Human Embryonic Stem Cell Lines Research Articles

Generation of a homozygous MYH7 gene knockout human embryonic stem cell line (WAe009-A-69) using an episomal vector-based CRISPR/Cas9 system

Myosin heavy chain 7 (MYH7) encodes the human heart myosin heavy chain subunit, which plays an important role in myocardial contraction. MYH7 is the main pathogenic gene that causes Hypertrophic cardiomyopathy (HCM) and Dilated cardiomyopathy (DCM). In this experiment, a MYH7 homozygous knockout human embryonic stem cell (hESC) line, WAe009-A-69, was generated using an episomal vector-based CRISPR/Cas9 system. It can be an ideal tool to further study the function of MYH7. The cell line was confirmed with pluripotency, normal karyotype and trilineage differentiation potential.

Generation of a heterozygous SOX10 knockout human embryonic stem cell line using CRISPR/Cas9 technology

SOX10 is one of the master transcription factors in neural crest development. Human SOX10 mutations are associated with Waardenburg syndrome type 4 (Waardenburg-Shah, WS4), which can be inherited in both autosomal dominant and recessive patterns. Here, the human embryonic stem cell (hESC) line, H9, was used to generate a heterozygous SOX10 knockout cell line as the in vitro model of WS4 by CRISPR/Cas9-mediated gene targeting. This cell line may represent a valuable tool for uncovering the pathogenesis of WS4.

Common Postzygotic Mutational Signatures in Healthy Adult Tissues Related to Embryonic Hypoxia

Postzygotic mutations are acquired in normal tissues throughout an individual’s lifetime and hold clues for identifying mutagenic factors. Here, we investigated postzygotic mutation spectra of healthy individuals using optimized ultra-deep exome sequencing of the time-series samples from the same volunteer as well as the samples from different individuals. In blood, sperm, and muscle cells, we resolved three common types of mutational signatures. Signatures A and B represent clock-like mutational processes, and the polymorphisms of epigenetic regulation genes influence the proportion of signature B in mutation profiles. Notably, signature C, characterized by C>T transitions at GpCpN sites, tends to be a feature of diverse normal tissues. Mutations of this type are likely to occur early during embryonic development, supported by their relatively high allelic frequencies, presence in multiple tissues, and decrease in occurrence with age. Almost none of the public datasets for tumors feature this signature, except for 19.6% of samples of clear cell renal cell carcinoma with increased activation of the hypoxia-inducible factor 1 (HIF-1) signaling pathway. Moreover, the accumulation of signature C in the mutation profile was accelerated in a human embryonic stem cell line with drug-induced activation of HIF-1α. Thus, embryonic hypoxia may explain this novel signature across multiple normal tissues. Our study suggests that hypoxic condition in an early stage of embryonic development is a crucial factor inducing C>T transitions at GpCpN sites; and individuals’ genetic background may also influence their postzygotic mutation profiles.

MFN2 Deficiency Impairs Mitochondrial Transport and Downregulates Motor Protein Expression in Human Spinal Motor Neurons.

Charcot-Marie-Tooth (CMT) disease is one of the most common genetically inherited neurological disorders and CMT type 2A (CMT 2A) is caused by dominant mutations in the mitofusin-2 (MFN2) gene. MFN2 is located in the outer mitochondrial membrane and is a mediator of mitochondrial fusion, with an essential role in maintaining normal neuronal functions. Although loss of MFN2 induces axonal neuropathy, the detailed mechanism by which MFN2 deficiency results in axonal degeneration of human spinal motor neurons remains largely unknown. In this study, we generated MFN2-knockdown human embryonic stem cell (hESC) lines using lentivirus expressing MFN2 short hairpin RNA (shRNA). Using these hESC lines, we found that MFN2 loss did not affect spinal motor neuron differentiation from hESCs but resulted in mitochondrial fragmentation and dysfunction as determined by live-cell imaging. Notably, MFN2-knockodwn spinal motor neurons exhibited CMT2A disease-related phenotypes, including extensive perikaryal inclusions of phosphorylated neurofilament heavy chain (pNfH), frequent axonal swellings, and increased pNfH levels in long-term cultures. Importantly, MFN2 deficit impaired anterograde and retrograde mitochondrial transport within axons, and reduced the mRNA and protein levels of kinesin and dynein, indicating the interfered motor protein expression induced by MFN2 deficiency. Our results reveal that MFN2 knockdown induced axonal degeneration of spinal motor neurons and defects in mitochondrial morphology and function. The impaired mitochondrial transport in MFN2-knockdown spinal motor neurons is mediated, at least partially, by the altered motor proteins, providing potential therapeutic targets for rescuing axonal degeneration of spinal motor neurons in CMT2A disease.

Abstract P305: Patient-specific Ipsc-derived Cardiomyocytes Reveal Abnormal Regulation Of Fgf16 In A Familial Atrial Septal Defect

Aims: Congenital heart disease (CHD) frequently occurs in newborns due to abnormal formation of the heart or major blood vessels. Mutations in the GATA4 gene, which encodes GATA binding protein 4, are responsible for atrial septal defect (ASD), a common CHD. This study aims to gain insights into the molecular mechanisms of CHD using human induced pluripotent stem cells (iPSCs) from a family cohort with ASD. Methods and Results: Patient-specific iPSCs possess the same genetic information as the donor and can differentiate into various cell types from all three germ layers in vitro , thus presenting a promising approach for disease modeling and molecular mechanism research. Here, we generated a patient-specific iPSC line (iPSC-G4 T280M ) from a family cohort carrying a hereditary ASD mutation in GATA4 gene (T280M), as well as a human embryonic stem cell line (ESC-G4 T280M ) carrying the isogenic T280M mutation using the CRISPR/Cas9 genome editing method. The GATA4-mutant iPSCs and ESCs were then differentiated into cardiomyocytes (CMs) to model GATA4 mutation-associated ASD. We observed an obvious defect in cell proliferation in cardiomyocytes derived from both GATA4 T280M -mutant iPSCs (iPSC-G4 T280M -CMs) and ESCs (ESC-G4 T280M -CMs), while the impaired proliferation ability of iPSC-G4 T280M -CMs could be restored by gene correction. Integrated analysis of RNA-Seq and ChIP-Seq data indicated that FGF16 is a direct target of wild-type GATA4. However, the T280M mutation obstructed GATA4 occupancy at the FGF16 promoter region, leading to impaired activation of FGF16 transcription. Overexpression of FGF16 in GATA4-mutant cardiomyocytes rescued the cell proliferation defect. The direct relationship between GATA4 T280M and ASD was demonstrated in a human iPSC model for the first time. Conclusions: In summary, our study revealed the molecular mechanism of the GATA4 T280M mutation in ASD. Understanding the roles of the GATA4-FGF16 axis in iPSC-CMs will shed light on heart development and provide novel insights for the treatment of ASD and other CHD disorders.

Global molecular features in transcription and chromatin accessibility in human extended pluripotent stem cells

Human extended pluripotent stem (hEPS) cell is a newly established human embryonic stem cell (hESC) line with the capacity of chimerizing both embryonic and extraembryonic tissues compared with primed hESCs which are inefficient to contribute to the inner cell mass (ICM). The molecular mechanism underlying the pluripotency of hEPS cells is still not clear. We conducted RNA-seq and ATAC-seq analysis to investigate the differential expression profiling and genomic chromatin accessibility features. According to our data, more than 2000 genes were specially up-regulated in hEPS cells. Furthermore, the open chromatin regions in these two human embryonic stem cell lines were quite different. In hEPS cells, transcriptional factors binding motifs associated with pluripotency maintenance were enriched in chromatin accessible regions. Integrating the results from ATAC-seq and RNA-seq, we identified new regulatory features which were important for pluripotency maintenance and cell development in hEPS cells. Together, these results provided a new perspective on the understanding of molecular features of hESCs in different pluripotent states and a novel resource for further studies on regenerative medicine by using hEPS cells.

Generation of a COL4A5 heterozygous mutation human embryonic stem cell line (WAe009-A-58) using an episomal vector-based CRISPR/Cas9 system

X-linked Alport syndrome (XLAS) is the second most common inherited kidney disease which pathogenic variants related to a mutation in the COL4A5 gene encoding the type IV collagen α5 chain. Here, we have generated a COL4A5 heterozygous mutant human embryonic stem cell (hESC) line (H9-COL4A5+/−) by an episomal vector-based CRISPR/Cas9 system. The generated H9-COL4A5+/− maintained a normal stem cell morphology, stably expressed pluripotent markers, and could differentiate into all three germ layers in vivo. This cell line offers an in vitro efficient platform to explore pathogenic mechanisms in XLAS and provides a cell-based disease model for drug testing.

Development of a Human Intestinal Organoid Model for In Vitro Studies on Gut Inflammation and Fibrosis.

Inflammatory Bowel Diseases (IBDs) are characterized by chronic intestinal inflammation and fibrosis, the latter being the predominant denominator for long-term complications. Epithelial and mesenchymal 2D cultures are highly utilized in vitro models for the preclinical evaluation of anti-inflammatory and antifibrotic therapies. More recently, human intestinal organoids (HIOs), a new 3D in vitro model derived from pluripotent stem cells, have the advantage to closely resemble the architecture of the intestinal mucosa. However, the appropriate timing for the study of inflammatory and fibrotic responses, during HIO development, has not been adequately investigated. We developed HIOs from the human embryonic stem cell line, H1, and examined the expression of mesenchymal markers during their maturation process. We also investigated the effect of inflammatory stimuli on the expression of fibrotic and immunological mediators. Serial evaluation of the expression of mesenchymal and extracellular matrix (ECM) markers revealed that HIOs have an adequately developed mesenchymal component, which gradually declines through culture passages. Specifically, CD90, collagen type I, collagen type III, and fibronectin were highly expressed in early passages but gradually diminished in late passages. The proinflammatory cytokines IL-1α and TNF-α induced the mRNA expression of fibronectin, collagen types I and III, tissue factor (TF), and alpha-smooth muscle actin (α-SMA) primarily in early passages. Similarly, HIOs elicited strong mRNA and protein mesenchymal (CXCL10) and epithelial (CXCL1, CCL2, CXCL8, and CCL20) chemokine responses in early but not late passages. In contrast, the epithelial tight junction components, CLDN1 and JAMA, responded to inflammatory stimulation independently of the culture passage. Our findings indicate that this HIO model contains a functional mesenchymal component, during early passages, and underline the significance of the mesenchymal cells' fitness in inflammatory and fibrotic responses. Therefore, we propose that this model is suitable for the study of epithelial-mesenchymal interactions in early passages when the mesenchymal component is active.

Establishment of a KCNQ1 homozygous knockout human embryonic stem cell line by episomal vector-based CRISPR/Cas9 system

As a member of the voltage-gated potassium ion channels, KCNQ1 plays an important role in heart physiological functions. Numerous mutations in KCNQ1 were identified as primary causes to hereditary long-QT syndrome. To further study the role of KCNQ1 in human cardiac functions, here we generated a homozygous KCNQ1 knockout human embryonic stem cell line (KCNQ1-KO) using episomal vector-based CRISPR/Cas9 system. This generated cell line presented typical stem cells colony morphology, maintained highly pluripotency and normal karyotype, also was able to differentiate into all three germ layers in vivo.

Generation of a TPM1 homozygous knockout embryonic stem cell line by CRISPR/Cas9 editing

Alpha-Tropomyosin (TPM1) plays a crucial role in actin regulation and stability and contributes fundamental functions to heart development: without TPM1 expressing, mice embryos will die early in embryogenesis. To further identify the role of TPM1 in human cardiac development, here we generated a homozygous TPM1 knockout (TPM1−/−) human embryonic stem cell (hESC) line using CRISPR/Cas9-based genome editing system. The generated TPM1−/− hESC line maintained normal karyotype, highly expressed pluripotency markers and was able to differentiate into all three germ layers in vivo. This cell line provides a powerful tool to investigate the role of TPM1 in heart development in future.

Generation of clinical-grade human embryonic stem cell line KthES11 according to Japanese regulations

The human embryonic stem cell line, KthES11, is derived from a normal healthy blastocyst donated for clinical research. The inner cell mass (ICM) was isolated using mechanical dissection and plated on laminin fragments. Cell line derivation, its propagation and storage were performed without feeders in an animal product-free environment according to current Good Manufacturing Practice (cGMP) standards. KthES11 shows a normal karyotype, pluripotent state and differentiation to the three germ layers. The cell line was further validated for sterility, mycoplasma-free, antibiotic residues and specific human pathogens.

MOCVD of Noble Metal Film Materials for Medical Implants: Microstructure and Biocompatibility of Ir and Au/Ir Coatings on TiNi

Noble metals such as Ir, Pt, Au are promising as coatings for metal medical implants to improve biocompatibility and corrosion resistance. Moreover, these coatings can be used as a basis for the further formation of bimetallic hetero-structures with enhanced antibacterial properties. In this work, we develop an approach to obtain such coatings by metal-organic chemical vapor deposition (MOCVD). We have been focused on the formation of Ir coating with developed morphology and subsequent discrete Au coating onto the titanium nickelide (TiNi) implant material. Iridium was deposited in an oxidizing atmosphere from the volatile precursor [Ir(cod)(acac)] (cod = cyclooctadiene-1,5, acac = acetylacetonate-anion). The effects of the deposition temperature (290–350 °C) and amount of introduced oxygen on the composition (Ir, Ir + IrO2) and microstructure of the samples were studied. Hetero-metallic Au/Ir coatings were obtained using [(CH3)2Au(thd)] precursor (thd = dpm = dipivaloylmethanate-anion) at a deposition temperature of 240 °C in the presence of oxygen. To assess the biocompatibility, the toxicity of Ir/TiNi, Au/Ir/TiNi, and uncoated TiNi in relation to human embryonic stem cell line Man-1 was examined after 1, 3, and 5 days of incubation. The results obtained were explained based on the coating microstructures.

Generation of human embryonic stem cell lines (WAe001-A-67,WAe001-A-68) with TEAD1 and TEAD4 expression by the PiggyBac transposon system

Transcription factors TEAD1 and TEAD4 play an important role in development, differentiation, cell growth and proliferation. To further understand the exact role of TEAD1 and TEAD4 in these processes. We generated TEAD1 and TEAD4 doxycycline-inducible expression human embryonic stem cell lines (WAe001-A-67 and WAe001-A-68) by PiggyBac transposon system. These cell lines retained normal morphology and karyotype, normal expression of pluripotent markers, and differentiation potential. These cell lines can be used to verify whether the TEAD1 and TEAD4 play a role in stem cell and cell lineage differentiation.

Using Advanced Cell Culture Techniques to Differentiate Pluripotent Stem Cells and Recreate Tissue Structures Representative of Teratoma Xenografts.

Various methods are currently used to investigate human tissue differentiation, including human embryo culture and studies utilising pluripotent stem cells (PSCs) such as in vitro embryoid body formation and in vivo teratoma assays. Each method has its own distinct advantages, yet many are limited due to being unable to achieve the complexity and maturity of tissue structures observed in the developed human. The teratoma xenograft assay allows maturation of more complex tissue derivatives, but this method has ethical issues surrounding animal usage and significant protocol variation. In this study, we have combined three-dimensional (3D) in vitro cell technologies including the common technique of embryoid body (EB) formation with a novel porous scaffold membrane, in order to prolong cell viability and extend the differentiation of PSC derived EBs. This approach enables the formation of more complex morphologically identifiable 3D tissue structures representative of all three primary germ layers. Preliminary in vitro work with the human embryonal carcinoma line TERA2.SP12 demonstrated improved EB viability and enhanced tissue structure formation, comparable to teratocarcinoma xenografts derived in vivo from the same cell line. This is thought to be due to reduced diffusion distances as the shape of the spherical EB transforms and flattens, allowing for improved nutritional/oxygen support to the developing structures over extended periods. Further work with EBs derived from murine embryonic stem cells demonstrated that the formation of a wide range of complex, recognisable tissue structures could be achieved within 2–3 weeks of culture. Rudimentary tissue structures from all three germ layers were present, including epidermal, cartilage and epithelial tissues, again, strongly resembling tissue structure of teratoma xenografts of the same cell line. Proof of concept work with EBs derived from the human embryonic stem cell line H9 also showed the ability to form complex tissue structures within this system. This novel yet simple model offers a controllable, reproducible method to achieve complex tissue formation in vitro. It has the potential to be used to study human developmental processes, as well as offering an animal free alternative method to the teratoma assay to assess the developmental potential of novel stem cell lines.

Patient-specific iPSC-derived cardiomyocytes reveal abnormal regulation of FGF16 in a familial atrial septal defect.

Congenital heart disease (CHD) frequently occurs in newborns due to abnormal formation of the heart or major blood vessels. Mutations in the GATA4 gene, which encodes GATA binding protein 4, are responsible for atrial septal defect (ASD), a common CHD. This study aims to gain insights into the molecular mechanisms of CHD using human-induced pluripotent stem cells (iPSCs) from a family cohort with ASD. Patient-specific iPSCs possess the same genetic information as the donor and can differentiate into various cell types from all three germ layers in vitro, thus presenting a promising approach for disease modelling and molecular mechanism research. Here, we generated a patient-specific iPSC line (iPSC-G4T280M) from a family cohort carrying a hereditary ASD mutation in GATA4 gene (T280M), as well as a human embryonic stem cell line (ESC-G4T280M) carrying the isogenic T280M mutation using the CRISPR/Cas9 genome editing method. The GATA4-mutant iPSCs and ESCs were then differentiated into cardiomyocytes (CMs) to model GATA4 mutation-associated ASD. We observed an obvious defect in cell proliferation in cardiomyocytes derived from both GATA4T280M-mutant iPSCs (iPSC-G4T280M-CMs) and ESCs (ESC-G4T280M-CMs), while the impaired proliferation ability of iPSC-G4T280M-CMs could be restored by gene correction. Integrated analysis of RNA-Seq and ChIP-Seq data indicated that FGF16 is a direct target of wild-type GATA4. However, the T280M mutation obstructed GATA4 occupancy at the FGF16 promoter region, leading to impaired activation of FGF16 transcription. Overexpression of FGF16 in GATA4-mutant cardiomyocytes rescued the cell proliferation defect. The direct relationship between GATA4T280M and ASD was demonstrated in a human iPSC model for the first time. In summary, our study revealed the molecular mechanism of the GATA4T280M mutation in ASD. Understanding the roles of the GATA4-FGF16 axis in iPSC-CMs will shed light on heart development and provide novel insights for the treatment of ASD and other CHD disorders.

Establishment and characterization of a human embryonic stem cell line carrying a heterozygous GATA4T280M mutation

Heterozygous T280M mutation in the GATA4 gene, encoding GATA binding protein 4, was recently identified in patients with congenital heart disease (CHD). Here, a human embryonic stem cell line carrying a heterozygous GATA4T280M mutation was generated by using the CRISPR/Cas9 and piggyBac technologies. This GATA4T280M cell line remains normal morphology, pluripotency and karyotype, and could differentiate into all three germ layers both in vivo and in vitro. Thus, the GATA4T280M cell line will be a useful platform to model GATA4-associated CHD through the induction of cardiomyocyte differentiation in vitro.

Generation of a heterozygous GAPDH-Luciferase human ESC line (HVRDe008-A-1) for in vivo monitoring of stem cells and their differentiated progeny

Human stem cell-derived beta (SC-β) cells are a candidate for cell replacement therapy for type 1 diabetes. Whilst refinements to the differentiation protocol have resulted in the production of SC-β cells that resemble adult beta cells, the unsolved challenge to protect transplanted SC-β cells from the host immune system remains. To monitor the survival of SC-β cells in vivo, we knocked-in the Firefly luciferase gene into the GAPDH locus of the HUES8 human embryonic stem cell (hESC) line, such that differentiated islet cells constitutively express luciferase.

Generation of a FTO gene knockout human embryonic stem cell line using CRISPR/Cas9 editing

Fat mass and obesity-associated protein (FTO) is the first protein found to have the activity of N6-methyladenosine (m6A) demethylation. It has been reported that FTO was involved in different physiological and pathological processes, including stem cell differentiation, sex determination, tumorigenesis, and progression. To further understand the exact role of FTO in these processes, we generated a FTO knockout human embryonic stem cell (hESC) line by CRISPR/Cas9 mediated gene editing method. This cell line maintained normal karyotype, pluripotency, and trilineage differentiation potential, which are considered as a model for function studies of the FTO protein in hESC self-renewal and differentiation.

Generation of an ELTD1 knockout human embryonic stem cell line by the iCRISPR/Cas9 system

Human ELTD1 (Epidermal growth factor, latrophilin and seven-transmembrane domain-containing 1), an orphan G-protein-coupled receptor (GPCR) belonging to the adhesion GPCR family, has been reported as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. However, little is known about the function of ELTD1, especially its undiscovered ligands. In this experiment, an ELTD1 homozygous knockout human embryonic stem cell line, FAHZUe001-A, was generated by the iCRISPR/Cas9 system to achieve a deeper understanding of ELTD1. The FAHZUe001-A was confirmed with normal karyotype, typical undifferentiated morphology, pluripotency and trilineage differentiation potential in vitro.

RB1 Depleted Pluripotent Stem Cell Retinal Organoids Recapitulate Cell State Transitions of Retinoblastoma Development

Retinoblastoma (Rb) is a childhood cancer of the developing retina in response to bi-allelic inactivation of RB1 or MYCN amplification, accounting for up to 17% of all tumours in infancy. To gain insights into the transcriptional events of cell state transitions during Rb development, we developed two models via retinal organoid differentiation of a RB1 depleted human embryonic stem cell line (RB1-null hESCs) and a RB1 patient-specific induced pluripotent (iPSC) line harbouring a RB1 biallelic mutation (c.2082delC). Both models were characterised by RB1 depletion and a significant increase in the fraction of proliferating cone precursors (RXRγ+Ki67+), which were defined as the cell of origin for Rb by single cell RNA-Seq analysis. The RB1 depleted retinal organoids displayed similar features to Rb tumours, including mitochondrial cristae aberrations and rosette-like structures and were able to undergo cell growth in an anchorage-independent manner, indicative of cell transformation in vitro. The patient-specific iPSC model displayed an enhanced reduction of amacrine, horizontal and retinal ganglion cells and an accelerated loss of cone photoreceptor markers during transition towards Rb, compared to the RB1-null hESC model. In both models, the Rb cells of origin, intermediate retinoma and/or Rb cells expressed retinal ganglion and horizontal cell markers in addition to cone markers, a novel finding, which could help to better characterise these tumours with possible therapeutic implications. Application of Melphalan, Topotecan and TW-37 led to a significant reduction in the fraction of proliferating cone precursors, validating the suitability of these in vitro models for testing novel therapeutics for Rb.