for hepatic progenitors [alpha-feto protein (AFP), HNF-4a, CK-19, albumin, alpha-1-antitrypsin (AAT)], followed by maturation into functional hepatocytes [tyrosine transferase (TAT), tryptophan-2, 3-dioxygenase (TDO), glucose 6-phosphate (G6P), CYP3A4, CYP7A1]. We witnessed that the gene expression profile during this differentiation process recapitulated in vivo liver development demonstrating a gradual down-regulation of extra embryonic endodermal markers (SOX-7, HNF-1b, SNAIL-1, LAMININ-1, CDX2), and the generated hepatic cells performed multiple liver functions. Since prenatal alcohol exposure is known to provoke irreversible abnormalities in the fetal cells and developing tissues, we exposed in vitro generated hepatocytes to ethanol (EtOH) and found that EtOH treatment not only impairs the survival and proliferation, but also induces apoptosis and perturbs differentiation of progenitor cells into hepatocytes. This disruption was accompanied by alterations in the expression of genes and proteins involved in hepatogenesis. Our results provide new insights into the wider range of destruction caused by alcohol on the dynamic process of liver organogenesis. J. Cell. Biochem. 113: 19–30, 2012.

In vitro models based on embryonic stem cells (ESC) are highly promising for improvement of predictive toxicology screening in humans. After the successful validation of embryonic stem cell test (EST) in 2001; concerns have been raised on the usage of mouse ESC and also the morphological evaluation of beating cell clusters. This requires specialized skill-sets and is highly prone to misjudgement and false positive results. To overcome these limitations, we undertook the present study incorporating improvisations over the conventional EST. Here, we explored the potential of a human ESC (hESC)-based assay to evaluate the potential toxicity of penicillin-G, caffeine, and hydroxyurea. Drug treatment inhibited hESC adhesion and substantially altered the morphology and viability (∼ 50%) of embryoid bodies (EBs). Flow cytometry analysis not only showed a significant increase of apoptotic cells in the highest doses but also induced a diverse pattern in DNA content and cell cycle distribution relative to control. Both semi-quantitative and quantitative RT-PCR studies revealed a selective down regulation of markers associated with stemness (Nanog, Rex1, SOX-2, and hTERT); cardiac mesoderm (Cripto1, MEF-2C, and Brachyury); hepatic endoderm (AFP, HNF-3β, HNF-4α, GATA-4, and SOX-17); and neuroectoderm (Nestin, SOX-1, NURR1, NEFH, Synaptophysin, TH, and Olig2) in a drug as well as dose dependent manner indicating abnormal differentiation. Furthermore, a decrease in the expression of AFP and GFAP proteins followed by a dose-dependent reduction in the levels of hCG-β, progesterone-II, and estradiol hormones was demonstrated by immunocytochemistry and ECLIA, respectively. This new and unique approach comprising of DNA cell cycle analysis, germ layer-specific marker expression and hormone levels as endpoints might offer a clinically relevant and commercially viable alternative for predicting in vivo developmental toxicity.

Human embryonic stem cells (hESCs) are able to stably maintain their characteristics for an unlimited period; nevertheless, substantial differences among cell lines in gene and protein expression not manifested during the undifferentiated state may appear when cells differentiate. It is widely accepted that developing an efficient protocol to control the differentiation of hESCs will enable us to produce adequate numbers of desired cell types with relative ease for diverse applications ranging from basic research to cell therapy and drug screening. Hence of late, there has been considerable interest in understanding whether and how hESC lines are equivalent or different to each other in their in vitro developmental tendencies. In this study, we compared the developmental competences of two hESC lines (HUES-9 and HUES-7) at molecular, cellular and functional levels, upon spontaneous differentiation without any added inducing agents. Both cell lines generated the three embryonic germ layers, extra-embryonic tissues and primordial germ cells during embryoid body (EB) formation. However HUES-9 showed a stronger propensity towards formation of neuroectodermal lineages, whereas HUES-7 differentiated preferentially into mesoderm and endoderm. Upon further differentiation, HUES-9 generated largely neural cells (neurons, oligodendrocytes, astrocytes and gangliosides) whereas HUES-7 formed mesendodermal derivatives, including cardiomyocytes, skeletal myocytes, endothelial cells, hepatocytes and pancreatic cells. Overall, our findings endorse the hypothesis that independently-derived hESCs biologically differ among themselves, thereby displaying varying differentiation propensity. These subtle differences not only highlight the importance of screening and deriving lines for lineage-specific differentiation but also indicate that individual lines may possess a repertoire of capabilities that is unique.

Human embryonic stem cells (hESCs) are an exceptionally useful tool for studies of human development and represent a potential source for transplantation therapies. At present, only a limited number of hESCs lines representing a very small sample of genetic diversity of the human populations are available. Here, we report the derivation and characterization of a new hESC line, ReliCell® hES1. These cells, established from the inner cell mass (ICM) on mouse embryonic feeder (MEF) layer, satisfy the criteria that characterize pluripotent hESCs: The cell line expresses high levels of cell surface markers (such as SSEA-3, SSAEA-4, TRA-1-60 and TRA-1-81), transcription factor Oct-4, alkaline phosphatase (AP) and telomerase. The cell line retains normal karyotype in long-term culture and has a distinct identity as revealed by DNA fingerprinting by short tandem repeat (STR) analysis. Further, upon examination of the in vitro differentiation potential, ReliCell®hES1 was found to be capable of giving rise to dopaminergic neurons, cardiomyocytes, pancreatic islets, and hepatocyte-like cells belonging to ectoderm, mesoderm, and endoderm lineages, respectively. To our knowledge, this is the first report of a well-characterized hES cell line from the Indian subcontinent.