Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats

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Abstract

We tested the hypothesis that bone marrow stromal cells (MSCs) transplanted into the ischemic boundary zone, survive, differentiate and improve functional recovery after middle cerebral artery occlusion (MCAo). MSCs were harvested from adult rats and cultured with or without nerve growth factor (NGF). For cellular identification, MSCs were prelabeled with bromodeoxyuridine (BrdU). Rats (n=24) were subjected to 2 h of MCAo, received grafts at 24 h and were euthanized at 14 days after MCAo. Test groups consisted of: (1) control-MCAo alone (n=8); (2) intracerebral transplantation of MSCs (n=8); (3) intracerebral transplantation of MSCs cultured with NGF (n=8). Immunohistochemistry was used to identify cells from MSCs. Behavioral tests (rotarod, adhesive-removal and modified neurological severity score [NSS]) were performed before and after MCAo. The data demonstrate that MSCs survive, migrate and differentiate into phenotypic neural cells. Significant recovery of somatosensory behavior (p<0.05) and NSS (p<0.05) were found in animals transplanted with MSCs compared with control animals. Animals that received MSCs cultured with NGF displayed significant recovery in motor (p<0.05), somatosensory (p<0.05) and NSS (p<0.05) behavioral tests compared with control animals. Our data suggest that intracerebral transplantation of MSCs may provide a powerful autoplastic therapy for stroke.

Introduction

Intracerebrally transplanted embryonic mesencephalic precursors have been shown to provide therapeutic benefit in Parkinsonian rats [1]. Embryonic cells can differentiate into various phenotypes when placed in different regions of the brain, suggesting a key role for the host-derived signals in directing differentiation [2], [3]. Grafted fetal tissue establishes extensive interconnections with the host when grafted into homologous regions of adult animals following cerebral ischemic infarction [4], [5]. Although transplantation is a promising treatment for neurodegeneration [6], [7], brain injury and brain ischemia [8], [9], its clinical use is restricted, because of ethical and logistical problems. Therefore, alternatives are being sought to human fetal tissue, and consideration is being given to bone marrow (BM) cells as a source for cell transplantation therapy [10], [11].

Adult BM contains stem and progenitor cells, which have multiple differentiation potentials [12]. BM contains marrow stromal cells (MSCs) [12], capable of differentiating into various cell types [13]. MSCs are chiefly regarded as hematopoietic support cells, and these marrow stromal cells appear to be a source for continual renewal of cells in a number of non-hematopoietic tissues [12]. Adult rat and human BM stromal cells can differentiate into neurons in vitro [14]. MSCs can be induced to differentiate into astrocytes, microglia and macroglia in the brain of adult mice [10], [15], [16], and MSCs differentiate into osteoblasts, chondrocytes and adipocytes [17] when placed into different microenvironments. MSCs migrate throughout forebrain and cerebellum, and they differentiate into astrocytes and neurons after injection into neonatal mouse brain [18]. Collectively, these results have led to the hypothesis that MSCs are capable of differentiating along multiple cell lineages, and hence they represent multipotent stem like cells for nonlymphohematopoietic tissues [19]. MSCs have been used as vehicles for both cell therapy and gene therapy [20], [21].

Neurotrophic factors have well established roles in neuronal development and adult synaptic plasticity, and culture. Nerve growth factor (NGF) is a well-characterized neurotrophic polypeptide for the survival, development, and function of basal forebrain cholinergic neurons in the central nervous system [22]. Administration of NGF by ex vivo gene transfer and grafting of neural stem cells reduces death of striatal projection neurons caused by transient focal ischemia [23], and NGF protects CA1–CA2 hippocampal neurons from ischemic damage by implantation of genetically engineered cells producing NGF [24]. NGF regulates synthesis of acetylcholine and promotes the differentiation and survival of cultured cholinergic neurons [25].

In the present study, we have two primary aims. Using a model of middle cerebral artery occlusion (MCAo) in rats, we test whether multipotential marrow stromal cells from BM transplanted into ischemic brain: (1) survive and express neural phenotypic proteins in an ischemic brain microenvironment; (2) reduce behavioral and functional deficits associated with cerebral infarction. As a secondary aim, we also test the hypothesis that MSCs cultured in NGF confers additional therapeutic benefit.

Section snippets

Animal MCAo model

Adult male Wistar rats (n=24) weighing 270–300 g were employed in all our experiments. Briefly, rats were initially anesthetized with 3.5% halothane and maintained with 1.0–2.0% halothane in 70% N2O and 30% O2 using a face mask. Rectal temperature was maintained at 37 °C throughout the surgical procedure using a feedback-regulated water heating system. We induced transient MCAo using a method of intraluminal vascular occlusion modified in our laboratory [26]. The right common carotid artery,

Results

Ischemic severity was balanced among groups with p-values of >0.47. Rats treated with MSC or MSC+NGF had significant improvement on NSS at 14 days compared to control animals (p<0.05, Fig. 1). No difference in means on NSS between MSC-treated and MSC+NGF treated groups (p=0.45) were detected at 14 days after MCAo.

The treatment of MSC or MSC+NGF significantly improved functional recovery on somatosensory adhesive-removal test (p<0.05). Motor deficit was significantly reduced in animals treated

Discussion

We have demonstrated that MSCs transplanted into the ischemic boundary zone of an ischemic lesion, survive, differentiate into phenotypic neural cells and yield improved functional recovery from stroke.

MSCs from BM survive and express neural phenotypic proteins when grafted into the ischemic brain microenvironment. The observation is consistent with others [44], [18] that grafted BM cells can adopt neural cell fate when exposed to the brain microenvironment and may differentiate into microglia,

Acknowledgements

The authors wish to thank Cecylia Powers, Cynthia Roberts and Xiuli Zhang for technical assistance, and Deb Jewell for secretarial support. This work was supported by NINDS grants PO1 NS23393 and RO1 NS35504.

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