17-P024 Nanog is a master regulator of axolotl development

1 2 6 ( 2 0 0 9 ) S 2 7 1 –S 2 8 4

MECHANISMS OF DEVELOPMENT

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not form neural precursors after Sox2 shRNA but acquired a more

Virus-free induction of pluripotency and subsequentexcision of

glial (astrocytic) phenotype, as demonstrated by increased levels

reprogramming factors

of Musashi1 and Gfap by day 14, with parallel decreased levels

Keisuke Kaji1, Katherine Norrby1, Agnieszka Paca1,

of Sox1, Beta III tubulin and Pax6, compared to mock-transfected

Maria Mileikovsky2, Paria Mohseni2,3, Knut Woltjen2

control ES cells. The most striking impact of knocking down Sox2

1

in mouse ES cells was observed within the first 3 days of the neu-

2

ral differentiation protocol with significant up-regulation of the

3

trophectoderm markers Cdx2, Eomes and Fgfr2, as well as of

MRC Centre for Regenerative Medicine, Edinburgh, United Kingdom Mount Sinai Hospital, Toronto, Canada University of Toronto, Toronto, Canada Reprogramming of somatic cells to pluripotent cells promises

to transform regenerative medicine. Recently many groups have achieved direct reprogramming of somatic cells by forced expression of defined factors using multiple viral vectors. However, such induced pluripotent stem (iPS) cells contain a number

the mesendoderm markers Mixl1, Goosecoid and Brachyury. Concomitant up-regulation of b-catenin and down-regulation of Lef1 in the absence of Sox2, suggests that Sox2 may normally prevent differentiation in ES cells via regulation of the canonical Wnt pathway. doi:10.1016/j.mod.2009.06.744

of viral vector integrations, any one of which could cause unpredictable genetic dysfunction. Moreover, viral vectors silenced in iPS cells can be re-activated when the cells differentiate. Here we show non-viral transfection with a single multiprotein

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expression vector can generate iPS cells from non-genetically

Nanog is a master regulator of axolotl development

modified mouse embryonic fibroblasts. This one vector system can achieve reprogramming from one integration site, enabling

James E. Dixon1, Catherine Redwood1, Marie-Anne O’Reilly1, Jodie Chatfield1, Matt Loose1, Ramiro Alberio1,2, Andrew D. Johnson1

subsequent elimination of the reprogramming cassette by

1

Cre-mediated excision. These non-viral iPS cells show robust

United Kingdom

Institute of Genetics, University of Nottingham, Nottingham,

expression of pluripotent markers and genuine pluripotency

2

was confirmed by invitro differentiation assays and formation

United Kingdom

School of Biosciences, University of Nottingham, Nottingham,

of adult chimeric mice. When the single vector reprogramming system was combined with a piggyBac transposon we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This non-viral single vector system minimizes genome modification

Cells in the animal cap of axolotl embryos can produce germ cells and somatic cells, sharing a rigorous definition of pluripotency with mammalian embryonic stem cells (ESC). In ESC pluripotency is governed by a network of transcription factors,

and eliminates the unpredictable reactivation of reprogramming

including Oct-4 and Nanog. We isolated a Nanog ortholog from

factors, providing iPS cells more applicable to regenerative med-

axolotls (axNanog), and show that it is co-expressed with axolotl

icine, reliable drug screening and establishment of trustworthy

(ax)Oct-4. axNanog and axOct-4 are physically associated in ani-

disease models.

mal caps, as are Oct-4 and Nanog in ESC.

doi:10.1016/j.mod.2009.06.743

Morpholinos against axNanog were used to knock down its expression. Morphant embryos arrest development prior to gastrulation, and gene expression suggests that these embryos cannot initiate somatic lineage specification. Experiments with

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animal caps show that the block to further development results

The role of Sox2 in regulation of self-renewal and early cell fate

from a failure to establish pluripotency, and that this requires

decisions in mouse embryonic stem cells

the synergistic effects of axNanog and Nodal signalling.

Maria Keramari, Christopher Ward, Susan Kimber

We investigated mesoderm specification in morphant caps. In response to activin (mimicking Nodal) stimulation, axNanog mor-

University of Manchester, Manchester, United Kingdom

phant caps produce mesendoderm similar to controls, however they can not specify mesoderm. These cells instead divert to

The transcription factor Sox2 (SRY-related HMG-box gene 2), in

endoderm. We show axNanog interacts physically with activated

synergy with Oct4, plays a pivotal role in maintaining embryonic

SMAD2, and this complex down-regulates responsiveness of

stem (ES) cells self-renewing and pluripotent. After knock-down

mesoderm specific promoters to Nodal signalling. Thus, axNanog

of Sox2 in mouse ES cells with three different shRNA constructs,

acts as a rheostat, governing the response to Nodal ignalling, par-

alterations in transcript and protein expression of genes associ-

titioning mesoderm from mesendodermal precursors.

ated with pluripotency, commitment to trophectoderm, mesen-

Our results identify mechanisms for Nanog dependent meso-

doderm, neuroectoderm and cell-cycle regulation were detected.

derm specification in axolotls that are conserved in mammals.

Absence of Sox2 resulted in loss of self-renewal (with down-reg-

Other work from our lab demonstrates that Xenopus evolved novel

ulation of Oct4, Nanog, Rex1 and Utf1), severe compromise of

functional redundancies within the mesendoderm gene regula-

ES cell growth rate and cell-cycle arrest (as indicated by increased

tory network that compensate for Nanog deletion from the frog

levels of p21 and p27, as well as decreased levels of Nucleostemin

genome.

and Cyclin D2). However, apoptosis in Sox2 shRNA ES cells was not significantly different to mock-transfected control ES cells. In a culture system designed to promote neurogenesis, cells did

doi:10.1016/j.mod.2009.06.745