Application of Human Umbilical Cord Blood-Derived Mononuclear Cells in Animal Models of Ischemic Stroke

Abstract

Complex pathology of ischemic stroke warrants a combination treatment approach that targets multiple pathways in order to provide a general effective therapy for stroke. Human umbilical cord blood-derived cells have been used in experimental models of injury and disease over the past several years and show encouraging leaps toward development of such an all-inclusive treatment. The purpose of this review is to highlight the potential use of human umbilical cord blood-derived cells in ischemic stroke and to discuss the animal studies which were conducted to date in that direction.

Keywords: Ischemic stroke; Umbilical cord blood; Stem cells; Infarct size; Neurological recovery

Abbreviations

HUCB: Human umbilical cord blood; HUCBCs: Human umbilical cord blood cells; HSCs: Hematopoietic stem cells; nHSCs: non-hematopoietic stem cells; MSCs: Mesenchymal stem cells; EPCs: Endothelial progenitor cells; SSEA: State-specific embryonic antigen; TRA: Tumor rejection antigen; MCAO: Middle cerebral artery occlusion; tPA: tissue plasminogen activator; ROA: Route of administration; BBB: Blood brain barrier; SH: Spontaneously hypertensive; BDNF: Brain-derived neurotrophic factor; VEGF: Vascular endothelial growth factor; IFN: Interferon.

Introduction

Globally, fifteen million people suffer from a stroke each year and five million stroke patients die with another five million left permanently disabled [1]. Stroke is the leading cause of disability and has become one of the major challenges to health. Approximately 85% of the strokes are ischemic and occur due to thrombosis, embolism or stenosis. However, many ischemic strokes occur without a welldefined etiology and are labeled as cryptogenic. Cryptogenic stroke accounts for 30 to 40 percent of ischemic strokes.

Stem cell transplantation offers a promising therapeutic strategy for ischemic stroke. In addition to preventing the ongoing damage, which has been the focus of conventional therapy, stem cell transplantation actually repairs the injured brain. It has emerged as a potential regenerative treatment to reduce post-stroke handicap. In addition to ethical and moral concerns, limited availability of embryonic, fetal, and adult brain-derived neural stem cells has prompted the search for alternative sources of stem cells. Human umbilical cord blood (HUCB) has emerged as an alternative stem cell sources. HUCB is the blood left over in the placenta and in the umbilical cord after the birth of the baby. HUCB contains a highly heterogeneous mixture of cells, which includes red blood cells (erythrocytes), white blood cells (leukocytes), thrombocytes (platelets), stem cells, etc. HUCB contains at least three types of stem cells; hematopoietic stem cells (HSCs), non-hematopoietic stem cells (nHSCs), and mesenchymal stem cells (MSCs). HSCs, nHSCs, MSCs, and agranulocytes of leukocytes (lymphocytes and monocytes) constitute the mononuclear cells of HUCB (HUCBCs).

HUCB-HSCs are characterized by their differential expression of hematopoietic markers CD34, CD45, and CD133 [2,3]. HUCBHSCs can be selectively induced into specific hematopoietic lineages. HUCB-nHSCs are characterized by their expression of pluripotency markers (Sox2, Oct4, and Nanog), state-specific embryonic antigen markers (SSEA-3 and SSEA-4), tumor rejection antigen markers (TRA1-60 and TRA1-80), and lacking of hematopoietic markers CD34 and CD45 [4]. HUCB-nHSCs possess multipotent differentiation potential and have been shown to differentiate in various cell types representing the three germ layers. HUCB-MSCs show high morphological and molecular similarities to bone marrow MSCs including the lacking of hematopoietic surface antigens CD34, CD45, and CD133 [5-8]. HUCB-MSCs are characterized by their expression of MSC-specific surface markers CD29, CD44, CD73, CD105 and vimentin [9]. HUCB-MSCs possess several advantages over other types of stem cells including those derived from bone marrow [10]. HUCB-MSCs possess multipotent differentiation potential and thus can be induced to differentiate into cells of multiple lineages such as adipocytes, osteocytes, chondrocytes, myocytes, hepatocytes, neurons, astrocytes and oligodendrocytes [10-17]. Recent investigations suggested that the HUCB-MSCs harbor a small unique population of cells that express pluripotent stem cell markers such as Sox2, Oct4, Nanog, ABCG2, and nestin along with MSC markers [9]. Although few stem cell types obtained from HUCBCs show the expression of pluripotency markers, these cells do not form teratomas after transplantation in immuno-compromised mice, the current gold standard for determining the pluripotency of human cell lines. Therefore, these cells do not satisfy the current criteria for defining them as pluripotent stem cells [18].

Why ischemic stroke is the primary disease target for stem cell therapy?

The goal of any cell-based therapy is to replace and/or repair dead or diseased cells. Therapy with stem cells can target multiple mechanisms/pathways associated with the pathology of a disease condition. After an ischemic stroke, the affected brain tissue can be described as having two regions, the ischemic core and the penumbra. The tissue damage that occurs in the ischemic core is irreversible and permanent. Therefore, the cells in the ischemic core are considered beyond rescue and the cells in the penumbra are potential targets for therapeutic intervention. To date, clinical treatment had not emerged from 1026 neuroprotective agents deemed successful in animals, reinforcing the perception that "everything works in animals but nothing works in people" [19]. Many neuroprotective agents that failed in clinical trials are aimed at excitotoxicity and oxidative stress. It is quite evident that targeting cell death in the ischemic core will have poor prospects compared to targeting cell death in the penumbra. Further, any approach targeting a single mechanism may not provide a general effective therapy for stroke because of its complex pathology. Hence, the best possible option to replace the dead tissue in the ischemic core and to reestablish the lost neurological function as well as inhibit cell death in the penumbra would be stem cell transplantation.

Current status of drug therapy for ischemic stroke

Although the US FDA approved the usage of certain antiplatelet agents such as aspirin, ticlopidine, clopidogrel and a combination of aspirin and dipyridamole for secondary ischemic stroke prevention, initial stroke therapy is limited to the FDA-approved clot-busting drug, tissue plasminogen activator (tPA), which must be administered within a four and half hour window from the onset of symptoms [20,21]. Unfortunately, only three to five percent of those who suffer a stroke reach the hospital in time to be considered for this treatment. Moreover, about half of the patients receiving tPA therapy show little or no improvement in functional outcome [22]. In addition, treatment with thrombolytics such as tPA present real safety concerns because of the increased incidence of secondary hemorrhagic transformation and the increased mortality rate in patients especially those who have bleeding disorders [23-25]. The major goal of clot-busting therapy is to reestablish the blood flow to the previously ischemic brain portions and not to address the injury that occurred due to ischemia. Reperfusion of the ischemic brain portion further damages the brain due to reperfusion injury. Despite decades of research, no clinically effective pharmacotherapies exist which can target both ischemia and reperfusion injury as well as facilitate cellular functional recovery after an ischemic stroke.

Rationale for the use of HUCBCs in ischemic stroke

HUCBCs possess several advantages over stem cells from other sources [10]. Recently, we showed that these cells survive, migrate and transdifferentiate to neuronal cells after their transplantation in animal spinal cords or brains [10,16,17,26]. We also demonstrated the potential of these cells in modulating the spinal cord microenvironment and improving the locomotor recovery of spinal cord injured rats [16,26- 29]. Based on our studies, we understand that HUCBSCs treatment inhibited apoptosis, inhibited myelin degradation, remyelinated the damaged axons and thereby contributed to the functional recovery of spinal cord injured rats. Moreover, intracranial implantation of these cells in shiverer mice myelinated the hypomyelinated axons and significantly reduced their shivering [10]. Supported by in vitro and pre-clinical studies, HUCBCs have been utilized in many different clinical trials aiming to treat a wide range of diseases and disorders [4]. The pathophysiology of ischemic stroke is extremely complex and involves numerous processes, including: energy failure, excitotoxicity, oxidative stress, disruption of the blood-brain barrier (BBB), inflammation, necrosis, apoptosis etc. Studies that utilized HUCBCs improved the stroke outcome in animal models of ischemic stroke by multiple mechanisms. All these studies are discussed in detail in the subsequent sections of this review.

How HUCBCs are recruited to the infarct site after their systemic administration?

Delivery of viable cells to the damaged brain tissue is the first thing to be considered with any cell-based therapy. Ideally these cells or the neurotrophic factors they secrete will re-establish the damaged host neural connections, either by forming new networks or reconstructing the old pathways.

After intravenous administration, HUCBCs are recruited to the infarct site possibly by passive diffusion across a damaged BBB. Early disruption of BBB begins within the first three hours after MCAO [30]. The second peak of BBB disruption is between 24 and 72 hours, in case of transient occlusion model, which is associated with reperfusion injury [31]. In case of permanent MCAO model, however, the second peak of BBB disruption peaks approximately at six days after MCAO [32]. HUCBCs are also recruited to the site of injury by chemokines when their expression reaches peak level, which usually occur at 48 hours after stroke [33-35]. Administration of HUCBCs at early time points after ischemia may compromise with the body's early natural attempt to fight against injury and lead to exacerbation of the damage. Treatment with cells that is initiated too early or too late may not help recovery. The timing of treatment with HUCBCs is more important than the dose. Based on the reports discussed above, it appears that the optimal timing of HUCBCs treatment is 48 hours after transient focal cerebral ischemia.

Effect of cell type, dose, route and time of administration on stroke outcome in animal models

Application of HUCBCs in animal models of ischemic stroke has been initiated more than a decade ago. Recent research reports indicated the usage of HUCBCs, which also include HSCs, nHSCs, and MSCs derived from HUCB. The summary of these studies, including the research outcome is detailed in Table 1. All the studies listed in Table 1 used a rat model of transient or permanent middle cerebral artery occlusion (MCAO) and administered the cells intravenously within seven days after MCAO procedure. The number of cells administered range from 1x104 to 5x107. Intravenously administered HUCBCs after ischemic stroke entered the rat brain, survived, migrated, improved the neurological/functional recovery, and reduced the infarct size [36-44]. In contrast, systemic administration of HUCBCs did not improve histological outcome or functional recovery in MCAO subjected rats [45]. These authors also have reported that only few of the administered cells were detected in the ipsilateral hemisphere. However, we cannot attribute this finding as a reason for the lack of improvement in histological outcome and functional recovery because several other research groups reported the presence of administered cells in ischemic brain regions. Reduced infarcts and improved behavioral function in the absence of administered cells in the ipsilateral hemisphere was also reported [36,42,44]. In addition, intravenous administration of HUCBCs in a spontaneously hypertensive rat MCAO model failed to reduce the infarct volume [46]. However, the absence of positive outcome could be due to the divergent pathophysiological sequences in these rats compared to commonly used rat strains [46]. Therefore, prior to initiating the clinical studies with HUCBCs in stroke patients, their therapeutic potential in animal models that mimic the most common comorbidities of stroke patients should be investigated.