Characterization of epidermal neural crest stem cell (EPI-NCSC) grafts in the lesioned spinal cord

https://doi.org/10.1016/j.mcn.2006.02.003Get rights and content

Abstract

We have characterized in the contusion-lesioned murine spinal cord the behavior of acutely implanted epidermal neural crest stem cells (EPI-NCSC, formerly eNCSC). EPI-NCSC, a novel type of multipotent adult stem cell, are remnants of the embryonic neural crest. They reside in the bulge of hair follicles and have the ability to differentiate into all major neural crest derivatives (Sieber-Blum, M., Grim, M., Hu, Y.F., Szeder, V., 2004. Pluripotent neural crest stem cells in the adult hair follicle. Dev. Dyn. 231, 258–269). Grafted EPI-NCSC survived, integrated, and intermingled with host neurites in the lesioned spinal cord. EPI-NCSC were non-migratory. They did not proliferate and did not form tumors. Significant subsets expressed neuron-specific β-III tubulin, the GABAergic marker glutamate decarboxylase 67 (GAD67), the oligodendrocyte marker, RIP, or myelin basic protein (MBP). Close physical association of non-neuronal EPI-NCSC with host neurites was observed. Glial fibrillary acidic protein (GFAP) immunofluorescence was not detected. Collectively, our data indicate that intraspinal EPI-NCSC demonstrate several desirable characteristics that may include local neural replacement and re-myelination.

Introduction

The objective of this study was to characterize in the injured spinal cord the behavior of implanted EPI-NCSC (formerly eNCSC; Sieber-Blum et al., 2004, Sieber-Blum and Grim, 2004). One rationale for this work relates to the notion that EPI-NCSC combine several advantages of embryonic stem cells and adult stem cells. Similar to embryonic stem cells, EPI-NCSC exhibit a high degree of physiological plasticity and they can be expanded in vitro. Similar to other types of adult stem cell, EPI-NCSC do not raise ethical concerns, and since the patients' own EPI-NCSC could be used for autologous transplants, there would be no graft rejection. Furthermore, EPI-NCSC are very accessible. They can be isolated from the bulge of hair follicles by minimal-invasive procedure as a virtually pure neural-crest-derived population (Sieber-Blum et al., 2004, Sieber-Blum and Grim, 2004). A second rationale for this work pertains to the close ontological relationship between neural crest stem cells and stem cells of the dorsal spinal cord as they share a common progenitor cell (Mujtaba et al., 1998). Based on these facts, we hypothesized that EPI-NCSC are likely to be compatible with the spinal cord environment and that they may acquire desirable traits that would establish them as candidates for future studies in cell replacement therapy. Our data support this notion. Acutely grafted EPI-NCSC survive and integrate in the lesioned spinal cord, are non-migratory, do not form tumors, some are in close contact with host neurites, and significant subsets express relevant neuronal and glial markers.

The embryonic neural crest, from which EPI-NCSC are derived, originates from the neural folds during neurulation, undergoes an epithelial-to-mesenchymal transformation, and subsequently invades the embryo where it generates numerous structures of the adult organism, including the autonomic and enteric nervous systems, most primary sensory neurons, peripheral glia, pigment cells of the skin and internal organs, Merkel cells, and the cranial mesenchyme (Le Douarin et al., 1999, Szeder et al., 2003). Neural crest cells invade the epidermis early in embryonic development, as early as day 3 of incubation in avian trunk skin (Richardson and Sieber-Blum, 1993) and day 9.5 of gestation in murine facial skin (Sieber-Blum and Grim, 2004).

In this study, we have isolated EPI-NCSC from the bulge of mouse whisker follicles. The bulge of the hair follicle is a multilayered region within the otherwise single-layered outer root sheath, which is continuous with the basal layer of the surface epidermis. The bulge is a known niche for keratinocyte/epidermal stem cells, which can form new epidermis, sebaceous gland, and hair (Rochat et al., 1994, Kobayashi et al., 1993, Lyle et al., 1998, Ito et al., 2004, Tumbar et al., 2004). Thus, the combination of these reports and our data (Sieber-Blum et al., 2004, Sieber-Blum and Grim, 2004) establish the fact that the bulge of hair follicles contains a mixed population of stem cells, epidermal stem cells, and neural crest stem cells. Our culture strategy favors the emigration of a pure neural-crest-derived cell population from bulge explants, whereas epidermal stem cells (keratinocyte stem cells) remain within the bulge explant.

Neuronal regeneration of the injured central nervous system is limited by the presence of inhibitory molecules, such as Nogo-A (Caroni and Schwab, 1988, Chen et al., 2000), chondroitin sulfate proteoglycans (Qui et al., 2000) and other myelin- and glial-scar-associated molecules (Fawcett and Asher, 1999, Grimpe and Silver, 2002, Grimpe et al., 2002, Jones et al., 2003a, Jones et al., 2003b, Tang et al., 2003, Schwab, 2004). This notion is illustrated by the observation that in mice with a deleted Nogo-A gene (Simonen et al., 2003) or in rats treated with anti-Nogo-A antibodies (Schnell and Schwab, 1990, Schwab, 2004, Liebscher et al., 2005), there is a remarkable regeneration of corticospinal axons and an important enhancement of compensatory sprouting in the lesioned spinal cord.

Main goals in spinal cord repair include reconnecting brain and lower spinal cord, building new circuits, re-myelination of demyelinated axons, providing trophic support, and bridging the gap of the lesion (reviewed by Enzmann et al., in press). Overcoming myelin-associated growth inhibition and glial-scar-associated growth inhibition are experimental approaches that have been most successful in vivo in recent studies. Further issues concern gray matter reconstitution and protecting neurons and glia from secondary death (Rosenzweig and McDonald, 2004, Enzmann et al., in press). Our present results in combination with our gene profiling data (Hu et al., submitted for publication) suggest that in the current experimental paradigm EPI-NCSC are candidates for local neural replacement and re-myelination and that they likely provide trophic support and facilitate re-vascularization.

Section snippets

Isolation, in vitro expansion and characterization of EPI-NCSC

Similar to wild type bulge explants, bulge explants from C57BL/6-TgN(ACTbEGFP)1Osb mouse whisker follicles adhered to the collagen substratum and within 3 to 4 days started to release highly migratory cells that proliferated rapidly (Fig. 1A). With compound transgenic Wnt1-cre/R26R mice, in which neural crest cells and their derivatives are marked permanently by the expression of β-galactosidase, we have shown previously that these cells consist of a pure population of neural-crest-derived

Isolation of EPI-NCSC

C57BL/6-TgN(ACTbEGFP)1Osb breeder mice were purchased from Jackson Laboratory (Bar Harbor, ME). They express enhanced green fluorescent protein (EGFP) ubiquitously under the control of the chicken actin promoter (Okabe et al., 1997). All cells, except hair and red blood cells, are intensely green-fluorescent. Anagen whisker follicles of 10-week-old heterozygous mice were dissected as described (Sieber-Blum et al., 2004). Briefly, the mice were sacrificed and disinfected with iodine/peroxide

Acknowledgments

We thank Drs. A. McKinney and A.G. Bittermann for their help with confocal microscopy and Eva Kluzákova for excellent technical assistance. MSB thanks Dr. Scott Whittemore for providing a preprint to the review by Enzmann et al. (in press). This work was supported by USPHS grant NS38500 from the National Institute of Neurological Disorders and Stroke, NIH (MSB); Charles E. Culpeper Biomedical Pilot Initiative Grant 03-128 from the Rockefeller Brothers Fund, New York (MSB); grants from the Swiss

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