Human Embryonic Stem Cell Lines Research Articles

Generation of three isogenic gene-edited Huntington’s disease human embryonic stem cell lines with DOX-inducible NGN2 expression cassette in the AAVS1 safe locus

Neurogenin 2 (NGN2), a neuronal transcription factor, can expedite differentiation of stem cells into mature glutamatergic neurons. We have utilized an allelic series of previously published and characterized isogenic Huntington's disease (IsoHD) human embryonic stem cell lines (Ooi et al., 2019), carrying different CAG repeat lengths in the first exon of the huntingtin gene. These IsoHDs were modified using CRISPR/Cas9 to insert NGN2 under the TET-ON doxycycline inducible promoter. The resulting IsoHD-NGN2 cell lines retained pluripotency in the absence of doxycycline (DOX), and via addition of DOX to the culturing media differentiation to neurons was achieved within 14 days.

Generation of a TSC2 knockout embryonic stem cell line by CRISPR/Cas9 editing

Tuberous Sclerosis Complex (TSC) is a severe developmental disorder with various clinical effects, primarily caused by TSC2 gene mutations, often involving loss of function(Henske,et al., 2016).To explore role of TSC2 in human heart development, we successfully developed a TSC2 knockout (TSC2-/-) human embryonic stem cells (hESCs) line using CRISPR/Cas9 gene editing. This TSC2-/- hESC line maintained a normal karyotype, expressed pluripotency markers strongly, and could differentiate into all three germ layers in vivo. This cell line will be a valuable tool for future research on the role of TSC2 in heart development.

Generation of SST-P2A-mCherry reporter human embryonic stem cell line using the CRISPR/Cas9 system (WAe001-A-2C)

Somatostatin (SST)-producing pancreatic delta-cells play an important role in maintaining the balance of insulin and glucagon secretion within the islets. This study aimed to generate a human embryonic stem cell (hESC) line with a SST-P2A-mCherry reporter using CRISPR/Cas9 system. The SST-P2A-mCherry reporter cell line was shown to maintain typical pluripotent characteristics and able to be induced into SST-producing pancreatic delta-cells. The generation of the cell line would provide useful platform for the characterization of stem cell-derived delta-cells, discovery of delta-cell surface markers and investigation of paracrine mechanisms, which will ultimately promote the drug discovery and cell therapy of diabetes mellitus.

Generation of a human embryonic stem cell line (SMUDHe010-A-1A) carrying Brainbow cassette in the AAVS1 gene by CRISPR/Cas9-mediated homologous recombination

Tracking single-cell lineages and their phenotypes longitudinally would help us better study skin development. Brainbow multicolor labeling approach is a genetic cell-labeling technique that tracks individual cells or analyzes cell lineages during development. Hence, we generated a stable Cre-inducible rainbow reporter human embryonic stem cell line (Named SMUDHe010-A-1A) by inserting the fluorescent protein gene (nGFP, YFP, RFP and CFP) at AAVS1 safe harbor locus using CRISPR/Cas9 technology. We verified that SMUDHe010-A-1A expressed the pluripotency markers and showed normal stem cell morphology. Furthermore, SMUDHe010-A-1A preserves normal karyotype and the ability to differentiate toward three germ layers. So the SMUDHe010-A-1A is effective for identifying and track cell populations.

Generation of a TBX20 homozygous knockout stem cell line (WAe009-A-1E) by episomal vector-based CRISPR/Cas9 system

The T-box family transcription factor gene TBX20 plays a crucial role in cardiac development and function. TBX20 mutations are associated with congenital heart disease, dilated cardiomyopathy, arrhythmias, and heart failure. To further study the role of TBX20 in human heart, here we generated a homozygous TBX20 knockout (TBX20-KO) human embryonic stem cell line using the CRISPR/Cas9 system. This TBX20-KO cell line maintains normal morphology, pluripotency, and karyotype, making it a valuable tool for investigating TBX20′s role in cardiac biology.

Generation of a ISL1 homozygous knockout stem cell line (WAe009-A-1G) by episomal vector-based CRISPR/Cas9 system

The ISL LIM homeobox 1 (ISL1) gene belongs to the LIM/homeodomain transcription factor family and plays a pivotal role in conveying multipotent and proliferative properties of cardiac precursor cells. Mutations in ISL1 are linked to congenital heart disease. To further explore ISL1′s role in the human heart, we have created a homozygous ISL1 knockout (ISL1-KO) human embryonic stem cell line using the CRISPR/Cas9 system. Notably, this ISL1-KO cell line retains normal morphology, pluripotency, and karyotype. This resource serves as a valuable tool for investigating ISL1′s function in cardiomyocyte differentiation.

The generation and validation of a dual cardiac HAND1-Tomato NKX2-5-GFP human embryonic stem cell line UMANe002-A-3

The transcription factor HAND1 is a critical regulator of cardiac development which is expressed in sub-populations of cardiac progenitors and cardiomyocytes. The transcription factor NKX2-5, in contrast, is expressed more widely in cardiac cells. Here we report the generation of a dual reporter hESC line where the expression of these genes can be simultaneously measured, enabling lineage analysis as well as studies of HAND1 and NKX2-5 gene regulation and protein function. This tool will have wide utility particularly for research on developmental biology and disease modelling.

Clinical-grade human embryonic stem cell–derived mesenchymal stromal cells ameliorate diabetic retinopathy in db/db mice

Background aimsMesenchymal stromal cells (MSCs) hold great promise in the treatment of diabetic retinopathy (DR), as evidenced by increasing preclinical and clinical studies. However, the absence of standardized and industrialized clinical-grade donor cells hampers the continued development and large-scale clinical application of MSCs-based therapies for DR. Previously, we have identified a unique population of MSCs generated from a clinical-grade human embryonic stem cell (hESC) line under Good Manufacturing Practice conditions that could be a potential source to address the issues. Here, we investigated the therapeutic potential of the clinical-grade hESC line–derived MSCs (hESC-MSCs) on db/db mice with DR. MethodshESC-MSCs were initially characterized by morphological assessment, flow cytometry analysis and trilineage differentiation assays. These cells (5 × 106 cells) were then transplanted intravenously into 12-week-old db/db mice via tail vein, with phosphate-buffered saline transplantation and untreated groups used as controls. The retinal alterations in neural functions and microvascular perfusions, and inflammatory responses in peripheral blood and retina were evaluated at 4 and 6 weeks after transplantation using electroretinography, optical coherence tomography angiography and flow cytometry, respectively. Body weight and fasting blood glucose (FBG) levels were also measured to investigate their systemic implications. ResultsCompared with controls, intravenous transplantation of hESC-MSCs could significantly: (i) enhance impaired retinal electroretinography functions (including amplitudes of a-, b-wave and oscillatory potentials) at 4 weeks after transplantation; (ii) alleviate microvascular dysfunctions, especially in the inner retina with significance (including reducing non-perfusion area and increasing vascular area density) at 4 weeks after transplantation; (iii) decrease FBG levels at 4 weeks after transplantation and induce weight loss up to 6 weeks after transplantation and (iv) increase both peripheral blood and retinal interleukin-10 levels at 4 weeks after transplantation and modulate peripheral blood inflammatory cytokines and chemokines levels, such as monocyte chemotactic protein-1, up to 6 weeks after transplantation. ConclusionsThe findings of our study indicated that intravenous transplantation of hESC-MSCs ameliorated retinal neural and microvascular dysfunctions, regulated body weight and FBG and modulated peripheral blood and retinal inflammation responses in a mouse model of DR. These results suggest that hESC-MSCs could be a potentially effective clinical-grade cell source for the treatment of DR.

Generation of a DMD loss-of-function mutant human embryonic stem cell lines by CRISPR base editing

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disorder, which is caused mostly by frame-disrupting, out-of-frame variation in the dystrophin (DMD) gene. Loss-of- function mutations are the most common type of mutation in DMD, accounting for approximately 60–90% of all DMD variations. In this study, we used adenine base editing to generate a human embryonic stem cell line with splice-site mutations to mimic exon deletion variants in clinical Duchenne muscular dystrophy patients. This cell line has differentiation potential and a normal karyotypic.

Generation of a gene-edited H9 embryonic stem cell line carrying a DOX-inducible NGN2 expression cassette in the CLYBL locus

The pro-neural transcription factor neurogenin-2 (NGN2) possesses the ability to rapidly and effectively transform stem cells into fully operational neurons. Here we report the successful generation of a modified H9 human embryonic H9 stem cell line containing a doxycycline (DOX) inducible NGN2 expression construct featuring a floxed Blasticidin/mApple selection module in the safe-harbor locus CLYBL. This cell line retains its pluripotent state in the absence of DOX, yet readily transitions into a neuronal state upon DOX introduction.

Differential Gene Regulation of the Human Blastocyst Trophectoderm and Inner Cell Mass by Progesterone.

Knowledge of action of progesterone (P4) on the human preimplantation embryo is lacking. The objective of this study was to determine expression of a mitochondrial P4 receptor (PR-M) in the trophectoderm (TE) and the inner cell mass (ICM) of the human blastocyst and to determine P4-induced gene expression during growth from the cleavage to the blastocyst stage. Previously cryopreserved cleavage stage embryos were treated with P4 (10-6 M) or vehicle until blastocyst development. Cells from the TE and the ICM of dissected euploid embryos underwent RNA-seq analysis, while other embryos were used for analysis of nuclear PR (nPR) and PR-M expression.PR-M expression was confirmed in the TE, the ICM, and a human embryonic stem cell line (HESC). Conversely, nPR expression was absent in the TE and the ICM with low expression in the HESC line. RNA-seq analysis revealed P4 effects greater in the TE with 183 significant pathway changes compared to 27 in the ICM. The TE response included significant upregulation of genes associated with DNA replication, cell cycle phase transition and others, exemplified by a 7.6-fold increase in the cell proliferation gene, F-Box Associated Domain Containing. The majority of ICM pathways were downregulated including chromosome separation, centromere complex assembly and chromatin remodeling at centromere. This study confirms that human blastocysts express PR-M in both the TE and the ICM, but lack expression of nPR. P4-induced gene regulation differs greatly in the two cell fractions with the predominant effect of cell proliferation in the TE and not the ICM.

Generation of a TAB2 knockout hESC line (WAe009-A-Z) derived from H9 using CRISPR/Cas9

TGF-β-activated kinase 1 binding protein 2 (TAB2) is an intermediate protein that connects TNFR1 and other receptor signals to the TGF-β-activated kinase 1 (TAK1) signaling complex. TAB2 has been proved clinically relevant to congenital heart defects (CHD) and cardiomyopathy. In this study, we created a TAB2 knockout human embryonic stem cell line by CRISPR/Cas9 technology. The WAe009-A-Z cell line displayed stem cell morphology, pluripotency and normal karyotype, which could develop into three germ layers in vitro.

Generation of a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette in the AAVS1 locus by CRISPR/Cas9-mediated homologous recombination

Diphtheria toxin A (DTA) is an exotoxin secreted by Corynebacterium diphtheriae. After entering the cell through receptor-mediated manner, DTA can trigger the programmed cell death mechanism and lead to cell death. In 2001, Michiko Saito established a Diphtheria toxin receptor–mediated cell knockout system, which can conditional deplete specific cell type in transgenic mice. This system is not only very useful in the pathogenesis study of human diseases, but also has a wide application prospect in the study of organ development and regeneration. In 2008, David Voehringer described a newly generated mouse strain that encodes DTA under control of a loxP-flanked stop cassette in the ubiquitously expressed ROSA26 locus. Thereby, it can be used in combination with tissue-specific and/or inducible Cre-expressing mouse strains to achieve toxin-mediated cell ablation in vivo. The application of DTA–mediated cell knockout system in mice has been widely reported, but it has rarely been used in human cells. Accordingly, we generated a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette (loxp-stop-loxp-DTA, LSL-DTA) using CRISPR/Cas9-mediated homologous recombination. The cell line preserves normal karyotype, pluripotency and the ability to differentiate into all three germ layers. Moreover, the cell line can be used to prepare human organoid, which may provide a model for achieving conditional cell ablation in human tissues and organs.

The generation and validation of two NKX2-5 fluorescent reporter human embryonic stem cell lines: UMANe002-A-1 and UMANe002-A-2

The transcription factor NKX2-5 is a highly conserved master regulator of heart development which is widely expressed in cardiac progenitors and cardiomyocytes. Fluorescent reporters of NKX2-5 that minimally perturb normal protein expression can enable the identification, quantification and isolation of NKX2-5-expressing cells in a normal physiological state. Here we report the generation of two new hESC lines with eGFP inserted upstream (5′) or downstream (3′) of NKX2-5, linked by a cleavable T2A peptide. These complementary reporters produce a robust fluorescent signal in cardiac cells and have wide utility particularly for research on developmental biology and disease modelling.

ELTD1 Suppression Enhances Human Embryonic Stem Cell Hematopoiesis By Facilitating Hemogenic Endothelial Progenitor Induction Via Wnt/β-Catenin Signaling Inhibition

Objectives Human embryonic stem cells (hESCs) serve as an attractive cell source for generating functional blood cells and hold great potential for cell-based therapy in future clinical applications. However, the underlying molecular mechanisms that regulate hESC differentiation into hematopoietic cells remain elusive. Therefore, understanding hESC differentiation is essential for the acquisition of sufficient and functional cells in vitro for successful clinical translation. Materials and Methods ELTD1 was identified as a potential mediator of hESC hematopoietic differentiation by genome-wide gene profiling. ELTD1-deleted human embryonic stem cell (hESC) lines were constructed by the iCRISPR/Cas9 technology and were used to undergo hematopoietic differentiation. Flow cytometry, quantitative RT-PCR, immunofluorescence, and RNA sequencing were used for phenotype and molecular mechanism assessment. Results Real-time PCR analysis and bioinformatic studies demonstrated that the dynamic expression of ELTD1 parallels the hemogenic endothelial progenitors-related genes expression and subsequently decreased as they differentiate into hematopoietic cells ( Figure A, C), leading us to speculate that it might play a role in hESC early hematopoietic differentiation. To further confirm these results, stage-specific cell populations at different time points were sorted to document the cell-type specificity of ELTD1. As shown in Figure B, ELTD1 was exclusively expressed in CD31 +CD34 + HEP populations. Loss-of-function studies revealed that ELTD1 deletion promoting the induction of CD31 +CD34 + HEPs at day 6, thereby promoting endothelial-to-hematopoietic transition to generate more CD43 +/CD45 + hematopoietic cells, and the results were confirmed by flow cytometry and immunofluorescence analyses ( Figure D, E and F). To dissect the molecular mechanism by which ELTD1 regulates hESC hematopoietic differentiation, the differentiated cells at day 6 were used to perform the RNA-Seq. KEGG analysis showed that hematopoietic cell lineage-related genes and Wnt signaling pathway were prominently enriched in ELTD1-deleted cells compared to WT-H1, consistent with the enhancement of HEPs and hematopoietic cells generation caused by ELTD1 deletion ( Figure G). Moreover, decreased expression of Wnt target genes: SOX17, TCF7L1, MYC, CCND1, BIRC5, and AXIN2 was further validated by quantitative RT-PCR analysis ( Figure H). To determine whether the downregulation of Wnt/β-catenin signaling is required for ELTD1 deletion-mediated enhancement of early hematopoietic differentiation from hESCs, we treated ELTD1-KO1# and ELTD1-KO2# hESCs with the Wnt agonist CHIR99021. As expected, treatment with CHIR99021 inhibited the generation of CD31 +CD34 + HEPs compared to ELTD1 deleted alone at day 6, which was validated by flow cytometry ( Figure I). Conclusions We identified ELTD1 as a novel regulator of hematopoietic differentiation, which may act via Wnt/β-catenin signaling, and provided new insights into understanding the mechanism of human hematopoiesis.

Generation of a human embryonic stem cell reporter line, TMEM119-EGFP, for the visualisation of in vitro differentiated human microglia

Transmembrane protein 119 (TMEM119) is a recently identified microglia marker that is not expressed by other immune cells. Using CRISPR/Cas9 technology, we introduced enhanced green fluorescence protein (EGFP), into the H9 WA-09 human embryonic stem cell line, directly before the TMEM119 stop codon. Sanger sequencing confirmed successful insertion of the EGFP sequence. The newly created cell line expressed a normal morphology and karyotype, several pluripotency markers, and the ability to differentiate into all three germ layers. H9-TMEM119-EGFP can be used to provide a deeper understanding of the role of TMEM119 in microglia by monitoring its expression under different experimental conditions.

Generation of an NSD2-deficient human embryonic stem cell line using CRISPR/Cas9 technology

NSD2 is a histone methyltransferase (HMT) and is involved in the epigenetic regulation of hematopoiesis and hematological cancers. To understand and illustrate the precise roles of NSD2 in hematopoietic development, here we constructed a human embryonic stem cell (hESC) line with knockout of NSD2 using CRISPR/Cas9-mediated gene targeting. The cell line maintained typical stem cell morphology and normal karyotype. Furthermore, the pluripotency of the cell line was evidenced by high expression level of pluripotency genes and differentiation potential into three germ layers. The cell line provides a good model for studying roles of NSD2 in embryonic development, especially hematopoiesis.

Functional interrogation of twenty type 2 diabetes-associated genes using isogenic human embryonic stem cell-derived β-like cells

Genetic studies have identified numerous loci associated with type 2 diabetes (T2D), but the functional roles of many loci remain unexplored. Here, we engineered isogenic knockout human embryonic stem cell lines for 20 genes associated with T2D risk. We examined the impacts of each knockout on β cell differentiation, functions, and survival. We generated gene expression and chromatin accessibility profiles on β cells derived from each knockout line. Analyses of T2D-association signals overlapping HNF4A-dependent ATAC peaks identified a likely causal variant at the FAIM2 T2D-association signal. Additionally, the integrative association analyses identified four genes (CP, RNASE1, PCSK1N, and GSTA2) associated with insulin production, and two genes (TAGLN3 and DHRS2) associated with β cell sensitivity to lipotoxicity. Finally, we leveraged deep ATAC-seq read coverage to assess allele-specific imbalance at variants heterozygous in the parental line and identified a single likely functional variant at each of 23 T2D-association signals.

Heterozygous deletion of the autism-associated gene CHD8 impairs synaptic function through widespread changes in gene expression and chromatin compaction

Whole-exome sequencing of autism spectrum disorder (ASD) probands and unaffected family members has identified many genes harboring de novo variants suspected to play a causal role in the disorder. Of these, chromodomain helicase DNA-binding protein 8 (CHD8) is the most recurrently mutated. Despite the prevalence of CHD8 mutations, we have little insight into how CHD8 loss affects genome organization or the functional consequences of these molecular alterations in neurons. Here, we engineered two isogenic human embryonic stem cell lines with CHD8 loss-of-function mutations and characterized differences in differentiated human cortical neurons. We identified hundreds of genes with altered expression, including many involved in neural development and excitatory synaptic transmission. Field recordings and single-cell electrophysiology revealed a 3-fold decrease in firing rates and synaptic activity in CHD8+/- neurons, as well as a similar firing-rate deficit in primary cortical neurons from Chd8+/- mice. These alterations in neuron and synapse function can be reversed by CHD8 overexpression. Moreover, CHD8+/- neurons displayed a large increase in open chromatin across the genome, where the greatest change in compaction was near autism susceptibility candidate 2 (AUTS2), which encodes a transcriptional regulator implicated in ASD. Genes with changes in chromatin accessibility and expression in CHD8+/- neurons have significant overlap with genes mutated in probands for ASD, intellectual disability, and schizophrenia but not with genes mutated in healthy controls or other disease cohorts. Overall, this study characterizes key molecular alterations in genome structure and expression in CHD8+/- neurons and links these changes to impaired neuronal and synaptic function.

Human pallial MGE-type GABAergic interneuron cell therapy for chronic focal epilepsy.

Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy. One-third of patients have drug-refractory seizures and are left with suboptimal therapeutic options such as brain tissue-destructive surgery. Here, we report the development and characterization of a cell therapy alternative for drug-resistant MTLE, which is derived from a human embryonic stem cell line and comprises cryopreserved, post-mitotic, medial ganglionic eminence (MGE) pallial-type GABAergic interneurons. Single-dose intrahippocampal delivery of the interneurons in a mouse model of chronic MTLE resulted in consistent mesiotemporal seizure suppression, with most animals becoming seizure-free and surviving longer. The grafted interneurons dispersed locally, functionally integrated, persisted long term, and significantly reduced dentate granule cell dispersion, a pathological hallmark of MTLE. These disease-modifying effects were dose-dependent, with a broad therapeutic range. No adverse effects were observed. These findings support an ongoing phase 1/2 clinical trial (NCT05135091) for drug-resistant MTLE.