scholarly journals Pipeline for the Generation and Characterization of Transgenic Human Pluripotent Stem Cells Using the CRISPR/Cas9 Technology

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1312 ◽  
Author(s):  
Joffrey Mianné ◽  
Chloé Bourguignon ◽  
Chloé Nguyen Van ◽  
Mathieu Fieldès ◽  
Amel Nasri ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Genome Engineering ◽  
Disease Modeling ◽  
Mutant Cell ◽  
Point Mutations ◽  
Human Pluripotent Stem Cells ◽  
Wide Range

Recent advances in genome engineering based on the CRISPR/Cas9 technology have revolutionized our ability to manipulate genomic DNA. Its use in human pluripotent stem cells (hPSCs) has allowed a wide range of mutant cell lines to be obtained at an unprecedented rate. The combination of these two groundbreaking technologies has tremendous potential, from disease modeling to stem cell-based therapies. However, the generation, screening and molecular characterization of these cell lines remain a cumbersome and multi-step endeavor. Here, we propose a pipeline of strategies to efficiently generate, sub-clone, and characterize CRISPR/Cas9-edited hPSC lines in the function of the introduced mutation (indels, point mutations, insertion of large constructs, deletions).

scholarly journals A dual cardiomyocyte reporter model derived from human pluripotent stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yuqian Jiang ◽  
Xiaoping Bao ◽  
Xiaojun Lance Lian
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Fluorescent Protein ◽  
Disease Modeling ◽  
Human Pluripotent Stem Cells ◽  
Purification And Characterization ◽  
Dual Reporter ◽  
The Usa ◽  
Voltage Indicator

AbstractCardiovascular diseases (CVD) remain the leading cause of death in the USA. Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) provide a valuable cell source for regenerative therapy, disease modeling, and drug screening. Here, we established a hPSC line integrated with a mCherry fluorescent protein driven by the alpha myosin heavy chain (aMHC) promoter, which could be used to purify CMs based on the aMHC promoter activity in these cells. Combined with a fluorescent voltage indicator, ASAP2f, we achieved a dual reporter CM platform, which enables purification and characterization of CM subtypes and holds great potential for disease modeling and drug discovery of CVD.

scholarly journals Efficient GNE myopathy disease modeling with mutation specific phenotypes in human pluripotent stem cells by base editors

2020 ◽  
Author(s):  
Ju-Chan Park ◽  
Jumee Kim ◽  
Hyun-Ki Jang ◽  
Seung-Yeon Lee ◽  
Keun-Tae Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Point Mutations ◽  
Human Pluripotent Stem Cells ◽  
Drug Candidate ◽  
Disease Models ◽  
Target Cells ◽  
Gne Myopathy ◽  
Low Efficiency

AbstractIsogenic pairs of cell lines derived from human pluripotent stem cells (hPSCs) enable the precise assessment of mutation-specific phenotypes through differentiation to target cells, as this method of disease modeling excludes the bias of genetic variation. However, the extremely low efficiency of precise gene editing based on homology-directed repair (HDR) with Cas9 in hPSCs remains a technical hurdle for this approach. Herein, we took advantage of currently available base editors (BEs) in hPSCs to epitomize the isogenic disease model from hPSCs with a pathophysiological indicator. Using this method, we established 14 hPSCs that harbor point mutations on the GNE gene, including four different mutations found in GNE myopathy patients. Because BEs activated p53 to a lesser degree than Cas9, we observed a higher editing efficiency with BEs. Four different mutations in the epimerase or kinase domains of GNE revealed mutation-specific hyposialylation, which was closely correlated to pathological clinical phenotypes. These mutation-specific hyposialylation patterns were evident in GNE protein structure modeling. Furthermore, treatment with a drug candidate currently under clinical trials showed a mutation-specific drug response in GNE myopathy disease models. These data suggest that isogenic disease models from hPSCs using BEs could serve as a useful tool for mimicking the pathophysiology of GNE myopathy and for predicting drug responses.

scholarly journals The Impact of Acquired Genetic Abnormalities on the Clinical Translation of Human Pluripotent Stem Cells

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3246
Author(s):  
Alexander Keller ◽  
Claudia Spits
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Chromosomal Abnormalities ◽  
Current Knowledge ◽  
Point Mutations ◽  
Human Pluripotent Stem Cells ◽  
Clinical Translation ◽  
Copy Number Changes ◽  
Clinical Potential ◽  
The Impact

Human pluripotent stem cells (hPSC) are known to acquire chromosomal abnormalities, which range from point mutations to large copy number changes, including full chromosome aneuploidy. These aberrations have a wide-ranging influence on the state of cells, in both the undifferentiated and differentiated state. Currently, very little is known on how these abnormalities will impact the clinical translation of hPSC, and particularly their potential to prime cells for oncogenic transformation. A further complication is that many of these abnormalities exist in a mosaic state in culture, which complicates their detection with conventional karyotyping methods. In this review we discuss current knowledge on how these aberrations influence the cell state and how this may impact the future of research and the cells’ clinical potential.

scholarly journals Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions

2016 ◽  
Vol 5 (22) ◽  
pp. 2951-2958 ◽  
Author(s):  
Ken-ichiro Kamei ◽  
Yoshie Koyama ◽  
Yumie Tokunaga ◽  
Yasumasa Mashimo ◽  
Momoko Yoshioka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
3D Culture ◽  
Culture Conditions ◽  
Human Pluripotent Stem Cells ◽  
2D And 3D

scholarly journals Chemical hypoxia induces apoptosis of human pluripotent stem cells by a NOXA-mediated HIF-1α and HIF-2α independent mechanism

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Luciana Isaja ◽  
Sofía Mucci ◽  
Jonathan Vera ◽  
María Soledad Rodríguez-Varela ◽  
Mariela Marazita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Cell Viability ◽  
Pluripotent Stem Cells ◽  
Chromatin Condensation ◽  
Human Pluripotent Stem Cells ◽  
Chemical Hypoxia ◽  
Wide Range ◽  
Independent Mechanism ◽  
Induced Pluripotent

AbstractHuman embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are self-renewing human pluripotent stem cells (hPSCs) that can differentiate to a wide range of specialized cells. Notably, hPSCs enhance their undifferentiated state and self-renewal properties in hypoxia (5% O2). Although thoroughly analyzed, hypoxia implication in hPSCs death is not fully determined. In order to evaluate the effect of chemically mimicked hypoxia on hPSCs cell survival, we analyzed changes in cell viability and several aspects of apoptosis triggered by CoCl2 and dimethyloxalylglycine (DMOG). Mitochondrial function assays revealed a decrease in cell viability at 24 h post-treatments. Moreover, we detected chromatin condensation, DNA fragmentation and CASPASE-9 and 3 cleavages. In this context, we observed that P53, BNIP-3, and NOXA protein expression levels were significantly up-regulated at different time points upon chemical hypoxia induction. However, only siRNA-mediated downregulation of NOXA but not HIF-1α, HIF-2α, BNIP-3, and P53 did significantly affect the extent of cell death triggered by CoCl2 and DMOG in hPSCs. In conclusion, chemically mimicked hypoxia induces hPSCs cell death by a NOXA-mediated HIF-1α and HIF-2α independent mechanism.

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