305 TRANSPOSON-MEDIATED REPROGRAMMING OF LIVESTOCK SOMATIC CELLS TO INDUCED PLURIPOTENT STEM CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 300
Author(s):  
T. R. Talluri ◽  
D. Hermann ◽  
B. Barg-Kues ◽  
K. Debowski ◽  
R. Behr ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Insertional Mutagenesis ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Sleeping Beauty ◽  
Major Step ◽  
Increased Risk ◽  
Induced Pluripotent

The elusive nature of embryonic stem cells in livestock makes reprogramming of somatic cells to induced pluripotent stem (iPS) cells a promising approach for targeted genetic modifications. The first attempts to produce iPS cells from livestock species were made using retro- and lentiviral vectors, which are associated with an increased risk of insertional mutagenesis and which are not easily removable after reprogramming. Here, we describe a nonviral method for the derivation of porcine and bovine iPS cells, using Sleeping Beauty (SB) and piggyBac (PB) transposon systems. The transposons encode the murine or primate reprogramming factors OCT4, SOX2, KLF4, MYC, and LIN28, separated by self-cleaving peptide sequences, respectively. In addition, the PB transposon cassette contains a NANOG-cDNA. The SB or PB transposon-reprogrammed porcine iPS cells expressed typical markers of embryonic stem cells (SSEA1, SSEA4, TRA-1-60, and endogenous stemness genes), showed long-term proliferation under feeder-free culture conditions, differentiated into cell types of the 3 germ layers in vitro, and formed teratomas after subcutaneous injection into immune-deficient nude mice. Both transposon systems are currently being tested in bovine fibroblasts. The results are a major step towards the derivation of authentic porcine and bovine iPS cells, in which the transposon transgenes can be eliminated after reprogramming.

332 DERIVATION OF BOVINE-INDUCED PLURIPOTENT STEM CELLS BY piggyBac-MEDIATED REPROGRAMMING

2015 ◽  
Vol 27 (1) ◽  
pp. 255
Author(s):  
T. T. Rao ◽  
K. Dharmendra ◽  
G. Silke ◽  
W. Garrels ◽  
H. Niemann ◽  
...  
Keyword(s):  
Stem Cell ◽  
Insertional Mutagenesis ◽  
Bovine Genome ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Major Step ◽  
Livestock Species ◽  
Increased Risk ◽  
Induced Pluripotent

Induced pluripotent stem (iPS) cells are a seminal breakthrough in stem cell research and are promising for the development of advanced regenerative therapies and farm animal biotechnology. Considering the potential of this technology for both basic and clinical research, it is tempting to extend this research to important livestock species, such as cattle, in which authentic embryonic stem cell lines are yet not available. The first attempts to produce iPS cells from livestock species were made using retro- and lentiviral vectors, which are associated with an increased risk of insertional mutagenesis and which are not removable after reprogramming. Here, we describe a nonviral method for the derivation of bovine iPS cells, employing a piggyBac (PB) transposon system. The reprogramming PB transposon encodes the primate cDNA of 6 core reprogramming factors, OCT4, SOX2, KLF4, MYC, LIN28, and NANOG, separated by self-cleaving 2A peptide sequences and driven by the chimeric CAGGS promoter. The derived bovine iPS line expressed typical endogenous genes (OCT4, SOX2, c-MYC, KLF4, NANOG, REX1, and ALP) by RT-PCR and OCT-4 as well as SSEA-1 and 4 pluripotency-related markers by immunostaining, and it exhibited silencing of exogenous reprograming factors. Moreover, the iPS line showed long-term proliferation (until the 40th passage) under feeder-free culture conditions, differentiated into derivatives of the 3 germ layers in vitro, and formed teratomas (4/6) after subcutaneous injection into immunodeficient nude mice. These results are a major step towards the derivation of authentic bovine iPS cells, and thus facilitate the genetic modifications of the bovine genome.

scholarly journals Induced Pluripotent Stem Cells: Hope in the Treatment of Diseases, including Muscular Dystrophies

2020 ◽  
Vol 21 (15) ◽  
pp. 5467
Author(s):  
Daniela Gois Beghini ◽  
Samuel Iwao Horita ◽  
Cynthia Machado Cascabulho ◽  
Luiz Anastácio Alves ◽  
Andrea Henriques-Pons
Keyword(s):  
Stem Cells ◽  
Clinical Trials ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Ips Cells ◽  
High Plasticity ◽  
Cell Based Therapy ◽  
Induced Pluripotent

Induced pluripotent stem (iPS) cells are laboratory-produced cells that combine the biological advantages of somatic adult and stem cells for cell-based therapy. The reprogramming of cells, such as fibroblasts, to an embryonic stem cell-like state is done by the ectopic expression of transcription factors responsible for generating embryonic stem cell properties. These primary factors are octamer-binding transcription factor 4 (Oct3/4), sex-determining region Y-box 2 (Sox2), Krüppel-like factor 4 (Klf4), and the proto-oncogene protein homolog of avian myelocytomatosis (c-Myc). The somatic cells can be easily obtained from the patient who will be subjected to cellular therapy and be reprogrammed to acquire the necessary high plasticity of embryonic stem cells. These cells have no ethical limitations involved, as in the case of embryonic stem cells, and display minimal immunological rejection risks after transplant. Currently, several clinical trials are in progress, most of them in phase I or II. Still, some inherent risks, such as chromosomal instability, insertional tumors, and teratoma formation, must be overcome to reach full clinical translation. However, with the clinical trials and extensive basic research studying the biology of these cells, a promising future for human cell-based therapies using iPS cells seems to be increasingly clear and close.

Reprogramming of Somatic Cells After Fusion With Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells

2010 ◽  
Vol 19 (2) ◽  
pp. 239-246 ◽  
Author(s):  
Huseyin Sumer ◽  
Karen L. Jones ◽  
Jun Liu ◽  
Corey Heffernan ◽  
Pollyanna A. Tat ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Induced Pluripotent

scholarly journals Investigating Differentiation Ability of Induced Pluripotent Stem (Ips) Cell and Endometrial Stromal Cells (Enscs) Toward Pre-Oligodendrocytes using Growth Factors In Vitro

2017 ◽  
Vol 14 (2) ◽  
pp. 697-707
Author(s):  
Behnam Younesi ◽  
Mahnaz Azarnia
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Therapy ◽  
Stromal Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Neural System ◽  
Main Function ◽  
Endometrial Stromal Cells ◽  
Induced Pluripotent

ABSTRACT: Oligodendrocytes are types of cells in central neural system (CNS). Their main function is generation of Myelin sheath in CNS, this sheath insulates the Axons. Any disorder in the function of these cells leads to demyelination of neurons and causes neural disorders including multiple sclerosis (MS). Nowadays, cell therapy provides plenty of hope for cure of MS. So far it has used different sources such as stem cells or progenitor for cell therapy of neural system. But each of them had some limitations, for instance using neural stem cells requires certain amount of CNS tissue. Embryonic stem cells also introduced as another candidate for cell therapy but due to some moral problems, such as necessity to creating a Blastocyst, using these cells accompanied many limitations. In cell therapy, the most important factor is facility to acquiring stem cells. iPS cells are kinds of Induced Pluripotent Stem cells which directly created by transferring of 4 transcription factors: oct4, sox2, klf2, and c-Myc into the differentiated cells. iPS cells are like pluripotent embryonic stem cells although they do not require demolition of Blastocyte. Endometrial Stromal cells are kinds of mesenchyme or adult cells which have been proven in human and mice’s uterine endometrial and they are easy to access. Both of these types of cells can be appropriate candidates for cell therapy. In this research we use these two types of cells for differentiate to Oligodendrocytes and we are able to differentiate iPS cells which are from human's eye and also human Endometrial Stromal cells to pre-Oligodendrocytes. Also we can compare their differentiation ability. These cells can be used for transplanting in MS patients.

197 INDUCED PLURIPOTENT STEM CELLS (iPS) DERIVED FROM EQUINE UMBILICAL CORD CELLS USING LENTIVIRUS VECTOR Stemcca

2014 ◽  
Vol 26 (1) ◽  
pp. 213
Author(s):  
M. Guastali ◽  
F. Bressan ◽  
R. Maziero ◽  
D. Paschoal ◽  
M. Sudano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Embryonic Stem Cells ◽  
Phosphatase Activity ◽  
Umbilical Cord ◽  
Alkaline Phosphatase Activity ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Induced Pluripotent

Research on induced pluripotent stem cells (iPS) emerged to overcome the limitations of embryonic stem cells, such as ethical issues, security, compatibility, and availability. The nuclear reprogramming induced by viral vectors aims to induce differentiated cells to an embryonic pluripotent state. The iPS cells can be generated using retroviral vector expressing Oct4, Sox2, Klf4 and c-Myc, but produces much genomic integration (GI) which limit its use for therapeutic purpose. Alternatively, lentiviral vectors have been used to be safe and equally effective in producing iPS. Despite several cell types can be reprogramed, there is no information of which is the best cell type to be used in the generation of iPS. The umbilical cord is a reserve of multipotent mesenchymal stem cells and may present a greater reprogramming efficiency compared with fibroblasts in the generation of iPS. Here we describe the use of a single lentiviral vector composed by the combination of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) for the generation of iPS cells using equine umbilical cord (UC) cells. Therefore, samples were collected from 5 equine UC at birth. The umbilical matrices were subjected to enzymatic digestion in a solution of 0.004% collagenase diluted in PBS, and the cells obtained by filtration were plated in plastic culture bottles with 5 mL of DMEM supplemented with 20% fetal calf serum, antibiotics, and antimycotics, followed by incubation at 37°C in a 100% humid atmosphere at 5% CO2 in air. When the cells reached 40% of confluence and a concentration of 105 cells, these cells were transduced with 50 μL Human Stemcca cre-excisable constitutive polycistronic (oskm) lentivirus (EMD Millipore Corp., Billerica, MA, USA) produced according manufacturer's protocol plus 8 ng mL–1 polybrene (hexadimethrine bromide, Sigma, St. Louis, MO, USA). The culture medium was renewed 12 h after incubation. Five days after transduction, cells were transferred to murine embryonic fibroblasts (MEF) feeder layer and cultured for 14 days in a specific medium for iPS. The morphologically similar colonies to the embryonic stem cells were visualised after two weeks of infection. When the clones were well established two mechanical and two enzymatic passages were performed. Cells were re-expanded under new MEFs and submitted to alkaline phosphatase activity detection (Leukocyte Alkaline Phosphatase Kit, Sigma) according to manufacturer's recommendations. Briefly, cell cultures were fixed, incubated with a mixture of alkaline naphthol AS-BI with fast red violet LB. Red labelling insoluble deposits indicated the sites of alkaline phosphatase activity. In all cultures tested (n = 10) the expression of alkaline phosphatase was detected. The cell culture samples will still be tested for gene expression of pluripotency factors. The combination of all factors in a single transcript was efficient for reprogramming cells from the umbilical cord and allowed the derivation of mesenchymal cells in equine iPS. The use of a single lentiviral reprogramming vector represents a powerful tool for the study of iPS technology and its possible therapeutic application.

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