Expression analysis of pluripotency factors in the undifferentiated porcine inner cell mass and epiblast during in vitro culture

Blomberg, Le Ann; Schreier, Lori L.; Talbot, Neil C.

doi:10.1002/mrd.20780

Cited by 60 publications

(59 citation statements)

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“…2B), but examination of the microarray data of others (23,54) revealed that a similar phenomenon has been observed in reprogramming of murine and human cells. Finally, pNANOG was up-regulated during reprogramming, although it was already expressed in the parental PFF cells, an observation consistent with previous reports that NANOG is not a reliable marker for pluripotency in pig (32).…”

Section: Discussionsupporting

confidence: 90%

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Derivation of induced pluripotent stem cells from pig somatic cells

Ezashi¹,

Telugu²,

Alexenko³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

439

399

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For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here, we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes, hOCT4, hSOX2, hKLF4, and hc-MYC, the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after Ϸ22 days, providing an overall reprogramming efficiency of Ϸ0.1%. The cells expressed porcine OCT4, NANOG, and SOX2 and had high telomerase activity, but also continued to express the 4 human transgenes. Unlike human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line, ID6, had a normal karyotype, a cell doubling time of Ϸ17 h, and has been maintained through >220 doublings. The ID6 line formed embryoid bodies, expressing genes representing all 3 germ layers when cultured under differentiating conditions, and teratomas containing tissues of ectoderm, mesoderm, and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age.iPS ͉ reprogramming ͉ OCT4

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Section: Discussionsupporting

confidence: 90%

“…Several genes associated with pluripotency and known to be expressed in the ICM of pig embryos, e.g., OCT4, SOX2, TDGF1 (CRIPTO) and TERT (32,33), were expressed only in the piPSC, and not in the founder PFF ( Fig. 2), providing evidence that reprogramming into a pluripotent state had been successful.…”

Section: Expression Of Pluripotent Genes In Pipscmentioning

confidence: 97%

Derivation of induced pluripotent stem cells from pig somatic cells

Ezashi¹,

Telugu²,

Alexenko³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

439

399

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“…OCT4/SOX2 proteins cooperate to activate the NANOG gene, suggesting that in pig blastocysts this interaction has not been established and therefore NANOG is not expressed. NANOG plays a critical role in establishing pluripotency in mammals [ 31 ]. In mouse blastocysts, NANOG is expressed in conjunction with OCT4 and SOX2 conferring pluripotent properties to the ICM [ 32 , 33 ].…”

Section: Discussionmentioning

confidence: 99%

Pig Epiblast Stem Cells Depend on Activin/Nodal Signaling for Pluripotency and Self-Renewal

Alberio

Croxall

Allegrucci

2010

Stem Cells and Development

108

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Activin/Nodal signaling is required for maintaining pluripotency and self-renewal of mouse epiblast stem cells and human embryonic stem cells (hESC). In this study, we investigated whether this signaling mechanism is also operative in cultured epiblasts derived from Days 10.5-12 pig embryos. Pig epiblast stem cell lines (pEpiSC) were established on mouse feeder layers and medium supplemented with basic fi broblast growth factor (bFGF). pEpiSC express the core pluripotency factors OCT4 (or POU5F1 ), NANOG , SOX2 , and NODAL , but they do not express REX1 or alkaline phosphatase activity. Blocking leukemia inhibitory factor (LIF)/JAK/STAT3 pathway by adding the specifi c JAK I inhibitor 420099 and an anti-LIF antibody over 3 passages did not affect pluripotency of pEpiSC. In contrast, cells grown with the Alk-5 inhibitor SB431542, which blocks Activin/Nodal pathway, differentiated readily toward the neural lineage. pEpiSC are pluripotent, as established by their differentiation potential to ectoderm, mesoderm, and endoderm. These cells can be induced to differentiate toward trophectoderm and to germ cell precursors in response to bone morphogenetic protein 4 (BMP-4). In conclusion, our study demonstrates that pig epiblasts express the core pluripotency genes and that the capacity for maintaining self-renewal in pEpiSC depends on Activin/Nodal signaling. This study provides further evidence that maintenance of pluripotency via Activin/Nodal signal is conserved in mammals. Introduction Mouse embryonic stem cells (mESC), conventionally derived from the inner cell mass (ICM) of preimplantation blastocysts, are pluripotent and can self-renew indefinitely in culture. mESC depend on the cytokines leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP-4) to maintain the undifferentiated state [ 1 , 2 ]. Human embryonic stem cells (hESC) are also derived from blastocysts; however, these cells depend on basic fi broblast growth factor (bFGF) and Activin A for pluripotency and selfrenewal [ 3 , 4 ]. Interestingly, pluripotent cells derived from mouse epiblasts, a part of the ICM, also require bFGF and Activin A for pluripotency and self-renewal [ 5 , 6 ]. hESC share multiple features with mouse epiblast stem cells (mEpiSC), such as growing as fl at and compact colonies [5][6][7], they respond to BMP-4 by differentiating to trophectoderm (TE) [ 8 ] and germ cell progenitors [ 9 ], and they do not require LIF/ JAK/STAT3 signaling for self-renewal [ 4 , 10 ]. The functional and phenotypic similarities between these cell types suggest a developmental relationship [ 5 , 6 ], and demonstrate that there are at least 2 signaling mechanisms involved in capturing pluripotency in vitro. Recent reports show that bFGF and Activin A are also necessary for maintaining rabbit ESC [ 11 , 12 ], indicating that this signaling pathway may be a conserved mechanism of pluripotency in mammals.Here we tested this hypothesis in pigs, a representative of ungulates. Attempts to derive stem cells from pig embryos have traditionally reli...

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“…Most of studies were merely confined to Nanog expression patterns by immunofluorescence and real time-PCR in porcine embryos and somatic stem cells, but the function of its role in cellular reprogramming and early embryo development by cooperating with other genes is still not defined. (Blomberg et al, 2008;Goel et al, 2008;Magnani and Cabot, 2008;Hall et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Overexpression Nanog Activates Pluripotent Genes in Porcine Fetal Fibroblasts and Nuclear Transfer Embryos

Zhang

Luo

Bou

et al. 2011

The Anatomical Record

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Nanog as an important transcription factor plays a pivotal role in maintaining pluripotency and in reprogramming the epigenome of somatic cells. Its ability to function on committed somatic cells and embryos has been well defined in mouse and human, but rarely in pig. To better understand Nanog's function on reprogramming in porcine fetal fibroblast (PFF) and nuclear transfer (NT) embryo, we cloned porcine Nanog CDS and constructed pcDNA3.1 (þ)/Nanog and pEGFP-C1/Nanog overexpression vectors and transfected them into PFFs. We studied the cell biological changes and the expression of Nanog, Oct4, Sox2, Klf4, C-myc, and Sall4 in transfected PFFs. We also detected the development potential of the cloned embryos harboring Nanog stably overexpressed fibroblasts and the expression of Oct4, Sox2, and both endogenous and exogenous Nanog in these embryos. The results showed that transient overexpression Nanog in PFF could activate the expression of Oct4 (5-fold), C-myc (2-fold), and Sall4 (5-fold) in somatic cells, but they could not be maintained during G418 selection. In NT embryos, although Nanog overexpression did not have a significant effect on blastocyst development rate and blastocyst cell number, it could significantly activate the expression of endogenous Nanog, Oct4, Sox2 to 160-fold, 93-fold, and 182-fold, respectively (P < 0.05). Our results demonstrate that Nanog could interact with and activate other pluripotent genes both in PFFs and embryos. Anat Rec, 294:1809Rec, 294: -1817Rec, 294: , 2011. V V C 2011 Wiley-Liss, Inc.

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Expression analysis of pluripotency factors in the undifferentiated porcine inner cell mass and epiblast during in vitro culture

Cited by 60 publications

References 59 publications

Derivation of induced pluripotent stem cells from pig somatic cells

Derivation of induced pluripotent stem cells from pig somatic cells

Pig Epiblast Stem Cells Depend on Activin/Nodal Signaling for Pluripotency and Self-Renewal

Overexpression Nanog Activates Pluripotent Genes in Porcine Fetal Fibroblasts and Nuclear Transfer Embryos

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