Adult stem cell therapy in stroke Sebastian Haas, Norbert Weidner and Jürgen Winkler

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1 Adult stem cell therapy in stroke Sebastian Haas, Norbert Weidner and Jürgen Winkler Purpose of review Acute cerebral infarction causes irreversible locally restricted loss of the neuronal circuitry and supporting glial cells with consecutive functional deficits and disabilities. The currently available and effective therapy targets fast vessel recanalization accompanied by symptomatic measures. Research activities focusing on stem cells, which represent a promising source for organotypic cell replacement and functional recovery after stroke, have gained momentum in recent years, making regenerative cellbased therapies a much more feasible realistic approach. This review provides an update about preclinical and clinical cell-based studies in stroke focusing on stem cells derived from the adult central nervous and hematopoetic systems. Recent findings Endogenous neural stem cells, which have been shown to reside throughout life in the central nervous system, have the capacity to replace lost neurons in models for numerous disorders, including cerebral ischemia. Considering adult neural stem cell transplantation as a regenerative strategy after stroke, progress has been made in isolating human adult neural stem cells and demonstrating the feasibility of autologous neural stem cell transplantation. An increasing number of studies provide evidence that hematopoietic stem cells, either after stimulation of endogenous stem cell pools or after exogenous hematopoietic stem cell application (transplantation), improve functional outcome after ischemic brain lesions. Various underlying mechanisms such as transdifferentiation into neural lineages, neuroprotection through trophic support, and cell fusion have been deciphered. Summary Many preclinical studies employing adult stem cell-based strategies hold great promise. For endogenous approaches the correlate of cell replacement underlying functional improvement needs to be demonstrated. Transplantation approaches on the experimental level need further development before clinical application can be considered. Keywords cerebral ischemia, hematopoietic stem cell, neurogenesis, transdifferentiation Curr Opin Neurol 18: # 2005 Lippincott Williams & Wilkins. Department of Neurology, University of Regensburg, Regensburg, Germany Correspondence to Jürgen Winkler, MD, Department of Neurology, University of Regensburg, Universitaetsstrasse 84, D Regenburg, Germany Tel: ; fax: ; juergen.winkler@klinik.uni-regensburg.de Current Opinion in Neurology 2005, 18:59 64 Abbreviations BDNF brain-derived neurotrophic factor CNS central nervous system G-CSF granulocyte colony stimulating factor HSC hematopoietic stem cell MCA-O middle cerebral artery occlusion NSC neural stem cell SVZ subventricular zone VEGF vascular endothelial growth factor # 2005 Lippincott Williams & Wilkins Introduction Acute ischemic stroke caused by cerebral artery occlusion leading to infarction of brain tissue with acute loss of neurons, astroglia and oligodendroglia, is the most important vascular central nervous system (CNS) disorder in industrial nations and remains a leading cause of death and disability. Despite significant clinical benefits after systemic thombolysis, only a minority of patients has timely access to this therapy. Therefore, it is crucial to develop new alternative therapeutic strategies with less time constraints. The most encouraging approach is directed towards cell replacement into ischemic regions. The main rationale to employ cell-based therapies in ischemic stroke is to replace infarcted CNS tissue in an organotypic appropriate manner. Lost neurons need to be replaced to allow the reestablishment of a functional neuronal circuitry. Glial cells such astroglia and oligodendroglia have to be replaced to maintain neuronal circuitry and to establish proper nerve conduction. Furthermore, cell-based approaches could provide trophic support to tissue at risk in the penumbra surrounding the infarct area or help promote survival, migration and differentiation of endogenous precursor cells. Potential sources to fulfill these requirements are various kinds of stem cells. Stem cells are immature cells with self-renewal capacity and repopulating properties for the life span of each individual. Different types of stem cells can be distinguished based on their developmental origin: embryonic, fetal and adult stem cells. Embryonic stem cells derive from the blastocyst, are totipotent and give rise to progenies of the embryonic germ layers. Fetal stem cells represent more restricted progenitor cells harvested from fetal organs still undergoing major developmental changes. The use of embryonic or fetal stem cells, however, raises serious biological, ethical and legal questions in the clinic limiting a widespread use of these 59

2 60 Cerebrovascular disease cells at present. Stem cells derived from the adult CNS or bone marrow are a very attractive alternative source for cell-based therapies for the CNS. Adult stem cells are a continuous source for a physiological organ-specific cell replacement during normal cell turnover as well as in disease/injury conditions. Adult stem cells have the capacity for self-renewal and differentiation into organspecific lineages. This review will focus on studies investigating the regenerative capacity of adult stem cells derived from the CNS adult neural stem cells (NSCs) or hematopoietic system hematopoietic stem cells (HSCs) after stroke. Adult stem cell therapy for stroke can be divided in an endogenous and exogeneous approach (Fig. 1) [1]. The aim of the endogenous stem cell therapy is to exploit the population of adult stem cells already physiologically present either in the CNS or hematopoetic system. Using the exogenous mode, CNS or hematopoetic system derived adult stem or precursor cells are administered locally or systemically after purification and propagation in culture. Adult neural stem cells During the last century, the dogma existed that the adult CNS was incapable of generating new neurons (neurogenesis). Over the past decades, convincing evidence emerged that neurogenesis in the adult CNS is a continuous physiological process. Neurogenesis is present in two regions: the subventricular zone (SVZ) and the subgranular zone of the dentate gyrus [2 4]. Additionally, recent studies [5 7] also indicated the existence of NSCs in other regions of the CNS, namely the striatum, spinal cord and neocortex [5 7]. SVZ and dentate gyrus derived NSCs are characterized by long-term, selfrenewal capacity and multipotency (Fig. 2). Adult SVZ and dentate gyrus derived NSCs persist throughout the life span of mammals including humans [3,8]. It is important to note that neurogenesis occurs in a physiological mode or is exogenously modulated by external Figure 1. The endogenous and the exogenous approaches of stem cell therapy in stroke G-CSF, granulocyte colony stimulating factor. Reproduced with permission [1].

3 Adult stem cell therapy in stroke Haas et al. 61 Figure 2. Localization and differentiation of adult neural stem cells Adapted with permission [1]. signals or pathophysiological processes. External global stimulants such as enriched environment, physical activity and stress [9] or application of defined molecules such as fibroblast growth factor-2 [10], vascular endothelial growth factor (VEGF) [11,12], brain-derived neurotrophic factor (BDNF) [13,14] and erythropoietin [15] differentially modulate adult neurogenesis. Finally, CNS disease conditions such as seizures and traumatic brain injury represented by respective animal models induce neurogenesis [16 18]. Spontaneous and pharmacologically stimulated neurogenesis after ischemia Both global forebrain ischemia and focal ischemia, or middle cerebral artery occlusion (MCA-O), have been shown to induce neurogenesis in the dentate gyrus in adult mammals [17 20]. The main questions are whether newborn neurons in these conditions are able to replace lost neurons at the lesion site in a specifically appropriate manner and whether neuronal replacement through endogenous neurogenesis can be correlated with functional recovery. Recent work by Nakatomi et al. [21] provides evidence that after intracerebroventricular infusion of epidermal growth factor and fibroblast growth factor-2 subventricular zone derived progenitor cells give rise to hippocampal pyramidal neurons in a global ischemia model, which restore long-term potentiation and can be correlated with an improved spatial orientation. Following local ischemia induced by MCA-O, neurons are specifically replaced only within the striatum [22]. However, the quantitative fraction of newborn neurons seems to be low. Experimental data suggest that over 80% of new neurons die within the first 6 weeks after stroke and only about 0.2% of destroyed cells are replaced by new generated neurons [22]. There is no conclusive evidence for appropriate endogenous neuronal replacement in cortical regions following focal stroke [23,24]. Whether neurogenesis following focal stroke is functionally relevant has yet to be clarified. Studies in intact animals provide evidence that various molecules can enhance the intrinsic neurogenic capacity and thus might be suitable to promote neuronal replacement following ischemic events in the CNS. Sun and co-workers [25] report that VEGF elicits a delayed response on neurogenesis following MCA-O in rats, which is attributed to a survival rather than a proliferation effect. Reduction of infarct volume and functional recovery point to an additional neuroprotective effect exerted by VEGF. Interestingly, BDNF, which enhances neurogenesis in intact animals, decreases neurogenesis in a global ischemia model following adeno-associated virusmediated BDNF overexpression in the hippocampus [26]. The intraperitoneal administration of erythropoietin, which promotes neural progenitor cell proliferation in vitro and in vivo, has been shown to induce neurogenesis and functional improvement following embolic stroke in adult rats [27]. Further analysis indicates that these effects are at least partially mediated through upregulation of VEGF and BDNF expression. None of the above-mentioned experiments can clearly attribute the observed functional benefit to the therapeutic agent induced effects on neurogenesis. Based on the above-mentioned preclinical findings a clinical study was conducted by Ehrenreich et al. [28] investigating effects of erythropoietin application in stroke patients. In a phase Ib study 13 patients received erythropoietin intravenously for the first 3 days after stroke. To show efficacy either erythropoietin or sialin was applied after double-blind randomization to 40 patients with stroke in the middle cerebral artery territory. No safety concerns were reported within the small group of erythropoietin treated stroke patients. A superior outcome in

4 62 Cerebrovascular disease erythropoietin treated patients versus the placebo control group was reported supported by improved functional recovery and reduction of infarct size on magnetic resonance imaging. These promising findings have to be validated in a larger number of stroke patients before erythropoietin application can be considered as add-on therapy for established acute stroke interventions. Exogenous neural stem cell therapy in stroke The rationale to transplant NSCs into the ischemic brain is primarily to reconstruct the destroyed neuronal circuit. In addition, cell grafts may exert a neuroprotective effect by secreting survival promoting neurotrophic factors either intrinsically or after introduction of therapeutic transgenes. After having shown that multipotent NSC can be isolated from the adult rodent CNS and propagated in culture [29,30], findings were reproduced using adult human CNS tissue. Recent studies [31 33] provide evidence that adult neural stem cells can be obtained from adult human subventricular zone, hippocampus, cortex and subcortical white matter. The autologous transplantation mode represents an important rationale to use adult stem cells. Not until recently was it determined that autologous transplantation is feasible in a rat model of spinal cord injury. Spinal cord injured rats underwent a minimal aspiration biopsy of the SVZ, which allowed the generation of sufficient quantities of multipotent neural stem cells within several weeks for transplantation without inducing noticeable functional deficits related to the biopsy procedure [34]. Very few preclinical data are available regarding adult neural stem cell transplantation in stroke models. Previous studies investigating the regenerative capacity of rodent or human embryonic or fetal derived neural stem/ progenitor cells reported differentiation of grafted neural stem cells into neurons and astroglia as well as functional recovery [35 39]. Regarding the survival of grafted cells an optimal time point for transplantation was determined around 3 weeks after the stroke. Only one preclinical study [40] investigated the effects of adult derived neural stem cell transplantation in the context of stroke in adult rats. Adult neural progenitor cells, transplanted intracisternally 48 h following MCA-O, survived and migrated toward the ischemic area as assessed by histological and radiological analysis (MRI). Some functional recovery was reported, but the underlying mechanisms need to be elucidated. Hematological precursor cells for stroke therapy The discovery that HSCs are capable of transdifferentiating into neural lineages [41], together with the accessibility of HSCs has shifted the focus of attention into the application of HSCs as a promising cell replacement strategy in the diseased CNS. Subsequently, convincing evidence accumulated, which questioned the occurrence of transdifferentiation. Recent studies discovered that the phenomenon of cell fusion and procedural artifacts in determining cell fate account for the expression of neural lineage markers rather than true transdifferentiation [42 45]. Nevertheless, a vast number of preclinical studies were conducted investigating the regenerative capacity of HSCs following ischemic brain damage either by mobilization of intrinsic HSC pools (endogenous approach) or by transplantation of HSC (exogenous approach) [46]. Various sources for HSCs have to be distinguished: directly bone marrow derived stem cells, umbilical cord blood and peripheral blood cells (CD34þ cells). Spontaneous hematopoietic stem cell mobilization and stimulation by granulocyte colony stimulating factor Interestingly, acute cerebral ischemia in human individuals leads spontaneously to a threefold increase in CD34þ cell count in the peripheral blood (B. Hennemann, unpublished data). Considering this change as an insufficient self-repair mechanism, it is a logical consequence to further promote CD34þ cell mobilization pharmacologically by the administration of granulocyte colony stimulating factor (G-CSF). In addition, G-CSF has been described to exert neuroprotective effects following cerebral ischemia [47]. A recent preclinical study [48] found functional improvement in rats with focal cerebral ischemia after subcutaneous administration of G-CSF. The underlying mechanisms have yet to be elucidated. Currently, an ongoing clinical trial investigating safety of G-CSF application in acute ischemic stroke patients has not reported any adverse side effects [49]. Exogenous hematopoetic stem cell therapy in stroke An alternative approach to increase the number of HSCs in the diseased CNS is to transplant cells either systemically or locally following acute cerebral ischemia. Various sources for HSCs have been used for preclinical studies: bone marrow stromal cells, umbilical cord blood cells and G-CSF mobilized peripheral blood cells (CD34þ cells). Allogenic marrow stromal cells, either after intravenous, intracarotid or local transplantation into the penumbra following transient intraluminal MCA-O in adult rats, yielded functional improvement [50 52]. Intracarotid marrow stromal cell application resulted in a wide distribution of transplanted cells lining the ischemic core, whereas local grafts were restricted to the injection site. Considering the feasibility for potential clinical application, intravenous transplantation is by far the safest means. The majority of intravenously administered

5 Adult stem cell therapy in stroke Haas et al. 63 marrow stromal cells, however, are harvested in the spleen and lung before reaching the CNS. Zhao et al. [53] replicated these findings transplanting human derived marrow stromal cells into acute MCA-O induced ischemic lesions. Umbilical cord blood represents an effective source for HSCs. The intravenous administration of human umbilical cord blood derived CD34þ cells following MCA-O in severe combined immunodeficient mice promoted neovascularization and neurogenesis with concomitant functional improvement [54]. The underlying mechanisms for functional recovery remains unclear. Human umbilical cord blood cell transplantation can be reproduced in rats following transient intraluminal MCA-O [55]. A substantial disadvantage of umbilical cord blood derived HSC transplantation is its allogenic nature, requiring permanent immunosuppressive treatment to avoid graft rejection. Alternatively, the number of HSCs in the peripheral blood can be increased by mobilizing cells from the bone marrow using G-CSF, thus serving as an effective source for autologous HSC transplantation. Isolating HSCs from peripheral blood can be performed without risk or potential side effects. The first preclinical data suggest that peripheral blood derived HSCs are as efficient as umbilical cord blood derived HSCs in promoting functional improvement following MCA-O and intravenous transplantation in rats [56]. Conclusion Considering the feasibility of adult stem cell-based therapeutic approaches, pharmacological stimulation of endogenous stem cell pools (neural or hematopoietic) is favorable. Several impressive studies have shown that neuronal replacement through endogenous stem cells pools occurs. Whether the magnitude of this phenomenon is sufficient to promote structural and functional recovery in stroke patients remains to be determined. The global improvement, which has been observed thus far in preclinical and clinical studies, cannot easily be attributed solely to neural cell replacement. Alternative underlying mechanisms, in particular neuroprotection (e.g. secretion of neurotrophic factors), have to be considered, which is extremely difficult to discern in clinical studies. The perspectives of transplantation-based cellular strategies for the treatment of stroke are much less defined. Neural stem cells can be harvested from human tissue and autologous transplantation might be feasible, but it is too early to draw conclusions regarding effective cell replacement and restoration of compromised neuronal circuitry in stroke patients. The discovery of cell fusion as well as procedural artifacts in the analysis of HSC differentiation make neural cell replacement by means of transdifferentiation highly unlikely as underlying mechanism for functional improvement following stroke. 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