4059 Introduction Proliferation of neural stem cells (NSC)/neural progenitors largely determines the final size of neuronal populations during development and their subsequent renewal in postnatal neurogenic areas, but mechanisms regulating cycling of neural progenitors are poorly understood. NSCs are endowed with unlimited self-renewal capacity as well as multipotency to generate progeny that are fated to differentiate into the three major neural lineages (Reynolds and Weiss, 1992). They can be isolated from both the embryonic and mature mammalian CNS and expanded ex vivo under mitogenic stimulation for extended periods of time (reviewed by Gage, 2000; Temple, 2001). In the embryonic ventricular zone, NSCs are likely to coexist with fate-restricted progenitors all along the embryonic neuraxis (Temple, 2001). Stem-like cells from adult brains have been isolated not only from postnatal neurogenic regions, such as the hippocampus and the subventricular zone (SVZ), but from some non-neurogenic regions as well, including the spinal cord (see Gage, 2000). Thus, provided mammalian brains have residing cells with NSC properties in multiple locations, hope has been raised by the prospect of cell therapy based on reactivation of endogenous NSCs or on transplantation of ex vivo-expanded NSCs to replace neurons lost in traumatic or degenerative processes (Gage, 2000). The development of strategies to efficiently drive the differentiation and integration in vivo of these cells needs to be preceded by devising methods to obtain homogeneous, well characterized, genetically stable populations of multipotential stem cells. Most studies aimed at understanding the nature of the signals involved in the regulation of NSC proliferation, self-renewal and differentiation have so far been concentrated on external signals, i.e. secreted growth factors, which induce NSCs to adopt specific decisions (see Johe et al., 1996; Gritti et al., 1996; Gritti et al., 1999; Tropepe et al., 1997; Taupin et al., 2000; Shimazaki et al., 2001; Lai et al., 2003). Fewer studies have addressed the role of intrinsic mechanisms in the regulation of NSC behavior (Groszer et al., 2001; Otshuka et al., 2001). Telomeres are specialized chromatin structures at the ends of eukaryotic chromosomes that consist of non-coding single G-rich DNA repeats (TTAGGG in all vertebrates), bound to an array of associated proteins, and that play an essential role in chromosome capping (Greider, 1998; Blackburn, 2000). In most somatic tissues, telomeric DNA undergoes progressive shortening with each round of DNA replication, at a rate between 50 and 200 bp per cell division, resulting from incomplete replication of linear chromosomes by cellular DNA polymerases (see Blackburn, 2000). Telomere dysfunction, caused by significant loss of TTAGGG sequences or of telomere-binding proteins, leads to disruption of the telomere structure resulting in end-to-end chromosome fusions and genomic instability (de Lange, 2002). The non-homologous end-joining pathway for double strand break repair has been recently shown to mediate these outcomes of telomere Chromosome integrity is essential for cell viability and, therefore, highly proliferative cell types require active telomere elongation mechanisms to grow indefinitely. Consistently, deletion of telomerase activity in a genetically modified mouse strain results in growth impairments in all highly proliferative cell populations analyzed so far. We show that telomere attrition dramatically impairs the in vitro proliferation of adult neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of telomerase- deficient adult mice. Reduced proliferation of postnatal neurogenic progenitors was also observed in vivo, in the absence of exogenous mitogenic stimulation. Strikingly, severe telomere erosion resulting in chromosomal abnormalities and nuclear accumulation of p53 did not affect the in vitro proliferative potential of embryonic NSCs. These results suggest that intrinsic differences exist between embryonic and adult neural progenitor cells in their response to telomere shortening, and that some populations of tissue-specific stem cells can bypass DNA damage check points. Key words: Telomerase knockout, Neural progenitor, Neurogenesis, Differentiation Summary Telomere shortening and chromosomal instability abrogates proliferation of adult but not embryonic neural stem cells Sacri Ferrón 1, *, Helena Mira 1, * ,† , Sonia Franco 2, *, Marifé Cano-Jaimez 1 , Elena Bellmunt 1 , Carmen Ramírez 1 , Isabel Fariñas 1,‡ and María A. Blasco 2 1 Departamento de Biología Celular, Universidad de Valencia, 46100 Burjassot, Spain 2 Spanish National Cancer Center (CNIO), 28029 Madrid, Spain *These authors contributed equally to this work † Present address: Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden ‡ Author for correspondence (e-mail: isabel.farinas@uv.es) Accepted 8 April 2004 Development 131, 4059-4070 Published by The Company of Biologists 2004 doi:10.1242/dev.01215 Research article