Showing papers on "Human embryonic stem cell line published in 2019"

Effect of the sonic hedgehog inhibitor GDC-0449 on an in vitro isogenic cellular model simulating odontogenic keratocysts.

Abstract: Odontogenic keratocysts (OKCs) are common cystic lesions of odontogenic epithelial origin that can occur sporadically or in association with naevoid basal cell carcinoma syndrome (NBCCS). OKCs are locally aggressive, cause marked destruction of the jaw bones and have a propensity to recur. PTCH1 mutations (at ∼80%) are frequently detected in the epithelia of both NBCCS-related and sporadic OKCs, suggesting that PTCH1 inactivation might constitutively activate sonic hedgehog (SHH) signalling and play a major role in disease pathogenesis. Thus, small molecule inhibitors of SHH signalling might represent a new treatment strategy for OKCs. However, studies on the molecular mechanisms associated with OKCs have been hampered by limited epithelial cell yields during OKC explant culture. Here, we constructed an isogenic PTCH1R135X/+ cellular model of PTCH1 inactivation by introducing a heterozygous mutation, namely, c.403C>T (p.R135X), which has been identified in OKC patients, into a human embryonic stem cell line using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. This was followed by the induction of epithelial differentiation. Using this in vitro isogenic cellular model, we verified that the PTCH1R135X/+ heterozygous mutation causes ligand-independent activation of SHH signalling due to PTCH1 haploinsufficiency. This activation was found to be downregulated in a dose-dependent manner by the SHH pathway inhibitor GDC-0449. In addition, through inhibition of activated SHH signalling, the enhanced proliferation observed in these induced cells was suppressed, suggesting that GDC-0449 might represent an effective inhibitor of the SHH pathway for use during OKC treatment.

Evolving Transcriptomic Signature of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Cells With Age

Abstract: We differentiated the human embryonic stem cell line H9 into retinal pigment epithelium (RPE) cells to assess temporal changes in transcriptomic profiles of cells. We performed single cell RNA-Sequencing of a total of 16,576 cells, and analysed the resulting data to access the molecular changes of RPE cells across two culture time points (1 and 12 months). Our results indicate the stability of the RPE transcriptomic signature over time in culture, with results indicating maturing populations of RPE could be observed over time, with no evidence of an epithelial mesenchymal transition. Assessment of gene ontology (GO) pathways reveals that as cell cultures age, RPE cells upregulate expression of genes involved in metal binding and antioxidant functions, which might reflect an increased ability to handle oxidative stress as cells mature. Comparison with the transcriptional profiles of native RPE identified a progression towards a maturing RPE profile. These results suggest that in vitro long-term culture of RPE cells allow the modelling of phenotypes observed in native mature tissue. Our work highlights the changing transcriptional landscape of hPSC-derived RPE as they age in culture, which provides a reference for native and patient- samples to be benchmarked against.

Antisense oligonucleotide therapy rescues aggresome formation in a novel Spinocerebellar Ataxia type 3 human embryonic stem cell line

Abstract: Spinocerebellar ataxia type 3 (SCA3) is a fatal, late-onset neurodegenerative disorder characterized by selective neuropathology in the brainstem, cerebellum, spinal cord, and substantia nigra. Here, we characterize the first NIH-approved human embryonic stem cell (hESC) line derived from an embryo harboring the SCA3 mutation. Referred here as SCA3-hESC, this line is heterozygous for the mutant polyglutamine-encoding CAG repeat expansion in the ATXN3 gene within the pathogenic repeat range for SCA3. We observed relevant molecular hallmarks of the human disease at all differentiation stages from stem cells to cortical neurons, including robust ATXN3 aggregation and altered expression of key components of the protein quality control machinery. Finally, antisense oligonucleotide-mediated reduction of ATXN3 prevented the formation of p62-positive aggresomes in SCA3-hESCs. The SCA3-hESC line offers a unique and highly relevant human disease model that holds strong potential to advance understanding of SCA3 disease mechanisms and facilitate the evaluation of possible SCA3 therapies. Highlights Generated first NIH-approved SCA3 human embryonic stem cell line (SCA3-hESC) SCA3–hESC exhibit robust ATXN3 aggregation pathology and form p62+ aggresomes Anti-ATXN3 antisense oligonucleotides rescue aggresome formation in SCA3-hESC Derived SCA3 neurons form aggregates and exhibit impaired protein quality control

One-Step Generation of Seamless Luciferase Gene Knockin Using CRISPR/Cas9 Genome Editing in Human Pluripotent Stem Cells.

Abstract: Human pluripotent stem cells (hPSCs) offer powerful platforms for studying mechanisms of human diseases and for evaluating potential treatments. Genome editing, particularly the CRISPR/Cas9-based method, is highly effective for generating cell and animal models to study genetic human diseases. However, the procedure for generating gene-edited hPSCs is laborious, time consuming and unintentional genetic changes may confound the consequent experiments and conclusions. Here we describe one-step knockin of the NanoLuc luciferase gene (Nluc) to the fragile X syndrome gene, FMR1, in a human embryonic stem cell line (hESC), H1, and a fragile X disease model human induced pluripotent stem cell line (hiPSC), FX-iPSC. The luciferase reporter cell lines provide new platforms for exploring potential treatments for fragile X syndrome. The shortened and scarless targeting method described here can be effectively applied to other genes.

Method of producing a population of mafa-expressing cells (versions)

Abstract: FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology, particularly to producing a population of MAFA expressing cells. Method involves differentiation of human cells expressing markers characteristic for line of formed endoderm, obtained from pluripotent stem cells, which are human pluripotent stem cells obtained without destruction of human embryo, human pluripotent stem cells of non-embryonic origin, cells from the human embryonic stem cell line H1, cells from a human embryonic stem cell line H7, cells from a human embryonic stem cell line H9, or cells from a human embryonic stem cell line SA002, into human cells expressing markers characteristic of the pancreatic endoderm line. That is followed by differentiating human cells expressing markers characteristic of a pancreatic endoderm line into human cells expressing markers characteristic of a line of pancreatic endocrine cells expressing at least one of NGN3, NEUROD, ISL1, PDX1, NKX6.1, PAX4 and PTF1-alpha; and treating human cells expressing markers specific to the pancreatic endocrine cell line, with a kinase inhibitor selected from the group consisting of 2-cyanoethyl alster-paullonom; SU9516; alster-pumpallon; Cdk1/2-inhibitor III; casein kinase I inhibitor; Akt-inhibitor IV; EGFR-inhibitor; MEK1/2-inhibitor; staurosporine N-benzoyl-, PKR-inhibitor and PDGF receptor IV tyrosine kinase inhibitor.EFFECT: invention widens the range of agents.21 cl, 16 dwg, 7 tbl, 9 ex

Preparation method and application of high-concentration composite regeneration factor derived from neural stem cells

Abstract: The invention provides a preparation method and application of a high-concentration composite regeneration factor derived from neural stem cells. The human neural stem cells are induced and differentiated by a human embryonic stem cell line, second to third generations of human neural stem cells in a logarithmic growth phase are selected, a stem cell culture medium is collected, and the high-concentration composite regeneration factor is prepared. The neural stem cell-derived regeneration factor prepared by the inventor has a very good application prospect in treatment of spinal cord injury, and provides a new choice for effective treatment of central nervous system injury, including traumatic brain injury, spinal cord injury and the like.