Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells

Wilfried A. Kues; Doris Herrmann; Brigitte Barg-Kues; Srividiameena Haridoss; Monika Nowak-Imialek; Thomas Buchholz; Miriam Streeck; Antonia Grebe; Ivana Grabundzija; Sylvia Merkert; Ulrich Martin; Vanessa Jane Hall; Mikkel Aabech Rasmussen; Zoltan Ivics; Poul Hyttel; Heiner Niemann

doi:10.1089/scd.2012.0382

Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells

Wilfried A. Kues, Doris Herrmann, Brigitte Barg-Kues, Srividiameena Haridoss, Monika Nowak-Imialek, Thomas Buchholz, Miriam Streeck, Antonia Grebe, Ivana Grabundzija, Sylvia Merkert, Ulrich Martin, Vanessa Jane Hall, Mikkel Aabech Rasmussen, Zoltan Ivics, Poul Hyttel, Heiner Niemann

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Abstract

The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed induced pluripotent stem (iPS) cells showed long-term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5, and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives of the 3 germ layers. Upon injection of putative iPS cells under the skin of immunodeficient mice, we observed teratomas in 3 of 6 cases. These results form the basis for in-depth studies toward the derivation of porcine iPS cells, which hold great promise for preclinical testing of novel cell therapies in the pig model.

Original language	English
Journal	Stem Cells and Development
Volume	22
Issue number	1
Pages (from-to)	124-135
Number of pages	12
ISSN	1547-3287
DOIs	https://doi.org/10.1089/scd.2012.0382
Publication status	Published - 1 Jan 2013

Keywords

Animals
Antigens, Differentiation
Cell Transformation, Neoplastic
Cells, Cultured
Coculture Techniques
DNA Transposable Elements
Fibroblasts
Green Fluorescent Proteins
Induced Pluripotent Stem Cells
Mice
Mice, Nude
Microscopy, Fluorescence
Neurogenesis
Octamer Transcription Factor-3
Promoter Regions, Genetic
SOXB1 Transcription Factors
Sus scrofa
Teratoma
Transcriptome
Transgenes

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Kues, W. A., Herrmann, D., Barg-Kues, B., Haridoss, S., Nowak-Imialek, M., Buchholz, T., Streeck, M., Grebe, A., Grabundzija, I., Merkert, S., Martin, U., Hall, V. J., Rasmussen, M. A., Ivics, Z., Hyttel, P., & Niemann, H. (2013). Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells. Stem Cells and Development, 22(1), 124-135. https://doi.org/10.1089/scd.2012.0382

Kues, WA, Herrmann, D, Barg-Kues, B, Haridoss, S, Nowak-Imialek, M, Buchholz, T, Streeck, M, Grebe, A, Grabundzija, I, Merkert, S, Martin, U, Hall, VJ , Rasmussen, MA, Ivics, Z, Hyttel, P & Niemann, H 2013, 'Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells', Stem Cells and Development, vol. 22, no. 1, pp. 124-135. https://doi.org/10.1089/scd.2012.0382

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abstract = "The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed induced pluripotent stem (iPS) cells showed long-term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5, and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives of the 3 germ layers. Upon injection of putative iPS cells under the skin of immunodeficient mice, we observed teratomas in 3 of 6 cases. These results form the basis for in-depth studies toward the derivation of porcine iPS cells, which hold great promise for preclinical testing of novel cell therapies in the pig model.",

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AU - Kues, Wilfried A.

AU - Herrmann, Doris

AU - Barg-Kues, Brigitte

AU - Haridoss, Srividiameena

AU - Nowak-Imialek, Monika

AU - Buchholz, Thomas

AU - Streeck, Miriam

AU - Grebe, Antonia

AU - Grabundzija, Ivana

AU - Merkert, Sylvia

AU - Martin, Ulrich

AU - Hall, Vanessa Jane

AU - Rasmussen, Mikkel Aabech

AU - Ivics, Zoltan

AU - Hyttel, Poul

AU - Niemann, Heiner

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N2 - The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed induced pluripotent stem (iPS) cells showed long-term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5, and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives of the 3 germ layers. Upon injection of putative iPS cells under the skin of immunodeficient mice, we observed teratomas in 3 of 6 cases. These results form the basis for in-depth studies toward the derivation of porcine iPS cells, which hold great promise for preclinical testing of novel cell therapies in the pig model.

AB - The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed induced pluripotent stem (iPS) cells showed long-term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5, and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives of the 3 germ layers. Upon injection of putative iPS cells under the skin of immunodeficient mice, we observed teratomas in 3 of 6 cases. These results form the basis for in-depth studies toward the derivation of porcine iPS cells, which hold great promise for preclinical testing of novel cell therapies in the pig model.

KW - Animals

KW - Antigens, Differentiation

KW - Cell Transformation, Neoplastic

KW - Cells, Cultured

KW - Coculture Techniques

KW - DNA Transposable Elements

KW - Fibroblasts

KW - Green Fluorescent Proteins

KW - Induced Pluripotent Stem Cells

KW - Mice

KW - Mice, Nude

KW - Microscopy, Fluorescence

KW - Neurogenesis

KW - Octamer Transcription Factor-3

KW - Promoter Regions, Genetic

KW - SOXB1 Transcription Factors

KW - Sus scrofa

KW - Teratoma

KW - Transcriptome

KW - Transgenes

U2 - 10.1089/scd.2012.0382

DO - 10.1089/scd.2012.0382

M3 - Journal article

C2 - 22989381

SN - 1547-3287

VL - 22

SP - 124

EP - 135

JO - Stem Cells and Development

JF - Stem Cells and Development

IS - 1

ER -

Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells

Abstract

Keywords

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