Differentiation of ES cells into cerebellar neurons

Enrique Salero* and
Mary E. Hatten^†

Laboratory of Developmental Neurobiology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399

Communicated by Torsten N. Wiesel, The Rockefeller University, New York, NY, December 15, 2006 (received for review September 15, 2006)

Abstract

The neuronal circuits of the cerebellar cortex are essential for motor and sensory learning, associative memory formation, and the vestibular ocular reflex. In children and young adults, tumors of the granule cell, the medulloblastomas, represent 40% of brain tumors. We report the differentiation of E14 ES cells into mature granule neurons by sequential treatment with secreted factors (WNT1, FGF8, and RA) that initiate patterning in the cerebellar region of the neural tube, bone morphogenic proteins (BMP6/7 and GDF7) that induce early granule cell progenitor markers (MATH1, MEIS1, ZIC1), mitogens (SHH, JAG1) that control proliferation and induce additional granule cell markers (Cyclin D2, PAX2/6), and culture in glial-conditioned medium to induce markers of mature granule neurons (GABAα₆r), including ZIC2, a unique marker for granule neurons. Differentiated ES cells formed classic “T-shaped” granule cell axons in vitro, and implantation of differentiated Pde1c-Egfp-BAC transgenic ES cells into the external granule cell layer of neonatal mice resulted in the extension of parallel fibers, migration across the molecular layer, incorporation into the internal granule cell layer, and extension of short dendrites, typical of young granule cells forming synaptic connections with afferent mossy fibers. These results underscore the utility of treating ES cells with local, inductive signals that regulate CNS neuronal development in vivo as a strategy for cell replacement therapy of defined neuronal populations.

Footnotes

^†To whom correspondence should be addressed. E-mail: hatten{at}rockefeller.edu
Author contributions: E.S. and M.E.H. designed research; E.S. performed research; E.S. and M.E.H. analyzed data; and E.S. and M.E.H. wrote the paper.
↵*Present address: Albany Medical College, Albany, NY 12208.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0610879104/DC1.
Abbreviations:

EB,

embryoid bodies;

EGL,

external granule cell layer;

GCP,

granule cell progenitor;

Pn,

postnatal day n;

ML,

molecular layer;

IGL,

internal granule cell layer;

DIV,

days in vivo;

RA,

retinoic acid;

SHH,

sonic hedgehog.
Freely available online through the PNAS open access option.