AXIN2 reduces the survival of porcine induced pluripotent stem cells (piPSCs)

Abstract BackgroundThe establishment of porcine pluripotent stem cells (piPSCs) is still a critical topic and challenging issue. However, all piPSCs are extremely sensitive to changes of the culture conditions.In addition, the side effect of inhibitors in culture medium confine the pluripotency and practicability. This study aimed to investigate the roles of AXIN in piPSCs and further explore the mechanism. Here, porcine AXIN1 gene and AXIN2 were knockdown, cloned, and overexpressed in piPSCs. Digital RNA-seq was performed to explore the mechanism of cell proliferation and anti-apoptosis. ResultsHere, we found (1): overexpression of the porcine AXIN2 gene significantly reduce the survivability of piPSCs, meanwhile wreck the pluripotency of piPSCs; (2): The Digital RNA-seq analysis reveals that AXIN2, as a negative effector of the WNT signaling pathway, whom after knockdown enhances the expression of genes involved in cell cycle such as CCND1, and reduced the expression of genes related to cell differentiation, cell death, and cell apoptosis. ConclusionAXIN2 could reduce the pluripotency and survival of piPSCs and also provided a potential to simplify the cultrue medium.

Download Full-text

AXIN2 Reduces the Survival of Porcine Induced Pluripotent Stem Cells (piPSCs)

International Journal of Molecular Sciences ◽

10.3390/ijms222312954 ◽

2021 ◽

Vol 22 (23) ◽

pp. 12954

Author(s):

Rui Zhang ◽

Shuai Yu ◽

Qiaoyan Shen ◽

Wenxu Zhao ◽

Juqing Zhang ◽

...

Keyword(s):

Stem Cells ◽

Wnt Signaling ◽

Signaling Pathway ◽

Pluripotent Stem Cells ◽

Wnt Signaling Pathway ◽

Culture Conditions ◽

Rna Seq ◽

Expression Of Genes ◽

Proliferation And Apoptosis ◽

Induced Pluripotent

The establishment of porcine pluripotent stem cells (piPSCs) is critical but remains challenging. All piPSCs are extremely sensitive to minor perturbations of culture conditions and signaling network. Inhibitors, such as CHIR99021 and XAV939 targeting the WNT signaling pathway, have been added in a culture medium to modify the cell regulatory network. However, potential side effects of inhibitors could confine the pluripotency and practicability of piPSCs. This study aimed to investigate the roles of AXIN, one component of the WNT pathway in piPSCs. Here, porcine AXIN1 and AXIN2 genes were knocked-down or overexpressed. Digital RNA-seq was performed to explore the mechanism of cell proliferation and apoptosis. We found that (1) overexpression of the porcine AXIN2 gene significantly reduced survival and negatively impacted the pluripotency of piPSCs, and (2) knockdown of AXIN2, a negative effector of the WNT signaling pathway, enhanced the expression of genes involved in cell cycle but reduced the expression of genes related to cell differentiation, death, and apoptosis.

Download Full-text

Genome‐wide hyper‐methylation is closely associated with abnormal expression of genes involved in neural development in induced pluripotent stem cells derived from a Down Syndrome mouse model

Cell Biology International ◽

10.1002/cbin.11560 ◽

2021 ◽

Author(s):

Jiao‐jiao Xi ◽

Guan‐heng Yang ◽

Yan‐na Liu ◽

Jia‐jun Qiu ◽

Xiu‐li Gong ◽

...

Keyword(s):

Stem Cells ◽

Down Syndrome ◽

Mouse Model ◽

Induced Pluripotent Stem Cells ◽

Neural Development ◽

Pluripotent Stem Cells ◽

Abnormal Expression ◽

Expression Of Genes ◽

Genome Wide ◽

Induced Pluripotent

Download Full-text

Abstract 41: Cell Cycle Activation of Cardiomyocytes Derived From Induced Pluripotent Stem Cells for Cardiac Regeneration

Circulation Research ◽

10.1161/res.119.suppl_1.41 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Wuqiang Zhu ◽

Meng Zhao ◽

Saidulu Mattapally ◽

Ling Gao ◽

Jianyi Zhang

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Cardiac Function ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Cell Mass ◽

Brdu Incorporation ◽

Myocardial Repair ◽

Cyclin D2 ◽

Induced Pluripotent

Transplantation of cardiomyocytes derived from induced pluripotent stem cells (iPSCs) improves cardiac function in animal models with myocardial infarction. However, the poor number of survived cells and the low proliferation capability of cardiomyocyte derived from iPSCs are bottlenecks in myocardial repair with cell therapy. We hypothesize that increasing the number of surviving iPSC-CMs in the engraftment via cell cycle induction may lead to a transmural replacement of scar tissue and a lasting restoration of cardiac function. Cyclin D2 is a protein that regulates cell cycle transition from G1 to S phase. We transfected MHC-cyclin D2 (designated as MHC-cycD2) cDNA into the iPSCs, and differentiated the iPSCs into cardiomyocytes. Comparing to non-expressing cells, MHC-cycD2-expressing cardiomyocytes displayed increased Brdu incorporation activity, suggesting the enhanced cell cycle in MHC-cycD2-expressing cardiomyocytes. Cell cycle activity was confirmed by increased number of Ki-67 and PCNA positive immunostaining cardiomyocytes and more contractile embryonic body cell mass in MHC-cycD2-expressing culture compared to non-expressing culture. Data from Q-PCR and histology suggested that expression of MHC-cycD2 didn’t alter the pluripotency or cardiomyogenic potential of iPSCs. Thus, we have successfully induced cell cycle in iPSC-derived cardiomyocytes via expression of cyclin D2. We are currently studying if MHC-cycD2-expressing iPSC-cardiomyocytes exhibit superior regenerative potential compared to their non-expressing counterparts following transplantation into chronically infarcted hearts.

Download Full-text

A Src inhibitor regulates the cell cycle of human pluripotent stem cells and improves directed differentiation

The Journal of Cell Biology ◽

10.1083/jcb.201502035 ◽

2015 ◽

Vol 210 (7) ◽

pp. 1257-1268 ◽

Cited By ~ 20

Author(s):

Sundari Chetty ◽

Elise N. Engquist ◽

Elie Mehanna ◽

Kathy O. Lui ◽

Alexander M. Tsankov ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Pluripotent Stem Cells ◽

Human Pluripotent Stem Cells ◽

Directed Differentiation ◽

Differentiation Potential ◽

Germ Layers ◽

Positive Effects ◽

Src Inhibitor ◽

Expression Of Genes

Driving human pluripotent stem cells (hPSCs) into specific lineages is an inefficient and challenging process. We show that a potent Src inhibitor, PP1, regulates expression of genes involved in the G1 to S phase transition of the cell cycle, activates proteins in the retinoblastoma family, and subsequently increases the differentiation propensities of hPSCs into all three germ layers. We further demonstrate that genetic suppression of Src regulates the activity of the retinoblastoma protein and enhances the differentiation potential of hPSCs across all germ layers. These positive effects extend beyond the initial germ layer specification and enable efficient differentiation at subsequent stages of differentiation.

Download Full-text

Analysis of genes involved in cell proliferation, adhesion, and control of apoptosis during embryonic neurogenesis in Induced Pluripotent Stem Cells (iPSCs) from patients with Focal Cortical Dysplasia

Brain Research Bulletin ◽

10.1016/j.brainresbull.2019.11.016 ◽

2020 ◽

Vol 155 ◽

pp. 112-118 ◽

Cited By ~ 2

Author(s):

Daniel Rodrigo Marinowic ◽

Fernanda Majolo ◽

Gabriele Goulart Zanirati ◽

Ismael Plentz ◽

Eliseu Paglioli Neto ◽

...

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Focal Cortical Dysplasia ◽

Cortical Dysplasia ◽

Embryonic Neurogenesis ◽

Induced Pluripotent ◽

And Control

Download Full-text

Knockdown of the Cell Cycle Inhibitor p21 Enhances Cartilage Formation by Induced Pluripotent Stem Cells

Tissue Engineering Part A ◽

10.1089/ten.tea.2014.0240 ◽

2015 ◽

Vol 21 (7-8) ◽

pp. 1261-1274 ◽

Cited By ~ 12

Author(s):

Brian O. Diekman ◽

Pratiksha I. Thakore ◽

Shannon K. O'Connor ◽

Vincent P. Willard ◽

Jonathan M. Brunger ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Cartilage Formation ◽

Cell Cycle Inhibitor ◽

Induced Pluripotent

Download Full-text

Modelling the developmental spliceosomal craniofacial disorder Burn-McKeown syndrome using induced pluripotent stem cells

10.1101/2020.05.13.094029 ◽

2020 ◽

Author(s):

Katherine A. Wood ◽

Charlie F. Rowlands ◽

Huw B. Thomas ◽

Steven Woods ◽

Julieta O’Flaherty ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Craniofacial Development ◽

Rna Seq ◽

Unrelated Control ◽

Mesenchymal Transition ◽

Base Pair Deletion ◽

Induced Pluripotent

ABSTRACTThe craniofacial developmental disorder Burn-McKeown Syndrome (BMKS) is caused by biallelic variants in the pre-messenger RNA splicing factor gene TXNL4A/DIB1. The majority of affected individuals with BMKS have a 34 base pair deletion in the promoter region of one allele of TXNL4A combined with a loss-of-function variant on the other allele, resulting in reduced TXNL4A expression. However, it is unclear how reduced expression of this ubiquitously expressed spliceosome protein results in craniofacial defects during development. Here we reprogrammed peripheral mononuclear blood cells from a BMKS patient and her unaffected mother into induced pluripotent stem cells (iPSCs) and differentiated the iPSCs into induced neural crest cells (iNCCs), the key cell type required for correct craniofacial development. BMKS patient-derived iPSCs proliferated more slowly than both mother- and unrelated control-derived iPSCs, and RNA-Seq analysis revealed significant differences in gene expression and alternative splicing. Patient iPSCs displayed defective differentiation into iNCCs compared to maternal and unrelated control iPSCs, in particular a delay in undergoing an epithelial-to-mesenchymal transition (EMT). RNA-Seq analysis of differentiated iNCCs revealed widespread gene expression changes and mis-splicing in genes relevant to craniofacial and embryonic development that highlight a dampened response to WNT signalling, the key pathway activated during iNCC differentiation. Furthermore, we identified the mis-splicing of TCF7L2 exon 4, a key gene in the WNT pathway, as a potential cause of the downregulated WNT response in patient cells. Additionally, mis-spliced genes shared common sequence properties such as length, splice site strengths and sequence motifs, suggesting that splicing of particular subsets of genes is particularly sensitive to changes in TXNL4A expression. Together, these data provide the first insight into how reduced TXNL4A expression in BMKS patients might compromise splicing and NCC function, resulting in defective craniofacial development in the embryo.

Download Full-text

Dysregulation of Cells Cycle and Apoptosis in Human Induced Pluripotent Stem Cells Chondrocytes Through p53 Pathway by HT-2 Toxin: An in vitro Study

Frontiers in Genetics ◽

10.3389/fgene.2021.677723 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yanan Zhang ◽

Huan Liu ◽

Xialu Lin ◽

Feng’e Zhang ◽

Peilin Meng ◽

...

Keyword(s):

Risk Factors ◽

Cell Cycle ◽

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Environmental Risk ◽

Pluripotent Stem Cells ◽

Environmental Risk Factors ◽

P53 Pathway ◽

Induced Pluripotent ◽

And Control

Kashin–Beck disease (KBD) mainly damages growth plate of adolescents and is susceptible to both gene and gene–environmental risk factors. HT-2 toxin, which is a primary metabolite of T-2 toxin, was regarded as one of the environmental risk factors of KBD. We used successfully generated KBD human induced pluripotent stem cells (hiPSCs) and control hiPSCs, which carry different genetic information. They have potential significance in exploring the effects of HT-2 toxin on hiPSC chondrocytes and interactive genes with HT-2 toxin for the purpose of providing a cellular disease model for KBD. In this study, we gave HT-2 toxin treatment to differentiating hiPSC chondrocytes in order to investigate the different responses of KBD hiPSC chondrocytes and control hiPSC chondrocytes to HT-2 toxin. The morphology of HT-2 toxin-treated hiPSC chondrocytes investigated by transmission electron microscope clearly showed that the ultrastructure of organelles was damaged and type II collagen expression in hiPSC chondrocytes was downregulated by HT-2 treatment. Moreover, dysregulation of cell cycle was observed; and p53, p21, and CKD6 gene expressions were dysregulated in hiPSC chondrocytes after T-2 toxin treatment. Flow cytometry also demonstrated that there were significantly increased amounts of late apoptotic cells in KBD hiPSC chondrocytes and that the mRNA expression level of Fas was upregulated. In addition, KBD hiPSC chondrocytes presented stronger responses to HT-2 toxin than control hiPSC chondrocytes. These findings confirmed that HT-2 is an environmental risk factor of KBD and that p53 pathway interacted with HT-2 toxin, causing damaged ultrastructure of organelles, accelerating cell cycle in G1 phase, and increasing late apoptosis in KBD hiPSC chondrocytes.

Download Full-text

Intermittent compressive force induces cell cycling and reduces apoptosis in embryoid bodies of mouse induced pluripotent stem cells

International Journal of Oral Science ◽

10.1038/s41368-021-00151-3 ◽

2022 ◽

Vol 14 (1) ◽

Author(s):

Jeeranan Manokawinchoke ◽

Phoonsuk Limraksasin ◽

Hiroko Okawa ◽

Prasit Pavasant ◽

Hiroshi Egusa ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Transforming Growth Factor ◽

Compressive Force ◽

Embryoid Bodies ◽

Sequencing Data ◽

Metabolic Processes ◽

Induced Pluripotent

AbstractIn vitro manipulation of induced pluripotent stem cells (iPSCs) by environmental factors is of great interest for three-dimensional (3D) tissue/organ induction. The effects of mechanical force depend on many factors, including force and cell type. However, information on such effects in iPSCs is lacking. The aim of this study was to identify a molecular mechanism in iPSCs responding to intermittent compressive force (ICF) by analyzing the global gene expression profile. Embryoid bodies of mouse iPSCs, attached on a tissue culture plate in 3D form, were subjected to ICF in serum-free culture medium for 24 h. Gene ontology analyses for RNA sequencing data demonstrated that genes differentially regulated by ICF were mainly associated with metabolic processes, membrane and protein binding. Topology-based analysis demonstrated that ICF induced genes in cell cycle categories and downregulated genes associated with metabolic processes. The Kyoto Encyclopedia of Genes and Genomes database revealed differentially regulated genes related to the p53 signaling pathway and cell cycle. qPCR analysis demonstrated significant upregulation of Ccnd1, Cdk6 and Ccng1. Flow cytometry showed that ICF induced cell cycle and proliferation, while reducing the number of apoptotic cells. ICF also upregulated transforming growth factor β1 (Tgfb1) at both mRNA and protein levels, and pretreatment with a TGF-β inhibitor (SB431542) prior to ICF abolished ICF-induced Ccnd1 and Cdk6 expression. Taken together, these findings show that TGF-β signaling in iPSCs enhances proliferation and decreases apoptosis in response to ICF, that could give rise to an efficient protocol to manipulate iPSCs for organoid fabrication.

Download Full-text