Showing papers by "Peter W. Andrews published in 2020"
TL;DR: 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, but the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when the authors also found alterations in imprint methylation and reversible DNA hypomethylation.
Abstract: 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.
52 citations
TL;DR: The types of mutations acquired by human PSCs and the mechanisms that lead to their accumulation are discussed, which suggest that the underlying mutation rate in P SCs is low, although they also seem to be particularly susceptible to genomic damage.
Abstract: 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. Cultured pluripotent stem cells (PSCs) acquire genetic changes — gains or losses of entire chromosomal regions, or point mutations, including in cancer-associated genes such as TP53. Recent work provides insights into the mechanisms of mutation and selection, which have implications for the use of human PSCs in regenerative medicine.
38 citations
TL;DR: It is 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.
Abstract: 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.
21 citations
TL;DR: The efficient and accelerated generation of ENS progenitors from hPSCs are described, revealing that retinoic acid is critical for the acquisition of vagal axial identity and early E NS progenitor specification.
Abstract: Summary 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.
16 citations
TL;DR: 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.
Abstract: 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.
11 citations
TL;DR: It is shown that combined WNT-FGF activities drive a posterior dorsal early neural state while suppression of TGFβ-BMP signalling pathways, combined with SHH stimulation, promotes a ventral identity.
Abstract: The anteroposterior axial identity of motor neurons (MNs) determines their functionality and vulnerability to neurodegeneration Thus it is a critical parameter in the design of strategies aiming to produce MNs from human pluripotent stem cells (hPSCs) for regenerative medicine and disease modelling applications However, the in vitro generation of posterior spinal cord MNs has been challenging Although the induction of cells resembling neuromesodermal progenitors (NMPs), the bona fide precursors of the mammalian spinal cord, offers a promising solution, the progressive specification of posterior MNs from these cells is not well-defined Here we determine the signals guiding the transition of human NMP-like cells toward posterior ventral spinal cord neurectoderm We show that combined WNT-FGF activities drive a posterior dorsal early neural state while suppression of TGFβ-BMP signalling pathways, combined with SHH stimulation, promotes a ventral identity Based on these results, we define an optimised protocol for the generation of posterior MNs that can efficiently integrate within the neural tube of chick embryos We expect that our findings will facilitate the functional comparison of hPSC-derived spinal cord cells of distinct axial identities
10 citations
TL;DR: 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 are presented.
Abstract: 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.
4 citations
TL;DR: Long-range Nanopore sequencing on two examples of this CNV found that breakpoint signatures point to a specific mechanism of template switching in CNV formation, with surrounding Alu sequences likely contributing to the instability of this genomic region.
Abstract: 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 surrounding Alu sequences likely contributing to the instability of this genomic region.
2 citations
TL;DR: Cancer-related microcalcifications may arise secondary to gonadal mineral alterations, which in combination with impaired Sertoli cell function and reduced PPi due to high alkaline phosphatase activity in GCNIS and TGCTs, facilitates osteogenic transdifferentiation of testicular germ cells and deposition of hydroxyapatite.
Abstract: Testicular microcalcifications consist of hydroxyapatite and their demonstration by ultrasound has been associated with increased risk of testicular germ cell cancer (TGCT) Here, we show that fibroblast growth factor 23 (FGF23), a bone-specific regulator of phosphate homeostasis, is expressed in testicular germ cell neoplasia in situ (GCNIS), embryonal carcinoma (EC), and human embryonic stem cells FGF23 is not glycosylated in TGCTs and thus rapidly cleaved into a C-terminal fragment that serves as a competitive antagonist for full-length FGF23 High levels of C-terminal FGF23 occupy the receptor formed by Klotho and FGF receptor 1 (FGFR1) in the germ cells facilitating a shift in the expression of phosphate transport proteins from SLC34A2 to SLC34A1 in seminiferous tubules adjacent to GCNIS Fgf23 knockout mice have a marked epididymal deposition of hydroxyapatite, while the testicular phenotype is milder with spermatogenic arrest and focal germ-cell-specific expression of the bone-like markers runt-related transcription factor 2 (RUNX2) and bone gamma-carboxyglutamic acid-containing protein (BGLAP) In accordance, mice with no functional androgen receptor and lack of circulating gonadotropins develop microcalcifications in 94% of cases and have lower Slc34a2, and higher Slc34a1 and Bglap expression In accordance, human testicular specimens with microcalcifications also have lower SLC34A2, and focally germ cells express SLC34A1, BGLAP, and RUNX2 Importantly, calcium or phosphate induced osteogenic transdifferentiation of a spermatogonial cell line in vitro demonstrated by induction of alkaline phosphatase activity and deposition of hydroxyapatite, which could be fully rescued by pyrophosphate (PPi) Severe microcalcifications were also found in a mouse model with Sertoli-cell ablation particularly when Sertoli-ablation was conducted prepubertally where the germ cells retain stem cell potential In conclusion, cancer-related microcalcifications may arise secondary to gonadal mineral alterations, which in combination with impaired Sertoli cell function and reduced PPi due to high alkaline phosphatase activity in GCNIS and TGCTs, facilitates osteogenic transdifferentiation of testicular germ cells and deposition of hydroxyapatite
2 citations
26 Aug 2020
TL;DR: A detailed study of the faunas was carried out by Bishop, who concluded that the derived Westbury 1 fauna is of early Pleistocene age, while the Westbury 2 and 3 fauna are post-Cromerian sensu stricto but pre-date those of the Hoxnian Stage as discussed by the authors.
Abstract: The Westbury-sub-Mendip cave deposits were first exposed during quarrying in 1969. Detailed study of the faunas was carried out by Bishop, who concluded that the derived Westbury 1 fauna is of early Pleistocene age, while the Westbury 2 and 3 faunas are post-Cromerian sensu stricto but pre-date those of the Hoxnian Stage. An alternative proposal by Stuart was that the main Westbury faunas are Cromerian, representing the end of that interglacial Stage. Systematic excavations at Westbury (1976–1984) led by a team from the Natural History Museum have demonstrated the complexity of the site and faunal sequences. The probable early Pleistocene ‘Siliceous Group’ (Unit 1) and its derived fauna is succeeded by two separate stratigraphic and faunal sequences at the western and eastern ends of the site. Palaeoecological reconstructions, using a Taxonomic Habitat Index derived from the small mammal faunas, indicate that the western sequence contains three temperate phases, separated by more boreal conditions and followed by a tundra phase. This tundra phase also appears to be represented in the upper parts of the eastern sequence. Since all of these faunas, including those indicating optimum interglacial conditions, contain Arvicola cantiana, they must be post-Cromerian sensu stricto and appear to pre-date the Hoxnian Stage.