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. N6-methyladenosine (m6A) is prevalent at RNA:DNA hybrids in human pluripotent stem cells. The m6A reader YTHDF2 interacts with R-loop-enriched loci in dividing cells, and YTHDF2 loss leads to increased R-loop levels and accumulation of γH2AX.

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.

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.

Improved biomaterials are required for application in regenerative medicine, biosensing, and as medical devices. The response of cells to the chemistry of polymers cultured in media is generally regarded as being dominated by proteins adsorbed to the surface. Here we use mass spectrometry to identify proteins adsorbed from a complex mouse embryonic fibroblast (MEF) conditioned medium found to support pluripotent human embryonic stem cell (hESC) expansion on a plasma etched tissue culture polystyrene surface. A total of 71 proteins were identified, of which 14 uniquely correlated with the surface on which pluripotent stem cell expansion was achieved. We have developed a microarray combinatorial protein spotting approach to test the potential of these 14 proteins to support expansion of a hESC cell line (HUES-7) and a human induced pluripotent stem cell line (ReBl-PAT) on a novel polymer (N-(4-Hydroxyphenyl) methacrylamide). These proteins were spotted to form a primary array yielding several protein mixture 'hits' that enhanced cell attachment to the polymer. A second array was generated to test the function of a refined set of protein mixtures. We found that a combination of heat shock protein 90 and heat shock protein-1 encourage elevated adherence of pluripotent stem cells at a level comparable to fibronectin pre-treatment.

In 1980 Ludwig et. al. was first to coin the term non-alcoholic steatohepatitis (NASH) to describe a syndrome in morbidly obese females with type 2 diabetes mellitus (DM) and no history of alcohol use, in whom the hepatic histology was consistent with alcoholic hepatitis. There are, however, other secondary causes of non-alcoholic fatty liver (NAFL), which can develop into non-alcoholic steatohepatitis (NASH), recognized in the years since, such as dramatic rapid weight loss and malnutrition. A number of cases have described either deterioration of NASH or development of NAFL within 2 years after gastric bypass surgery (GBS). However, there are no case reports describing NAFL as a long term complication of GBS, which is related to malnutrition and protein deficiencies. We report a case of a post GBS patient with malnutrition who developed NAFL, which resolved after nutritional support.

A scalable and cost-effective synthetic polymer substrate that supports robust expansion and subsequent multilineage differentiation of human pluripotent stem cells (hPSCs) with defined commercial media is presented. This substrate can be applied to common cultureware and used off-the-shelf after long-term storage. Expansion and differentiation of hPSCs are performed entirely on the polymeric surface, enabling the clinical potential of hPSC-derived cells to be realized.

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.

Background: Limited data on nutrition support practices in the hospital setting are available in the country of Vietnam.Methods: From October 2011‐December 2011, a collaboration between investigators from the U.S. and the Vietnamese National Institute of Nutrition enrolled 72 subjects admitted for elective GI surgery for this observational study at Bach Mai Hospital, a major Hanoi teaching hospital. Baseline height, weight, BMI, mid upper arm circumference (MUAC), Subjective Global Assessment (SGA), body weight status, and daily kcal and protein/amino acid intake from oral diet, tube feeding, and IV feedings from admission until discharge were recorded.Results: Mean age of subjects was 56±15 years and length of stay 12±5 days. 50% of subjects scored a B or C (moderate to severe malnutrition) on the SGA. BMI range was 13‐28; 48% had a BMI < 18.5. MUAC was low normal (24±4 cm). All subjects lost weight during admission. Almost all patients (98%) were fed using parenteral nutrition (PN) postoperatively with oral feeding starting on postoperative day 4. Tube feedings were used in only one patient. Mean daily total calorie intake was 15 kcal/kg/day and protein intake was 0.61g/kg/day. Minimal or no micronutrient supplementation was given to most patients.Conclusions: Study findings support the development of a nutrition intervention with feeding protocols to increase postoperative energy and micronutrient intake.

Objective: To determine the utility of MUAC to screen underweight hospitalized patients in a major Hanoi teaching hospital.Methods: Data from a one‐day study of patients 蠅19 years old admitted at Bach Mai Hospital, Hanoi, Vietnam (2010) were utilized. Data included actual weight and height, MUAC, age, sex, presence of edema, reason for admission, ward, and prior subjective weight loss (major, >10%) over six months. BMI was calculated and underweight defined as BMI < 18.5 (kg/m²; WHO standards). Receiver operating characteristic curve (ROC) analyses was performed.Results: A total of 420 inpatients were studied (51.4 ± 0.8 years old; 177 (42%) female. Patients were admitted for acute (n=174, 41%), chronic (n=186, 44%), elective (n=31, 7%), or other (n=29, 7%) reasons. Mean ± SD for BMI was 20.0 ± 0.2 kg/m² and for MUAC 24.5 ± 0.2 cm. Rates of underweight status, and moderate and severe, or severe thinness were 34%, 17%, and 8%. A total of 22% subjects exhibited edema and 15% reported major weight loss prior to admission. For underweight status, sensitivity of MUAC <23 cm was 71%, specificity 90%, (+) predictive value 79%, and (‐) predictive value 86%. Area Under Curve (AUC) for MUAC<23 cm was 0.81.Conclusion: MUAC <23 cm appears to be a good method to identify undernutrition in hospitalized adults in Vietnam (AUC 0.80‐0.90 is good). Further evaluation of MUAC in larger samples and association with hospital morbidity and mortality in Vietnam are needed.Grant Funding Source: Supported by the Abbott Fund

5-hydroxymethyl-cytosine (5-hmC) is a cytosine modification that is relatively abundant in mammalian pre-implantation embryos and embryonic stem cells (ESC) derived from mammalian blastocysts. Recent observations imply that both 5-hmC and Tet1/2/3 proteins, catalyzing the conversion of 5-methyl-cytosine to 5-hmC, may play an important role in self renewal and differentiation of ESCs. Here we assessed the distribution of 5-hmC in zebrafish and chick embryos and found that, unlike in mammals, 5-hmC is immunochemically undetectable in these systems before the onset of organogenesis. In addition, Tet1/2/3 transcripts are either low or undetectable at corresponding stages of zebrafish development. However, 5-hmC is enriched in later zebrafish and chick embryos and exhibits tissue-specific distribution in adult zebrafish. Our findings show that 5-hmC enrichment of non-committed cells is not a universal feature of vertebrate development and give insights both into evolution of embryonic pluripotency and the potential role of 5-hmC in its regulation.

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. MACS and MiniMACS are registered trademarks or trademarks of Miltenyi Biotec GmbH. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2012 Miltenyi Biotec GmbH. All rights reserved. Excerpt from MACS&more Vol 14 – 2/2012 Isolation of human induced pluripotent stem cell lines

The UK Stem Cell Bank (UKSCB) is located at the National Institute for Biological Standards and Control (NIBSC). NIBSC has a primary remit to develop, establish and perform biological assays for the control and standardisation of biological medicines. To fulfil this activity, NIBSC has a number of programmes where human embryonic stem cell lines (hES) are being adapted for use in functional cell based bioassays. The availability and suitability of hES lines distributed from the UKSCB, for use in these “novel” assays underpins much of this work. Any successful cell bank must address challenges associated with: receipt of new cell lines (possibly requiring non-standard culture conditions), their expansion (to generate sufficient material for generation of master and working cell banks), cell banking with appropriate characterisation and implementation of suitable procedures for further onward distribution of cells. Human ES lines condense a number of these variables into their banking. Human ES cell lines commonly require complex “specialist” culture regimes employing manual cutting of “blocks” of cells from feeder layers. Alternative, enzymatic subculture strategies may be employed, however, they have not been traditionally used by academic or commercial groups deriving hES lines. The UKSCB has considerable expertise in the expansion of a wide range of hES lines from both commercial and academic depositors. This experience has allowed UKSCB to overcome many of the challenges associated with culturing hES lines, whilst maintaining genetic stability and pluripotency. In addition, the UKSCB is able to maintain their deposits within the stringent controls imposed on banking by numerous end user and regulatory demands whilst maintaining cells consistent with the characteristics of the depositor’s original material. Here we review post-thaw viability and the retention of pluripotency from 40 hES lines cryopreserved by either vitrification or two step cryopreservation (10% Me2SO, −1 °C min−1 to −80 °C followed by LN plunge). Cells were assessed for viability, pluripotency, time to first passage and karyology. In addition, batch to batch variation along with possible freeze-recalcitrance were assessed following recovery from original depositors’ cryopreserved material and following in house cryopreservation of expanded hES lines produced as part of the banking activities of the UKSCB. A key observation was a high degree of variability in vitrified material (0–100% post thaw viability as measured by a colony establishing and expanding as a pluripotent hES line). That initial poor vitrification survival (<70%) of hES lines by “non-cryospecialist” depositors was not due to inherent freeze-recalcitrance on the part of the hES line. Instead, cryopreservation post thaw viability (with maintenance of pluripotency) was readily improved following cryopreservation by experienced UKSCB staff (>80%). Post thaw viability, as a measure of expansion and retention of pluripotency was less variable when two step cryopreservation was employed. That adaptation of hES lines to a TrypLE (trypsinisation) passaging had no adverse impact on post thaw viability or on maintenance of pluripotency. Instead trypsinisation of hES lines allowed robust two-step cryopreservation of a wide range of hES with high >85% post thaw viability.

There are two critical stages in the retroviral reprogramming of somatic cells to produce human induced pluripotent stem cell (hiPSC) lines. One is the production of high titer virus required to reprogram somatic cells; the other is identification of true hiPSC colonies from heterogeneous cell populations, and their isolation and expansion to generate a sustainable, pluripotent stem cell line. Here we describe simple, time-saving methods to address the current difficulties at these two critical junctures. First, we have developed a method to increase the number of infectious viral units 600-fold. Second, we have developed a TRA-1-81-based positive selection column method for isolating “true” hiPSCs from the heterogeneous cell populations, which overcomes the labor-intensive and highly subjective method of manual selection of hiPSC colonies. We have used these techniques to produce 8 hiPSC lines from human fibroblasts and we believe that they are of considerable utility to researchers in the hiPSC field.

The protocols described here address methods used in two crucial stages in the retroviral reprogramming of somatic cells to produce human induced pluripotent stem cell (hiPSC) lines. The first is an optimized method for producing lentivirus at an efficiency 600-fold greater than previously published, and it includes conjugation of the lentivirus to streptavidin superparamagnetic particles; this process takes 8 d. The second method enables the isolation of true hiPSCs immediately after somatic cell reprogramming and involves column-based positive selection of cells expressing the pluripotency marker TRA-1-81. This process takes 2 h and, as it is directly compatible with feeder-free culture, the time burden of manually identifying and mechanically propagating hiPSC colonies is reduced drastically. Taken together, these methods accelerate the production of hiPSCs and enable lines to be isolated, expanded to ∼107 cells and cryopreserved within 6–8 weeks.

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 IKr 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 IKr 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 β-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.

The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.

Research into the behavior, efficacy, and biosafety of stem cells with a view to clinical transplantation requires the development of noninvasive methods for in vivo imaging of cells transplanted into animal models. This is particularly relevant for human embryonic stem cells (hESCs), because transplantation of undifferentiated hESCs leads to tumor formation. The present study aimed to monitor hESCs in real time when injected in vivo. hESCs were stably transfected to express luciferase, and luciferase expression was clearly detected in the undifferentiated and differentiated state. When transfected hESCs were injected into chick embryos, bioluminescence could be detected both ex and in ovo. In the SCID mouse model, undifferentiated hESCs were detectable after injection either into the muscle layer of the peritoneum or the kidney capsule. Tumors became detectable between days 10-30, with approximately a 3 log increase in the luminescence signal by day 75. The growth phase occurred earlier in the kidney capsule and then reached a plateau, whilst tumors in the peritoneal wall grew steadily throughout the period analysed. These results show the widespread utility of bioluminescent for in vivo imaging of hESCs in a variety of model systems for preclinical research into regenerative medicine and cancer biology.

Unlike the mouse embryo, where loss of DNA methylation in the embryonic nucleus leaves cleavage stage embryos globally hypomethylated, sheep preimplantation embryos retain high levels of methylation until the blastocyst stage. We have cloned and sequenced sheep Dnmt1 and found it to be highly conserved with both the human and mouse homologues. Furthermore, we observed that the transcript normally expressed in adult somatic tissues is highly abundant in sheep oocytes. Throughout sheep preimplantation development the protein is retained in the cytoplasm whereas Dnmt1 transcript production declines after the embryonic genome activation at the 8-16 cell stage. Attempts to clone oocyte-specific 5' regions of Dnmt1, known to be present in the mouse and human gene, were unsuccessful. However, a novel ovine Dnmt1 exon, theoretically encoding 13 amino acids, was found to be expressed in sheep oocytes, preimplantation embryos and early fetal lineages, but not in the adult tissue. RNAi-mediated knockdown of this novel transcript resulted in embryonic developmental arrest at the late morula stage, suggesting an essential role for this isoform in sheep blastocyst formation.