CRISPR activation enables high-fidelity reprogramming into human pluripotent stem cells

Sokka, Joonas; Yoshihara, Masahito; Kvist, Jouni; Laiho, Laura; Andrew, Warren; Stadelmann, Christian; Jouhilahti, Eeva-Mari; Kilpinen, Helena; Balboa, Diego; Katayama, Shintaro; Kyttälä, Aija; Kere, Juha; Otonkoski, Timo; Weltner, Jere; Trokovic, Ras

doi:10.1016/j.stemcr.2021.12.017

Cited by 20 publications

(17 citation statements)

References 52 publications

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“…To better understand how EBV in infected cells spontaneously enter the lytic life cycle, we analyzed publicly available single cell RNA‐sequencing (scRNA‐seq) data from nine LCLs from several independent sources (see Section 5 ). 34 , 40 , 41 , 42 , 43 , 44 Briefly, we performed an unbiased integrative analysis across all these LCLs after regressing for potential batch effects, doublets and/or artifacts and known sources of heterogeneity, such as the stage of cell cycle using the Seurat platform 45 (Supporting Information: Figure S1A–C see Section 5 ). Unsupervised clustering of all 46 205 cells according to expressions of both host and viral genes at three different resolutions yielded several clusters (Supporting Information: Figure S1D ).…”

Section: Resultsmentioning

confidence: 99%

A comprehensive single cell data analysis of lymphoblastoid cells reveals the role of super‐enhancers in maintaining EBV latency

Yan

Wang

Chakravorty

et al. 2022

Journal of Medical Virology

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We probed the lifecycle of Epstein‐Barr virus (EBV) on a cell‐by‐cell basis using single cell RNA sequencing (scRNA‐seq) data from nine publicly available lymphoblastoid cell lines (LCLs). While the majority of LCLs comprised cells containing EBV in the latent phase, two other clusters of cells were clearly evident and were distinguished by distinct expression of host and viral genes. Notably, both were high expressors of EBV LMP1/BNLF2 and BZLF1 compared to another cluster that expressed neither gene. The two novel clusters differed from each other in their expression of EBV lytic genes, including glycoprotein gene GP350. The first cluster, comprising GP350–LMP1hi cells, expressed high levels of HIF1A and was transcriptionally regulated by HIF1‐α. Treatment of LCLs with Pevonedistat, a drug that enhances HIF1‐α signaling, markedly induced this cluster. The second cluster, containing GP350+LMP1hi cells, expressed EBV lytic genes. Host genes that are controlled by super‐enhancers (SEs), such as transcription factors MYC and IRF4, had the lowest expression in this cluster. Functionally, the expression of genes regulated by MYC and IRF4 in GP350+LMP1hi cells were lower compared to other cells. Indeed, induction of EBV lytic reactivation in EBV+ AKATA reduced the expression of these SE‐regulated genes. Furthermore, CRISPR‐mediated perturbation of the MYC or IRF4 SEs in LCLs induced the lytic EBV gene expression, suggesting that host SEs and/or SE target genes are required for maintenance of EBV latency. Collectively, our study revealed EBV‐associated heterogeneity among LCLs that may have functional consequence on host and viral biology.

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Section: Resultsmentioning

confidence: 99%

A comprehensive single cell data analysis of lymphoblastoid cells reveals the role of super‐enhancers in maintaining EBV latency

Yan

Wang

Chakravorty

et al. 2022

Journal of Medical Virology

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“… 24 This approach could be further improved by targeting the microRNA-302/367 locus, showing that both human lymphoblastoid cell lines and primary skin fibroblasts could be efficiently reprogrammed. 25 All these recent advancements may make the use of iPSCs in personalized cell replacement therapy feasible and more reliable ( Fig. 1 ).…”

Section: Somatic Origin Of Human Ipscs and Reprogramming Methodsmentioning

confidence: 99%

Human Induced Pluripotent Stem Cells: From Cell Origin, Genomic Stability, and Epigenetic Memory to Translational Medicine

2022

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The potential of human induced pluripotent stem cells (iPSCs) to self-renew indefinitely and to differentiate virtually into any cell type in unlimited quantities makes them attractive for in-vitro disease modeling, drug screening, personalized medicine, and regenerative therapies. As the genome of iPSCs thoroughly reproduces that of the somatic cells from which they are derived, they may possess genetic abnormalities, which would seriously compromise their utility and safety. Genetic aberrations could be present in donor somatic cells and then transferred during iPSC generation, or they could occur as de novo mutations during reprogramming or prolonged cell culture. Therefore, to warrant safety of human iPSCs for clinical applications, analysis of genetic integrity, particularly during iPSC generation and differentiation, should be carried out on a regular basis. On the other hand, reprogramming of somatic cells to iPSCs requires profound modifications in the epigenetic landscape. Changes in chromatin structure by DNA methylations and histone tail modifications aim to reset the gene expression pattern of somatic cells to facilitate and establish self-renewal and pluripotency. However, residual epigenetic memory influences the iPSC phenotype, which may affect their application in disease therapeutics. The present review discusses the somatic cell origin, genetic stability, and epigenetic memory of iPSCs and their impact on basic and translational research.

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“…However, chromatin context, presence of co-factors, excessive expression level and epigenetic barriers can limit the activity of exogenous transcription factors. We hypothesized that direct CRISPR activation of endogenous regulatory elements can overcome epigenetic barriers associated with differentiation of embryonic stem cells [27][28][29][30]. To test our hypothesis, we implemented a CRISPRa-based strategy to generate major cell types that comprise the preimplantation embryo: epiblast stem cells (Epi), trophoblast stem cells (TS), and primitive endoderm cells (PrE), also known as hypoblast cells in human development.…”

Section: Generation Of Crispra-induced Primitive Endoderm-like Cells ...mentioning

confidence: 99%

Self-organization of embryonic stem cells into a reproducible embryo model through epigenome editing

Lodewijk,

Kozuki,

Han

et al. 2024

Preprint

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Embryonic stem cells (ESCs) can self-organize in vitro into developmental patterns with spatial organization and molecular similarity to that of early embryonic stages. This self-organization of ESCs requires transmission of signaling cues, via addition of small molecule chemicals or recombinant proteins, to induce distinct embryonic cellular fates and subsequent assembly into structures that can mimic aspects of early embryonic development. During natural embryonic development, different embryonic cell types co-develop together, where each cell type expresses specific fate-inducing transcription factors through activation of non-coding regulatory elements and interactions with neighboring cells. However, previous studies have not fully explored the possibility of engineering endogenous regulatory elements to shape self-organization of ESCs into spatially-ordered embryo models. Here, we hypothesized that cell-intrinsic activation of a minimum number of such endogenous regulatory elements is sufficient to self-organize ESCs into early embryonic models. Our results show that CRISPR-based activation (CRISPRa) of only two endogenous regulatory elements in the genome of pluripotent stem cells is sufficient to generate embryonic patterns that show spatial and molecular resemblance to that of pre-gastrulation mouse embryonic development. Quantitative single-cell live fluorescent imaging showed that the emergence of spatially-ordered embryonic patterns happens through the intrinsic induction of cell fate that leads to an orchestrated collective cellular motion. Based on these results, we propose a straightforward approach to efficiently form 3D embryo models through intrinsic CRISPRa-based epigenome editing and independent of external signaling cues. CRISPRa-Programmed Embryo Models (CPEMs) show highly consistent composition of major embryonic cell types that are spatially-organized, with nearly 80% of the structures forming an embryonic cavity. Single cell transcriptomics confirmed the presence of main embryonic cell types in CPEMs with transcriptional similarity to pre-gastrulation mouse embryos and revealed novel signaling communication links between different embryonic cell types. Our findings offer a programmable embryo model and demonstrate that minimum intrinsic epigenome editing is sufficient to self-organize ESCs into highly consistent pre-gastrulation embryo models.

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CRISPR activation enables high-fidelity reprogramming into human pluripotent stem cells

Cited by 20 publications

References 52 publications

A comprehensive single cell data analysis of lymphoblastoid cells reveals the role of super‐enhancers in maintaining EBV latency

A comprehensive single cell data analysis of lymphoblastoid cells reveals the role of super‐enhancers in maintaining EBV latency

Human Induced Pluripotent Stem Cells: From Cell Origin, Genomic Stability, and Epigenetic Memory to Translational Medicine

Self-organization of embryonic stem cells into a reproducible embryo model through epigenome editing

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