Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth, LacZ activity was detected in most endothelial cells within the embryo and of extra-embryonic tissues such as yolk sac and chorioallantoic placenta. Ectopic expression of Cre was observed in approximately 12–20% of the adult erythroid, myeloid and lymphoid cells and in subregions of the adult brain. These results show that the tie-1-Cre transgenic strain can efficiently direct deletion of floxed genes in endothelial cells in vivo.

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182 Establishment of expanded potential embryonic stem cell lines from porcine embryos

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab182 ◽

2019 ◽

Vol 31 (1) ◽

pp. 215

Author(s):

M. Nowak-Imialek ◽

X. Gao ◽

P. Liu ◽

H. Niemann

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Cell Lines ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Differentiation Potential ◽

Germ Layers ◽

Embryonic Tissues

The domestic pig is an excellent large animal in biomedical medicine and holds great potential for testing the clinical safety and efficacy of stem cell therapies. Previously, numerous studies reported the derivation of porcine embryonic stem cell (ESC)-like lines, but none of these lines fulfilled the stringent criteria for true pluripotent germline competent ESC. Here, we report the first establishment of porcine expanded potential stem cells (pEPSC) from parthenogenetic and in vivo-derived blastocysts. A total of 12 cell lines from parthenogenetic blastocysts from Day 7 (12/24) and 26 cell lines from in vivo-derived blastocysts from Day 5 (26/27) were established using defined stem cell culture conditions. These cells closely resembled mouse ESC with regard to morphology, formed compact colonies with high nuclear/cytoplasmic ratios, and could be maintained in vitro for more than 40 passages with a normal karyotype. The pEPSC expressed key pluripotency genes, including OCT4, NANOG, SOX2, and SALL4 at similar levels as porcine blastocysts. Immunostaining analysis confirmed expression of critical cell surface markers SSEA-1 and SSEA-4 in pEPSC. The EPSC differentiated in vitro into tissues expressing markers of the 3 germ layers: SOX7, AFP, T, DES, CRABP2, α-SMA, β-tubulin, PAX6, and, notably, the trophoblast markers HAND1, GATA3, PGF, and KRT7. After injection into immunocompromised mice, the pEPSC formed teratomas with derivatives of the 3 germ layers and placental lactogen-1 (PL-1)-positive trophoblast-like cells. Additionally, pEPSC cultured in vitro under conditions specific for germ cells formed embryoid bodies, which contained ~9% primordial germ cell (PGC)-like cells (PGCLC) that expressed PGC-specific genes, including NANOS3, BLIMP1, TFAP2C, CD38, DND1, KIT, and OCT4 as detected by quantitative RT-PCR and immunostaining. Next, we examined the in vivo differentiation potential of pEPSC and injected pEPSC stably expressing the CAG-H2B-mCherry transgene reporter into porcine embryos. The donor cells proliferated and were localised in both the trophectoderm and inner cell mass of the blastocysts cultured in vitro. After transfer to 3 recipient sows, chimeric embryos implanted and a total of 45 fetuses were recovered on Days 26 to 28. Flow cytometry of single cells collected from embryonic and extraembryonic tissues of the fetuses revealed mCherry+ cells in 7 conceptuses, in both the placenta and embryonic tissues; in 3 chimeric conceptuses, mCherry+ cells were exclusively found in embryonic tissues; and in 2 conceptuses, mCherry+ cells were exclusively localised in the placenta. The contribution of the mCherry+ cells was low (0.4-1.7%), but they were found and co-detected in multiple porcine embryonic tissues using tissue lineage-specific markers, including SOX2, TUJ1, GATA4, SOX17, AFP, α-SMA, and trophoblast markers PL-1 and KRT7 in the placental cells. The successful establishment of pEPSC represents a major step forward in stem cell research and provides cell lines with the unique state of cellular potency useful for genetic engineering and unravelling pluripotency regulation in pigs.

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Monoisoamyl dimercaptosuccinic acid abrogates arsenic-induced developmental toxicity in human embryonic stem cell-derived embryoid bodies: Comparison with in vivo studies

Biochemical Pharmacology ◽

10.1016/j.bcp.2009.07.003 ◽

2009 ◽

Vol 78 (10) ◽

pp. 1340-1349 ◽

Cited By ~ 42

Author(s):

S.J.S. Flora ◽

Ashish Mehta

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Human Embryonic Stem Cell ◽

Developmental Toxicity ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Dimercaptosuccinic Acid ◽

In Vivo Studies

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ROCK suppression promotes differentiation and expansion of endothelial cells from embryonic stem cell–derived Flk1+ mesodermal precursor cells

Blood ◽

10.1182/blood-2012-04-421610 ◽

2012 ◽

Vol 120 (13) ◽

pp. 2733-2744 ◽

Cited By ~ 29

Author(s):

Hyung Joon Joo ◽

Dong-Kyu Choi ◽

Joon Seo Lim ◽

Jin-Sung Park ◽

Seung-Hun Lee ◽

...

Keyword(s):

Endothelial Cells ◽

Stem Cell ◽

Embryonic Stem Cell ◽

Embryonic Stem ◽

Precursor Cells ◽

Rock Inhibitor ◽

Downstream Signaling ◽

Matrigel Culture

Abstract Successful differentiation and expansion of endothelial cells (ECs) from embryonic stem cell (ESC)–derived Flk1+ mesodermal precursor cells (MPCs) requires supplementation of vascular endothelial growth factor-A (VEGF-A). While analyzing VEGF-A/VEGFR2 downstream signaling pathway that underlies the VEGF-A-induced differentiation and expansion of ECs, we fortuitously found that Rho-associated protein kinase (ROCK) inhibitor Y27632 profoundly promoted the differentiation and expansion of ECs from Flk1+ MPCs while reducing the differentiation and expansion of mural cells. The ROCK suppression-induced expansion of ECs appears to have resulted from promotion of proliferation of ECs via activation of PI3-kinase-Akt signaling. The ECs obtained by the combination of ROCK suppression and VEGF-A supplementation faithfully expressed most pan-EC surface makers, and phenotypic analyses revealed that they were differentiated toward arterial EC. Further incubation of the ICAM2+ ECs with Y27632 and VEGF-A for 2 days promoted expansion of ECs by 6.5-fold compared with those incubated with only VEGF-A. Importantly, the ROCK suppression-induced ECs displayed neovasculogenic abilities in vitro and in vivo. Thus, supplementation of ROCK inhibitor Y27632 along with VEGF-A in 2D Matrigel culture system provides a simple, efficient, and versatile method for obtaining ample amount of ESC-derived ECs at high purity suitable for use in therapeutic neovascularization.

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