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

Tailored for Real-World: A Whole Slide Image Classification System Validated on Uncurated Multi-Site Data Emulating the Prospective Pathology Workload.

Standard of care diagnostic procedure for suspected skin cancer is microscopic examination of hematoxylin & eosin stained tissue by a pathologist. Areas of high inter-pathologist discordance and rising biopsy rates necessitate higher efficiency and diagnostic reproducibility. We present and validate a deep learning system which classifies digitized dermatopathology slides into 4 categories. The system is developed using 5,070 images from a single lab, and tested on an uncurated set of 13,537 images from 3 test labs, using whole slide scanners manufactured by 3 different vendors. The system\u27s use of deep-learning-based confidence scoring as a criterion to consider the result as accurate yields an accuracy of up to 98%, and makes it adoptable in a real-world setting. Without confidence scoring, the system achieved an accuracy of 78%. We anticipate that our deep learning system will serve as a foundation enabling faster diagnosis of skin cancer, identification of cases for specialist review, and targeted diagnostic classifications

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

Time-Lapse Analysis of Human Embryonic Stem Cells Reveals Multiple Bottlenecks Restricting Colony Formation and Their Relief upon Culture Adaptation

Summary 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

Generation and trapping of a mesoderm biased state of human pluripotency

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

Identification and single-cell functional characterization of an endodermally biased pluripotent substate in human embryonic stem cells

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

Genetically variant human pluripotent stem cells selectively eliminate wild-type counterparts through YAP-mediated cell competition

The appearance of genetic changes in human pluripotent stem cells (hPSCs) presents a concern for their use in research and regenerative medicine. Variant hPSCs that harbor recurrent culture-acquired aneuploidies display growth advantages over wild-type diploid cells, but the mechanisms that yield a drift from predominantly wild-type to variant cell populations remain poorly understood. Here, we show that the dominance of variant clones in mosaic cultures is enhanced through competitive interactions that result in the elimination of wild-type cells. This elimination occurs through corralling and mechanical compression by faster-growing variants, causing a redistribution of F-actin and sequestration of yes-associated protein (YAP) in the cytoplasm that induces apoptosis in wild-type cells. YAP overexpression or promotion of YAP nuclear localization in wild-type cells alleviates their “loser” phenotype. Our results demonstrate that hPSC fate is coupled to mechanical cues imposed by neighboring cells and reveal that hijacking this mechanism allows variants to achieve clonal dominance in cultures