scholarly journals Cardiomyocyte differentiation from iPS cells is delayed following knockout of Bcl-2

2022 ◽  
Author(s):  
Tim Vervliet ◽  
Robin Duelen ◽  
lLewelyn H Roderick ◽  
Maurilio Sampaolesi
Keyword(s):  
Cell Death ◽  
Pluripotent Stem Cells ◽  
Direct Interaction ◽  
B Cell Lymphoma ◽  
Ips Cells ◽  
Cardiomyocyte Differentiation ◽  
Cellular Functions ◽  
Post Translational Modifications ◽  
Induced Pluripotent

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) regulates a wide array of cellular functions involved in cell death, cell survival decisions and autophagy. Bcl-2 acts by both direct interaction with different components of the pathways involved and by intervening in intracellular Ca2+ signalling. The function of Bcl-2 is in turn regulated by post-translational modifications including phosphorylation at different sites by various kinases. Besides functions in cell death and apoptosis, Bcl-2 regulates cell differentiation processes, including of cardiomyocytes, although the signalling pathways involved are not fully elucidated. To further address the role of Bcl-2 during cardiomyocyte differentiation, we investigated the effect of its genetic knockout by CRISPR/Cas9 on the differentiation and functioning of human induced pluripotent stem cells to cardiomyocytes. Our results indicate that differentiation of iPS cells to cardiomyocytes is delayed by Bcl-2 KO, resulting in reduced size of spontaneously beating cells and reduced expression of cardiomyocyte Ca2+ toolkit and functionality. These data thus indicate that Bcl-2 an essential protein for cardiomyocyte generation.

scholarly journals NAMPT/SIRT2-mediated inhibition of the p53-p21 signaling pathway is indispensable for maintenance and hematopoietic differentiation of human iPS cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yun Xu ◽  
Masoud Nasri ◽  
Benjamin Dannenmann ◽  
Perihan Mir ◽  
Azadeh Zahabi ◽  
...  
Keyword(s):  
Embryoid Body ◽  
P53 Protein ◽  
Ips Cells ◽  
Hematopoietic Differentiation ◽  
Cellular Functions ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Signaling Axis ◽  
Human Ips Cells ◽  
Induced Pluripotent

Abstract Background Nicotinamide phosphoribosyltransferase (NAMPT) regulates cellular functions through the protein deacetylation activity of nicotinamide adenine dinucleotide (NAD+)-dependent sirtuins (SIRTs). SIRTs regulate functions of histones and none-histone proteins. The role of NAMPT/SIRT pathway in the regulation of maintenance and differentiation of human-induced pluripotent stem (iPS) cells is not fully elucidated. Methods We evaluated the effects of specific inhibitors of NAMPT or SIRT2 on the pluripotency, proliferation, survival, and hematopoietic differentiation of human iPS cells. We also studied the molecular mechanism downstream of NAMPT/SIRTs in iPS cells. Results We demonstrated that NAMPT is indispensable for the maintenance, survival, and hematopoietic differentiation of iPS cells. We found that inhibition of NAMPT or SIRT2 in iPS cells induces p53 protein by promoting its lysine acetylation. This leads to activation of the p53 target, p21, with subsequent cell cycle arrest and induction of apoptosis in iPS cells. NAMPT and SIRT2 inhibition also affect hematopoietic differentiation of iPS cells in an embryoid body (EB)-based cell culture system. Conclusions Our data demonstrate the essential role of the NAMPT/SIRT2/p53/p21 signaling axis in the maintenance and hematopoietic differentiation of iPS cells.

scholarly journals CRISPRi mediated Down regulation of SFPQ Gene Expression in Human induced Pluripotent Stem Cells Results in Massive Cell Death

2021 ◽  
Author(s):  
Navya Lam ◽  
Shinya Yamanaka ◽  
Samuel Perli
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cell Death ◽  
Human Cell ◽  
Pluripotent Stem Cells ◽  
Cell Biology ◽  
Splicing Factor ◽  
Core Component ◽  
Induced Pluripotent

Abstract CRISPR-Cas9 is widely used for targeted genome editing for a wide range of organisms. In this study, we used CRISPR interference (CRISPRi) which employs a catalytically inactive version of Cas9 (known as Dead Cas9 or dCas9) fused to KRAB a chromatin modifier. Similar to Cas9, the dCas9 protein forms a complex along with single guide RNA (sgRNA) and binds desired DNA sequences in the presence of a protospacer adjacent motif (PAM). However, unlike Cas9, dCas9 doesn’t cleave the DNA sequence but can repress gene expression when fused with KRAB. The role of paraspeckles in human cell biology is relatively unknown. In this study, we used CRISPRi to investigate the role of splicing factor proline and glutamine rich (SFPQ), also known as PSF (PTB-associated splicing factor), which is a conserved and core component of paraspeckles. We designed and constructed six different sgRNAs to target different locations of the SFPQ gene in Human induced Pluripotent Stem Cells (HiPSCs). The most effective sgRNA (sgRNA #5) knocked down the expression of SFPQ up to > 99%. We also observed that the knockdown of SFPQ exhibits severe cell-death phenotype in HiPSCs. This finding suggests that CRISPRi based SFPQ repression can modulate stem cell proliferation and maintenance. This study also suggests that SFPQ could play a vital role in proliferating cells such as cancer cells. Most importantly, these results are significant because in mice, knocking out another core component of paraspeckles results in no apparent phenotype despite the complete loss of paraspeckles. Given that traditional drug testing approaches rely on animal models, it is consequential to consider that human cell biology is more distinct than previously imagined. This study provides a new insight for role and function of SFPQ in iPSCs.

scholarly journals Irradiation strongly reduces tumorigenesis of human induced pluripotent stem cells

2017 ◽  
Vol 58 (4) ◽  
pp. 430-438 ◽  
Author(s):  
Shoki Inui ◽  
Kazumasa Minami ◽  
Emiko Ito ◽  
Hiromasa Imaizumi ◽  
Seiji Mori ◽  
...  
Keyword(s):  
Cell Death ◽  
Pluripotent Stem Cells ◽  
Ips Cells ◽  
Tumor Formation ◽  
Significant Suppression ◽  
Survival Fraction ◽  
Self Renewal ◽  
Clinical Transplantation ◽  
Induced Pluripotent

Abstract Induced pluripotent stem (iPS) cells have demonstrated they can undergo self-renewal, attain pluripotency, and differentiate into various types of functional cells. In clinical transplantation of iPS cells, however, a major problem is the prevention of tumorigenesis. We speculated that tumor formation could be inhibited by means of irradiation. Since the main purpose of this study was to explore the prevention of tumor formation in human iPS (hiPS) cells, we tested the effects of irradiation on tumor-associated factors such as radiosensitivity, pluripotency and cell death in hiPS cells. The irradiated hiPS cells showed much higher radiosensitivity, because the survival fraction of hiPS cells irradiated with 2 Gy was < 10%, and there was no change of pluripotency. Irradiation with 2 and 4 Gy caused substantial cell death, which was mostly the result of apoptosis. Irradiation with 2 Gy was detrimental enough to cause loss of proliferation capability and trigger substantial cell death in vitro. The hiPS cells irradiated with 2 Gy were injected into NOG mice (NOD/Shi-scid, IL-2 Rγnull) for the analysis of tumor formation. The group of mice into which hiPS cells irradiated with 2 Gy was transplanted showed significant suppression of tumor formation in comparison with that of the group into which non-irradiated hiPS cells were transplanted. It can be presumed that this diminished rate of tumor formation was due to loss of proliferation and cell death caused by irradiation. Our findings suggest that tumor formation following cell therapy or organ transplantation induced by hiPS cells may be prevented by irradiation.

scholarly journals NAMPT/SIRT2–mediated inhibition of the p53-p21 signaling pathway is indispensable for maintenance and hematopoietic differentiation of human iPS cells

2020 ◽  
Author(s):  
Julia Skokowa ◽  
Yun Xu ◽  
Masoud Nasri ◽  
Benjamin Dannenmann ◽  
Perihan Mir ◽  
...  
Keyword(s):  
Embryoid Body ◽  
P53 Protein ◽  
Ips Cells ◽  
Hematopoietic Differentiation ◽  
Cellular Functions ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Signaling Axis ◽  
Human Ips Cells ◽  
Induced Pluripotent

Abstract Background: Nicotinamide phosphoribosyltransferase (NAMPT) regulates cellular functions through the protein deacetylation activity of nicotinamide adenine dinucleotide (NAD+)-dependent sirtuins (SIRTs). SIRTs regulate functions of histones and none-histone proteins. The role of NAMPT/SIRTs pathway in the regulation of maintenance and differentiation of human induced pluripotent stem (iPS) cells is not fully elucidated. Methods: We evaluated the effects of specific inhibitors of NAMPT-, or SIRT2 on the pluripotency, proliferation, survival and hematopoietic differentiation of human iPS cells. We also studied the molecular mechanism downstream of NAMPT/SIRTs in iPS cells. Results: We demonstrated that NAMPT is indispensable for the maintenance, survival and hematopoietic differentiation of induced pluripotent stem (iPS) cells. We found that inhibition of NAMPT or SIRT2 in iPS cells induces p53 protein by promoting its lysine acetylation. This leads to activation of the p53 target, p21, with subsequent cell cycle arrest and induction of apoptosis in iPS cells. NAMPT and SIRT2 inhibition also affects hematopoietic differentiation of iPS cells in an embryoid body (EB)-based cell culture system. Conclusions: Our data demonstrate the essential role of the NAMPT/SIRT2/p53/p21 signaling axis in the maintenance and hematopoietic differentiation of iPS cells.

scholarly journals NAMPT/SIRT2–mediated inhibition of the p53-p21 signaling pathway is indispensable for maintenance and hematopoietic differentiation of human iPS cells

2020 ◽  
Author(s):  
Yun Xu ◽  
Masoud Nasri ◽  
Benjamin Dannenmann ◽  
Perihan Mir ◽  
Azadeh Zahabi ◽  
...  
Keyword(s):  
Embryoid Body ◽  
P53 Protein ◽  
Ips Cells ◽  
Hematopoietic Differentiation ◽  
Cellular Functions ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Signaling Axis ◽  
Human Ips Cells ◽  
Induced Pluripotent

Abstract Background: Nicotinamide phosphoribosyltransferase (NAMPT) regulates cellular functions through the protein deacetylation activity of nicotinamide adenine dinucleotide (NAD+)-dependent sirtuins (SIRTs). SIRTs regulate functions of histones and none-histone proteins. The role of NAMPT/SIRTs pathway in the regulation of maintenance and differentiation of human induced pluripotent stem (iPS) cells is not fully elucidated.Methods: We evaluated the effects of specific inhibitors of NAMPT-, or SIRT2 on the pluripotency, proliferation, survival and hematopoietic differentiation of human iPS cells. We also studied the molecular mechanism downstream of NAMPT/SIRTs in iPS cells.Results: We demonstrated that NAMPT is indispensable for the maintenance, survival and hematopoietic differentiation of induced pluripotent stem (iPS) cells. We found that inhibition of NAMPT or SIRT2 in iPS cells induces p53 protein by promoting its lysine acetylation. This leads to activation of the p53 target, p21, with subsequent cell cycle arrest and induction of apoptosis in iPS cells. NAMPT and SIRT2 inhibition also affect hematopoietic differentiation of iPS cells in an embryoid body (EB)-based cell culture system.Conclusions: Our data demonstrate the essential role of the NAMPT/SIRT2/p53/p21 signaling axis in the maintenance and hematopoietic differentiation of iPS cells.

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