Publications by authors named "Peter W Andrews"

108 Publications

Human pluripotent stem cells: genetic instability or stability?

Authors:
Peter W Andrews

Regen Med 2021 Mar 2. Epub 2021 Mar 2.

Department of Biomedical Science, Centre for Stem Cell Biology, The University of Sheffield, Sheffield S10 2TN, United Kingdom.

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http://dx.doi.org/10.2217/rme-2021-0013DOI Listing
March 2021

Generation and trapping of a mesoderm biased state of human pluripotency.

Nat Commun 2020 10 5;11(1):4989. Epub 2020 Oct 5.

Stem Cell Laboratory, Department of Cancer Biology, University College London Cancer Institute, 72 Huntley St, London, WC1E 6AG, UK.

We postulate that exit from pluripotency involves intermediates that retain pluripotency while simultaneously exhibiting lineage-bias. Using a MIXL1 reporter, we explore mesoderm lineage-bias within the human pluripotent stem cell compartment. We identify a substate, which at the single cell level coexpresses pluripotent and mesodermal gene expression programmes. Functionally these cells initiate stem cell cultures and exhibit mesodermal bias in differentiation assays. By promoting mesodermal identity through manipulation of WNT signalling while preventing exit from pluripotency using lysophosphatidic acid, we 'trap' and maintain cells in a lineage-biased stem cell state through multiple passages. These cells correspond to a normal state on the differentiation trajectory, the plasticity of which is evidenced by their reacquisition of an unbiased state upon removal of differentiation cues. The use of 'cross-antagonistic' signalling to trap pluripotent stem cell intermediates with different lineage-bias may have general applicability in the efficient production of cells for regenerative medicine.
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http://dx.doi.org/10.1038/s41467-020-18727-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536399PMC
October 2020

Acquired genetic changes in human pluripotent stem cells: origins and consequences.

Nat Rev Mol Cell Biol 2020 12 23;21(12):715-728. Epub 2020 Sep 23.

Centre for Stem Cell Biology, Department of Biomedical Science, The University of Sheffield, Sheffield, UK.

In the 20 years since human embryonic stem cells, and subsequently induced pluripotent stem cells, were first described, it has become apparent that during long-term culture these cells (collectively referred to as 'pluripotent stem cells' (PSCs)) can acquire genetic changes, which commonly include gains or losses of particular chromosomal regions, or mutations in certain cancer-associated genes, especially TP53. Such changes raise concerns for the safety of PSC-derived cellular therapies for regenerative medicine. Although acquired genetic changes may not be present in a cell line at the start of a research programme, the low sensitivity of current detection methods means that mutations may be difficult to detect if they arise but are present in only a small proportion of the cells. In this Review, we discuss the types of mutations acquired by human PSCs and the mechanisms that lead to their accumulation. Recent work suggests that the underlying mutation rate in PSCs is low, although they also seem to be particularly susceptible to genomic damage. This apparent contradiction can be reconciled by the observations that, in contrast to somatic cells, PSCs are programmed to die in response to genomic damage, which may reflect the requirements of early embryogenesis. Thus, the common genetic variants that are observed are probably rare events that give the cells with a selective growth advantage.
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http://dx.doi.org/10.1038/s41580-020-00292-zDOI Listing
December 2020

Retinoic Acid Accelerates the Specification of Enteric Neural Progenitors from In-Vitro-Derived Neural Crest.

Stem Cell Reports 2020 09 27;15(3):557-565. Epub 2020 Aug 27.

Stem Cells and Regenerative Medicine, UCL Great Ormond Street Institute of Child Health, London, UK. Electronic address:

The enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1 and 7. Defects in the development and function of the ENS cause a range of enteric neuropathies, including Hirschsprung disease. Little is known about the signals that specify early ENS progenitors, limiting progress in the generation of enteric neurons from human pluripotent stem cells (hPSCs) to provide tools for disease modeling and regenerative medicine for enteric neuropathies. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neurons in vitro and, following in vivo transplantation, achieved long-term colonization of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS.
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http://dx.doi.org/10.1016/j.stemcr.2020.07.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486303PMC
September 2020

Frequent copy number gains of SLC2A3 and ETV1 in testicular embryonal carcinomas.

Endocr Relat Cancer 2020 09;27(9):457-468

Department of Molecular Oncology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

Testicular germ cell tumours (TGCTs) appear as different histological subtypes or mixtures of these. They show similar, multiple DNA copy number changes, where gain of 12p is pathognomonic. However, few high-resolution analyses have been performed and focal DNA copy number changes with corresponding candidate target genes remain poorly described for individual subtypes. We present the first high-resolution DNA copy number aberration (CNA) analysis on the subtype embryonal carcinomas (ECs), including 13 primary ECs and 5 EC cell lines. We identified recurrent gains and losses and allele-specific CNAs. Within these regions, we nominate 30 genes that may be of interest to the EC subtype. By in silico analysis of data from 150 TGCTs from The Cancer Genome Atlas (TCGA), we further investigated CNAs, RNA expression, somatic mutations and fusion transcripts of these genes. Among primary ECs, ploidy ranged between 2.3 and 5.0, and the most common aberrations were DNA copy number gains at chromosome (arm) 7, 8, 12p, and 17, losses at 4, 10, 11, and 18, replicating known TGCT genome characteristics. Gain of whole or parts of 12p was found in all samples, including a highly amplified 100 kbp segment at 12p13.31, containing SLC2A3. Gain at 7p21, encompassing ETV1, was the second most frequent aberration. In conclusion, we present novel CNAs and the genes located within these regions, where the copy number gain of SLC2A3 and ETV1 are of interest, and which copy number levels also correlate with expression in TGCTs.
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http://dx.doi.org/10.1530/ERC-20-0064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424350PMC
September 2020

Nucleosides Rescue Replication-Mediated Genome Instability of Human Pluripotent Stem Cells.

Stem Cell Reports 2020 06 14;14(6):1009-1017. Epub 2020 May 14.

Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK. Electronic address:

Human pluripotent stem cells (PSCs) are subject to the appearance of recurrent genetic variants on prolonged culture. We have now found that, compared with isogenic differentiated cells, PSCs exhibit evidence of considerably more DNA damage during the S phase of the cell cycle, apparently as a consequence of DNA replication stress marked by slower progression of DNA replication, activation of latent origins of replication, and collapse of replication forks. As in many cancers, which, like PSCs, exhibit a shortened G1 phase and DNA replication stress, the resulting DNA damage may underlie the higher incidence of abnormal and abortive mitoses in PSCs, resulting in chromosomal non-dysjunction or cell death. However, we have found that the extent of DNA replication stress, DNA damage, and consequent aberrant mitoses can be substantially reduced by culturing PSCs in the presence of exogenous nucleosides, resulting in improved survival, clonogenicity, and population growth.
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http://dx.doi.org/10.1016/j.stemcr.2020.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355123PMC
June 2020

Low rates of mutation in clinical grade human pluripotent stem cells under different culture conditions.

Nat Commun 2020 03 23;11(1):1528. Epub 2020 Mar 23.

The Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK.

The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, commonly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations.
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http://dx.doi.org/10.1038/s41467-020-15271-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089967PMC
March 2020

Fully Defined and Xeno-Free Induction of hPSCs into Neural Crest Using Top-Down Inhibition of BMP Signaling.

Methods Mol Biol 2019 ;1976:49-54

Department of Biomedical Science, University of Sheffield, Sheffield, UK.

The neural crest is a transient embryonic tissue that originates from the border of the neural plate prior to delamination and migration throughout the developing embryo, where it contributes to a wide range of different tissues. Defects in neural crest development have been implicated in a variety of different disorders (neurocristopathies) including cancers, neuropathies, craniofacial malformations, and pigment disorders. The differentiation of human pluripotent stem cells (hPSCs) into an in vitro counterpart to neural crest cells holds huge potential for the study of neural crest development, as well as modeling neurocristopathy, carrying out drug discovery experiments and eventually cell replacement therapy. Here we describe a method for generating human neural crest cells from hPSCs that is fully defined and free from animal-derived components. We found that in the absence of serum or bovine serum albumin (BSA), variability in endogenous BMP expression leads to unpredictable differentiation efficiency. In order to control against this issue, we have developed a system termed "top-down inhibition" (TDi) that allows robust neural crest induction as described below.
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http://dx.doi.org/10.1007/978-1-4939-9412-0_4DOI Listing
August 2019

Stem cell culture conditions and stability: a joint workshop of the PluriMes Consortium and Pluripotent Stem Cell Platform.

Regen Med 2019 03 2;14(3):243-255. Epub 2019 Apr 2.

STEMCELL Technologies, Vancouver, BC, V6A 1B6, Canada.

Human stem cells have the potential to transform medicine. However, hurdles remain to ensure that manufacturing processes produce safe and effective products. A thorough understanding of the biological processes occurring during manufacture is fundamental to assuring these qualities and thus, their acceptability to regulators and clinicians. Leaders in both human pluripotent and somatic stem cells, were brought together with experts in clinical translation, biomanufacturing and regulation, to discuss key issues in assuring appropriate manufacturing conditions for delivery of effective and safe products from these cell types. This report summarizes the key issues discussed and records consensus reached by delegates and emphasizes the need for accurate language and nomenclature in the scientific discourse around stem cells.
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http://dx.doi.org/10.2217/rme-2019-0001DOI Listing
March 2019

Anti-apoptotic Mutations Desensitize Human Pluripotent Stem Cells to Mitotic Stress and Enable Aneuploid Cell Survival.

Stem Cell Reports 2019 03 14;12(3):557-571. Epub 2019 Feb 14.

Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing 100084, China. Electronic address:

Human pluripotent stem cells (hPSCs) are susceptible to numerical and structural chromosomal alterations during long-term culture. We show that mitotic errors occur frequently in hPSCs and that prometaphase arrest leads to very rapid apoptosis in undifferentiated but not in differentiated cells. hPSCs express high levels of proapoptotic protein NOXA in undifferentiated state. Knocking out NOXA by CRISPR or upregulation of the anti-apoptosis gene BCL-XL significantly reduced mitotic cell death, allowing the survival of aneuploid cells and the formation of teratomas significantly larger than their wild-type parental hPSCs. These results indicate that the normally low threshold of apoptosis in hPSCs can safeguard their genome integrity by clearing cells undergoing abnormal division. The amplification of BCL2L1 on chromosome 20q11.21, a frequent mutation in hPSCs, although not directly oncogenic, reduces the sensitivity of hPSCs to damage caused by erroneous mitosis and increases the risk of gaining aneuploidy.
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http://dx.doi.org/10.1016/j.stemcr.2019.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411485PMC
March 2019

Human axial progenitors generate trunk neural crest cells in vitro.

Elife 2018 08 10;7. Epub 2018 Aug 10.

Centre for Stem Cell Biology, Department of Biomedical Science, The University of Sheffield, Sheffield, United Kingdom.

The neural crest (NC) is a multipotent embryonic cell population that generates distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.
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http://dx.doi.org/10.7554/eLife.35786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101942PMC
August 2018

Identification and Single-Cell Functional Characterization of an Endodermally Biased Pluripotent Substate in Human Embryonic Stem Cells.

Stem Cell Reports 2018 06 17;10(6):1895-1907. Epub 2018 May 17.

Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.

Human embryonic stem cells (hESCs) display substantial heterogeneity in gene expression, implying the existence of discrete substates within the stem cell compartment. To determine whether these substates impact fate decisions of hESCs we used a GFP reporter line to investigate the properties of fractions of putative undifferentiated cells defined by their differential expression of the endoderm transcription factor, GATA6, together with the hESC surface marker, SSEA3. By single-cell cloning, we confirmed that substates characterized by expression of GATA6 and SSEA3 include pluripotent stem cells capable of long-term self-renewal. When clonal stem cell colonies were formed from GATA6-positive and GATA6-negative cells, more of those derived from GATA6-positive cells contained spontaneously differentiated endoderm cells than similar colonies derived from the GATA6-negative cells. We characterized these discrete cellular states using single-cell transcriptomic analysis, identifying a potential role for SOX17 in the establishment of the endoderm-biased stem cell state.
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http://dx.doi.org/10.1016/j.stemcr.2018.04.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993559PMC
June 2018

Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xeno-free Conditions.

Stem Cell Reports 2017 10 14;9(4):1043-1052. Epub 2017 Sep 14.

University of Sheffield, Department of Biomedical Science, Sheffield S10 2TN, UK.

Defects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.
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http://dx.doi.org/10.1016/j.stemcr.2017.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639211PMC
October 2017

Assessing the Safety of Human Pluripotent Stem Cells and Their Derivatives for Clinical Applications.

Stem Cell Reports 2017 07;9(1):1-4

UK Stem Cell Bank, Advanced Therapies Division, NIBSC-MHRA, London EN6 3QG, UK.

Pluripotent stem cells may acquire genetic and epigenetic variants during culture following their derivation. At a conference organized by the International Stem Cell Initiative, and held at The Jackson Laboratory, Bar Harbor, Maine, October 2016, participants discussed how the appearance of such variants can be monitored and minimized and, crucially, how their significance for the safety of therapeutic applications of these cells can be assessed. A strong recommendation from the meeting was that an international advisory group should be set up to review the genetic and epigenetic changes observed in human pluripotent stem cell lines and establish a framework for evaluating the risks that they may pose for clinical use.
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http://dx.doi.org/10.1016/j.stemcr.2017.05.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206225PMC
July 2017

Teratomas produced from human pluripotent stem cells xenografted into immunodeficient mice - a histopathology atlas.

Int J Dev Biol 2016 ;60(10-11-12):337-419

Department of Pathology, The University of Kansas School of Medicine, Kansas City, Kansas, USA.

This atlas illustrates the microscopic features of tumors produced from human pluripotent stem cells (hPSCs) xenografted into immunosuppressed mice, according to the generally accepted protocols for performing this teratoma assay of stem cell pluripotency. Microphotographs depict various hematoxylin and eosin (H&E) stained tissues derived from all three embryonic germ layers (ectoderm, mesoderm and endoderm). The appearance of persistent hPSC in teratomas is also described with special emphasis on the morphogenesis of embryoid bodies and yolk sac components surrounding them. The use of immunohistochemistry for analyzing hPSC-derived teratomas is also illustrated.
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http://dx.doi.org/10.1387/ijdb.160274idDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338735PMC
January 2018

Pluripotent human stem cells: Standing on the shoulders of giants.

Int J Dev Biol 2016 ;60(10-11-12):321-325

Department of Pathology, The University of Kansas School of Medicine, Kansas City, Kansas, USA.

The advent of human pluripotent stem cells, with the first derivation of human embryonic stem cells in 1998, and of human induced pluripotent stem cells in 2007, has ushered in an era of considerable excitement about the prospects of using these cells to develop new opportunities for healthcare, from their potential for regenerative medicine to their use as tools for studying the cellular basis of many diseases and the discovery of new drugs. But as with the flowering of many new areas in science, the biology of human pluripotent stem cells has its roots in a long history of, sometimes, less fêted research. In a period when research funding is frequently driven by a desire to meet specific clinical or economic goals, it is salutary to remember that the opportunities offered by human pluripotent stem cells have their origins in curiosity driven research without any of those goals in mind. In this case, that research focused on the relatively rare gonadal cancers known as teratomas, tumors that have fascinated people since antiquity because their sometime grotesque manifestations with haphazard collections of tissues and sometimes recognizable body parts. Although well known to clinical pathologists it was the pioneering work of Leroy Stevens, who first discovered that teratomas occur at a significant rate in the 129 strain of the laboratory mouse and could be produced experimentally, that laid the foundations for our understanding of the biology of these tumors and the central role of the embryonal carcinoma cell, one of the archetypal tumor stem cells.
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http://dx.doi.org/10.1387/ijdb.160437idDOI Listing
January 2018

Detecting Genetic Mosaicism in Cultures of Human Pluripotent Stem Cells.

Stem Cell Reports 2016 11;7(5):998-1012

Department of Biomedical Science, Centre for Stem Cell Biology, The University of Sheffield, Sheffield S10 2TN, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.stemcr.2016.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5106530PMC
November 2016

Current Biosafety Considerations in Stem Cell Therapy.

Cell J 2016 Jul-Sep;18(2):281-7. Epub 2016 May 30.

Department of Biological Sciences, Faculty of Science, Tehran Kharazmi University, Tehran, Iran.

Stem cells can be valuable model systems for drug discovery and modelling human diseases as well as to investigate cellular interactions and molecular events in the early stages of development. Controlling the differentiation of stem cells into specific germ layers provides a potential source of highly specialized cells for therapeutic applications. In recent years, finding individual properties of stem cells such as their ultimate self-renewal capacity and the generation of particular cell lines by differentiation under specific culture conditions underpins the development of regenerative therapies. These futures make stem cells a leading candidate to treat a wide range of diseases. Nevertheless, as with all novel treatments, safety issues are one of the barriers that should be overcome to guarantee the quality of a patient's life after stem cell therapy. Many studies have pointed to a large gap in our knowledge about the therapeutic applications of these cells. This gap clearly shows the importance of biosafety concerns for the current status of cell-based therapies, even more than their therapeutic efficacy. Currently, scientists report that tumorigenicity and immunogenicity are the two most important associated cell-based therapy risks. In principle, intrinsic factors such as cell characteristics and extrinsic elements introduced by manufacturing of stem cells can result in tumor formation and immunological reactions after stem cell transplantation. Therapeutic research shows there are many biological questions regarding safety issues of stem cell clinical applications. Stem cell therapy is a rapidly advancing field that needs to focus more on finding a comprehensive technology for assessing risk. A variety of risk factors (from intrinsic to extrinsic) should be considered for safe clinical stem cell therapies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988427PMC
http://dx.doi.org/10.22074/cellj.2016.4324DOI Listing
August 2016

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies.

Dev Biol 2016 09 5;417(2):229-51. Epub 2016 Apr 5.

Laboratory for Enteric NeuroScience (LENS), TARGID, University of Leuven, Belgium.

Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts' views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.
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http://dx.doi.org/10.1016/j.ydbio.2016.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026875PMC
September 2016

The origins of stem cells as tools for regenerative medicine.

Biochem Biophys Res Commun 2016 05 10;473(3):663-4. Epub 2016 Mar 10.

Department of Industrial Engineering, University of Padova, Via Marzolo 9, Padova 35131, Italy.

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http://dx.doi.org/10.1016/j.bbrc.2016.03.032DOI Listing
May 2016

Identification of Novel Fusion Genes in Testicular Germ Cell Tumors.

Cancer Res 2016 Jan 9;76(1):108-16. Epub 2015 Dec 9.

Department of Molecular Oncology, Institute for Cancer Research, Oslo University Hospital-Norwegian Radium Hospital, Oslo, Norway. Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway.

Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men ages 15 to 44 years. Embryonal carcinomas (EC) comprise a subset of TGCTs that exhibit pluripotent characteristics similar to embryonic stem (ES) cells, but the genetic drivers underlying malignant transformation of ECs are unknown. To elucidate the abnormal genetic events potentially contributing to TGCT malignancy, such as the existence of fusion genes or aberrant fusion transcript expression, we performed RNA sequencing of EC cell lines and their nonmalignant ES cell line counterparts. We identified eight novel fusion transcripts and one gene with alternative promoter usage, ETV6. Four out of nine transcripts were found recurrently expressed in an extended panel of primary TGCTs and additional EC cell lines, but not in normal parenchyma of the testis, implying tumor-specific expression. Two of the recurrent transcripts involved an intrachromosomal fusion between RCC1 and HENMT1 located 80 Mbp apart and an interchromosomal fusion between RCC1 and ABHD12B. RCC1-ABHD12B and the ETV6 transcript variant were found to be preferentially expressed in the more undifferentiated TGCT subtypes. In vitro differentiation of the NTERA2 EC cell line resulted in significantly reduced expression of both fusion transcripts involving RCC1 and the ETV6 transcript variant, indicating that they are markers of pluripotency in a malignant setting. In conclusion, we identified eight novel fusion transcripts that, to our knowledge, are the first fusion genes described in TGCT and may therefore potentially serve as genomic biomarkers of malignant progression.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-1790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4704135PMC
January 2016

Evidence for bystander signalling between human trophoblast cells and human embryonic stem cells.

Sci Rep 2015 Jul 14;5:11694. Epub 2015 Jul 14.

Musculoskeletal Research Unit, School of Clinical Sciences (North Bristol), University of Bristol, Bristol BS10 5NB.

Maternal exposure during pregnancy to toxins can occasionally lead to miscarriage and malformation. It is currently thought that toxins pass through the placental barrier, albeit bi-layered in the first trimester, and damage the fetus directly, albeit at low concentration. Here we examined the responses of human embryonic stem (hES) cells in tissue culture to two metals at low concentration. We compared direct exposures with indirect exposures across a bi-layered model of the placenta cell barrier. Direct exposure caused increased DNA damage without apoptosis or a loss of cell number but with some evidence of altered differentiation. Indirect exposure caused increased DNA damage and apoptosis but without loss of pluripotency. This was not caused by metal ions passing through the barrier. Instead the hES cells responded to signalling molecules (including TNF-α) secreted by the barrier cells. This mechanism was dependent on connexin 43 mediated intercellular 'bystander signalling' both within and between the trophoblast barrier and the hES colonies. These results highlight key differences between direct and indirect exposure of hES cells across a trophoblast barrier to metal toxins. It offers a theoretical possibility that an indirectly mediated toxicity of hES cells might have biological relevance to fetal development.
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http://dx.doi.org/10.1038/srep11694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501009PMC
July 2015

Culture adaptation alters transcriptional hierarchies among single human embryonic stem cells reflecting altered patterns of differentiation.

PLoS One 2015 14;10(4):e0123467. Epub 2015 Apr 14.

Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom.

We have used single cell transcriptome analysis to re-examine the substates of early passage, karyotypically Normal, and late passage, karyotypically Abnormal ('Culture Adapted') human embryonic stem cells characterized by differential expression of the cell surface marker antigen, SSEA3. The results confirmed that culture adaptation is associated with alterations to the dynamics of the SSEA3(+) and SSEA3(-) substates of these cells, with SSEA3(-) Adapted cells remaining within the stem cell compartment whereas the SSEA3(-) Normal cells appear to have differentiated. However, the single cell data reveal that these substates are characterized by further heterogeneity that changes on culture adaptation. Notably the Adapted population includes cells with a transcriptome substate suggestive of a shift to a more naïve-like phenotype in contrast to the cells of the Normal population. Further, a subset of the Normal SSEA3(+) cells expresses genes typical of endoderm differentiation, despite also expressing the undifferentiated stem cell genes, POU5F1 (OCT4) and NANOG, whereas such apparently lineage-primed cells are absent from the Adapted population. These results suggest that the selective growth advantage gained by genetically variant, culture adapted human embryonic stem cells may derive in part from a changed substate structure that influences their propensity for differentiation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123467PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4397016PMC
April 2016

Concise review: workshop review: understanding and assessing the risks of stem cell-based therapies.

Stem Cells Transl Med 2015 Apr 26;4(4):389-400. Epub 2015 Feb 26.

Medical Research Council Centre for Drug Safety Science, Division of Molecular & Clinical Pharmacology, Institute of Translational Medicine, and Institute of Integrative Biology, University of Liverpool, Liverpool, U.K.; Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, Pharmazentrum, University of Basel, Basel, Switzerland; Laboratorios Almirall, S.A. Laurea Miro, Sant Feliu de Llobregat, Spain; SANOFI-Research & Development, Disposition, Safety and Animal Research, Alfortville, France; Uppsala Biomedicinska Centrum BMC, Husarg, Uppsala University, Uppsala, Sweden; Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Sheffield, U.K.; Developmental & Regenerative Biomedicine, School of Biomedicine, Faculty of Human and Medical Sciences, University of Manchester, Manchester, U.K.; Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, U.K.; Department of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany; OakMore Solutions Ltd., Faversham, U.K.; ReNeuron Limited, Guildford, U.K.; Medicines and Healthcare Products Regulatory Agency, London, U.K.; Paul Ehrlich Institut, Langen, Germany; Faculty of Science, Leiden Academic Centre for Drug Research, Toxicology, Leiden, The Netherlands; Stem Cell Unit, Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Givat-Ram, Jerusalem, Israel; U.K. Stem Cell Bank, National Institute for Biological Standards and Control, Medicines and Healthcare Products Regulatory Agency, Potters Bar, U.K.; Cellectis AB, Göteborg, Sweden; GSK, David Jack Centre for Research & Development, Ware, U.K.; Personalised Healthcare and Biomarkers, AstraZeneca, Macclesfield, U.K.; CN-bio Innovations Limited, Centre for Innovation & Enterprise, Begbroke, U.K.; Institute of Veterinary Pathology, University of Zurich, Zurich, Switzerland; School of Bio Scien

The field of stem cell therapeutics is moving ever closer to widespread application in the clinic. However, despite the undoubted potential held by these therapies, the balance between risk and benefit remains difficult to predict. As in any new field, a lack of previous application in man and gaps in the underlying science mean that regulators and investigators continue to look for a balance between minimizing potential risk and ensuring therapies are not needlessly kept from patients. Here, we attempt to identify the important safety issues, assessing the current advances in scientific knowledge and how they may translate to clinical therapeutic strategies in the identification and management of these risks. We also investigate the tools and techniques currently available to researchers during preclinical and clinical development of stem cell products, their utility and limitations, and how these tools may be strategically used in the development of these therapies. We conclude that ensuring safety through cutting-edge science and robust assays, coupled with regular and open discussions between regulators and academic/industrial investigators, is likely to prove the most fruitful route to ensuring the safest possible development of new products.
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http://dx.doi.org/10.5966/sctm.2014-0110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4367503PMC
April 2015

Aneuploidy induces profound changes in gene expression, proliferation and tumorigenicity of human pluripotent stem cells.

Nat Commun 2014 Sep 8;5:4825. Epub 2014 Sep 8.

1] Department of Biomedical Sciences and Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA [2] Zilkha Neurogenetic Institute, Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.

Human pluripotent stem cells (hPSCs) tend to acquire genomic aberrations in culture, the most common of which is trisomy of chromosome 12. Here we dissect the cellular and molecular implications of this trisomy in hPSCs. Global gene expression analyses reveal that trisomy 12 profoundly affects the gene expression profile of hPSCs, inducing a transcriptional programme similar to that of germ cell tumours. Comparison of proliferation, differentiation and apoptosis between diploid and aneuploid hPSCs shows that trisomy 12 significantly increases the proliferation rate of hPSCs, mainly as a consequence of increased replication. Furthermore, trisomy 12 increases the tumorigenicity of hPSCs in vivo, inducing transcriptionally distinct teratomas from which pluripotent cells can be recovered. Last, a chemical screen of 89 anticancer drugs discovers that trisomy 12 raises the sensitivity of hPSCs to several replication inhibitors. Together, these findings demonstrate the extensive effect of trisomy 12 and highlight its perils for successful hPSC applications.
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http://dx.doi.org/10.1038/ncomms5825DOI Listing
September 2014

Harmonizing standards for producing clinical-grade therapies from pluripotent stem cells.

Nat Biotechnol 2014 Aug;32(8):724-6

1] Kyoto Center for iPS Cell Research and Application, Kyoto University, Shogoin, Sakyo-ku, Kyoto, Japan. [2] International Society for Stem Cell Research at the time of submission and acceptance of this work.

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http://dx.doi.org/10.1038/nbt.2973DOI Listing
August 2014

Induced pluripotency enables differentiation of human nullipotent embryonal carcinoma cells N2102Ep.

Biochim Biophys Acta 2014 Nov 30;1843(11):2611-9. Epub 2014 Jul 30.

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA; Centre for Stem Cell Biology, University of Sheffield, S10 2TN, UK. Electronic address:

Embryonal carcinoma (EC) cells, which are considered to be malignant counterparts of embryonic stem cells, comprise the pluripotent stem cell component of teratocarcinomas, a form of testicular germ cell tumors (GCTs). Nevertheless, many established human EC cell lines are nullipotent with limited or no capacity to differentiate under normal circumstances. In this study, we tested whether an over-expression of Yamanaka's reprogramming factors OCT4, SOX2, c-MYC and KLF4 might enable differentiation of the human nullipotent EC cells N2102Ep. Using OCT4 knockdown differentiated N2102Ep cells, we are able to derive reprogrammed N2102Ep cell lines. The induced pluripotency of N2102Ep allows the cells to differentiate toward neural lineage by retinoic acid; the expression of SSEA3 and SSEA4 is down-regulated, whereas that of neural surface markers is up-regulated. Consistent with the up-regulation of neural surface markers, the expression of the master neuroectodermal transcription factor PAX6 is also induced in reprogrammed N2102Ep. We next investigated whether PAX6 might induce spontaneous differentiation of nullipotent stem cells N2102Ep. However, while an ectopic expression of PAX6 promotes differentiation of NTERA2, it induces cell death in N2102Ep. We nevertheless find that upon induction of retinoic acid, the reprogrammed N2102Ep cells form mature neuronal morphology similar to differentiated pluripotent stem cells NTERA2 as determined by TUJ1 expression, which is absent in N2102Ep parental cells. Altogether, we conclude that the nullipotent state of human EC cells can be reprogrammed to acquire a more relaxed state of differentiation potential by Yamanaka's factors.
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http://dx.doi.org/10.1016/j.bbamcr.2014.07.013DOI Listing
November 2014

Time-lapse analysis of human embryonic stem cells reveals multiple bottlenecks restricting colony formation and their relief upon culture adaptation.

Stem Cell Reports 2014 Jul 12;3(1):142-55. Epub 2014 Jun 12.

Centre for Stem Cell Biology, Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK.

Using time-lapse imaging, we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells (hESCs) and that are relieved by karyotypically abnormal variants that are selected by prolonged culture. Only a minority of karyotypically normal cells divided after plating, and these were mainly cells in the later stages of cell cycle at the time of plating. Furthermore, the daughter cells showed a continued pattern of cell death after division, so that few formed long-term proliferating colonies. These colony-forming cells showed distinct patterns of cell movement. Increasing cell density enhanced cell movement facilitating cell:cell contact, which resulted in increased proportion of dividing cells and improved survival postplating of normal hESCs. In contrast, most of the karyotypically abnormal cells reentered the cell cycle on plating and gave rise to healthy progeny, without the need for cell:cell contacts and independent of their motility patterns.
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http://dx.doi.org/10.1016/j.stemcr.2014.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4110749PMC
July 2014

Aneuploidy in pluripotent stem cells and implications for cancerous transformation.

Protein Cell 2014 5;5(8):569-79. Epub 2014 Jun 5.

School of Medicine, Tsinghua University, Beijing, 100084, China,

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.
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http://dx.doi.org/10.1007/s13238-014-0073-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4130921PMC
March 2015

Identifiability and privacy in pluripotent stem cell research.

Cell Stem Cell 2014 Apr;14(4):427-30

Centre of Genomics and Policy, Faculty of Medicine, Department of Human Genetics, McGill University, Montreal, QC H3A 0G1, Canada.

Data sharing is an essential element of research; however, recent scientific and social developments have challenged conventional methods for protecting privacy. Here we provide guidance for determining data sharing thresholds for human pluripotent stem cell research aimed at a wide range of stakeholders, including research consortia, biorepositories, policy-makers, and funders.
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http://dx.doi.org/10.1016/j.stem.2014.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815907PMC
April 2014