Research reportAxon growth and recovery of function supported by human bone marrow stromal cells in the injured spinal cord exhibit donor variations
Introduction
Treatments that enhance axonal growth and regeneration of damaged axons in the central nervous system (CNS) have a potential for improving recovery following spinal cord injury (SCI). The adult, and especially the injured CNS, is inhibitory to axonal growth. Therefore, effective repair strategies for SCI require the creation of a permissive environment within the injured spinal cord that protects damaged neurons from the effects of secondary injury and also facilitates axonal regeneration. Cell transplantation is among the most promising therapeutic approaches for treating SCI. Ideally, cell transplants would be readily obtainable, easy to expand and bank, and capable of surviving long enough to facilitate sufficient and appropriate axonal regeneration [14].
Bone marrow stromal cells (MSC) are connective tissue progenitor cells that are distinct from hematopoietic stem cells [45]. While MSC can be easily expanded ex vivo from raw bone marrow, there is no generally accepted method for MSC isolation, propagation, and characterization. As a result, the phenotype of culture-expanded MSC can vary considerably when derived by different methods [44] or from different sources [42], [43].
Recent studies proposed a more extensive differentiation potential of MSC showing phenotypic plasticity that appears to cross the boundaries of the traditional germ layers including cardiac cells [41], skeletal muscle [31], and neural cells [30]. Whether this apparent plasticity represents transdifferentiation, a pool of persistent pluripotent stem cells, cell fusion, or artifacts of culturing remains controversial [21], [25], [34], [53].
Because of their ability to differentiate into a variety of cells, the ease of their isolation and expansion, and their potential use for clinical application, efforts have increased to better understand the biology of MSC. In the injured CNS, MSC transplantation has been shown to improve recovery after stroke or traumatic brain injury [8]. In animal models of SCI, grafts of MSC have been shown to promote remyelination [1] as well as partial recovery of function [9], [23], [60]. While previous studies have suggested that MSC can differentiate into cells with neural characteristics in vitro [11], [28], [47] and in vivo [9], [23], [30], it is unclear whether such differentiation contributes to recovery of function in animal models of neurotrauma.
There is growing evidence that MSC produce a variety of neurotrophic factors as well as chemokines and cytokines in vitro and in vivo (for review, see [8]). Kinnaird et al. [29] found that paracrine signaling of MSC is an important therapeutic mechanism in the treatment of ischemia. A recent study [54] showed that MSC secrete brain natriuretic peptide (BNP), a peptide with diuretic and vasodilatory effects in vitro, suggesting that MSC could facilitate recovery by reducing edema and improving perfusion. In addition, Chen et al. [6] showed that the secretion profile of MSC is responsive to the environment with increased secretion of certain growth factors (e.g., BDNF, NGF) in the injured brain. Zhong et al. [62] demonstrated that neural cell death in response to oxygen-glucose deprivation was reduced in hippocampal slices co-cultured with MSC, suggesting a neuroprotective effect possibly mediated by diffusible factors released by MSC. Thus, the cells may create a permissive environment for axon outgrowth and axonal guidance mediated by their release of trophic factors, thereby improving self-repair in the damaged CNS.
In the present study, we investigated the efficacy of different lots of MSC, each obtained from the bone marrow aspirate of a different donor, by evaluating their ability to support axonal growth following engraftment in a rat model of subtotal cervical hemisection. Functional recovery was evaluated by an array of motor and sensory tests. In addition, we showed variations in the secretion profiles for selected growth factors and cytokines of MSC from different donors, and the ability of MSC-conditioned medium to promote axon outgrowth in an in vitro dorsal root ganglion (DRG) culture system independent of the donor.
Section snippets
Isolation and expansion of human MSC
Human MSC were isolated from bone marrow aspirates taken from the iliac crest of four healthy adult human volunteers under informed consent. Donors were tested for various chronic diseases (heart, kidney or liver disease, ulcer, cancer, diabetes, epilepsy) as well as for bacterial or viral infections. Vital signs, hematological lab values and donor weight were within normal range and donors were not currently taking prescription medication. Donor age ranged between 18 and 45 years. We took
Grafts of human MSC support axon outgrowth in a donor aspirate-dependent manner
Two weeks after grafting, MSC were identified by labeling with PKH26 and antibodies against human mitochondria. The lesion cavity was filled with MSC (Figs. 1a and b) regardless of which donor was used. Most of the grafted cells remained at the lesion site but some of them migrated into the penumbra of surrounding host tissue. In no instances were the grafted MSC observed more than 500 μm from the lesion site. Grafts of MSC supported extensive axonal growth, as evidenced by GAP43 and
Discussion
In this study, we have demonstrated for the first time that axon growth and recovery of function in response to a human MSC graft in the injured rat spinal cord is donor-dependent. Examination of the secretion profile for certain growth factors and cytokines revealed major differences between four human MSC donors. While this secretion profile did not seem to greatly affect axon growth in vitro, axon outgrowth into MSC grafts in a subtotal cervical hemisection differed significantly depending
Acknowledgments
Human marrow stromal cells and ELISA data for this study were provided by Neuronyx Inc., Malvern, PA. We thank Drs. Gene Kopen and Joseph Wagner for helpful suggestions and critical review of the manuscript, and Dr. Scott Stackhouse for help with the statistical analysis. We thank Dr. Masata Shibata, Maryla Obrocka, Lee Silver, Maureen Tumolo, and Guillermo Samper for excellent technical support. The hybridoma used to produce the neurofilament antibody was developed by Dr. John Wood and
References (62)
- et al.
Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy
Lancet
(2003) - et al.
Endogenous repair after spinal cord contusion injuries in the rat
Exp. Neurol.
(1997) - et al.
Treatment of neural injury with marrow stromal cells
Lancet Neurol.
(2002) - et al.
Mechanical and thermal allodynia in chronic central pain following spinal cord injury
Pain
(1996) - et al.
In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP
Biochem. Biophys. Res. Commun.
(2001) - et al.
Neuropathic pain from an experimental neuritis of the rat sciatic nerve
Pain
(1999) - et al.
Transplants of adrenal medullary chromaffin cells reduce forelimb and hindlimb allodynia in a rodent model of chronic central pain after spinal cord hemisection injury
Exp. Neurol.
(2000) - et al.
Engraftment of serotonergic precursors enhances locomotor function and attenuates chronic central pain behavior following spinal hemisection injury in the rat
Exp. Neurol.
(2001) - et al.
Changes in serotonin, serotonin transporter expression and serotonin denervation supersensitivity: involvement in chronic central pain after spinal hemisection in the rat
Exp. Neurol.
(2002) - et al.
A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia
Pain
(1988)
Plasticity of marrow-derived stem cells
Blood
Tales of transdifferentiation
Exp. Neurol.
Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury
Cell
Grafts of BDNF-producing fibroblasts rescue axotomized rubrospinal neurons and prevent their atrophy
Exp. Neurol.
The effect of bone marrow stromal cells on neuronal differentiation of mesencephalic neural stem cells in Sprague–Dawley rats
Brain Res.
Bone marrow stromal cells infused into the cerebrospinal fluid promote functional recovery of the injured rat spinal cord with reduced cavity formation
Exp. Neurol.
Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential
Blood
Manipulating neuroinflammatory reactions in the injured spinal cord: back to basics
Trends Pharmacol. Sci.
Adult bone marrow stromal cells differentiate into neural cells in vitro
Exp. Neurol.
Ex vivo MR determined apparent diffusion coefficients correlate with motor recovery mediated by intraspinal transplants of fibroblasts genetically modified to express BDNF
Exp. Neurol.
Delayed transplantation of fibroblasts genetically modified to secrete BDNF and NT-3 into a spinal cord injury site is associated with limited recovery of function
Exp. Neurol.
Brain as the sea of marrow
Exp. Neurol.
Expression of brain natriuretic peptide by human bone marrow stromal cells
Exp. Neurol.
Neurotrophic effects on dorsal root regeneration into the spinal cord
Prog. Brain Res.
Delayed grafting of BDNF and NT-3 producing fibroblasts into the injured spinal cord stimulates sprouting, partially rescues axotomized red nucleus neurons from loss and atrophy, and provides limited regeneration
Exp. Neurol.
Ischemic cerebral tissue and MCP-1 enhance rat bone marrow stromal cell migration in interface culture
Exp. Hematol.
Vascular endothelial growth factor improves functional outcome and decreases secondary degeneration in experimental spinal cord contusion injury
Neuroscience
Excitotoxic spinal cord injury: behavioral and morphological characteristics of a central pain model
Pain
Neuroprotective effects of bone marrow stromal cells on rat organotypic hippocampal slice culture model of cerebral ischemia
Neurosci. Lett.
Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells
J. Neurosci.
A sensitive and reliable locomotor rating scale for open field testing in rats
J. Neurotrauma
Cited by (0)
- 1
These authors contributed equally to the paper.