Elsevier

Experimental Neurology

Volume 190, Issue 2, December 2004, Pages 289-310
Experimental Neurology

Acute transplantation of glial-restricted precursor cells into spinal cord contusion injuries: survival, differentiation, and effects on lesion environment and axonal regeneration

https://doi.org/10.1016/j.expneurol.2004.05.043Get rights and content

Abstract

Transplantation of stem cells and immature cells has been reported to ameliorate tissue damage, induce axonal regeneration, and improve locomotion following spinal cord injury. However, unless these cells are pushed down a neuronal lineage, the majority of cells become glia, suggesting that the alterations observed may be potentially glially mediated. Transplantation of glial-restricted precursor (GRP) cells—a precursor cell population restricted to oligodendrocyte and astrocyte lineages—offers a novel way to examine the effects of glial cells on injury processes and repair. This study examines the survival and differentiation of GRP cells, and their ability to modulate the development of the lesion when transplanted immediately after a moderate contusion injury of the rat spinal cord. GRP cells isolated from a transgenic rat that ubiquitously expresses heat-stable human placental alkaline phosphatase (PLAP) were used to unambiguously detect transplanted GRP cells. Following transplantation, some GRP cells differentiated into oligodendrocytes and astrocytes, retaining their differentiation potential after injury. Transplanted GRP cells altered the lesion environment, reducing astrocytic scarring and the expression of inhibitory proteoglycans. Transplanted GRP cells did not induce long-distance regeneration from corticospinal tract (CST) and raphe-spinal axons when compared to control animals. However, GRP cell transplants did alter the morphology of CST axons toward that of growth cones, and CST fibers were found within GRP cell transplants, suggesting that GRP cells may be able to support axonal growth in vivo after injury.

Introduction

The growing promise for the use of cell transplantation to repair damage associated with spinal cord injury and other forms of CNS trauma must contend with an ever increasing number of cellular populations that are interesting transplant candidates. Neuroepithelial stem cells, Schwann cells, olfactory ensheathing cells, astrocytes, oligodendrocyte precursor cells, and a variety of genetically modified neural and nonneural cells have all been applied in multiple injury paradigms, and many have provided some degree of encouraging results (Fischer, 2000, Kocsis, 1999, McDonald et al., 1999, Ramon-Cueto et al., 2000, Rao and Mayer-Proschel, 2000, Takami et al., 2002). Moreover, the continuing discovery of new neural precursor cells is providing new potentially interesting transplant candidates (Rao and Mayer-Proschel, 2000).

There are several reasons to be particularly interested in glial cells and their precursors as transplantation candidates. In addition to neuronal damage, traumatic injury to the CNS is associated with death of oligodendrocytes (Beattie et al., 2000), leading to loss of myelin with its consequent impairment of axonal impulse conduction in many remaining axons (Loy et al., 2002). Astrocytes, the other major glial cell type of the CNS, can provide appropriate surfaces for the promotion of axonal elongation and are an important source of trophic factors for the survival of neurons and oligodendrocytes (Bush et al., 1999, Noble and Murray, 1984, Noble et al., 1988, Patel et al., 1996, Richardson et al., 1988). However, following CNS injury, astrocytes have a dichotomous role. They hypertrophy and deposit proteoglycans (Amat et al., 1996, Bush et al., 1999, Fawcett and Asher, 1999, Reier and Houle, 1988), which seal off the injury site and consequently limit tissue damage and allow for tissue repair by reestablishing the connective tissue substrate and the blood–brain barrier (Bush et al., 1999, Faulkner et al., 2004). The physical barrier produced by the hypertrophy of astrocytes and the molecular barrier produced by proteoglycans, particularly chondroitin sulphate proteoglycans (CSPGs), have also been implicated in the failure of axons to regrow following spinal cord injury (Bradbury et al., 2002, Davies et al., 1997, Fawcett and Asher, 1999, Fitch et al., 1999, McKeon et al., 1991, Reier and Houle, 1988).

Unlike their adult counterpart, immature glial cells can modulate the lesion environment to make it less inhibitory (Houle and Reier, 1988, Olby and Blakemore, 1996, Reier and Houle, 1988, Smith and Miller, 1991, Smith and Silver, 1988, Smith et al., 1986, Wang et al., 1995) and can promote axonal regeneration in vitro and in vivo (Smith and Silver, 1988, Smith et al., 1986, Smith et al., 1987, Smith et al., 1990). Varying degrees of benefit have been observed following transplantation of glial cells (Schwann cells, olfactory ensheathing cells or immature astrocytes) into the injured spinal cord. Indeed, even though neural stem cells can differentiate into both neurons and glia in vitro and in vivo (Chiasson et al., 1999, Chow et al., 2000, Doetsch et al., 1999, Shihabuddin et al., 2000, Temple and Alvarez-Buylla, 1999, Wu et al., 2001), and have been shown to improve locomotor performance in rats when transplanted into contusive spinal cord injuries (McDonald et al., 1999), the small proportion of neurons formed following transplantation (Cao et al., 2001, McDonald et al., 1999) suggests that benefits accrued from such transplantation may be mediated by glia. The extent to which benefit is derived from remyelination (Liu et al., 2000), from alterations of the lesion environment to make it more permissive for axonal growth (Smith and Miller, 1991, Smith and Silver, 1988) or from other effects, is not known.

The tripotential glial-restricted precursor (GRP) cell that has been isolated from the developing rat spinal cord (Rao et al., 1998) is of substantial potential interest as a transplantation candidate as GRP cells are the earliest precursor cell type restricted to the generation of glia that has thus far been isolated from the developing CNS (Mayer-Proschel et al., 1997, Noble et al., 2004, Rao and Mayer-Proschel, 1997, Rao et al., 1998). They are able to generate oligodendrocytes and two types of astrocytes in vitro (Rao et al., 1998), and readily generate both oligodendrocytes and astrocytes following transplantation into the uninjured CNS (Herrera et al., 2001). Moreover, GRP cells are able to generate oligodendrocyte type-2 astrocyte progenitor/oligodendrocyte precursor cells (O-2A/OPCs) (Gregori et al., 2002) and astrocyte-restricted precursor cells in vitro, as well as being able to generate cells that express antigens associated with radial glia (Liu et al., 2002). GRP cells show extensive self-renewal in vitro (Rao et al., 1998) and can be isolated directly from developing spinal cord (Rao et al., 1998), from neural stem cells, and from embryonic stem cells (Mayer-Proschel et al., 1997, Rao and Mayer-Proschel, 1997). Cells with these properties have also been isolated from mouse, rat, and human CNS (Dietrich et al., 2002, Mujtaba et al., 1999).

As GRP cells appear to be a critical contributor to the development of the spinal cord and are able to generate all the glial cell types and more restricted intermediate precursor cells, they appear to be an ideal candidate for restoring the glial environment following traumatic CNS injury (see review Rao and Mayer-Proschel, 2000). Early transplantation may allow GRP cells to modulate the lesion environment to make it less inhibitory, similar to the changes observed following transplantation of other immature cells (Houle and Reier, 1988, Olby and Blakemore, 1996, Reier and Houle, 1988, Smith and Miller, 1991, Smith and Silver, 1988, Smith et al., 1986, Wang et al., 1995). GRP cell-derived astrocytes, like immature astrocytes and fetal tissue, permit axonal growth in vitro (Dr. Mark Noble, unpublished data); thus GRP cells could form a growth-promoting substrate via immature astrocytes after injury (Smith and Miller, 1991, Smith and Silver, 1988, Smith et al., 1986, Smith et al., 1987, Smith et al., 1990). There are, however, considerable concerns regarding the transplantation of such cells that must be addressed before GRP cells can be used therapeutically. It is not known whether cells that may be so critical in embryonic development would even survive or differentiate following transplantation into the injured adult environment. It is also possible that these cells would undergo uncontrolled proliferation in such an environment.

The present study assesses the biological properties of GRP cells by examining the ability of GRP cells to survive, integrate, and differentiate into oligodendrocytes and immature astrocytes following transplantation immediately after spinal cord injury. Using GRP cells derived from transgenic rats harboring the heat-stable human placental alkaline phosphatase gene (PLAP), transplanted cells were readily detected after transplantation. Transplanted GRP cells were able to survive immediate transplantation following contusive SCI and retained the ability to differentiate into oligodendrocytes and astrocytes after injury. In addition, transplantation of GRP cells was able to alter the formation of the glial scar and the deposition of inhibitory molecules, and was also associated with modest axonal sprouting. Together, this study and the study of Han et al. (2004) indicate that GRP cells are capable of integrating into the spinal cord after injury and modulating the environment early after injury to potentially make it more permissive for axonal growth.

Section snippets

Materials and methods

The present study (1) compares three different markers (Hoechst (HO), green fluorescent protein (GFP)-transfection, and transgenic PLAP rat cells) to optimize identification of transplanted GRP cells; (2) assesses the properties of GRP cells by examining the ability of GRP cells to survive, migrate, and differentiate into oligodendrocytes and immature astrocytes following transplantation immediately after a spinal cord injury; (3) examines the ability of transplanted GRP cell to alter the

GRP cell identification

Initial GRP cell transplants used GFP-transfected GRP cells prelabeled with Hoechst 33342 for detection. Due to difficulty detecting the GFP and concerns about nonspecific Hoechst labeling, three additional animals with GRP cells derived from PLAP rat embryos prelabeled with Hoechst were examined 8 days after transplantation. Although prelabeling with Hoechst has been used to detect transplanted cells (Harvey and Plant, 1995, Menei et al., 1998, Ramon-Cueto et al., 1998, Vignais et al., 1993),

Discussion

Transplantation of embryonic GRP cells into acutely injured rat spinal cord was studied to begin evaluating the potential utility of this cell population in the repair of SCI. The studies we report were focused on survival and integration of these cells into the damaged cord, and on the effects of such transplants on the injured tissue. Our results demonstrate that GRP cells can be successfully applied in these transplantation paradigms, where they integrate into host tissue. In particular, GRP

Acknowledgments

We would like to thank our colleagues for useful discussion of the manuscript. This research was supported by NIH grants NS38079 (JB and MB), NS37166 (MN), and NS42820 (MMP), and a grant from the New York State Spinal Cord Injury Research Board (MMP).

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