Cardiomyocytes from human pluripotent stem cells: From laboratory curiosity to industrial biomedical platform.

Cardiomyocytes from human pluripotent stem cells (hPSCs-CMs) could revolutionise biomedicine. Global burden of heart failure will soon reach USD $90bn, while unexpected cardiotoxicity underlies 28% of drug withdrawals. Advances in hPSC isolation, Cas9/CRISPR genome engineering and hPSC-CM differentiation have improved patient care, progressed drugs to clinic and opened a new era in safety pharmacology. Nevertheless, predictive cardiotoxicity using hPSC-CMs contrasts from failure to almost total success. Since this likely relates to cell immaturity, efforts are underway to use biochemical and biophysical cues to improve many of the ~30 structural and functional properties of hPSC-CMs towards those seen in adult CMs. Other developments needed for widespread hPSC-CM utility include subtype specification, cost reduction of large scale differentiation and elimination of the phenotyping bottleneck. This review will consider these factors in the evolution of hPSC-CM technologies, as well as their integration into high content industrial platforms that assess structure, mitochondrial function, electrophysiology, calcium transients and contractility. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

A hypomorphic Egfr allele does not ameliorate the palmoplantar keratoderma caused by SLURP1 deficiency.

Mutations in SLURP1, a secreted protein of keratinocytes, cause a palmoplantar keratoderma (PPK) known as mal de Meleda. Slurp1 deficiency in mice faithfully recapitulates the human disease, with increased keratinocyte proliferation and thickening of the epidermis on the volar surface of the paws. There has long been speculation that SLURP1 serves as a ligand for a receptor that regulates keratinocyte growth and differentiation. We were intrigued that mutations leading to increased signalling through the epidermal growth factor receptor (EGFR) cause PPK. Here, we sought to determine whether reducing EGFR signalling would ameliorate the PPK associated with SLURP1 deficiency. To address this issue, we bred Slurp1-deficient mice that were homozygous for a hypomorphic Egfr allele. The hypomorphic Egfr allele, which leads to reduced EGFR signalling in keratinocytes, did not ameliorate the PPK elicited by SLURP1 deficiency, suggesting that SLURP1 deficiency causes PPK independently (or downstream) from the EGFR pathway.

Safety and Efficacy of Teduglutide (Gattex) in Patients With Crohn's Disease and Need for Parenteral Support Due to Short Bowel Syndrome-associated Intestinal Failure.

BACKGROUND: Teduglutide is a GLP-2 analogue indicated for treatment of adults with short bowel syndrome (SBS). Because of the rarity of SBS, real-world safety or efficacy data are not available in patients with Crohn's disease (CD) and SBS treated with teduglutide. AIM: To evaluate teduglutide's safety and efficacy in CD patients with SBS. METHODS: We conducted a retrospective cohort study at 3 tertiary centers in the United States between 2012 and 2014. Demographic, clinical, and therapeutic data were retrieved from medical record systems. RESULTS: Thirteen CD patients were included, 8 (62%) of whom were on concomitant immunosuppression. Median duration of teduglutide therapy was 365 days [interquartile range (IQR), 122 to 482 d] and 9/13 patients (69%) remain on therapy. At teduglutide initiation, 69% were on parenteral nutrition. At conclusion of follow-up, 1 patient was on parenteral nutrition. All patients were on intravenous fluids (IVF) before teduglutide; median IVF were 9000 mL/wk (IQR, 7000 to 14,000 mL/wk). IVF requirements decreased by a median of 3100 mL/wk (IQR, 2400 to 8400 mL/wk). Six patients (46%) ceased IVF. Adverse events attributed to teduglutide were obstructive symptoms (n=1), pancreatitis (n=1), asymptomatic lipase and amylase elevation (n=1), nausea (n=1), and abdominal pain (n=1). Catheter-related sepsis occurred in 4 patients. CONCLUSIONS: This is the first report evaluating the safety and efficacy of teduglutide in a cohort of CD patients with SBS requiring parenteral support. More of half the cohort was on concomitant immunosuppression. Teduglutide seemed to be safe and the majority of patients were weaned off parenteral support.

IODINE CONTENT OF ENTERAL AND PARENTERAL NUTRITION SOLUTIONS.

OBJECTIVE: Iodine is essential for thyroid hormone synthesis, and iodine deficiency may result in thyroid disorders including goiter and hypothyroidism. Patients on long-term enteral nutrition (EN) or parenteral nutrition (PN) may be at risk for micronutrient deficiencies. The recommended daily allowance for iodine intake is 150 µg for nonpregnant adults. However, there is no current consensus among scientific societies regarding the quantity of iodine to be added in adult EN and PN formulations. The objective of this study was to determine the iodine content of U.S. adult enteral and parenteral nutrition solutions. This study also aimed to determine whether adult patients in the United States who are receiving long-term artificial nutrition may be at risk for iodine deficiency. METHODS: Ten enteral nutrition solutions and 4 parenteral nutrition solutions were evaluated. The iodine contents of these solutions were measured spectrophotometrically and compared to the labeled contents. RESULTS: Measured and labeled EN iodine contents were similar (range 131-176 µg/L and 106-160 µg/L, respectively). In contrast, PN formulas were found to contain small, unlabeled amounts of iodine, averaging 27 µg/L. CONCLUSION: Typical fluid requirements are 30 to 40 mL/kg/day for adults receiving either total EN (TEN) or total PN (TPN). Adults on long-term TEN likely consume enough servings to meet their daily iodine requirements. However, patients on long-term TPN would require on average 5.6 L PN/day to meet the recommended daily allowance of iodine. This volume of PN is far in excess of typical consumption. Thus, U.S. patients requiring long-term TPN may be at risk for iodine deficiency. ABBREVIATIONS: EN = enteral nutrition; PN = parenteral nutrition; TEN = total enteral nutrition; TPN = total parenteral nutrition; UIC = urinary iodine concentration.

Materials for stem cell factories of the future.

Polymeric substrates are being identified that could permit translation of human pluripotent stem cells from laboratory-based research to industrial-scale biomedicine. Well-defined materials are required to allow cell banking and to provide the raw material for reproducible differentiation into lineages for large-scale drug-screening programs and clinical use. Yet more than 1 billion cells for each patient are needed to replace losses during heart attack, multiple sclerosis and diabetes. Producing this number of cells is challenging, and a rethink of the current predominant cell-derived substrates is needed to provide technology that can be scaled to meet the needs of millions of patients a year. In this Review, we consider the role of materials discovery, an emerging area of materials chemistry that is in large part driven by the challenges posed by biologists to materials scientists.

Sclerodermoid lesions in a patient with multiple transplants and porphyria cutanea tarda.

Patients with chronic graft versus host disease may exhibit a range of sclerotic features. Herein we present a patient with confirmed porphyria cutanea tarda who subsequently developed chronic graft versus host disease.

Nutrition: the future of melanoma prevention?

BACKGROUND: Melanoma is one of the deadliest forms of skin cancer, having a high metastatic potential and afflicting all age groups. The need for successful preventative measures is particularly urgent as metastatic melanoma is largely incurable. The beneficial role of nutrition and other natural compounds in the prevention and treatment of melanoma has been clearly demonstrated in the past, and is an exciting source for potential therapies in the future. OBJECTIVE: We sought to review updates in the current literature regarding new developments in the relationship between nutrition and melanoma risk and treatment. METHODS: Articles in the public domain regarding the impact of diet, grape seed proanthocyanidins, selenium, vitamin D, vitamin E, epigallocatechin-3-gallate, resveratrol, rosmarinic acid, lycopene, and fig latex on melanoma were included. RESULTS: Grape seed proanthocyanidins, epigallocatechin-3-gallate, resveratrol, rosmarinic acid, lycopene, and fig latex have demonstrated clear anticancer effects toward melanoma. The roles of selenium, vitamin D, and vitamin E, however, have been more controversial. LIMITATIONS: None. CONCLUSIONS: The role of natural compounds in the future of melanoma prevention and treatment is promising and one that is worthy of further exploration.

Aspirin use and melanoma risk: a review of the literature.

In view of the increasing incidence of melanoma, it is critical to find effective preventive approaches. Contradictory evidence has been reported with regard to the possible association of aspirin use and the risk of melanoma. We review these studies and seek to elucidate the mechanism by which aspirin may produce a chemoprotective effect against melanoma.

An unusual presentation and distribution of generalized eruptive syringomas.

A 19-year-old Caucasian man presented with numerous erythematous to flesh-colored papules that appeared in crops on his neck, axillae, buttocks, and lower back. The lesions started on his anterior neck at age 12. At 18 years, new crops of papules appeared on his axillae, back, and buttocks over several months. He reported pruritus in the lesions following exercise and perspiration. He denied any family history of similar lesions. His primary care physician treated him with topical triamcinolone 0.1% cream, which made the lesions smaller, less erythematous, and less pruritic; however, the papules never fully resolved. After discontinuation of the steroids, these erythematous pruritic papules gradually recurred in the same areas of his body. The patient denied any other medical complaints.

Application of creatinine height index in patients with trauma for the evaluation of psoas muscle mass: A clinical validation study.

BACKGROUND: The creatinine height index (CHI) is an estimate of lean body mass. We hypothesize that a modified CHI estimate using serum creatinine (sCr) levels in patients with normal renal function when performed soon after injury would reflect preinjury protein nutrition status. METHODS: The urine CHI (uCHI) was calculated using the 24-h urine sample. The serum-derived estimated CHI (sCHI) was calculated using the sCr on admission. Correlation between abdominal computed tomography images at specific lumbar vertebral levels and total body fat and muscle content was used for comparison as an independent measurement of nutrition status unlikely to be substantially altered by trauma. RESULTS: A total of 45 patients were enrolled, all with a significant injury burden (median injury severity score [ISS] = 25; interquartile range, 17-35). The calculated sCHI on admission was 71.0% (SD = 26.9%) and likely underestimates the CHI when compared with uCHI (mean = 112.5%, SD = 32.6%). Stratifying by degree of stress demonstrated that in a group of 23 moderately and severely stressed patients, uCHI (mean = 112.7%, SD = 5.7%) and sCHI (mean = 60.8%, SD = 1.9%) were significantly different and without correlation (r = -0.26, P = 0.91). In patients without stress, there was a significant negative correlation between sCHI and psoas muscle area (r = -0.869, P = 0.03), and in patients with severe stress there was a significant positive correlation between uCHI and psoas muscle area (r = 0.733, P = 0.016). CONCLUSION: The CHI calculated from the initial sCr is not an appropriate estimate of uCHI in critically ill trauma patients and is not a valid measure of psoas muscle mass in this setting.

Patients' expectations of screening and preventive treatments.

PURPOSE: An informed decision to accept a health care intervention requires an understanding of its likely benefit. This study assessed participants' estimates of the benefit, as well as minimum acceptable benefit, of screening for breast and bowel cancer and medication to prevent hip fracture and cardiovascular disease. METHODS: Three general practitioners sent questionnaires to all registered patients aged 50 to 70 years. Patients agreeing to participate in the study were asked to estimate the number of events (fractures or deaths) prevented in a group of 5,000 patients undergoing each intervention over a period of 10 years, and to indicate the minimum number of events avoided by the intervention that they considered justified its use. The proportions of participants that overestimated each intervention's benefit were calculated, and univariate and multivariable analyses of predictors of response were performed. RESULTS: The participation rate was 36%: 977 patients were invited to participate in the study, and 354 returned a completed questionnaire. Participants overestimated the degree of benefit conferred by all interventions: 90% of participants overestimated the effect of breast cancer screening, 94% overestimated the effect of bowel cancer screening, 82% overestimated the effect of hip fracture preventive medication, and 69% overestimated the effect of preventive medication for cardiovascular disease. Estimates of minimum acceptable benefit were more conservative, but other than for cardiovascular disease mortality prevention, most respondents indicated a minimum benefit greater than these interventions achieve. A lower level of education was associated with higher estimates of minimum acceptable benefit for all interventions. CONCLUSION: Patients overestimated the risk reduction achieved with 4 examples of screening and preventive medications. A lower level of education was associated with higher minimum benefit to justify intervention use. This tendency to overestimate benefits may affect patients' decisions to use such interventions, and practitioners should be aware of this tendency when discussing these interventions with patients.

Drug evaluation in cardiomyocytes derived from human induced pluripotent stem cells carrying a long QT syndrome type 2 mutation.

AIMS: Congenital long QT syndromes (LQTSs) are associated with prolonged ventricular repolarization and sudden cardiac death. Limitations to existing clinical therapeutic management strategies prompted us to develop a novel human in vitro drug-evaluation system for LQTS type 2 (LQT2) that will complement the existing in vitro and in vivo models. METHODS AND RESULTS: Skin fibroblasts from a patient with a KCNH2 G1681A mutation (encodes I(Kr) potassium ion channel) were reprogrammed to human induced pluripotent stem cells (hiPSCs), which were subsequently differentiated to functional cardiomyocytes. Relative to controls (including the patient's mother), multi-electrode array and patch-clamp electrophysiology of LQT2-hiPSC cardiomyocytes showed prolonged field/action potential duration. When LQT2-hiPSC cardiomyocytes were exposed to E4031 (an I(Kr) blocker), arrhythmias developed and these presented as early after depolarizations (EADs) in the action potentials. In contrast to control cardiomyocytes, LQT2-hiPSC cardiomyocytes also developed EADs when challenged with the clinically used stressor, isoprenaline. This effect was reversed by ß-blockers, propranolol, and nadolol, the latter being used for the patient's therapy. Treatment of cardiomyocytes with experimental potassium channel enhancers, nicorandil and PD118057, caused action potential shortening and in some cases could abolish EADs. Notably, combined treatment with isoprenaline (enhancers/isoprenaline) caused EADs, but this effect was reversed by nadolol. CONCLUSIONS: Findings from this paper demonstrate that patient LQT2-hiPSC cardiomyocytes respond appropriately to clinically relevant pharmacology and will be a valuable human in vitro model for testing experimental drug combinations.

Early metabolic support for critically ill trauma patients: A prospective randomized controlled trial.

BACKGROUND: There is a lack of consensus regarding the optimal nutritional support for trauma patients. We hypothesize that early postinjury metabolic support focusing on adequate protein would modify the metabolic signature and alter the inflammatory environment for critically ill trauma patients. METHODS: We conducted a prospective randomized controlled pilot trial for adult patients admitted to the surgical intensive care unit following traumatic injury. Patients were randomized to receive early metabolic support (EMS) (peripheral amino acid infusions) or standard of care (enteral nutrition as soon as feasible). Routine laboratory assessments, nitrogen balance, cytokines, and metabolomic analyses were assessed at baseline and day 5 after intervention. RESULTS: A total of 42 trauma patients were randomized into well-balanced groups with similar age (32 years), Injury Severity Score (25), and body mass index (27.4 kg/m2). Early metabolic support provided significantly more protein (1.43 g/kg vs. 0.35 g/kg; p < 0.0001) and more calories (12.6 kcal/kg vs. 7.5 g/kg; p = 0.0012) over the first 5 days as compared with the standard of care. Early metabolic support modified protein catabolism and synthesis as demonstrated by a larger median negative nitrogen balance (-16.3 g vs. -5.3 g; p = 0.03) and a unique metabolomic profile at day 5. The biochemical profile of patients who received EMS was defined by greater declines in circulating levels of stress hormone precursors and increased levels of amino acids. The inflammatory response following EMS resulted in a greater decrease in interleukin-1B (p = 0.02) and increase in soluble interleukin-6 receptor (p = 0.01) between baseline and day 5 as compared with the standard of care. The EMS group had a decreased length of stay (15 vs. 22 days) and decreased surgical intensive care unit length of stay (8 vs. 9 days); however, this disappeared after adjustment for Injury Severity Score in this small population. CONCLUSIONS: Early metabolic support with amino acid is safe, modifies metabolism, and may downregulate the inflammatory state associated with significant trauma, warranting a larger trial to assess for improved outcomes. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level II.

A case of lichen planus following Tdap vaccination.

Lichen planus (LP) is an inflammatory dermatosis of unknown etiology that is primarily associated with liver disease. Recently, there have been several reports of LP developing after administration of the hepatitis B, influenza and combined MMR-DTaP-IPV vaccines. Here we report the first case of LP developing on the lower extremities of an otherwise healthy adult male after administration of the Tdap vaccine. We present this case to draw awareness to this observation in light of recently updated Tdap vaccination recommendations.

Chemical burn from povidone-iodine: case and review.

Chemical burn is a rare complication of topical polyvinylpyrrolidone-iodine (PVP-I), commonly called povidone-iodine (trade name Betadine, Purdue, Stamford, NJ). This adverse reaction occurred on the buttocks of an eight-year-old male after undergoing a laparoscopic appendectomy involving antiseptic skin preparation using a 10% PVP-I solution. This case is consistent with previous reports in which a chemical burn develops when PVP-I does not adequately dry, pools beneath a dependent body part during surgery, or is placed under an occlusive device. Symptoms develop immediately to one day after surgery. The proposed mechanism is irritation from iodine coupled with maceration, pressure and friction. While patients typically heal without significant scarring, the burn subjects the patient to unnecessary pain, prolongs hospitalization and increases the risk for infection. Physicians should be aware of this complication and therefore take preventative measures. These include allowing PVP-I to completely dry, preventing dripping and pooling and avoiding occlusion.

DNA methylation, insulin resistance, and blood pressure in offspring determined by maternal periconceptional B vitamin and methionine status.

A complex combination of adult health-related disorders can originate from developmental events that occur in utero. The periconceptional period may also be programmable. We report on the effects of restricting the supply of specific B vitamins (i.e., B(12) and folate) and methionine, within normal physiological ranges, from the periconceptional diet of mature female sheep. We hypothesized this would lead to epigenetic modifications to DNA methylation in the preovulatory oocyte and/or preimplantation embryo, with long-term health implications for offspring. DNA methylation is a key epigenetic contributor to maintenance of gene silencing that relies on a dietary supply of methyl groups. We observed no effects on pregnancy establishment or birth weight, but this modest early dietary intervention led to adult offspring that were both heavier and fatter, elicited altered immune responses to antigenic challenge, were insulin-resistant, and had elevated blood pressure-effects that were most obvious in males. The altered methylation status of 4% of 1,400 CpG islands examined by restriction landmark genome scanning in the fetal liver revealed compelling evidence of a widespread epigenetic mechanism associated with this nutritionally programmed effect. Intriguingly, more than half of the affected loci were specific to males. The data provide the first evidence that clinically relevant reductions in specific dietary inputs to the methionine/folate cycles during the periconceptional period can lead to widespread epigenetic alterations to DNA methylation in offspring, and modify adult health-related phenotypes.

N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells.

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells1-4. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m6A-containing R-loops accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle, and that the m6A reader promoting mRNA degradation, YTHDF2 (ref. 7), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m6A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability.