Single Nucleotide Polymorphism (SNP) Arrays and Their Sensitivity for Detection of Genetic Changes in Human Pluripotent Stem Cell Cultures.

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.

First come, first served: Mammalian recombination is timed to replication.

Meiotic recombination drives the formation of new chromosomes in germ cells and is essential for fertility in mammals. In this issue of Cell, Pratto et al. have developed a method to map replication origins directly in mammalian tissue for the first time, revealing evolutionary conservation between replication timing and meiotic recombination in males.

Nanopore Sequencing Indicates That Tandem Amplification of Chromosome 20q11.21 in Human Pluripotent Stem Cells Is Driven by Break-Induced Replication.

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 (PSCs) has prompted concerns for their use in regenerative medicine. 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 duplication and as 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.

DNA Fiber Assay for the Analysis of DNA Replication Progression in Human Pluripotent Stem Cells.

Human pluripotent stem cells (PSC) acquire recurrent chromosomal instabilities during prolonged in vitro culture that threaten to preclude their use in cell-based regenerative medicine. The rapid proliferation of pluripotent cells leads to constitutive replication stress, hindering the progression of DNA replication forks and in some cases leading to replication-fork collapse. Failure to overcome replication stress can result in incomplete genome duplication, which, if left to persist into the subsequent mitosis, can result in structural and numerical chromosomal instability. We have recently applied the DNA fiber assay to the study of replication stress in human PSC and found that, in comparison to somatic cells states, these cells display features of DNA replication stress that include slower replication fork speeds, evidence of stalled forks, and replication initiation from dormant replication origins. These findings have expanded on previous work demonstrating that extensive DNA damage in human PSC is replication associated. In this capacity, the DNA fiber assay has enabled the development of an advanced nucleoside-enriched culture medium that increases replication fork progression and decreases DNA damage and mitotic errors in human PSC cultures. The DNA fiber assay allows for the study of replication fork dynamics at single-molecule resolution. The assay relies on cells incorporating nucleotide analogs into nascent DNA during replication, which are then measured to monitor several replication parameters. Here we provide an optimized protocol for the fiber assay intended for use with human PSC, and describe the methods employed to analyze replication fork parameters. © 2020 Wiley Periodicals LLC. Basic Protocol 1: DNA fiber labeling Basic Protocol 2: DNA fiber spreading Basic Protocol 3: Immunostaining Support Protocol 1: Microscopy/data acquisition Support Protocol 2: Data analysis.

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

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.

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

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.

The IPD and IMGT/HLA database: allele variant databases.

The Immuno Polymorphism Database (IPD) was developed to provide a centralized system for the study of polymorphism in genes of the immune system. Through the IPD project we have established a central platform for the curation and publication of locus-specific databases involved either directly or related to the function of the Major Histocompatibility Complex in a number of different species. We have collaborated with specialist groups or nomenclature committees that curate the individual sections before they are submitted to IPD for online publication. IPD consists of five core databases, with the IMGT/HLA Database as the primary database. Through the work of the various nomenclature committees, the HLA Informatics Group and in collaboration with the European Bioinformatics Institute we are able to provide public access to this data through the website http://www.ebi.ac.uk/ipd/. The IPD project continues to develop with new tools being added to address scientific developments, such as Next Generation Sequencing, and to address user feedback and requests. Regular updates to the website ensure that new and confirmatory sequences are dispersed to the immunogenetics community, and the wider research and clinical communities.

IMGT/HLA and the Immuno Polymorphism Database.

The IMGT/HLA Database (http://www.ebi.ac.uk/ipd/imgt/hla/) was first released over 15 years ago, providing the HLA community with a searchable repository of highly curated HLA sequences. The HLA complex is located within the 6p21.3 region of human chromosome 6 and contains more than 220 genes of diverse function. Many of the genes encode proteins of the immune system and are highly polymorphic, with some genes currently having over 3,000 known allelic variants. The Immuno Polymorphism Database (IPD) (http://www.ebi.ac.uk/ipd/) expands on this model, with a further set of specialist databases related to the study of polymorphic genes in the immune system. The IPD project works with specialist groups or nomenclature committees who provide and curate individual sections before they are submitted to IPD for online publication. IPD currently consists of four databases: IPD-KIR contains the allelic sequences of killer-cell immunoglobulin-like receptors; IPD-MHC is a database of sequences of the major histocompatibility complex of different species; IPD-HPA, alloantigens expressed only on platelets; and IPD-ESTDAB, which provides access to the European Searchable Tumour Cell-Line Database, a cell bank of immunologically characterized melanoma cell lines. Through the work of the HLA Informatics Group and in collaboration with the European Bioinformatics Institute we are able to provide public access to this data through the website http://www.ebi.ac.uk/ipd/.

IPD--the Immuno Polymorphism Database.

The Immuno Polymorphism Database (IPD), http://www.ebi.ac.uk/ipd/ is a set of specialist databases related to the study of polymorphic genes in the immune system. The IPD project works with specialist groups or nomenclature committees who provide and curate individual sections before they are submitted to IPD for online publication. The IPD project stores all the data in a set of related databases. IPD currently consists of four databases: IPD-KIR, contains the allelic sequences of killer-cell immunoglobulin-like receptors, IPD-MHC, a database of sequences of the major histocompatibility complex of different species; IPD-HPA, alloantigens expressed only on platelets; and IPD-ESTDAB, which provides access to the European Searchable Tumour Cell-Line Database, a cell bank of immunologically characterized melanoma cell lines. The data is currently available online from the website and FTP directory. This article describes the latest updates and additional tools added to the IPD project.

The IMGT/HLA database.

It is 14 years since the IMGT/HLA database was first released, providing the HLA community with a searchable repository of highly curated HLA sequences. The HLA complex is located within the 6p21.3 region of human chromosome 6 and contains more than 220 genes of diverse function. Of these, 21 genes encode proteins of the immune system that are highly polymorphic. The naming of these HLA genes and alleles and their quality control is the responsibility of the World Health Organization Nomenclature Committee for Factors of the HLA System. Through the work of the HLA Informatics Group and in collaboration with the European Bioinformatics Institute, we are able to provide public access to these data through the website http://www.ebi.ac.uk/imgt/hla/. Regular updates to the website ensure that new and confirmatory sequences are dispersed to the HLA community and the wider research and clinical communities. This article describes the latest updates and additional tools added to the IMGT/HLA project.