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 Analysis of Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells as a Means to Assess Pluripotency

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Steven D. Sheridan ◽  
Vasudha Surampudi ◽  
Raj R. Rao
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Embryoid Bodies ◽  
Functional Assay ◽  
Differentiation Potential ◽  
Disease Etiology ◽  
Induced Pluripotent

Human induced pluripotent stem cells (hiPSCs) have core properties of unlimited self-renewal and differentiation potential and have emerged as exciting cell sources for applications in regenerative medicine, drug discovery, understanding of development, and disease etiology. Key among numerous criteria to assess pluripotency includes thein vivoteratoma assay that has been widely proposed as a standard functional assay to demonstrate the pluripotency of hiPSCs. Yet, the lack of reliability across methodologies, lack of definitive clinical significance, and associated expenses bring into question use of the teratoma assay as the “gold standard” for determining pluripotency. We propose use of thein vitroembryoid body (EB) assay as an important alternative to the teratoma assay. This paper summarizes the methodologies for creating EBs from hiPSCs and the subsequent analyses to assess pluripotency and proposes its use as a cost-effective, controlled, and reproducible approach that can easily be adopted to determine pluripotency of generated hiPSCs.

scholarly journals Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ratchapong Netsrithong ◽  
Siriwal Suwanpitak ◽  
Bootsakorn Boonkaew ◽  
Kongtana Trakarnsanga ◽  
Lung-Ji Chang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Drug Screening ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Hematopoietic Cells ◽  
Erythroid Differentiation ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Induced Pluripotent

Abstract Background Human induced pluripotent stem cells (hiPSCs) offer a renewable source of cells for the generation of hematopoietic cells for cell-based therapy, disease modeling, and drug screening. However, current serum/feeder-free differentiation protocols rely on the use of various cytokines, which makes the process very costly or the generation of embryoid bodies (EBs), which are labor-intensive and can cause heterogeneity during differentiation. Here, we report a simple feeder and serum-free monolayer protocol for efficient generation of iPSC-derived multipotent hematoendothelial progenitors (HEPs), which can further differentiate into endothelial and hematopoietic cells including erythroid and T lineages. Methods Formation of HEPs from iPSCs was initiated by inhibition of GSK3 signaling for 2 days followed by the addition of VEGF and FGF2 for 3 days. The HEPs were further induced toward mature endothelial cells (ECs) in an angiogenic condition and toward T cells by co-culturing with OP9-DL1 feeder cells. Endothelial-to-hematopoietic transition (EHT) of the HEPs was further promoted by supplementation with the TGF-β signaling inhibitor. Erythroid differentiation was performed by culturing the hematopoietic stem/progenitor cells (HSPCs) in a three-stage erythroid liquid culture system. Results Our protocol significantly enhanced the number of KDR+ CD34+ CD31+ HEPs on day 5 of differentiation. Further culture of HEPs in angiogenic conditions promoted the formation of mature ECs, which expressed CD34, CD31, CD144, vWF, and ICAM-1, and could exhibit the formation of vascular-like network and acetylated low-density lipoprotein (Ac-LDL) uptake. In addition, the HEPs were differentiated into CD8+ T lymphocytes, which could be expanded up to 34-fold upon TCR stimulation. Inhibition of TGF-β signaling at the HEP stage promoted EHT and yielded a large number of HSPCs expressing CD34 and CD43. Upon erythroid differentiation, these HSPCs were expanded up to 40-fold and displayed morphological changes following stages of erythroid development. Conclusion This protocol offers an efficient and simple approach for the generation of multipotent HEPs and could be adapted to generate desired blood cells in large numbers for applications in basic research including developmental study, disease modeling, and drug screening as well as in regenerative medicine.

scholarly journals Collagen coated electrospun polyethersulfon nanofibers improved insulin producing cells differentiation potential of human induced pluripotent stem cells

2018 ◽  
Vol 46 (sup3) ◽  
pp. S734-S739 ◽  
Author(s):  
Reyhaneh Nassiri Mansour ◽  
Fatemeh Soleimanifar ◽  
Mohamad Foad Abazari ◽  
Sepehr Torabinejad ◽  
Abdolreza Ardeshirylajimi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Differentiation Potential ◽  
Insulin Producing Cells ◽  
Induced Pluripotent

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).

Electrospun silk nanofibers improve differentiation potential of human induced pluripotent stem cells to insulin producing cells

2020 ◽  
Vol 108 ◽  
pp. 110398 ◽  
Author(s):  
Seyed Ehsan Enderami ◽  
Seyedeh Fatemeh Ahmadi ◽  
Reyhaneh Nassiri Mansour ◽  
Saeid Abediankenari ◽  
Hossein Ranjbaran ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Differentiation Potential ◽  
Insulin Producing Cells ◽  
Induced Pluripotent

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.

Sign in / Sign up

Export Citation Format

Share Document