Improving the safety of human pluripotent stem cell therapies using genome-edited orthogonal safeguards.
Ontology highlight
ABSTRACT:
SUBMITTER: Martin RM
PROVIDER: S-EPMC7264334 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Chromosomes have an intrinsic tendency to segregate into compartments, forming long-distance contacts between loci of similar chromatin states. How genome compartmentalization is regulated remains elusive. Here, comparison of mouse ground-state embryonic stem cells (ESCs) characterised by open and active chromatin, and advanced serum ESCs with a more closed and repressed genome, reveals distinct regulation of their genome organization due to differential dependency on BAZ2A/TIP5, a component of the chromatin remodeling complex NoRC. On ESC chromatin, BAZ2A interacts with SNF2H, DNA topoisomerase 2? (TOP2A) and cohesin. BAZ2A associates with chromatin sub-domains within the active A compartment, which intersect through long-range contacts. We found that ground-state chromatin selectively requires BAZ2A to limit the invasion of active domains into repressive compartments. BAZ2A depletion increases chromatin accessibility at B compartments. Furthermore, BAZ2A regulates H3K27me3 genome occupancy in a TOP2A-dependent manner. Finally, ground-state ESCs require BAZ2A for growth, differentiation, and correct expression of developmental genes. Our results uncover the propensity of open chromatin domains to invade repressive domains, which is counteracted by chromatin remodeling to establish genome partitioning and preserve cell identity.
| S-SCDT-EMBOJ-2020-105606 | biostudies-other
Project description:Chromosomes have an intrinsic tendency to segregate into compartments, forming long-distance contacts between loci of similar chromatin states. How genome compartmentalization is regulated remains elusive. Here, comparison of mouse ground-state embryonic stem cells (ESCs) characterized by open and active chromatin, and advanced serum ESCs with a more closed and repressed genome, reveals distinct regulation of their genome organization due to differential dependency on BAZ2A/TIP5, a component of the chromatin remodeling complex NoRC. On ESC chromatin, BAZ2A interacts with SNF2H, DNA topoisomerase 2A (TOP2A) and cohesin. BAZ2A associates with chromatin sub-domains within the active A compartment, which intersect through long-range contacts. We found that ground-state chromatin selectively requires BAZ2A to limit the invasion of active domains into repressive compartments. BAZ2A depletion increases chromatin accessibility at B compartments. Furthermore, BAZ2A regulates H3K27me3 genome occupancy in a TOP2A-dependent manner. Finally, ground-state ESCs require BAZ2A for growth, differentiation, and correct expression of developmental genes. Our results uncover the propensity of open chromatin domains to invade repressive domains, which is counteracted by chromatin remodeling to establish genome partitioning and preserve cell identity.
| S-EPMC7705451 | biostudies-literature
Project description:Purpose of reviewAlternative approaches to conventional drug-based cancer treatments have seen T cell therapies deployed more widely over the last decade. This is largely due to their ability to target and kill specific cell types based on receptor recognition. Introduction of recombinant T cell receptors (TCRs) using viral vectors and HLA-independent T cell therapies using chimeric antigen receptors (CARs) are discussed. This article reviews the tools used for genome editing, with particular emphasis on the applications of site-specific DNA nuclease mediated editing for T cell therapies.Recent findingsGenetic engineering of T cells using TCRs and CARs with redirected antigen-targeting specificity has resulted in clinical success of several immunotherapies. In conjunction, the application of genome editing technologies has resulted in the generation of HLA-independent universal T cells for allogeneic transplantation, improved T cell sustainability through knockout of the checkpoint inhibitor, programmed cell death protein-1 (PD-1), and has shown efficacy as an antiviral therapy through direct targeting of viral genomic sequences and entry receptors.SummaryThe combined use of engineered antigen-targeting moieties and innovative genome editing technologies have recently shown success in a small number of clinical trials targeting HIV and hematological malignancies and are now being incorporated into existing strategies for other immunotherapies.
| S-EPMC5445182 | biostudies-literature
Project description:To create isogenic human pluripotent stem cell-derived macrophages with and without ABCA1 expression as a model for reverse cholesterol transport.The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) genome-editing system was used to introduce frameshift mutations into the coding sequence of ATP-binding cassette, subfamily A, member 1. Individual human pluripotent stem cell clones with deleterious mutations were identified, expanded, and differentiated into mature macrophages with a cytokine-based, feeder-free differentiation protocol. Wild-type cells demonstrated effective cholesterol efflux to apoAI acceptor, whereas ABCA1(-/-) cells displayed significantly reduced efflux ability and increased expression of proinflammatory cytokines.Human pluripotent stem cell-derived macrophages capable of reverse cholesterol transport can be rapidly generated and genetically edited with CRISPR/Cas9. Introduction of homozygous frameshift mutations results in loss of ABCA1 expression in differentiated macrophages and subsequent reduction of cholesterol efflux capability. This facile genome-editing approach and differentiation protocol pave the way for future studies of the molecular determinants of reverse cholesterol transport and other macrophage properties.
| S-EPMC4690765 | biostudies-other
Project description:Induced pluripotent stem cell (iPSC) therapies offer a promising path for patient-specific regenerative medicine. However, tumor formation from residual undifferentiated iPSC or transformation of iPSC or their derivatives is a risk. Inclusion of a suicide gene is one approach to risk mitigation. We introduced a dimerizable-"inducible caspase-9" (iCasp9) suicide gene into mouse iPSC (miPSC) and rhesus iPSC (RhiPSC) via a lentivirus, driving expression from either a cytomegalovirus (CMV), elongation factor-1 α (EF1α) or pluripotency-specific EOS-C(3+) promoter. Exposure of the iPSC to the synthetic chemical dimerizer, AP1903, in vitro induced effective apoptosis in EF1α-iCasp9-expressing (EF1α)-iPSC, with less effective killing of EOS-C(3+)-iPSC and CMV-iPSC, proportional to transgene expression in these cells. AP1903 treatment of EF1α-iCasp9 miPSC in vitro delayed or prevented teratomas. AP1903 administration following subcutaneous or intravenous delivery of EF1α-iPSC resulted in delayed teratoma progression but did not ablate tumors. EF1α-iCasp9 expression was downregulated during in vitro and in vivo differentiation due to DNA methylation at CpG islands within the promoter, and methylation, and thus decreased expression, could be reversed by 5-azacytidine treatment. The level and stability of suicide gene expression will be important for the development of suicide gene strategies in iPSC regenerative medicine.
| S-EPMC4448736 | biostudies-literature
Project description:Extended pluripotent stem cells (EPS cells) have unlimited self-renewal ability and the potential to differentiate into mesodermal, ectodermal, and endodermal cells. Notably, in addition to developing the embryonic (Em) lineages, it can also make an effective contribution to extraembryonic (ExEm) lineages both in vitro and in vivo. However, multiple mysteries still remain about the underlying molecular mechanism of EPS cells' maintenance and developmental potential. WDR36 (WD Repeat Domain 36), a protein of 105 kDa with 14 WD40 repeats, which may fold into two β-propellers, participates in 18sRNA synthesis and P53 stress response. Though WDR36 safeguards mouse early embryonic development, that is, homozygous knockout of WDR36 can result in embryonic lethality, what role does WDR36 plays in self-renewal and differentiation developmental potential of human EPS cells is still a subject of concern. Here, our findings suggested that the expression of WDR36 was downregulated during human hEPS cells lost self-renewal. Through constructing inducible knockdown or overexpressing WDR36-human EPS cell lines, we found that WDR36 knockdown disrupted self-renewal but promoted the mesodermal differentiation of human EPS cells; however, overexpressing of WDR36 had little effect. Additionally, P53 inhibition could reverse the effects of WDR36 knockdown, on both self-renewal maintenance and differentiation potential of human EPS cells. These data implied that WDR36 safeguards self-renewal and pluripotency of human EPS cells, which would extend our understanding of the molecular mechanisms of human EPS cells' self-renewal and differentiation.
| S-EPMC9353684 | biostudies-literature
Project description:Natural killer (NK) cells play a crucial role in host immunity by detecting cells that downregulate MHC class I presentation and upregulate stress ligands, as commonly seen in cancers. Current NK therapies using primary NK cells are prone to manufacturing issues related to expansion and storage. Alternative cell sources utilizing immortalized NK cell lines require irradiation and are dependent on systemic IL-2 administration, which has been associated with adverse effects. In contrast, NK cells differentiated from induced pluripotent stem cells (iPSC-NK cells) offer an off-the-shelf alternative that may overcome these bottlenecks. The development of a serum-free and feeder-free differentiation protocol allows for the manufacturing of clinically adaptable iPSC-NK cells that are equally as effective as primary NK cells and the NK-92 cell line for many indications. Moreover, genetic modifications targeting NK-mediated antibody-dependent cellular cytotoxicity capabilities, cytotoxicity, and checkpoint inhibitors may increase the therapeutic potential of iPSC-NK products. This review will highlight the current sources for NK therapies and their respective constraints, discuss recent developments in the manufacturing and genetic engineering of iPSC-NK cells, and provide an overview of ongoing clinical trials using NK cells.
| S-EPMC7298853 | biostudies-literature
Project description:BackgroundBrugada syndrome (BrS), a disorder associated with characteristic electrocardiogram precordial ST-segment elevation, predisposes afflicted patients to ventricular fibrillation and sudden cardiac death. Despite marked achievements in outlining the organ level pathophysiology of the disorder, the understanding of human cellular phenotype has lagged due to a lack of adequate human cellular models of the disorder.ObjectivesThe objective of this study was to examine single cell mechanism of Brugada syndrome using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).MethodsThis study recruited 2 patients with type 1 BrS carrying 2 different sodium voltage-gated channel alpha subunit 5 variants as well as 2 healthy control subjects. We generated iPSCs from their skin fibroblasts by using integration-free Sendai virus. We used directed differentiation to create purified populations of iPSC-CMs.ResultsBrS iPSC-CMs showed reductions in inward sodium current density and reduced maximal upstroke velocity of action potential compared with healthy control iPSC-CMs. Furthermore, BrS iPSC-CMs demonstrated increased burden of triggered activity, abnormal calcium (Ca2+) transients, and beating interval variation. Correction of the causative variant by genome editing was performed, and resultant iPSC-CMs showed resolution of triggered activity and abnormal Ca2+ transients. Gene expression profiling of iPSC-CMs showed clustering of BrS compared with control subjects. Furthermore, BrS iPSC-CM gene expression correlated with gene expression from BrS human cardiac tissue gene expression.ConclusionsPatient-specific iPSC-CMs were able to recapitulate single-cell phenotype features of BrS, including blunted inward sodium current, increased triggered activity, and abnormal Ca2+ handling. This novel human cellular model creates future opportunities to further elucidate the cellular disease mechanism and identify novel therapeutic targets.
| S-EPMC5373649 | biostudies-literature
Project description:The photoreceptor cells in the retina have a highly specialised sensory cilium, the outer segment (OS), which is important for detecting light. Mutations in cilia-related genes often result in retinal degeneration. The ability to reprogramme human cells into induced pluripotent stem cells and then differentiate them into a wide range of different cell types has revolutionised our ability to study human disease. To date, however, the challenge of producing fully differentiated photoreceptors in vitro has limited the application of this technology in studying retinal degeneration. In this review, we will discuss recent advances in stem cell technology and photoreceptor differentiation. In particular, the development of photoreceptors with rudimentary OS that can be used to understand disease mechanisms and as an important model to test potential new therapies for inherited retinal ciliopathies.
| S-EPMC5238943 | biostudies-literature
Project description:A major challenge in using human pluripotent stem cells (hPSCs) in therapy is the risk of teratoma formation due to contaminating undifferentiated stem cells. We used CRISPR-Cas9 for in-frame insertion of a suicide gene, iC9, into the endogenous SOX2 locus in human embryonic stem cell (ESC) line H1 for specific eradication of undifferentiated cells without affecting differentiated cells. This locus was chosen over NANOG and OCT4, two other well-characterized stem cell loci, due to significantly reduced off-target effect. We showed that undifferentiated H1-iC9 cells were induced to apoptosis by iC9 inducer AP1903, whereas differentiated cell lineages including hematopoietic cells, neurons, and islet beta-like cells were not affected. We also showed that AP1903 selectively removed undifferentiated H1-iC9 cells from a mixed cell population. This strategy therefore provides a layer of safety control before transplantation of a stem-cell-derived product in therapy.
| S-EPMC6909005 | biostudies-literature