Elsevier

Experimental Neurology

Volume 213, Issue 1, September 2008, Pages 176-190
Experimental Neurology

Transplanted neural stem/progenitor cells generate myelinating oligodendrocytes and Schwann cells in spinal cord demyelination and dysmyelination

https://doi.org/10.1016/j.expneurol.2008.05.024Get rights and content

Abstract

Stem cell therapy is a promising approach for remyelination strategies in demyelinating and traumatic disorders of the spinal cord. Self-renewing neural stem/progenitor cells (NSPCs) reside in the adult mammalian brain and spinal cord. We transplanted NSPCs derived from the adult spinal cord of transgenic rats into two models of focal demyelination and congenital dysmyelination. Focal demyelination was induced by X-irradiation and ethidium bromide injection (X-EB); and dysmyelination was in adult shiverer mutant mice, which lack compact CNS myelin. We examined the differentiation potential and myelinogenic capacity of NSPCs transplanted into the spinal cord. In X-EB lesions, the transplanted cells primarily differentiated along an oligodendrocyte lineage but only some of the oligodendrocytic progeny remyelinated host axons. In this glial-free lesion, NSPCs also differentiated into cells with Schwann-like features based on ultrastructure, expression of Schwann cell markers, and generation of peripheral myelin. In contrast, after transplantation into the spinal cord of adult shiverer mice, the majority of the NSPCs expressed an oligodendrocytic phenotype which myelinated the dysmyelinated CNS axons forming compact myelin, and none had Schwann cell-like features. This is the first study to examine the differentiation and myelinogenic capacity of adult spinal cord stem/progenitors in focal demyelination and dysmyelination of the adult rodent spinal cord. Our findings demonstrate that these NSPCs have the inherent plasticity to differentiate into oligodendrocytes or Schwann-like cells depending on the host environment, and that both cell types are capable of myelinating axons in the demyelinated and dysmyelinated adult spinal cord.

Introduction

There is considerable interest in stem cell therapy for the treatment of the injured or diseased nervous system. The presence of neural stem/progenitor cells in the adult mammalian brain and spinal cord (Reynolds and Weiss, 1992, Weiss et al., 1996) has suggested their potential therapeutic application. Adult neural stem/progenitor cells (NSPCs) are self-renewing and multipotent, capable of generating both neurons and glia in vitro (Reynolds and Weiss, 1992, Weiss et al., 1996). When cultured in the presence of growth factors they form neurospheres which are free-floating colonies of cells primarily composed of progenitor cells and< 1% stem cells (Morshead et al., 1994). Transplantation of NSPCs derived from the adult rodent spinal cord or subventricular zone of the forebrain produced limited functional recovery after spinal cord injury (Vacanti et al., 2001, Hofstetter et al., 2005, Karimi-Abdolrezaee et al., 2006, Pfeifer et al., 2006). In the spinal cord, NSPCs reside close to the ependymal/periventricular region since multipotential self-renewing neurospheres were generated only when the cultured tissue included parts of the central canal (Martens et al., 2002). In lower vertebrates, ependymal cells rapidly proliferate and differentiate into neurons and glia to regenerate the transected cord (Nordlander and Singer, 1978, Dervan and Roberts, 2003). In adult mammals, ependymal cells proliferate in response to several types of spinal cord trauma (Vaquero et al., 1981, Bruni and Anderson, 1987, Wallace et al., 1987, Beattie et al., 1997, Johansson et al., 1999, Namiki and Tator, 1999, Takahashi et al., 2003, Mothe and Tator, 2005, Horky et al., 2006). However, endogenous NSPCs appear to have only limited regenerative capacity for repair. Increasing cell numbers through transplantation may enhance this regenerative potential.

Stem cell therapy may be particularly effective for remyelination in diseases or injuries associated with demyelination. Recently, we reported that NSPCs derived from the periventricular region of the adult spinal cord showed an intrinsic capacity for predominant oligodendrocytic differentiation without targeted manipulation by neurotrophic factors or other agents (Kulbatski et al., 2007). The current study examines the differentiation potential and myelinogenic capacity of adult spinal cord NSPCs transplanted into the spinal cord of two complementary models of demyelination, focal demyelination induced by X-irradiation/ethidium bromide (X-EB) which leaves a population of demyelinated axons in a glial-free environment (Blakemore, 1982, Crang et al., 1992), and adult shiverer mutant mice which carry a spontaneous mutation of myelin basic protein (MBP) resulting in a lack of central myelin, a model of congenital dysmyelination with a long-term irreversible myelin deficiency (Privat et al., 1979, Chernoff, 1981). Although the myelination potential of many cell types has been examined in experimental studies (Radtke et al., 2007), to our knowledge, this is the first study to examine the differentiation and myelinogenic capacity of adult spinal cord stem/progenitors in focal demyelination and dysmyelination of the adult rodent spinal cord. Our findings demonstrate that spinal cord NSPCs differentiate into oligodendrocytes or Schwann-like cells depending on the host environment, and that these cells are capable of myelinating axons in the demyelinated and dysmyelinated adult rodent spinal cord.

Section snippets

Isolation and culture of adult periventricular spinal cord NSPCs

NSPCs were isolated from the spinal cords of transgenic adult Wistar rats expressing enhanced green fluorescent protein (GFP) (Wistar-TgN(CAG-GFP)184ys) (YS Institute Inc., Utsunomiya, Tochigi, Japan). The GFP transgene is driven by chicken β-actin promoter and cytomegalovirus enhancer (Hakamata et al., 2001). NSPCs isolated from these rats stably express the transgene long term both in vitro and in vivo (Mothe et al., 2005). The isolation and generation of periventricular neurospheres were

Adult spinal cord periventricular neurospheres are multipotent

NSPCs were isolated from the periventricular region of the spinal cord from adult GFP transgenic rats and grown as free-floating neurospheres in uncoated tissue culture flasks. The neurospheres were cultured in the presence of the growth factors EGF and FGF2 and passaged weekly for expansion. Neurospheres from passages 3 or 4 were used in both the in vitro and in vivo experiments.

Figs. 1A–C shows GFP expression of a single sphere with high levels of nestin, a marker for neural/stem progenitor

Discussion

In the present study, we show that transplanted adult spinal cord NSPCs preferentially differentiate along an oligodendroglial lineage in both the focal demyelination lesion and the dysmyelinated shiverer spinal cord. However, in both EB and X-EB lesions, some of the oligodendrocytic progeny remyelinate axons, yet a significant proportion of the transplanted cells express a Schwann cell phenotype and generate peripheral myelin. In culture, we show that spinal cord NSPCs do not express a Schwann

Acknowledgments

This work was supported by operating grants to C.H.T from the Christopher Reeve Paralysis Foundation, MS Society of Canada, International Foundation of Research in Paraplegia, and in part by CIHR NET team grant. A. J. M. was supported by fellowships from the Ontario Neurotrauma Foundation and CIHR. We thank Linda Lee and Rita van Bendegem for the technical assistance and Iris Kulbatski for culturing some of the cells used for the focal demyelination transplantation experiments. We thank Dr.

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