Knockout Human Embryonic Stem Cell Research Articles

Microglia regulate GABAergic neurogenesis in prenatal human brain through IGF1.

GABAergic neurons are an essential cellular component of neural circuits. Their abundance and diversity have enlarged significantly in the human brain, contributing to the expanded cognitive capacity of humans. However, the developmental mechanism of the extended production of GABAergic neurons in the human brain remains elusive. Here, we use single-cell transcriptomics, bioinformatics, and histological analyses to uncover microglial regulation of the sustained proliferation of GABAergic progenitors and neuroblasts in the human medial ganglionic eminence (hMGE). We show that insulin-like growth factor 1 (IGF1) and its receptor IGR1R as the top ligand-receptor pair underlying microglia-progenitor communication in the prenatal human brain. Using our newly developed neuroimmune hMGE organoids, which mimics hMGE cytoarchitecture and developmental trajectory, we demonstrate that microglia-derived IGF1 promotes progenitor proliferation and the production of GABAergic neurons. Conversely, IGF1-neutralizing antibodies and IGF1 knockout human embryonic stem cells (hESC)-induced microglia (iMG) completely abolished iMG-mediated progenitor proliferation. Together, these findings reveal a previously unappreciated role of microglia-derived IGF1 in promoting proliferation of neural progenitors and the development of GABAergic neurons.

Establishment of a CIB1 knockout human pluripotent stem cell line via CRISPR/Cas9 genome editing technology

Calcium- and integrin-binding protein 1 (CIB1) has a diverse role in many different cell types and processes, including calcium signaling, migration, adhesion, proliferation, and survival. It is associated with cancer, cardiovascular disease and male infertility. Here, CRISPR/Cas9 genome-editing technology was employed to establish a CIB1 knockout human embryonic stem cell line, which exhibited normal pluripotency and karyotype.

Generation of ID1/3 knockout human embryonic stem cell lines (WAe009-A-2A and WAe009-A-2B) derived from H9 using CRISPR/Cas9

ID1 and ID3 are the members of the Inhibitor of DNA Binding (ID) protein family, which negatively regulates the basic helix-loop-helix (bHLH) transcription factors by forming heterodimers, are involved in neurodevelopment, cardiovascular development, and tumor metastasis. We created twoID1/3knockout cell lines from a human embryonic stem cell (hESC) line (H9) by CRISPR/Cas9-mediated gene targeting. These cell lines maintain stem cell morphology, a normal karyotype, and the expression of pluripotent marker genes. Additionally, they retain their in vivo differentiation potential. Thecell lines are valuable tools for studying the roles of ID1/3 in neurodevelopment and cardiovascular diseases.

Biallelic variants in CSMD1 are implicated in a neurodevelopmental disorder with intellectual disability and variable cortical malformations

CSMD1 (Cub and Sushi Multiple Domains 1) is a well-recognized regulator of the complement cascade, an important component of the innate immune response. CSMD1 is highly expressed in the central nervous system (CNS) where emergent functions of the complement pathway modulate neural development and synaptic activity. While a genetic risk factor for neuropsychiatric disorders, the role of CSMD1 in neurodevelopmental disorders is unclear. Through international variant sharing, we identified inherited biallelic CSMD1 variants in eight individuals from six families of diverse ancestry who present with global developmental delay, intellectual disability, microcephaly, and polymicrogyria. We modeled CSMD1 loss-of-function (LOF) pathogenesis in early-stage forebrain organoids differentiated from CSMD1 knockout human embryonic stem cells (hESCs). We show that CSMD1 is necessary for neuroepithelial cytoarchitecture and synchronous differentiation. In summary, we identified a critical role for CSMD1 in brain development and biallelic CSMD1 variants as the molecular basis of a previously undefined neurodevelopmental disorder.

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 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.

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.

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.

Generation of a TRPV1 knockout human pluripotent stem cell line (WAe009-A-U) using CRISPR/Cas9

The transient receptor potential vanilloid subfamily 1 (TRPV1) is a polymodal nociceptor that is highly expressed in sensory nerves. Activation of TRPV1 receptors excites primary afferent nociceptors by opening cation channels, allowing the influx of Na+ and Ca2+ ions into the cytoplasm. Here, a TRPV1 knockout human embryonic stem cell line was generated using the CRISPR/Cas9 genome-editing technology to further study the function of TRPV1. The cell line confirmed with normal pluripotency and karyotype.

Chenodeoxycholic acid rescues axonal degeneration in induced pluripotent stem cell-derived neurons from spastic paraplegia type 5 and cerebrotendinous xanthomatosis patients

BackgroundBiallelic mutations in CYP27A1 and CYP7B1, two critical genes regulating cholesterol and bile acid metabolism, cause cerebrotendinous xanthomatosis (CTX) and hereditary spastic paraplegia type 5 (SPG5), respectively. These rare diseases are characterized by progressive degeneration of corticospinal motor neuron axons, yet the underlying pathogenic mechanisms and strategies to mitigate axonal degeneration remain elusive.MethodsTo generate induced pluripotent stem cell (iPSC)-based models for CTX and SPG5, we reprogrammed patient skin fibroblasts into iPSCs by transducing fibroblast cells with episomal vectors containing pluripotency factors. These patient-specific iPSCs, as well as control iPSCs, were differentiated into cortical projection neurons (PNs) and examined for biochemical alterations and disease-related phenotypes.ResultsCTX and SPG5 patient iPSC-derived cortical PNs recapitulated several disease-specific biochemical changes and axonal defects of both diseases. Notably, the bile acid chenodeoxycholic acid (CDCA) effectively mitigated the biochemical alterations and rescued axonal degeneration in patient iPSC-derived neurons. To further examine underlying disease mechanisms, we developed CYP7B1 knockout human embryonic stem cell (hESC) lines using CRISPR-cas9-mediated gene editing and, following differentiation, examined hESC-derived cortical PNs. Knockout of CYP7B1 resulted in similar axonal vesiculation and degeneration in human cortical PN axons, confirming a cause-effect relationship between gene deficiency and axonal degeneration. Interestingly, CYP7B1 deficiency led to impaired neurofilament expression and organization as well as axonal degeneration, which could be rescued with CDCA, establishing a new disease mechanism and therapeutic target to mitigate axonal degeneration.ConclusionsOur data demonstrate disease-specific lipid disturbances and axonopathy mechanisms in human pluripotent stem cell-based neuronal models of CTX and SPG5 and identify CDCA, an established treatment of CTX, as a potential pharmacotherapy for SPG5. We propose this novel treatment strategy to rescue axonal degeneration in SPG5, a currently incurable condition.

Generation of a PPP1CA knockout human pluripotent stem cell line via CRISPR/Cas9

The Protein phosphatase 1 catalytic subunit alpha (PPP1CA) is a alpha subunit of the PP1 complex, which known to be involved in the regulation of a variety of cellular processes, such as cell division, glycogen metabolism, muscle contractility, protein synthesis, and HIV-1 viral transcription. Increased PP1 activity has been observed in the end stage of heart failure. Here, a PPP1CA knockout human embryonic stem cell line, WAe009-A-B, was generated using the CRISPR/Cas9 system to further study the function of PPP1CA. The cell line confirmed with pluripotency, normal karyotype and differentiation potential.

Establishment of a PPP2CA homozygous knockout human embryonic stem cell line via CRISPR/Cas9 system

Protein phosphatase 2A (PPP2CA) is one of the four main Ser/Thr phosphatase enzymes, which involved in the negative control of cell growth and division. PPP2CA is the main protein phosphatase in the heart, which regulates Ca (2+) through a series of ion channels and transporters. In this study, we generated a PPP2CA homozygous knockout human embryonic stem cell line WAe009-A-25 based on the transient expression CRISPR/Cas9 system to investigate functional effect of PP1 deficiency. This cell line has multidirectional differentiation potential, normal karyotypic and trilineage differentiation potential in vivo.

Generation of OCIAD2 homozygous knockout (BJNhem20-OCIAD2-CRISPR-33) and heterozygous knockout (BJNhem20-OCIAD2-CRISPR-40) human embryonic stem cell lines using CRISPR-Cas9 mediated targeting

Ovarian Carcinoma Immunoreactive Antigen domain containing 2 (OCIAD2) was knocked out by targeting its exon 4 through CRISPR-Cas9 paired nickase strategy to generate two OCIAD2 knockout human embryonic stem cell lines- one homozygous (BJNhem20-OCIAD2-CRISPR-33) and one heterozygous (BJNhem20-OCIAD2-CRISPR-40) for mutant ociad2. Both lines maintain pluripotency, normal karyotype, and trilineage differentiation potential.

Generation of a homozygous FIS1 knockout human embryonic stem cell line GIBHe015-B by CRISPR/Cas9 system

The mitochondrial fission protein 1 (FIS1) is essential for mitochondrial division or fission and has been determined to mediate mitochondrial and peroxisomal fission. Other studies also found that FIS1 functions as an essential component of the mitophagy and apoptosis pathways in mammalian cells, suggesting that FIS1 has multiple important roles. Here, we generated homozygous FIS1 knockout human embryonic stem cells (hESCs) using the CRISPR/Cas9 system. This cell line exhibits normal karyotype, pluripotency, and trilineage differentiation potential, which could provide a useful cellular resource for exploring the functions of FIS1 and their implications in human health and diseases.

Generation of CD16A gene knockout human embryonic stem cell line using CRISPR/Cas9

CD16A is a receptor for the Fc portion of immunoglobulin G, and is involved in the antibody dependent cellular cytotoxicity (ADCC) of nature killer cells(Zhu et al., 2020) and antibody dependent enhancement (ADE) of virus infections(Wan et al., 2020). However, the role of CD16A in human embryonic stem cell modeled development has been merely documented. Hence, to illustrate the role of CD16A in the human cell development, we reported a CD16A knockout human embryonic stem(hESC) cell line via CRISPR/Cas9 mediated gene knockout. The CD16A mutated cell line displayed normal karyotype, pluripotent stem cell marker gene expression and differentiation potential.

Generation of a homozygous TAZ knockout hESCs line by CRISPR/Cas9 system

Tafazzin (TAZ), a mitochondrial transacylase located on chromosome X, is required for the production of the mitochondrial phospholipid cardiolipin. Mutations occurring in the TAZ gene will lead to Barth syndrome, an X-linked recessive disease generally presenting as cardiomyopathy affecting males. Disease modeling strategies based on pluripotent stem cells (PSCs) provide an unprecedented and powerful platform to study Barth Syndrome. However, current studies were mostly based on male PSCs, the results and conclusions of which neglected the potential distinctions existing in disease phenotypes and mechanisms between gender. In this study, based on the H9 cell line (Female), we generated a homozygous TAZ knockout (TAZ-KO) human embryonic stem cell (hESC) line by employing CRISPR/Cas9 genome editing tools. This female TAZ-KO cell line, with normal karyotype, robust pluripotency and remarkably reduced TAZ expression, would be a useful tool for further deeply studying the pathogenesis of Barth syndrome cardiomyopathy in females.

Generation of a human embryonic stem cell line (WAe009-A-78) carrying homozygous TBX18 knockout

T-Box Transcription Factor 18 is a member of the T-box family, encoding TBX18 protein. As a transcriptional repressor, it related to developmental processes of a majority of tissues and organs and plays crucial part in the embryonic development of sinoatrial node. Using an episomal vector-based CRISPR/Cas9 system, we have established a homozygous TBX18 knockout (TBX18-KO) human embryonic stem cell (hESC) line. This newly TBX18-/- hESC line display normal pluripotency, morphology, karyotype and trilineage differentiating capacity. This cell line may provide a powerful tool to investigate the role of TBX18 gene in sinoatrial node development in future.

CRISPR/Cas9-engineered human ES cells harboring heterozygous and homozygous c-KIT knockout

The receptor tyrosine kinase c-KIT (CD117) has a key role in hematopoiesis and is a marker for endothelial and cardiac progenitor cells. In vivo, deficiency of c-KIT is lethal and therefore using CRISPR/Cas9 editing we generated heterozygous and homozygous c-KIT knockout human embryonic stem cell (ES cell) lines. The c-KIT knockout left ES cell pluripotency unaffected as shown by immunofluorescence and trilineage differentiation potential. Heterozygous and homozygous c-KIT knockouts showed complete loss of exon 17, resulting in ablation of c-KIT protein from the cell surface. c-KIT knockout ES cells provide a valuable tool for further investigating c-KIT biology.

Transcriptional programs regulating neuronal differentiation are disrupted in DLG2 knockout human embryonic stem cells and enriched for schizophrenia and related disorders risk variants

Coordinated programs of gene expression drive brain development. It is unclear which transcriptional programs, in which cell-types, are affected in neuropsychiatric disorders such as schizophrenia. Here we integrate human genetics with transcriptomic data from differentiation of human embryonic stem cells into cortical excitatory neurons. We identify transcriptional programs expressed during early neurogenesis in vitro and in human foetal cortex that are down-regulated in DLG2−/− lines. Down-regulation impacted neuronal differentiation and maturation, impairing migration, morphology and action potential generation. Genetic variation in these programs is associated with neuropsychiatric disorders and cognitive function, with associated variants predominantly concentrated in loss-of-function intolerant genes. Neurogenic programs also overlap schizophrenia GWAS enrichment previously identified in mature excitatory neurons, suggesting that pathways active during prenatal cortical development may also be associated with mature neuronal dysfunction. Our data from human embryonic stem cells, when combined with analysis of available foetal cortical gene expression data, de novo rare variants and GWAS statistics for neuropsychiatric disorders and cognition, reveal a convergence on transcriptional programs regulating excitatory cortical neurogenesis.