Direct derivation of neural rosettes from cloned bovine blastocysts: a model of early neurulation events and neural crest specification in vitro

¹ Laboratorio di Tecnologie della Riproduzione, Istituto Sperimentale Italiano Lazzaro Spallanzani, Cremona, Italy
² Stem Cell Research Department, San Raffaele Scientific Institute, Milan, Italy
³ Laboratorio di Tecnologie della Riproduzione, Istituto Sperimentale Italiano Lazzaro Spallanzani, Cremona, Italy; Dipartimento Clinico Veterinario, Università di Bologna, Ozzano Emilia, Italy

^* To whom correspondence should be addressed. E-mail: giovannalazzari{at}galli2.it.

	Abstract

Embryonic stem cells differentiate into neuroectodermal cells under specific culture conditions. In primates, these cells are organised into rosettes expressing Pax6 and Sox1 and are responsive to inductive signals such as Sonic hedgehog (Shh) and retinoic acid. However, direct derivation of organised neuroectoderm in vitro from preimplantation mammalian embryos has never been reported. Here we show that bovine inner cell masses, from nuclear transfer and fertilised embryos, grown on feeders in serum-free medium, form polarised rosette structures expressing nestin, Pax6, Pax7, Sox1, Otx2 and exhibiting interkinetic nuclear migration activity and cell junctions distribution as in the developing neural tube. Following in vitro expansion neural rosettes give rise to p75 positive neural crest precursor cell lines capable of long-term proliferation and differentiation in autonomic and sensory peripheral neurons, glial cells, melanocytes, smooth muscle cells and chondrocytes recapitulating in vitro the unique plasticity of the neural crest lineage. Challenging the rosette dorsal fate by early exposure to Shh induces the expression of ventral markers Isl1, Nkx2.2 and Nkx6.1, and differentiation of mature astrocytes and neurons of CNS ventral identity, demonstrating appropriate response to inductive signals. All together these findings indicate that neural rosettes directly derived from cloned and fertilised bovine embryos represent an in vitro model of early neural specification and differentiation events. Moreover, this study provides a source of highly proliferative neural crest precursor cell lines of wide differentiation potential for cell therapy and tissue engineering applications.

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