Analysis of the neurogenic potential of multipotent skin-derived precursors

Abstract

Multipotent precursors similar to stem cells of the embryonic neural crest (NC) have been identified in several postnatal tissues, and are potentially useful for research and therapeutic purposes. However, their neurogenic potential, including their ability to produce electrophysiologically active neurons, is largely unexplored. We investigated this issue with regard to skin-derived precursors (SKPs), multipotent NC-related precursors isolated from the dermis of skin. SKP cultures follow an appropriate pattern and time-course of neuronal differentiation, with proliferating nestin-expressing SKPs generating post-mitotic neuronal cells that co-express pan-neuronal and peripheral autonomic lineage markers. These SKP-derived neuron-like cells survive and maintain their peripheral phenotype for at least 5 weeks when transplanted into the CNS environment of normal or kainate-injured hippocampal slices. Undifferentiated SKPs retain key neural precursor properties after multi-passage expansion, including growth factor dependence, nestin expression, neurogenic potential, and responsiveness to embryonic neural crest fate determinants. Despite undergoing an apparently appropriate neurogenic process, however, SKP-derived neuron-like cells possess an immature electrophysiological profile. These findings indicate that SKPs retain latent neurogenic properties after residing in a non-neural tissue, but that additional measures will be necessary to promote their differentiation into electrophysiologically active neurons.

Introduction

The embryonic neural crest (NC) is a population of ectodermally-derived precursors that has unique migratory properties and differentiation characteristics. These multipotent stem cells originate near the boundary between the primitive neural plate and adjacent epidermis along most of the vertebrate rostro-caudal axis. They subsequently migrate throughout the body to produce diverse neural and mesodermal cell types. Among the known derivatives of the neural crest are the neurons, glia, fibroblasts, and endocrine cells of the autonomic, sensory, and enteric nervous systems, the bone, cartilage, meninges, connective tissue, pericytes, and dermis of the head, smooth muscle of the outflow tract of the heart, and melanocytes and sensory receptors of the skin (Le Douarin and Dupin, 2003, Le Douarin and Kalcheim, 1999).

Multipotent cells that have characteristics reminiscent of embryonic NC stem cells have been isolated from several postnatal tissues, including skin, gut, dental pulp, and the heart (Fernandes et al., 2004, Kruger et al., 2002, Miura et al., 2003, Tomita et al., 2005). We recently reported that multipotent NC-related cells can be isolated from skin, a particularly abundant and accessible tissue (Fernandes et al., 2004). These skin-derived precursors, or SKPs, can be cultured from the dermis of rodent (Fernandes et al., 2004, Toma et al., 2001) and human (Toma et al., 2005) skin. Recent studies have revealed two important features of SKPs. First, SKPs are multipotential. When colonies were generated from single rodent (Toma et al., 2001, Fernandes et al., 2004) or human (Toma et al., 2005) SKPs, using both limiting dilution and methylcellulose-based methods of clonal analysis, they contained distinct sub-populations of cells with the properties of neurons, glial cells, smooth muscle cells, and adipocytes. Second, SKPs are a type of NC-related precursor. Cultured SKPs expressed a variety of markers of primitive embryonic NC precursors, including characteristic NC transcription factors, and they behaved similarly to host NC precursors when transplanted into the embryonic chick neural crest migratory stream. Fate mapping studies with Wnt1-cre;R26R compound transgenic mice, which express β-galactosidase in neural crest-derived cells, confirmed that at least the SKPs derived from facial skin were neural crest-derived (Fernandes et al., 2004). Moreover, SKPs from dorsal back skin can differentiate into functional myelinating Schwann cells (McKenzie and Miller, 2002), a cell type only derived from the neural crest.

The persistence of NC-related precursors within accessible postnatal tissues raises the possibility of their use for a variety of research and therapeutic purposes. For example, NC-related precursors have been useful for modeling how mutations in genetic diseases, such as Hirschprung's Disease (Iwashita et al., 2003), or in NC cancers, such as melanomas (Fang et al., 2005), affect the fate decisions, properties, and functions of embryonic NC cells. NC-related precursors could also potentially be expanded in vitro to produce larger numbers of NC cells for high throughput screening or for cell replacement transplantation.

In the present study, we investigated questions relating to the feasibility of using multipotent NC-related SKPs for such practical applications. Since skin is a non-neurogenic tissue, we specifically asked to what extent SKPs have neurogenic potential and whether they produce electrophysiologically active neuronal progeny.