Elsevier

Brain Research

Volume 1035, Issue 1, 21 February 2005, Pages 73-85
Brain Research

Research report
Axon growth and recovery of function supported by human bone marrow stromal cells in the injured spinal cord exhibit donor variations

https://doi.org/10.1016/j.brainres.2004.11.055Get rights and content

Abstract

Bone marrow stromal cells (MSC) are non-hematopoietic support cells that can be easily derived from bone marrow aspirates. Human MSC are clinically attractive because they can be expanded to large numbers in culture and reintroduced into patients as autografts or allografts. We grafted human MSC derived from aspirates of four different donors into a subtotal cervical hemisection in adult female rats and found that cells integrated well into the injury site, with little migration away from the graft. Immunocytochemical analysis demonstrated robust axonal growth through the grafts of animals treated with MSC, suggesting that MSC support axonal growth after spinal cord injury (SCI). However, the amount of axon growth through the graft site varied considerably between groups of animals treated with different MSC lots, suggesting that efficacy may be donor-dependent. Similarly, a battery of behavioral tests showed partial recovery in some treatment groups but not others. Using ELISA, we found variations in secretion patterns of selected growth factors and cytokines between different MSC lots. In a dorsal root ganglion explant culture system, we tested efficacy of conditioned medium from three donors and found that average axon lengths increased for all groups compared to control. These results suggest that human MSC produce factors important for mediating axon outgrowth and recovery after SCI but that MSC lots from different donors vary considerably. To qualify MSC lots for future clinical application, such notable differences in donor or lot–lot efficacy highlight the need for establishing adequate characterization, including the development of relevant efficacy assays.

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

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