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The UK National Diagnostic Service for Ehlers-Danlos Syndromes (EDS) was established in 2009 for the rare types of EDS. Vascular EDS (vEDS) is an inherited connective tissue disorder caused by pathogenic variants in the COL3A1 gene. Associated tissue fragility affects multiple organ systems, increasing the risk of blood vessel dissection and rupture, with potentially fatal consequences. The diagnosis of vEDS has improved with advances in genetic testing, however this is most often suspected following an acute event. We provide data on the clinical features of vEDS for 180 patients (full cohort) seen in our service with confirmed molecular diagnoses. Increased awareness of this rare condition will prompt genetic testing essential to confirm the diagnosis. Outcomes are improved by early diagnosis followed by appropriate management. Fragile connective tissues make invasive procedures potentially dangerous, particularly in an emergency setting. Lifestyle advice from a young age can help acceptance and understanding of the diagnosis and inform choices. There is currently limited evidence for the use of drug therapy to reduce vascular events. We report on the incidence of vascular events in 126 patients (statistical analysis cohort) in our care and the use of medication. Our retrospective data showed that those patients on a long-term angiotensin II receptor blocker and/or beta-blocker had fewer vascular events than those not on cardiac medication who received the same lifestyle and emergency care advice.

Background/ObjectivesIn England, children (0–18 years) with severe, complex and atypical osteogenesis imperfecta (OI) are managed by four centres (Birmingham, Bristol, London, Sheffield) in a ‘Highly Specialised Service’ (HSS OI); affected children with a genetic origin for their disease that is not in COL1A1 or COL1A2 form the majority of the ‘atypical’ group, which has set criteria for entry into the service. We have used the data from the service to assess the range and frequency of non-collagen pathogenic variants resulting in OI in a single country.MethodsChildren with atypical OI were identified through the HSS OI service database. All genetic testing for children with OI in the service were undertaken at the Sheffield Diagnostic Genetics Service. Variant data were extracted and matched to individual patients. This study was done as part of a service evaluation project registered with the Sheffield Children’s Hospital Clinical Governance Department.ResultsOne hundred of 337 children in the HSS met the ‘atypical’ criteria. Eighty have had genetic testing undertaken; 72 had genetic changes detected, 67 in 13 genes known to be causative for OI. The most frequently affected genes were IFITM5 (22), P3H1 (12), SERPINF1 (8) and BMP1 (6).ConclusionAmong children with more severe forms of OI (approximately one-third of all children with OI), around 20% have pathogenic variants in non-collagen genes. IFITM5 was the most commonly affected gene, followed by genes within the P3H1 complex. These data provide additional information regarding the likelihood of different genetic origins of the disease in children with OI, which may influence clinical care.

Human pluripotent stem cells (hPSCs) can be grown in culture indefinitely, making them a valuable tool for use in basic biology, disease modeling, and regenerative medicine. However, over prolonged periods in culture, hPSCs tend to acquire genomic aberrations that confer growth advantages, similar to those seen in some cancers. Monitoring the genomic stability of cultured hPSCs is critical to ensuring their efficacy and safety as a therapeutic tool. Most commonly employed methods for monitoring of hPSC genomes are cytogenetic methods, such as G-banding. Nonetheless, such methods have limited resolution and sensitivity for detecting mosaicism. Single nucleotide polymorphism (SNP) array platforms are a potential alternative that could improve detection of abnormalities. Here, we outline protocols for SNP array whole-genome screening of hPSCs. Moreover, we detail the procedure for assessing the SNP array's sensitivity in detecting low-level mosaic copy-number changes. We show that mosaicism can be confidently identified in samples only once they contain 20% variants, although samples containing 10% variants typically display enough variation to warrant further investigation and confirmation, for example by using a more sensitive targeted method. Finally, we highlight the advantages and limitations of SNP arrays, including a cost comparison of SNP arrays versus other commonly employed methods for detection of genetic changes in hPSC cultures. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA sample preparation for SNP arrays Basic Protocol 2: SNP array hybridization, washing, and scanning Basic Protocol 3: SNP array data analysis Support Protocol: Assessment of SNP array sensitivity for detection of mosaicism.

Purpose Currently, 31 patients with classical-like EDS (clEDS) due to tenascin-X deficiency have been reported in the literature. We report on the clinical and molecular characteristics of 20 additional patients with clEDS to expand knowledge and to enable improved management of this rare genetic disorder. Methods Patients diagnosed with clEDS by the national EDS service in the UK (n = 21) and abroad (n = 1) were asked for consent for publication of their clinical and molecular data. Results Of 22 patients, 20 consented. All patients had typical features of clEDS: joint hypermobility, easy bruising, and skin hyperextensibility without atrophic scars. Importantly, 3/20 patients experienced gastrointestinal complications consisting of small or large bowel ruptures and one esophageal rupture. Other notable observations included two separate occurrences of spontaneous compartment syndrome, suspicion of nonaccidental injury due to significant bruising, and significant clinical variability regarding the debilitating effect of joint dislocations. Conclusions We propose a predisposition to tissue fragility, particularly of the gastrointestinal tract in patients with clEDS. As such, clinical and molecular confirmation of this diagnosis is essential. It is recommended to follow up these patients closely to understand the natural history to develop better recommendations for management.

BackgroundThe Ehlers-Danlos syndromes (EDS) are a group of connective tissue disorders with several recognised types. Patients with a type of EDS have connective tissue abnormalities resulting in a varying degree of joint hypermobility, skin and vascular fragility and generalised tissue friability. Classical EDS (cEDS) typically occurs as a result of dominant pathogenic variants in COL5A1 or COL5A2. The cardinal features of cEDS are hyperextensible skin, atrophic scarring and joint hypermobility. Arterial complications are more characteristically a feature of vascular EDS although individual cases of arterial events in cEDS have been reported.MethodsA cohort of 154 patients with a clinical diagnosis of cEDS from the UK was analysed.ResultsSeven patients (4.5%) with a diagnosis of cEDS (four pathogenic, one likely pathogenic and two variants of uncertain significance in COL5A1) who had experienced arterial complications were identified. Arterial complications mostly involved medium-sized vessels and also two abdominal aortic aneurysms. No unique clinical features were identified in this group of patients.ConclusionThere is a possible increased risk of arterial complications in patients with cEDS, although not well-defined. Clinicians need to be aware of this possibility when presented with a patient with an arterial complication and features of cEDS. Long-term management in families with cEDS and a vascular complication should be individually tailored to the patient’s history and their family’s history of vascular events.

Arthrochalasia Ehlers-Danlos syndrome (aEDS) is a rare autosomal dominant connective tissue disorder that is characterized by congenital bilateral hip dislocations, severe generalized joint hypermobility, recurrent joint (sub)luxations, and skin hyperextensibility. To date, 42 patients with aEDS have been published. We report 12 patients with aEDS from 10 families with 6 unpublished individuals and follow-up data on 6 adult patients. The clinical features are largely comparable with patients reported in the literature. Most (n = 10) patients had variants leading to (partial) loss of exon 6 of the COL1A1 or COL1A2 genes. One patient did not have a previously reported likely pathogenic COL1A1 variant. Data regarding management were retrieved. Hip surgery was performed in 5/12 patients and 3/12 patients underwent spinal surgery. As much as 4/12 patients were wheelchair-bound or unable to walk unaided. Fractures were present in 9/12 individuals with 1 patient requiring bisphosphonate treatment. Echocardiograms were performed in 10 patients and 2 individuals showed an abnormality likely unrelated to aEDS. One patient gave birth to two affected children and went through preterm labor requiring medication but had no additional complications. Of the eight adults in our cohort, the majority entered a career. Our data point toward a genotype-phenotype relationship with individuals with aEDS due to pathogenic COL1A1 variants causing complete or partial loss of exon 6 being more severely affected regarding musculoskeletal features. There is a significant lack of knowledge with regard to management of aEDS, particularly in adulthood. As such, systematic follow-up and multidisciplinary treatment is essential.

The presence of genetic changes in human pluripotent stem cells (hPSCs) can affect their behavior and impact on the utility of hPSC-based applications in research and clinic. The spectrum of spontaneously arising genetic abnormalities in hPSCs is wide and ranges from numerical and structural chromosomal anomalies down to point mutations. The detection of genetic changes in hPSCs is confounded by the fact that no single method detects all types of abnormalities with the same accuracy and sensitivity, therefore necessitating the use of a combination of different methods. Here, we provide detailed protocols for two methods commonly utilized for the detection of genetic changes in naïve and primed hPSCs: karyotyping by G-banding and fluorescent in situ hybridization (FISH).

Vascular Ehlers-Danlos Syndrome (vEDS) and Osteogenesis Imperfecta (OI) are two forms of connective tissue disorders. Previously, transmission electron microscopy of skin biopsies was routinely performed on all patients who were clinically suspected to have vEDS. At present, molecular genetics using genomic DNA extracted from a blood sample is the first line investigation for these patients. However, when variants of uncertain clinical significance are identified on genetic testing and individuals do not have the classical features of OI or vEDS, additional phenotypic information obtained from a skin biopsy can be valuable for contributing to the evidence for re-classifying pathogenicity of variants.We present a cohort of six patients with molecularly confirmed vEDS and one patient with a severe form of OI, who each had expanded (or dilated), protein-filled, rough endoplasmic reticulum identified on transmission electron microscopy. The patients were identified through retrospective screening of medical records, and biopsies were taken between 1999-2016. We discuss the potential role for assessing rough endoplasmic reticulum expansion as a useful tool to allow further phenotyping of these individuals.

The Ehlers–Danlos syndromes (EDS) are a group of rare inherited connective tissue disorders. Vascular EDS (vEDS) is caused by pathogenic variants in COL3A1, most frequently glycine substitutions. We describe the phenotype of the largest series of vEDS patients with glutamic acid to lysine substitutions (Glu>Lys) in COL3A1, which were all previously considered to be variants of unknown significance. Clinical and molecular data for seven families with three different Glu>Lys substitutions in COL3A1 were analyzed. These Glu>Lys variants were reclassified from variants of unknown significance to either pathogenic or likely pathogenic in accordance with American College of Medical Genetics and Genomics guidelines. All individuals with these atypical variants exhibited skin hyperextensibility as seen in individuals with classical EDS and classical-like EDS and evidence of tissue fragility as seen in individuals with vEDS. The clinical data demonstrate the overlap between the different EDS subtypes and underline the importance of next-generation sequencing gene panel analysis. The three different Glu>Lys variants point toward a new variant type in COL3A1 causative of vEDS, which has consistent clinical features. This is important knowledge for COL3A1 variant interpretation. Further follow-up data are required to establish the severity of tissue fragility complications compared with patients with other recognized molecular causes of vEDS.

Secreted protein, acidic, cysteine rich (We describe a further two patients with previously unreported homozygousFrom the data we have obtained, common clinical features in individuals with OI type XVII caused byCommon phenotypic expressions include delayed motor development with neuromuscular weakness, scoliosis and multiple fractures. The data presented here broaden the phenotypic spectrum establishing similar patterns of neuromuscular presentation with a presumed diagnosis of 'myopathy'.

Copy number variants (CNVs) are genomic rearrangements implicated in numerous congenital and acquired diseases, including cancer. The appearance of culture-acquired CNVs in human pluripotent stem cells (PSC) has prompted concerns for their use in regenerative medicine applications. A particular problem in PSC is the frequent occurrence of CNVs in the q11.21 region of chromosome 20. However, the exact mechanism of origin of this amplicon remains elusive due to the difficulty in delineating its sequence and breakpoints. Here, we have addressed this problem using long-read Nanopore sequencing of two examples of this CNV, present as a duplication and as a triplication. In both cases, the CNVs were arranged in a head-to-tail orientation, with microhomology sequences flanking or overlapping the proximal and distal breakpoints. These breakpoint signatures point to a mechanism of microhomology-mediated break-induced replication in CNV formation, with surrounding Alu sequences likely contributing to the instability of this genomic region.

Copy number variants (CNVs) are genomic rearrangements implicated in numerous congenital and acquired diseases, including cancer. In human pluripotent stem cells (PSC), the appearance of culture-acquired CNVs prompted concerns for their use in regenerative medicine applications. A particularly common problem in PSC is the occurrence of CNVs in the q11.21 region of chromosome 20. However, the exact mechanisms of origin of this amplicon remains elusive due to the difficulty in delineating its sequence and breakpoints. Here, we used long-range Nanopore sequencing on two examples of this CNV, present as a duplication in one and a triplication in another line. The CNVs were arranged in a head-to-tail orientation in both lines, with sequences of microhomologies flanking or overlapping both the proximal and distal breakpoints. These breakpoint signatures point to a specific mechanism of template switching in CNV formation, with surroundingAlusequences likely contributing to the instability of this genomic region.

For more than 25 years Anne Lacaton and Jean Philippe Vassal have been practising together from their studio in Paris. They have a pragmatic approach towards issues of climate change and sustainability as a whole, never relying on expensive technological solutions, rather considering challenges in a genuinely holistic manner. They are, however, perhaps best known for having an acute awareness of how to make generous, beautiful spaces affordable: they make clients’ money go further than most. Lacaton and Vassal’s challenge was to complete three-quarters of the works with one-quarter of the original budget. Lacaton and Vassal have an extensive knowledge of construction materials and systems new and old, and they understand where to apply additional fabric and when to leave it alone. By undertaking research at an almost forensic level, such as working with the original architect on the Tour Bois-le-Pretre, Lacaton and Vassal unearth unrealised potentials.

The University of East Anglia (UEA) was established in 1963. Architype has a long-established record of delivering buildings with authentic ‘green’ credentials and is perhaps the UK’s most successful exponent of sustainable design solutions. The team behind the project took advantage of this unusual commission to test the viability of constructing a large university office building using locally sourced materials, whether that meant organic and grown, secondhand or material from local waste streams. The team also specified the reuse of local second-hand materials discarded by others, and complemented this with a policy of using local tradespeople and suppliers whenever possible. Architype located other local material sources for the project. The Enterprise Centre at UEA will, over its 100-year life, treat the planet with more kindness than most buildings occupied in the UK and beyond.

Professor Dirk Hebel, Assistant Professor of Architecture and Construction at ETH Zurich, has been pushing the boundaries of architectural teaching. His work considers ways to ‘activate’ unusual building materials, which over the years have included air, water, bamboo, and, most recently, locating sources of waste material. The project, in partnership with housing cooperative GMBZ, involves the design and construction of 140 apartments plus a kindergarten, common areas for residents and a shared laundry. The project highlights that many natural resources are becoming increasingly scarce, even aggregates such as sand and gravel for concrete production. It also focuses on the potentials of other material sources or ‘flows’ that have piled up over centuries – the materials that constitute our towns and cities that can now be conceived as our future ‘mines’.

German-born Cyrill Gutsch set up New-York-based Parley for the Oceans in 2012. By April 2015 Parley announced its first commercial partnership – with sportswear giant Adidas, initially making training shoes out of ocean plastic waste. Parley has attempted to ‘take ownership of the supply chain’ with Adidas. Adidas used the chemistry and innovative manufacturing processes of ‘tailored fibre technology’, which allows a more flexible combination of yarns, fibres and threads. Consumers will hopefully change their behaviour and return their shoes back to Adidas to reprocess, and avoid the product becoming waste. Parley creates products with a clear narrative; a story to tell that is enticing and intriguing. Its products allow consumers to learn as much as they care to about the environmental issues associated with the consumable, while knowing that they are 'doing their bit' for the environment by choosing Adidas and Parley training shoes over 'normal' ones.

Interface is a large multinational company that has been producing carpet tiles for the interior design industry since 1973. Since 1994 the company has been considering how to make the process of producing carpet tiles less harmful to the natural environment. The World Business Council for Sustainable Development had been working on ‘The Sustainable Livelihoods Business Model’, which broadly supported what we now refer to as ‘inclusive business’. The principle suggested that there is a way of connecting some of the poorest people in the world to our supply chain in a transparent and fair way so that we and our supply chain all benefit in the long term.

Every day around 5 million tonnes of waste is generated globally. Designing out waste can, in the process, eliminate greedy consumption patterns, prevent untold pollution, lower carbon emissions and enable greater equality locally and globally. Waste is a result of misappropriated scientific and business ingenuity that’s focused on product creation and has not been held responsible for long-term impacts of all those products, of six-plus decades of instant gratification, of an advertising industry heralding the ownership of more stuff as a barometer of status and pride. The UK’s Wood Recycling Network diverted 8,500 tonnes of wood from the waste stream in 2012. The UK generated 200 million tonnes of waste in 2012. Shockingly, 3.5 billion people have no or very poor waste management infrastructure, which leads to further poisoning of their soil, air and water, especially because increasingly discards are petroleum-based products – plastics.

In 2011 a group of architects working for the Danish practice Schmidt Hammer Lassen Architects enrolled on a week-long course arranged by ‘Vugge til Vugge Denmark’, the Danish representative for the Environment Protection Encouragement Agency. Planning approval has not yet been granted by city planning authorities in Oslo because of issues relating to the overall development of the urban area. Recycling or reusing 90% of material removed from the existing building is ambitious enough. Creating networks with suppliers and contractors to ensure that 80% of this product ends up back on the site it was taken from is even more ambitious. The team at Schmidt Hammer Lassen Architects were committed to exploring ‘Cradle to Cradle (C2C)’ principles further and applying them to their own architectural projects. To summarise the team’s list of C2C strategies, some materials will be directly reused, but most salvaged materials will be recycled and then returned to site as ‘new products’.

Silo, in Brighton, was conceived in 2014 as a direct response to the huge amount of waste involved in the production and consumption of food around the world. Founder Douglas McMaster looks for alternative sources of nutritious foodstuffs from varied local and regional sources. Silo doesn’t accept plastic or non-biodegradable packaging from its suppliers, and reduces road, sea and air miles associated with the transporting of food by growing as much as it can on site. Tables are made from galvanised steel ‘tiles’, formerly a raised floor in a commercial office space. There is a bit of reprocessing involved in the manufacture of Silo’s plates: they are formed from old plastic bags and surprisingly look great and function perfectly. Described by McMaster as supporting ‘a pre-industrial food system’, Silo aspires to reacquaint us with sources of foodstuff that have been neglected for centuries or longer.

Summary Genetic changes in human pluripotent stem cells (hPSCs) gained during culture can confound experimental results and potentially jeopardize the outcome of clinical therapies. Particularly common changes in hPSCs are trisomies of chromosomes 1, 12, 17, and 20. Thus, hPSCs should be regularly screened for such aberrations. Although a number of methods are used to assess hPSC genotypes, there has been no systematic evaluation of the sensitivity of the commonly used techniques in detecting low-level mosaicism in hPSC cultures. We have performed mixing experiments to mimic the naturally occurring mosaicism and have assessed the sensitivity of chromosome banding, qPCR, fluorescence in situ hybridization, and digital droplet PCR in detecting variants. Our analysis highlights the limits of mosaicism detection by the commonly employed methods, a pivotal requirement for interpreting the genetic status of hPSCs and for setting standards for safe applications of hPSCs in regenerative medicine., Highlights • hPSCs conform to random sampling rules used for karyotyping • Excluding mosaicism at 500 metaphases • qPCR is a rapid assay for detection of commonly amplified regions in hPSCs • Cultures scored as normal by commonly used methods could harbor up to 10% variants, Barbaric and colleagues tested how many metaphases need to be scored to detect different levels of mosaicism in hPSC cultures. They also devised qPCR assays as a rapid means of detecting common chromosomal abnormalities. Testing of the sensitivity of qPCR, digital droplet PCR, and FISH revealed that these methods can miss as many as 10% abnormal cells in the population.

Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation.

Genomic abnormalities may accumulate in human embryonic stem cells (hESCs) during in vitro maintenance. Characterization of the mechanisms enabling survival and expansion of abnormal hESCs is important due to consequences of genetic changes for the therapeutic utilization of stem cells. Furthermore, these cells provide an excellent model to study transformation in vitro. We report here that the histone deacetylase proteins, HDAC1 and HDAC2, are increased in karyotypically abnormal hESCs when compared to their normal counterparts. Importantly, similar to many cancer cell lines, we found that HDAC inhibitors repress proliferation of the karyotypically abnormal hESCs, whereas normal cells are more resistant to the treatment. The decreased proliferation correlates with downregulation of HDAC1 and HDAC2 proteins, induction of the proliferation inhibitor, cyclin-dependent kinase inhibitor 1A (CDKN1A), and altered regulation of tumor suppressor protein Retinoblastoma 1 (RB1). Through genome-wide transcriptome analysis we have identified genes with altered expression and responsiveness to HDAC inhibition in abnormal cells. Most of these genes are linked to severe developmental and neurological diseases and cancers. Our results highlight the importance of epigenetic mechanisms in the regulation of genomic stability of hESCs, and provide valuable candidates for targeted and selective growth inhibition of karyotypically abnormal cells.

Summary Human embryonic stem cells (hESCs) regularly acquire nonrandom genomic aberrations during culture, raising concerns about their safe therapeutic application. The International Stem Cell Initiative identified a copy number variant (CNV) amplification of chromosome 20q11.21 in 25% of hESC lines displaying a normal karyotype. By comparing four cell lines paired for the presence or absence of this CNV, we show that those containing this amplicon have higher population doubling rates, attributable to enhanced cell survival through resistance to apoptosis. Of the three genes encoded within the minimal amplicon and expressed in hESCs, only overexpression of BCL2L1 (BCL-XL isoform) provides control cells with growth characteristics similar to those of CNV-containing cells, whereas inhibition of BCL-XL suppresses the growth advantage of CNV cells, establishing BCL2L1 as a driver mutation. Amplification of the 20q11.21 region is also detectable in human embryonal carcinoma cell lines and some teratocarcinomas, linking this mutation with malignant transformation., Graphical Abstract, Highlights • The presence of the 20q11.21 CNV protects hESCs against apoptosis • 20q11.21 CNV cells have increased levels of antiapoptotic BCL-XL, driving selection • hECCs and primary embryonal carcinoma samples also display the 20q11.21 CNV • 20q11.21 CNV could be a feature of neoplastic progression, Avery and colleagues report that BCL2L1 (gene product BCL-XL) is the driver mutation for the copy number variant (CNV) amplification of chromosome 20q11.21 (present in 25% of normal-karyotype hESC lines). CNV cells exhibit enhanced cell survival through BCL-XL-associated resistance to apoptosis and rapidly outcompete nonmutant cells. Routine screening for this mutation should be considered for hESCs to be used in therapy.

Genomic integrity of human pluripotent stem cell (hPSC) lines requires routine monitoring. We report here that novel karyotyping assay, utilizing bead-bound bacterial artificial chromosome probes, provides a fast and easy tool for detection of chromosomal abnormalities in hPSC lines. The analysis can be performed from low amounts of DNA isolated from whole cell pools with simple data analysis interface. The method enables routine screening of stem cell lines in a cost-efficient high-throughput manner., Highlights ► We report a novel high-throughput karyotyping assay for human pluripotent cells. ► This assay provides fast and easy tool for detection of chromosomal abnormalities. ► The analysis can be run from low DNA amounts with simple data analysis interface. ► The results are in good concordance with the data from G-banding and SNP arrays. ► The assay enables routine screening of stem cell lines in a cost-efficient manner.

Understanding the complex mechanisms that govern the fate decisions of human embryonic stem cells (hESCs) is fundamental to their use in cell replacement therapies. The progress of dissecting these mechanisms will be facilitated by the availability of robust high-throughput screening assays on hESCs. In this study, we report an image-based high-content assay for detecting compounds that affect hESC survival or pluripotency. Our assay was designed to detect changes in the phenotype of hESC colonies by quantifying multiple parameters, including the number of cells in a colony, colony area and shape, intensity of nuclear staining, and the percentage of cells in the colony that express a marker of pluripotency (TRA-1-60), as well as the number of colonies per well. We used this assay to screen 1040 compounds from two commercial compound libraries, and identified 17 that promoted differentiation, as well as 5 that promoted survival of hESCs. Among the novel small compounds we identified with activity on hESC are several steroids that promote hESC differentiation and the antihypertensive drug, pinacidil, which affects hESC survival. The analysis of overlapping targets of pinacidil and the other survival compounds revealed that activity of PRK2, ROCK, MNK1, RSK1, and MSK1 kinases may contribute to the survival of hESCs.

Prolonged culture of human embryonic stem cells (hESCs) can lead to adaptation and the acquisition of chromosomal abnormalities, underscoring the need for rigorous genetic analysis of these cells. Here we report the highest-resolution study of hESCs to date using an Affymetrix SNP 6.0 array containing 906,600 probes for single nucleotide polymorphisms (SNPs) and 946,000 probes for copy number variations (CNVs). Analysis of 17 different hESC lines maintained in different laboratories identified 843 CNVs of 50 kb-3 Mb in size. We identified, on average, 24% of the loss of heterozygosity (LOH) sites and 66% of the CNVs changed in culture between early and late passages of the same lines. Thirty percent of the genes detected within CNV sites had altered expression compared to samples with normal copy number states, of which >44% were functionally linked to cancer. Furthermore, LOH of the q arm of chromosome 16, which has not been observed previously in hESCs, was detected.

background: Both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) bear a great potential in regenerative medicine. In addition to optimized clinical grade culture conditions, efficient clinical grade cryopreservation methods for these cells are needed. Obtaining good survival after thawing has been problematic. methods: We used a novel, chemically defined effective xeno-free cryopreservation system for cryostorage and banking of hESCs and iPSCs. The earlier established slow freezing protocols have, even after recent improvements, resulted in low viability and thawed cells had a high tendency to differentiate. The medium is a completely serum and animal substance free product containing dimethylsulfoxide, anhydrous dextrose and a polymer as cryoprotectants. The cells were directly frozen at 2708C, without a programmed freezer. results: The number of frozen colonies versus the number of surviving colonies differed significantly for both HS293 (x 2 ¼ 9.616 with one degree of freedom and two-tailed P ¼ 0.0019) and HS306 (x 2 ¼ 8.801 with one degree of freedom and two-tailed P ¼ 0.0030). After thawing, the cells had a high viability (90– 96%) without any impact on proliferation and differentiation, compared with the standard freezing procedure where viability was much lower (49%). The frozen –thawed hESCs and iPSCs had normal karyotype and maintained properties of pluripotent cells with corresponding morphological characteristics, and expressed pluripotency markers after 10 passages in culture. They formed teratomas containing tissue components of the three germ layers. conclusion: The defined freezing –thawing system described here offers an excellent simple option for banking of hESCs and iPSCs.

The long-term culture of human embryonic stem (ES) cells is inevitably subject to evolution, since any mutant that arises with a growth advantage will be selectively amplified. However, the evolutionary influences of population size, mutation rate, and selection pressure are frequently overlooked. We have constructed a Monte Carlo simulation model to predict how changes in these factors can influence the appearance and spread of mutant ES cells, and verified its applicability by comparison with in vitro data. This simulation provides an estimate for the expected rate of generation of culture-adapted ES cells under different assumptions for the key parameters. In particular, it highlights the effect of population size, suggesting that the maintenance of cells in small populations reduces the likelihood that abnormal cultures will develop.