Generation of human embryonic stem cell line expressing green fluorescent protein

Human embryonic stem cell line BJNhem20 pCAG-EGFP was generated using non-viral method (Fig. 1). The construct pCAG-EGFP was transfected using microporation procedure

Generation of transgenic human embryonic stem cell line BJNhem20–OCIAD1-OV

Ovarian Carcinoma Immuno-reactive Antigen domain containing protein 1 (OCIAD1) was overexpressed in BJNhem20 human embryonic stem cell line (hESC) using plasmid transfection, followed by stable cell line generation. The construct pCAG-OCIAD1 was introduced into hESCs by microporation

Aortic “Disease-in-a-Dish”: Mechanistic Insights and Drug Development Using iPSC-Based Disease Modeling

Thoracic aortic diseases, whether sporadic or due to a genetic disorder such as Marfan syndrome, lack effective medical therapies, with limited translation of treatments that are highly successful in mouse models into the clinic. Patient-derived induced pluripotent stem cells (iPSCs) offer the opportunity to establish new human models of aortic diseases. Here we review the power and potential of these systems to identify cellular and molecular mechanisms underlying disease and discuss recent advances, such as gene editing, and smooth muscle cell embryonic lineage. In particular, we discuss the practical aspects of vascular smooth muscle cell derivation and characterization, and provide our personal insights into the challenges and limitations of this approach. Future applications, such as genotype-phenotype association, drug screening, and precision medicine are discussed. We propose that iPSC-derived aortic disease models could guide future clinical trials via “clinical-trials-in-a-dish”, thus paving the way for new and improved therapies for patients

Understanding genomic medicine for thoracic aortic disease through the lens of induced pluripotent stem cells

Thoracic aortic disease (TAD) is often silent until a life-threatening complication occurs. However, genetic information can inform both identification and treatment at an early stage. Indeed, a diagnosis is important for personalised surveillance and intervention plans, as well as cascade screening of family members. Currently, only 20% of heritable TAD patients have a causative mutation identified and, consequently, further advances in genetic coverage are required to define the remaining molecular landscape. The rapid expansion of next generation sequencing technologies is providing a huge resource of genetic data, but a critical issue remains in functionally validating these findings. Induced pluripotent stem cells (iPSCs) are patient-derived, reprogrammed cell lines which allow mechanistic insights, complex modelling of genetic disease and a platform to study aortic genetic variants. This review will address the need for iPSCs as a frontline diagnostic tool to evaluate variants identified by genomic discovery studies and explore their evolving role in biological insight through to drug discovery

Generation of OCIAD1 inducible overexpression human embryonic stem cell line: BJNhem20-OCIAD1-Tet-On

AbstractHuman embryonic stem cell line BJNhem20-OCIAD1-Tet-On was generated using non-viral method. The constructs pCAG-Tet-On and pTRE-Tight vector driving OCIAD1 expression were transfected using microporation procedure. pCAG-Tet-On cells can be used for inducible expression of any coding sequence cloned into pTRE-Tight vector. For example, in human embryonic stem cells, Tet-On system has been used to generate SOX2 overexpression cell line (Adachi et al., 2010)

Aortic “Disease-in-a-Dish”: Mechanistic Insights and Drug Development Using iPSC-Based Disease Modeling

Thoracic aortic diseases, whether sporadic or due to a genetic disorder such as Marfan syndrome, lack effective medical therapies, with limited translation of treatments that are highly successful in mouse models into the clinic. Patient-derived induced pluripotent stem cells (iPSCs) offer the opportunity to establish new human models of aortic diseases. Here we review the power and potential of these systems to identify cellular and molecular mechanisms underlying disease and discuss recent advances, such as gene editing, and smooth muscle cell embryonic lineage. In particular, we discuss the practical aspects of vascular smooth muscle cell derivation and characterization, and provide our personal insights into the challenges and limitations of this approach. Future applications, such as genotype-phenotype association, drug screening, and precision medicine are discussed. We propose that iPSC-derived aortic disease models could guide future clinical trials via “clinical-trials-in-a-dish”, thus paving the way for new and improved therapies for patients

Generation of human embryonic stem cell line expressing a red fluorescent protein: BJNhem20-pCAG-tdTomato

Human embryonic stem cell line BJNhem20-pCAG-tdTomato was generated using non-viral method. The construct pCAG-tdTomato was transfected using microporation procedure. This fluorescent hESC line can help to study heterogeneity within individual cells in hESC colonies by enabling live tracking of their growth, migration and differentiation properties. This cell line also serves as a resource for additional transgene introduction/knock-out/knock-in generation in a fluorescent background and allows ease of analysis in studies involving cell mixing

Generation of a heterozygous knockout human embryonic stem cell line for the OCIAD1 locus using CRISPR/CAS9 mediated targeting: BJNhem20-OCIAD1-CRISPR-39

Ovarian carcinoma immuno-reactive antigen domain containing 1 (OCIAD1) single copy was knocked out generating an OCIAD1 heterozygous knockout human embryonic stem line named BJNhem20-OCIAD1-CRISPR-39. The line was generated using CRISPR-Cas9D10A double nickase knockout strategy (Mali et al., 2013)

Generation of a transgenic human embryonic stem cell line ectopically expressing the endosomal protein Asrij that regulates pluripotency in mouse embryonic stem cells: BJNhem20-Asrij

Asrij is an endocytic protein expressed in mouse embryonic stem cells (mESCs) and is essential for maintenance of stemness of mESCs (Mukhopadhyay et al., 2003; Sinha et al., 2013). Its human ortholog named Ovarian Carcinoma Immuno-reactive Antigen domain containing protein 1 (OCIAD1) is 85% identical. We ectopically expressed Asrij in epiblast stage equivalent-human embryonic stem cells (hESCs) to test for induction of naive pluripotency in primed pluripotent cells. The construct pCAG-Asrij was introduced into hESCs by microporation. Ectopic expression of Asrij in BJNhem20 hESC line was performed by selecting for plasmid transfection, followed by stable cell line generation