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

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.

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

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.

scholarly journals Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Laís Vicari de Figueiredo Pessôa ◽  
Pedro Ratto Lisboa Pires ◽  
Maite del Collado ◽  
Naira Caroline Godoy Pieri ◽  
Kaiana Recchia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Embryoid Bodies ◽  
Mirna Profile ◽  
Patient Specific ◽  
Induced Pluripotent

Introduction. Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. Objectives. We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. Methods. Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. Results. Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. Conclusions. We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

308 Tet1 PLAYS IMPORTANT ROLES IN MAINTAINING CHARACTERIZATIONS OF PORCINE INDUCED PLURIPOTENT STEM CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 301
Author(s):  
A. R. Fan ◽  
K. Y. Ma ◽  
T. C. Zhao ◽  
P. P. An ◽  
B. Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Self Renewal ◽  
Qrt Pcr ◽  
Post Transfection ◽  
Fetal Fibroblasts ◽  
Induced Pluripotent

It was recently found that the ten-eleven-translocation (TET) family of Fe(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and thus promotes active demethylation of genomes. Tet1 is highly expressed in mouse embryonic stem cells (mESC) and has been demonstrated to involve in mESC maintenance. Here we used small interference RNA (siRNA) to transiently knockdown expression of Tet1 in porcine induced pluripotent stem cells (iPSC) in order to identify its functions. The fetal fibroblasts were isolated from a 30-day-old porcine fetus and induced into iPSC with defined transcription factors, namely Oct-4, Sox-2, Klf-4, and C-myc. The colonies appeared on Day 12 and were picked up on Day 14. These colonies had normal ES-like morphology and alkaline phosphatase activity. Specifically, they were positively stained for pluripotency-specific markers, including Oct4, Sox2, Nanog, Rex1, and SSEA1. When cultured in vitro, the cells formed embryoid bodies (EB), and all 3 germ layer markers (endoderm: AFP, alphaAT; mesoderm: BMP4, Enolase; ectoderm: GFAP, Neurod) were detected positively in EB. For siRNA transfections, iPSC from the colonies were transfected with 40 pmol of siRNA and 2 µL of Lipofectamine 2000 in 1 well of a 24-well plate. After transfection, iPSC were subjected to fluorescence-activated cell sorting to determine the fraction of FAM-positive cells in order to confirm transfection efficiency; the percentage of positive cells reached 48 ± 4.96. We observed obvious knockdown of Tet1 after short-term transfection of siRNA, and the knockdown efficiency was confirmed using qRT-PCR and immunofluorescence staining. Notably, knockdown of Tet1 resulted in morphological abnormality and loss of undifferentiated state of porcine iPSC. However, no obvious morphological changes were observed in the negative control (transfected with nonsense-siRNA), positive control (transfected with GAPDH-siRNA), or mock control (transfected with DEPC-treated water). To gain insight into the molecular mechanism underlying the self-renewal defect, we analysed the effects of Tet1 knockdown on the expression of key stem cell factors and differentiation markers of different embryonic layers using qRT-PCR. We found that knockdown of Tet1 resulted in downregulated expression of pluripotency-related genes, such as Lefty-2, Klf-2, and Sox-2 (the expression ratios of post-transfection to pre-transfection were 0.31 ± 0.21, 0.48 ± 0.072, and 0.65 ± 0.046, respectively), and upregulated expression of differentiation-related genes, including Pitx-2, Hand-1, Gata-6, and Lef-1 (the expression ratios of post-transfection to pre-transfection were 4.35 ± 1.36, 2.56 ± 0.68, 2.91 ± 1.47, and 2.33 ± 1.11, respectively). However, Oct-4, C-myc, Klf-4, and Nanog were not downregulated (the expression ratios of post-transfection to pre-transfection were 0.91 ± 0.15, 1.12 ± 0.26, 1.15 ± 0.21, and 1.08 ± 0.08, respectively). Taken together, Tet1 plays important roles in porcine iPSC self-renewal and characterization maintenance. This study was financed by National Basic Research Program of China (NO.2009CB941001).

283 GENERATION AND CHARACTERIZATION OF BOVINE INDUCED PLURIPOTENT STEM CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 289
Author(s):  
O. J. Koo ◽  
H. S. Kwon ◽  
D. K. Kwon ◽  
K. S. Kang ◽  
B. C. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Transgenic Animals ◽  
Mouse Embryonic Fibroblast ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Serum Replacement ◽  
Induced Pluripotent ◽  
Large Animals

Stem cells in large animals are an excellent model for cell therapy research and fine resources for producing transgenic animals. However, there are only few reports of stem cells in large animals because of technical differences between species. In this report, we successfully generate bovine induced pluripotent stem cells (iPSC) using 4 human reprogramming factors (Oct4, Sox2, Klf4, and c-myc) under control of PiggyBac transposition vector. Fibroblasts derived from bovine fetuses were transfected using FugeneHD agent. After 21 days, colony-shaped structures on the culture plates were mechanically detached and then seeded on a mouse embryonic fibroblast (MEF) feeder layer pretreated with mitomycin C. The culture medium was DMEM/F12 supplemented with 20% serum replacement, 5 ng mL–1 basic fibroblast growth factor (bFGF), 0.1 mM β-mercaptoethanol, 1% NEAA, and 1% penicillin-streptomycin antibiotics. The iPSC colonies showed alkaline phosphatase activity and expressed several pluripotency markers (Oct4, Sox2, SSEA1, and SSEA4). To confirm differentiation potential, the iPSC were cultured as embryoid bodies and then plated again. βIII-tubulin (ectoderm) and GFAP or α-SMA (mesoderm) were well expressed on the attached cells. The results revealed that the bovine fibroblasts were well inducted to iPSC that had potential of multilineage differentiation. We hope this technology contributes to improving transgenic cattle production. This study was financially supported by IPET (grant # 109023-05-3-CG000, 111078-03-1-CG000) and the BK21 program for Veterinary Science.

scholarly journals Electrical Stimulation Promotes Cardiac Differentiation of Human Induced Pluripotent Stem Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Damián Hernández ◽  
Rodney Millard ◽  
Priyadharshini Sivakumaran ◽  
Raymond C. B. Wong ◽  
Duncan E. Crombie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Electrical Stimulation ◽  
Induced Pluripotent Stem Cells ◽  
Cell Line ◽  
Pluripotent Stem Cells ◽  
Embryoid Bodies ◽  
Cardiac Differentiation ◽  
Dependent Manner ◽  
Human Ipscs ◽  
Induced Pluripotent

Background.Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes.Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs) for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days.Results. In iPS(Foreskin)-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression ofACTC1,TNNT2,MYH7, andMYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner.Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used.

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

2015 ◽  
Vol 21 (7-8) ◽  
pp. 1261-1274 ◽  
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
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