One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations. These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes. This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered. However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient. This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson's disease modeling or cell replacement therapy.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758769 | PMC |
| http://dx.doi.org/10.3791/51764 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Antidiabetic potency of glimepiride and naringin: an and investigation.
J Biomol Struct Dyn
December 2024
School of Pharmacy and Life Sciences, Centurion University of Technology and Management, Bhubaneswar, India.
Glimepiride (GLM) is one of the potential antidiabetic drugs used in clinics for a long time. It is currently used in combination with metformin along with other drugs, but has shown various complications in patients from long-term use. Thus, the hypothesis is to use a lower dose of GLM with a non-toxic class of flavonoid, naringin (NARN), for better therapy with minimal side-effects.
View Article and Find Full Text PDFICAM-1 expression is upregulated in the umbilical vein endothelial cells in acute chorioamnionitis with foetal inflammatory response.
Malays J Pathol
December 2024
Universiti Kebangsaan Malaysia, Faculty of Medicine, Department of Pathology, 56000 Kuala Lumpur, Malaysia.
Introduction: ICAM-1 is an adhesion molecule expressed on the endothelial cells and is involved in regulating leukocyte recruitment to the site of inflammation. Elevated ICAM-1 mRNA expression was found in the serum of mothers with chorioamnionitis. This study aimed to determine the expression of ICAM-1 in the placenta and umbilical cord of pregnancy with chorioamnionitis, and its association with adverse neonatal outcome.
View Article and Find Full Text PDFThe interaction of isoquinoline alkaloid crebanine with immunoglobulin G and cytotoxic effects toward MCF-7 breast cancer cell line.
Int J Biol Macromol
December 2024
The Third Department of Breast Cancer, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China; Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China. Electronic address:
In this study, the interaction of crebanine, an isoquinoline alkaloid, with immunoglobulin G (IgG) was evaluated. Subsequently, the anticancer effects of crebanine in MCF-7 breast cancer cells were assessed. The results demonstrate that static quenching plays a key role in the fluorescence quenching of the IgG by crebanine, and some embedded hydrophobic patches of the IgG are exposed upon interaction with crebanine, while the characteristic β-sheet conformation of the IgG was almost preserved.
View Article and Find Full Text PDFNucleolar protein PEXF controls ribosomal RNA synthesis and pluripotency exit.
Dev Cell
December 2024
Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China; Guangdong Province Key Laboratory of Orthopaedics and Traumatology, Guangzhou, Guangdong, China. Electronic address:
Maintenance and exit from pluripotency of embryonic stem cells (ESCs) are controlled by highly coordinated processes of protein synthesis and ribosome biogenesis (RiBi). ESCs are characterized by low rates of global protein synthesis and high levels of RiBi. Transient reduction of RiBi is a characteristic molecular event during the exit from pluripotency, of which the regulatory mechanism is unclear.
View Article and Find Full Text PDFBisphenol S exposure interrupted human embryonic stem cell derived cardiomyocytes differentiation through ER-NF-κB/ERK signaling pathway.
Ecotoxicol Environ Saf
December 2024
Department of Nutrition and Food Safety, West China School of Public Health/West China Fourth Hospital, Sichuan University, Chengdu, China; Food Safety Monitoring and Risk Assessment Key Laboratory of Sichuan Province, Chengdu, China. Electronic address:
Bisphenol S (BPS) has been put into production as a wide range of Bisphenol A (BPA) alternatives, while little is known regarding its cardiac developmental toxicity. To explore the effect of BPS on cardiomyocyte differentiation and its mechanism, our study established the human embryonic stem cell-cardiomyocyte differentiation model (hESC-CM), which was divided into early period of differentiation (DP1:1-8d), anaphase period of differentiation (DP2:9-16d) and whole stage of differentiation (DP3:1-16d) exposed to human-related levels of BPS. We found that the survival rate of cardiomyocytes was more sensitive to BPS at the early stage of differentiation than at the anaphase stage of differentiation, and exposure to higher than 30 µg/mL BPS throughout the differentiation period decreased the expression of cTnT.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!