Generation and characterization of retinal pigment epithelium from patient iPSC line to model oculocutaneous albinism (OCA)1A disease.

Oculocutaneous albinism (OCA) is characterized by reduced melanin biosynthesis affecting the retina, thus impairing visual function. The disease pathology of OCA is poorly understood at the cellular level due to unavailability of suitable biological model systems. This study aimed to develop a disease-specific in vitro model for OCA type 1A, the most severe form caused by TYR (tyrosinase) gene mutations, using retinal pigment epithelium (RPE) differentiated from patient-derived human-induced pluripotent stem cells (hiPSCs). A comparative study between healthy and OCA1A RPE cells revealed that while healthy RPE cells exhibited timely onest of pigmentation during differentiation, OCA1A RPE cells failed to pigment even after an extended culture period. This observation was validated by ultrastructural studies using electron microscopy, hinting at melanosome-specific defects. Immunocytochemistry demonstrated abnormal expression patterns of melanogenesis-specific protein markers in OCA1A RPE cells, indicating reduced or absence of melanin synthesis. Next, a quantitative assay was performed to confirm the absence of melanin production in OCA1A RPE cells. Tyrosinase assay showed no activity in OCA1A compared with healthy RPE, suggesting non-functionality of TYR, further corroborated by western blot analysis showing complete absence of the protein. Gene expression by RNA sequencing of healthy and OCA1A RPE cells uncovered differential gene expression associated with lens development, visual perception, transmembrane transporter activity, and key signaling pathways. This disease-in-a-dish model of OCA1A provides an excellent platform to understand disease mechanism, identify potential therapeutic targets, and facilitate gene therapy or gene correction.

An improved protocol for generation and characterization of human-induced pluripotent stem cell-derived retinal pigment epithelium cells.

We present an optimized protocol for guided differentiation of retinal pigment epithelium (RPE) cells from human-induced pluripotent stem cells (iPSC). De novo-generated RPE cells are mature, polarized, and mimic the cellular and molecular profile of primary RPE; they are also suitable for in vivo cell transplantation studies. The protocol includes an enrichment step, making it useful for large-scale GMP manufacturing. RPE cells produced following this protocol are appropriate for cell replacement therapy for macular degeneration and disease modeling. For complete details on the use and execution of this protocol, please refer to Surendran et al. (2021).

SARS-CoV-2 infection of human-induced pluripotent stem cells-derived lung lineage cells evokes inflammatory and chemosensory responses by targeting mitochondrial pathways.

The COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily affects the lung, particularly the proximal airway and distal alveolar cells. NKX2.1+ primordial lung progenitors of the foregut (anterior) endoderm are the developmental precursors to all adult lung epithelial lineages and are postulated to play an important role in viral tropism. Here, we show that SARS-CoV-2 readily infected and replicated in human-induced pluripotent stem cell-derived proximal airway cells, distal alveolar cells, and lung progenitors. In addition to the upregulation of antiviral defense and immune responses, transcriptomics data uncovered a robust epithelial cell-specific response, including perturbation of metabolic processes and disruption in the alveolar maturation program. We also identified spatiotemporal dysregulation of mitochondrial heme oxygenase 1 (HMOX1), which is associated with defense against antioxidant-induced lung injury. Cytokines, such as TNF-α, INF-γ, IL-6, and IL-13, were upregulated in infected cells sparking mitochondrial ROS production and change in electron transport chain complexes. Increased mitochondrial ROS then activated additional proinflammatory cytokines leading to an aberrant cell cycle resulting in apoptosis. Notably, we are the first to report a chemosensory response resulting from SARS-CoV-2 infection similar to that seen in COVID-19 patients. Some of our key findings were validated using COVID-19-affected postmortem lung tissue sections. These results suggest that our in vitro system could serve as a suitable model to investigate the pathogenetic mechanisms of SARS-CoV-2 infection and to discover and test therapeutic drugs against COVID-19 or its consequences.

Derivation of Induced Pluripotent Stem Cell (iPSC) Lines from Patient-Specific Peripheral Blood Mononuclear Cells (PBMC) Using Episomal Vectors.

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.

Implementation of an adapted Sepsis Risk Calculator algorithm to reduce antibiotic usage in the management of early onset neonatal sepsis: a multicentre initiative in Wales, UK.

OBJECTIVE: Assess 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. DESIGN: Multicentre prospective study SETTING: Ten perinatal hospitals in Wales, UK. PATIENTS: All 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. METHODS: The 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 MEASURES: Proportion of antibiotic use in infants ≥34 weeks' gestation. RESULTS: 4304 (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. CONCLUSIONS: This 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.

Neuronal and cardiac toxicity of pharmacological compounds identified through transcriptomic analysis of human pluripotent stem cell-derived embryoid bodies.

Concurrent with the '3R' principle, the embryonic stem cell test (EST) using mouse embryonic stem cells, developed in 2000, remains the solely accepted in vitro method for embryotoxicity testing. However, the scope and implementation of EST for embryotoxicity screening, compliant with regulatory requirements, are limited. This is due to its technical complexity, long testing period, labor-intensive methodology, and limited endpoint data, leading to misclassification of embryotoxic potential. In this study, we used human induced pluripotent stem cell (hiPSC)-derived embryoid bodies (EB) as an in vitro model to investigate the embryotoxic effects of a carefully selected set of pharmacological compounds. Morphology, viability, and differentiation potential were investigated after exposing EBs to folic acid, all-trans-retinoic acid, dexamethasone, and valproic acid for 15 days. The results showed that the compounds differentially repressed cell growth, compromised morphology, and triggered apoptosis in the EBs. Further, transcriptomics was employed to compare subtle temporal changes between treated and untreated cultures. Gene ontology and pathway analysis revealed that dysregulation of a large number of genes strongly correlated with impaired neuroectoderm and cardiac mesoderm formation. This aberrant gene expression pattern was associated with several disorders of the brain like mental retardation, multiple sclerosis, stroke and of the heart like dilated cardiomyopathy, ventricular tachycardia, and ventricular arrhythmia. Lastly, these in vitro findings were validated using in ovo chick embryo model. Taken together, pharmacological compound or drug-induced defective EB development from hiPSCs could potentially be used as a suitable in vitro platform for embryotoxicity screening.

Interventional Strategies for Parkinson Disease: Can Neural Precursor Cells Forge a Path Ahead?

Neural precursor cells (NPCs), derived from pluripotent stem cells (PSCs), with their unique ability to generate multiple neuronal and glial cell types are extremely useful for understanding biological mechanisms in normal and diseased states. However, generation of specific neuronal subtypes (mature) from NPCs in large numbers adequate for cell therapy is challenging due to lack of a thorough understanding of the cues that govern their differentiation. Interestingly, neural stem cells (NSCs) themselves are in consideration for therapy given their potency to form different neural cell types, release of trophic factors, and immunomodulatory effects that confer neuroprotection. With the recent COVID-19 outbreak and its accompanying neurological indications, the immunomodulatory role of NSCs may gain additional significance in the prevention of disease progression in vulnerable populations. In this regard, small-molecule mediated NPC generation from PSCs via NSC formation has become an important strategy that ensures consistency and robustness of the process. The development of the mammalian brain occurs along the rostro-caudal axis, and the establishment of anterior identity is an early event. Wnt signaling, along with fibroblast growth factor and retinoic acid, acts as a caudalization signal. Further, the increasing amount of epigenetic data available from human fetal brain development has enhanced both our understanding of and ability to experimentally manipulate these developmental regulatory programs in vitro. However, the impact on homing and engraftment after transplantation and subsequently on therapeutic efficacy of NPCs based on their derivation strategy is not yet clear. Another formidable challenge in cell replacement therapy for neurodegenerative disorders is the mode of delivery. In this Perspective, we discuss these core ideas with insights from our preliminary studies exploring the role of PSC-derived NPCs in rat models of MPTP-induced Parkinson's disease following intranasal injections.

Transplantation of retinal pigment epithelium and photoreceptors generated concomitantly via small molecule-mediated differentiation rescues visual function in rodent models of retinal degeneration.

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.

Secretome studies of mesenchymal stromal cells (MSCs) isolated from three tissue sources reveal subtle differences in potency.

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-ß3, 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.

Human Induced Pluripotent Stem Cell-Derived Lung Epithelial System for SARS-CoV-2 Infection Modeling and Its Potential in Drug Repurposing.

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.

Derivation of three induced pluripotent stem cell lines under feeder-free culture conditions from peripheral blood mononuclear cells (PBMC) of Indian patients suffering from inherited retinal diseases carrying different mutations.

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.

Differentiating Human Induced Pluripotent Stem Cells (iPSCs) Into Lung Epithelial Cells.

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.

Induced pluripotent stem cells (iPSC)-derived retinal cells in disease modeling and regenerative medicine.

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.

Generation of Transplantable Retinal Pigmented Epithelial (RPE) Cells for Treatment of Age-Related Macular Degeneration (AMD).

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.

Primary Cilium-Mediated Retinal Pigment Epithelium Maturation Is Disrupted in Ciliopathy Patient Cells.

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.

Long Noncoding RNA RP11-380D23.2 Drives Distal-Proximal Patterning of the Lung by Regulating PITX2 Expression.

Early lung development is a tightly orchestrated process encompassing (a) formation of definitive endoderm, (b) anteriorization of definitive endoderm, followed by (c) specification and maturation of both proximal and distal lung precursors. Several reports detailing the interaction of genes and proteins during lung development are available; however, studies reporting the role(s) of long noncoding RNAs (lncRNA) in lung morphogenesis are limited. To investigate this, we tailored a protocol for differentiation of human-induced pluripotent stem cells into distal and proximal lung progenitors to mimic in vivo lung development. The authenticity of differentiated cells was confirmed by expression of key lung markers such as FoxA2, Sox-17, Nkx2.1, Pitx2, FoxJ1, CC10, SPC, and via scanning as well as transmission electron microscopy. We employed next generation sequencing to identify lncRNAs and categorized them based on their proximity to genes essential for lung morphogenesis. In-depth bioinformatical analysis of the sequencing data enabled identification of a novel lncRNA, RP11-380D23.2, which is located upstream of PITX2 and includes a binding site for PARP1. Chromatin immunoprecipitation and other relevant studies revealed that PARP1 is a repressor for PITX2. Whole genome microarray analysis of RP11-380D23.2/PITX2 knockdown populations of progenitors demonstrated enrichment in proximal progenitors and indicated altered distal-proximal patterning. Dysregulation of WNT effectors in both knockdowns highlighted direct modulation of PITX2 by RP11-380D23.2. Most of these results were validated in four independent hiPSC lines (including a patient-specific CFTR mutant line). Taken together, these findings offer a mechanistic explanation underpinning the role of RP11-380D23.2 during lung morphogenesis via WNT signaling. Stem Cells 2018;36:218-229.

Transcriptional profiling of human neural precursors post alcohol exposure reveals impaired neurogenesis via dysregulation of ERK signaling and miR-145.

Gestational alcohol exposure causes a range of neuropsychological disorders by modulating neurodevelopmental genes and proteins. The extent of damage depends on the stage of the embryo as well as dosage, duration and frequency of exposure. Here, we investigated the neurotoxic effects of alcohol using human embryonic stem cells. Multiple read-outs were engaged to assess the proliferation and differentiation capacity of neural precursor cells upon exposure to 100 mM ethanol for 48 h corresponding to the blood alcohol levels for binge drinkers. Whole-genome analysis revealed a spatiotemporal dysregulation of neuronal- and glial-specific gene expression that play critical roles in central nervous system (CNS) development. Alterations observed in the transcriptome may be attributed to epigenetic constitution witnessed by differential histone H3 Lys-4/Lys-27 modifications and acetylation status. In-depth mRNA and protein expression studies revealed abrogated extracellular signal-regulated kinases signaling in alcohol-treated cells. Consistent with this finding, ingenuity pathway analysis and micro-RNA profiling demonstrated up-regulation of miR-145 by targeting the neural specifier Sox-2. We also show that the neurite branching complexity of tubulin, beta 3 class III+ neurons was greatly reduced in response to alcohol. Finally, in vivo studies using zebrafish embryos reconfirmed the in vitro findings. Employing molecular endpoints in a human model, this report indicates for the first time that acute alcohol exposure could lead to impaired brain development via perturbation of extracellular signal-regulated kinases pathway and miR-145. However, it still needs to be addressed whether these modulations sustain throughout development, compromising the ability of the individual during adulthood and aging.

Metformin mediated reversal of epithelial to mesenchymal transition is triggered by epigenetic changes in E-cadherin promoter.

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-ß 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. KEY MESSAGE: 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.