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.

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 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 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 Nox4-IGF2 Axis Promotes Differentiation of Embryoid Body Cells Into Derivatives of the Three Embryonic Germ Layers

2021 ◽  
Author(s):  
Jusong Kim ◽  
Jaewon Kim ◽  
Hee Jung Lim ◽  
Sanghyuk Lee ◽  
Yun Soo Bae ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Second Messengers ◽  
Differentiation Markers ◽  
Germ Layers ◽  
Cellular Processes ◽  
Signaling Axis ◽  
Induced Pluripotent

AbstractReactive oxygen species (ROS) play important roles as second messengers in a wide array of cellular processes including differentiation of stem cells. We identified Nox4 as the major ROS-generating enzyme whose expression is induced during differentiation of embryoid body (EB) into cells of all three germ layers. The role of Nox4 was examined using induced pluripotent stem cells (iPSCs) generated from Nox4 knockout (Nox4−/−) mouse. Differentiation markers showed significantly reduced expression levels consistent with the importance of Nox4-generated ROS during this process. From transcriptomic analyses, we found insulin-like growth factor 2 (IGF2), a member of a gene family extensively involved in embryonic development, as one of the most down-regulated genes in Nox4−/− cells. Indeed, addition of IGF2 to culture partly restored the differentiation competence of Nox4−/− iPSCs. Our results reveal an important signaling axis mediated by ROS in control of crucial events during differentiation of pluripotent stem cells. Graphical Abstract

scholarly journals Bone Morphogenetic Protein 4 (BMP4) Enhances the Differentiation of Human Induced Pluripotent Stem Cells into Limbal Progenitor Cells

2021 ◽  
Vol 43 (3) ◽  
pp. 2124-2134
Author(s):  
Hyun Soo Lee ◽  
Jeewon Mok ◽  
Choun-Ki Joo
Keyword(s):  
Progenitor Cells ◽  
Dermal Fibroblast ◽  
Ips Cells ◽  
Human Dermal Fibroblast ◽  
Corneal Epithelial ◽  
Specific Medium ◽  
Morphogenetic Protein ◽  
Human Ips Cells ◽  
Limbal Stem Cell ◽  
Induced Pluripotent

Corneal epithelium maintains visual acuity and is regenerated by the proliferation and differentiation of limbal progenitor cells. Transplantation of human limbal progenitor cells could restore the integrity and functionality of the corneal surface in patients with limbal stem cell deficiency. However, multiple protocols are employed to differentiate human induced pluripotent stem (iPS) cells into corneal epithelium or limbal progenitor cells. The aim of this study was to optimize a protocol that uses bone morphogenetic protein 4 (BMP4) and limbal cell-specific medium. Human dermal fibroblast-derived iPS cells were differentiated into limbal progenitor cells using limbal cell-specific (PI) medium and varying doses (1, 10, and 50 ng/mL) and durations (1, 3, and 10 days) of BMP4 treatment. Differentiated human iPS cells were analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemical studies at 2 or 4 weeks after BMP4 treatment. Culturing human dermal fibroblast-derived iPS cells in limbal cell-specific medium and BMP4 gave rise to limbal progenitor and corneal epithelial-like cells. The optimal protocol of 10 ng/mL and three days of BMP4 treatment elicited significantly higher limbal progenitor marker (ABCG2, ∆Np63α) expression and less corneal epithelial cell marker (CK3, CK12) expression than the other combinations of BMP4 dose and duration. In conclusion, this study identified a successful reprogramming strategy to induce limbal progenitor cells from human iPS cells using limbal cell-specific medium and BMP4. Additionally, our experiments indicate that the optimal BMP4 dose and duration favor limbal progenitor cell differentiation over corneal epithelial cells and maintain the phenotype of limbal stem cells. These findings contribute to the development of therapies for limbal stem cell deficiency disorders.

Human induced pluripotent stem cells are capable of B-cell lymphopoiesis

Blood ◽  
2011 ◽  
Vol 117 (15) ◽  
pp. 4008-4011 ◽  
Author(s):  
Lee Carpenter ◽  
Ram Malladi ◽  
Cheng-Tao Yang ◽  
Anna French ◽  
Katherine J. Pilkington ◽  
...  
Keyword(s):  
Stem Cells ◽  
B Cell ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Ips Cell ◽  
Lymphoid Lineage ◽  
Human Ips Cells ◽  
Induced Pluripotent ◽  
First Time ◽  
Unique Potential

Abstract Induced pluripotent stem (iPS) cells offer a unique potential for understanding the molecular basis of disease and development. Here we have generated several human iPS cell lines, and we describe their pluripotent phenotype and ability to differentiate into erythroid cells, monocytes, and endothelial cells. More significantly, however, when these iPS cells were differentiated under conditions that promote lympho-hematopoiesis from human embryonic stem cells, we observed the formation of pre-B cells. These cells were CD45+CD19+CD10+ and were positive for transcripts Pax5, IL7αR, λ-like, and VpreB receptor. Although they were negative for surface IgM and CD5 expression, iPS-derived CD45+CD19+ cells also exhibited multiple genomic D-JH rearrangements, which supports a pre–B-cell identity. We therefore have been able to demonstrate, for the first time, that human iPS cells are able to undergo hematopoiesis that contributes to the B-cell lymphoid lineage.

scholarly journals p53 Is a Crucial Regulator of Hemogenic Endothelial Cell Differentiation from Human Induced Pluripotent Stem Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1085-1085
Author(s):  
Jitendra K. Kanaujiya ◽  
Elizabeth G. Lingenheld ◽  
William C. Skarnes ◽  
Hideyuki Oguro
Keyword(s):  
Stem Cells ◽  
Cell Surface ◽  
Pluripotent Stem Cells ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Scientific Advisory ◽  
Induced Pluripotent

Abstract De novo generation of hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (hiPSCs) could provide a virtually unlimited supply of autologous HSCs for clinical transplantation, and offer various approaches that enable gene therapy, drug discovery, disease modeling, and in vitro modeling of human hematopoietic development. However, the derivation of long-term self-renewing HSCs from hiPSCs in culture remains elusive. The tumor suppressor protein p53 plays important roles in normal and malignant hematopoiesis, and Trp53-deficient mice exhibit increased number of HSCs. Although activation of p53 is known to promote differentiation of hPSCs and hPSCs recurrently acquire TP53 dominant negative mutations, its role in hematopoietic differentiation of hiPSCs has not been explored. To differentiate hiPSCs into hematopoietic stem and progenitor cells (HSPCs), we used embryoid body (EB) formation method to first differentiate hiPSCs into hemogenic endothelial (HE) cells that express the CD34 highCD144 +CD73 -CD184 -CD43 -CD235a - cell-surface markers. HE cells were then transferred onto a Matrigel-coated plate to undergo endothelial-to-hematopoietic transition (EHT) to generate HSPCs that express the CD34 midCD45 mid cell-surface markers. Developed HSPCs were functionally evaluated by colony forming assay. We observed that the expression of CDKN1A, a p53 target gene, was upregulated in hiPSC-derived EBs and HSPCs over the course of differentiation. To investigate the role of p53 in the generation of HSPCs from hiPSCs, we genetically deleted TP53 in hiPSCs followed by hematopoietic differentiation. While TP53 deletion increased the growth of EBs, it resulted in severe impairment of differentiation into HE cells and overall production of HSPCs that can form colonies. During HE differentiation from hiPSCs, TP53-deficient EBs showed significant reduction of endothelial-lineage gene expression, such as ETV2, CDH5, and PECAM1, as well as expression of RUNX1, a master transcription factor required for HE specification. These results indicate the indispensable role of p53 in HE differentiation from hiPSCs. We then examined the effect of p53 activation on HE differentiation from hiPSCs by pharmacological activation of p53 in hiPSC-derived cells. Transient activation of p53 by Nutlin-3, a small molecule that inhibits the p53-HDM2 interaction and protects p53 from proteasomal degradation, only during HE differentiation but not during EHT significantly promoted HSPC generation as compared to the vehicle treated control. Our findings shed light on the importance of selecting hiPSC lines that retain normal p53 activity for HE differentiation, and provide an approach to promote hematopoietic differentiation of hiPSCs by transiently activating p53 during HE differentiation. Disclosures Kanaujiya: Synthego: Other: Scientific Advisory; eGenesis: Other: Scientific Advisory.

scholarly journals Micro-RNA Profiling Reveals the Key Role of miR-206 in the Hematopoietic Potential of Human Embryonic and Induced Pluripotent Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1275-1275
Author(s):  
Stephane Flamant ◽  
Jean-Claude Chomel ◽  
Christophe Desterke ◽  
Olivier Feraud ◽  
Emilie Gobbo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Target Genes ◽  
Hematopoietic Differentiation ◽  
Induced Pluripotent

Abstract Although human pluripotent stem cells (hPSCs) can theoretically be differentiated into any cell type, their ability to generate hematopoietic cells shows a major variability from one cell line to another. The reasons of this variable differentiation potential, which is constant and reproducible in a given hPSC line, are not clearly established. In order to study this phenomenon, we comparatively studied 4 human embryonic stem cell lines (hESC) and 11 human induced pluripotent stem cell (hiPSC) lines using transcriptome assays. These cell lines exhibited a significant variability to generate in vitro hematopoiesis as evaluated by day-16 embryoid body (EB) formation followed by clonogenic (CFC) assays. Four out of 11 iPSC lines (PB6, PB9, PB12.1, and PB14.3) were found to lack any hematopoietic differentiation ability whereas 7 cell lines showed variable hematopoietic potential. Among hESC lines, H9 and CL0 had low H1 and SA01 exhibited high hematopoietic potential using the above assays. Among hESC and hIPSC displaying hematopoietic potential, two sub-groups were further defined based on their hematopoietic CFC efficiency: a group of poor (generation of less than 100 CFC/105 cells, PB4 / PB10 /H9 /CL01), and high hematopoietic competency (more than 120 CFC/105 cells, PB3/ PB6.1 /PB7 /PB13 /PB17 /SA01/H1). Using global miRNome analysis performed at the pluripotency stage, the expression of 754 individual miRNAs was analyzed from 15 hPSC lines in order to explore a potential predictive marker between both sub-groups of pluripotent cells according to their hematopoietic potency. Using this approach, 27 miRNAs out of 754 appeared differentially expressed allowing the identification of a miRNA signature associated with hematopoietic-competency. The hematopoietic competency was associated with down-regulation of miR-206, miR-135b, miR-105, miR-492, miR-622 and upregulation of miR-520a, miR-296, miR-122, miR-515, miR-335. Amongst these, miR-206 harbored the most significant variation (0.04-Fold change). To explore the role of miRNA-206 in this phenomenon, we have generated a miR-206-eFGP-Puro lentiviral vector which was transfected in hESC line H1 followed by puromycin selection. As a control, H1 cell line was transfected with a Arabidopsis thaliana microRNA sequence (ath-miR-159a), which has no specific targets in mammalian cells. The correct expression of the transgenes were evaluated by flow cytometry (using GFP) and q-RT-PCR for miR-206 expression. The hematopoietic potential of H1 cell line and its miR-206-overexpressing counterpart was then tested using standard in vitro assays via d16-EB generation. We found that both CFC numbers and percentage of CD34+ were significantly lower in H1-mir-206-derived day-16 EB cells than in H1-ath- derived day-16 EB cells (p < 0.05). Thus, over-expression of miR-206 in this blood-competent hESC appeared to repress its hematopoietic potential at very early stage, since a similar lower CFC efficiency was observed in day-3 EB cells derived from miR-206 overexpressing H1 cell line. We then conducted an integrative bioinformatics analysis on miR-206 predicted target genes. To this end, 773 mRNA target transcripts of the broadly conserved (across vertebrates) miR-1-3p/206 family were identified in the TargetScan database and were integrated into the global transcriptomic analysis performed by microarray on day-16 EB cells. Using supervised ranking product analysis, 62 predicted gene targets of the miR-1-3p/206 family were found to be significantly up-regulated in hematopoietic-competent EB samples including the transcription factors RUNX1 and TAL1. Hierarchical unsupervised clustering, based on this subset of 62 predicted mir-206 target genes, fully discriminated hematopoietic-deficient from hematopoietic-competent cells. In conclusion, miRNA profiling performed at pluripotency stage could be useful to predict the ability to human iPSC to give rise to blood cell progenitors. This work emphasizes for the first time the critical role of the muscle-specific miR-206 in hematopoietic differentiation. Finally, these results suggest that genetic manipulation of hESC/iPSC could be used to enhance their hematopoietic potential and to design protocols for generation of hPSC-derived hematopoietic stem cells with long-term reconstitution ability. Disclosures No relevant conflicts of interest to declare.

scholarly journals Regulated expression of microRNAs-126/126* inhibits erythropoiesis from human embryonic stem cells

Blood ◽  
2011 ◽  
Vol 117 (7) ◽  
pp. 2157-2165 ◽  
Author(s):  
Xinqiang Huang ◽  
Eric Gschweng ◽  
Ben Van Handel ◽  
Donghui Cheng ◽  
Hanna K. A. Mikkola ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Protein Tyrosine Phosphatase ◽  
Embryoid Body ◽  
Tyrosine Phosphatase ◽  
Embryonic Stem ◽  
Hematopoietic Differentiation ◽  
Protein Tyrosine

Abstract MicroRNAs (miRs) play an important role in cell differentiation and maintenance of cell identity, but relatively little is known of their functional role in modulating human hematopoietic lineage differentiation. Human embryonic stem cells (hESCs) provide a model system to study early human hematopoiesis. We differentiated hESCs by embryoid body (EB) formation and compared the miR expression profile of undifferentiated hESCs to CD34+ EB cells. miRs-126/126* were the most enriched of the 7 miRs that were up-regulated in CD34+ cells, and their expression paralleled the kinetics of hematopoietic transcription factors RUNX1, SCL, and PU.1. To define the role of miRs-126/126* in hematopoiesis, we created hESCs overexpressing doxycycline-regulated miRs-126/126* and analyzed their hematopoietic differentiation. Induction of miRs-126/126* during both EB differentiation and colony formation reduced the number of erythroid colonies, suggesting an inhibitory role of miRs-126/126* in erythropoiesis. Protein tyrosine phosphatase, nonreceptor type 9 (PTPN9), a protein tyrosine phosphatase that is required for growth and expansion of erythroid cells, is one target of miR-126. PTPN9 restoration partially relieved the suppressed erythropoiesis caused by miRs-126/126*. Our results define an important function of miRs-126/126* in negative regulation of erythropoiesis, providing the first evidence for a role of miR in hematopoietic differentiation of hESCs.

Sign in / Sign up

Export Citation Format

Share Document