Acute transplantation of glial-restricted precursor cells into spinal cord contusion injuries: survival, differentiation, and effects on lesion environment and axonal regeneration
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).
References (102)
- et al.
Regenerating and sprouting axons differ in their requirements for growth after injury
Exp. Neurol.
(1997) - et al.
Neurotrophic factors increase axonal growth after spinal cord injury and transplantation in the adult rat
Exp. Neurol.
(1997) - et al.
Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice
Neuron
(1999) - et al.
Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage
Exp. Neurol.
(2001) - et al.
Methylprednisolone administration improves axonal regeneration into Schwann cell grafts in transected adult rat thoracic spinal cord
Exp. Neurol.
(1996) - et al.
Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord
Brain Res.
(2000) - et al.
Subventricular zone astrocytes are neural stem cells in the adult mammalian brain
Cell
(1999) - et al.
The glial scar and central nervous system repair
Brain Res. Bull.
(1999) Candidate cells for transplantation into the injured CNS
Prog. Brain Res.
(2000)- et al.
Temporal-spatial pattern of acute neuronal and glial loss after spinal cord contusion
Exp. Neurol.
(2001)
Methylprednisolone preservation of motor-nerve function during early degeneration
Exp. Neurol.
Grafted lineage-restricted precursors differentiate exclusively into neurons in the adult spinal cord
Exp. Neurol.
Schwann cells and fetal tectal tissue cografted to the midbrain of newborn rats: fate of Schwann cells and their influence on host retinal innervation of grafts
Exp. Neurol.
Embryonic-derived glial-restricted precursor cells (GRP cells) can differentiate into astrocytes and oligodendrocytes in vivo
Exp. Neurol.
Degeneration and sprouting of identified descending supraspinal axons after contusive spinal cord injury in the rat
Exp. Neurol.
Ubiquitous expression of marker transgenes in mice and rats
Dev. Biol.
Chondroitin sulfate proteoglycan immunoreactivity increases following spinal cord injury and transplantation
Exp. Neurol.
Isolation of lineage-restricted neuronal precursors from multipotent neuroepithelial stem cells
Neuron
Lineage-restricted neural precursors can be isolated from both the mouse neural tube and cultured ES cells
Dev. Biol.
Stable expression of the alkaline phosphatase marker gene by neural cells in culture and after transplantation into the CNS using cells derived from a transgenic rat
Exp. Neurol.
Getting a GR(i)P on oligodendrocyte development
Dev. Biol.
Glial cell derived neurotrophic factors and Alzheimer's disease
Neurodegeneration
Functional recovery of paraplegic rats and motor axon regeneration in their spinal cords by olfactory ensheathing glia
Neuron
Glial-restricted precursors are derived from multipotent neuroepithelial stem cells
Dev. Biol.
Precursor cells for transplantation
Prog. Brain Res.
A role for platelet-derived growth-factor in normal gliogenesis in the central nervous-system
Cell
An 'oligarchy' rules neural development
Trends Neurosci.
The recovery of 5-HT immunoreactivity in lumbosacral spinal cord and locomotor function after thoracic hemisection
Exp. Neurol.
Immature type-1 astrocytes suppress glial scar formation, are motile and interact with blood-vessels
Brain Res.
Transplantation of immature and mature astrocytes and their effect on scar formation in the lesioned central nervous-system
Prog. Brain Res.
Maturation of astrocytes in vitro alters the extent and molecular-basis of neurite outgrowth
Dev. Biol.
Stem cells in the adult mammalian central nervous system
Curr. Opin. Neurobiol.
Transplantation of oligodendrocyte precursors in the adult demyelinated spinal cord: migration and remyelination
Int. J. Dev. Neurosci.
Regeneration and sprouting of chronically injured corticospinal tract fibers in adult rats promoted by NT-3 and the mAb IN-1, which neutralizes myelin-associated neurite growth inhibitors
Exp. Neurol.
Effects of astrocyte implantation into the hemisected adult-rat spinal-cord
Neuroscience
Elimination of basal lamina and the collagen “scar” after spinal cord injury fails to augment corticospinal tract regeneration
Exp. Neurol.
Migration, integration, and differentiation of hippocampus-derived neurosphere cells after transplantation into injured rat spinal cord
Neurosci. Lett.
A combination of BDNF and NT-3 promotes supraspinal axonal regeneration into Schwann cell grafts in adult rat thoracic spinal cord
Exp. Neurol.
Phenotypic diversity and kinetics of proliferating microglia and astrocytes following cortical stab wounds
Glia
Alterations in temporal/spatial distribution of GFAP- and vimentin-positive astrocytes after spinal cord contusion with the New York University spinal cord injury device
J. Neurotrauma
Review of current evidence for apoptosis after spinal cord injury
J. Neurotrauma
Robust growth of chronically injured spinal cord axons induced by grafts of genetically modified NGF-secreting cells
Exp. Neurol.
Chondroitinase ABC promotes functional recovery after spinal cord injury
Nature
Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors
Nature
Spinal cord repair in adult paraplegic rats: partial restoration of hind limb function
Science
Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics
J. Neurosci.
The effects of methypredisolone and the gaglioside GM1 on acute spinal cord injury in rats
J. Neurosurg.
Axonal regeneration and functional recovery after complete spinal cord transection in rats by delayed treatment with transplants and neurotrophins
J. Neurosci.
Axonal elongation into peripheral nervous system “bridges” after central nervous system injury in adult rats
Science
Cited by (0)
- 1
Current address: The Miami Project to Cure Paralysis, University of Miami School of Medicine, Lois Pope LIFE Center, 1095 NW 14th Terrace, Miami, FL 33136, United States.