69 - 75: Experimental Evidence and Early Translational Steps Using Bone Marrow Derived Stem Cells after Human Stroke

Stroke is the third leading cause of death in developed countries after heart disease and cancer [1], and the leading cause of disability worldwide. More than 50% of stroke survivors are unable to completely recover and 20% of stroke patients require assistance with their daily activities [2]. Although thrombolysis can improve the functional outcomes of stroke patients, patients must be treated within 3 h (or 4.5) of the onset of a stroke [3] and no definitive treatment exists after that period other than rehabilitation. To improve functional recovery after stroke, clinical trials of various drugs have been conducted but have achieved either only mild or nonsignificant therapeutic effects, or have sometimes even had serious adverse effects [4,5]. Thus, development of novel and safe therapies is eagerly awaited.

Recently, a number of studies have focused on cell-based therapies to promote the neuronal regeneration in the ischemic brain [6,7,8]. In this chapter, we present current basic and clinical findings that focus on therapeutic neurogenesis after stroke. We also refer to a novel cell-based therapy that may enable regeneration of the aged brain.

Neuronal tissue in the central nervous system is well known for its limited reparative/regenerative capacity. Physiologically speaking, neurogenesis is principally restricted to the subventricular zone of the lateral ventricle wall and the subgranular zone of the hippocampal dentate gyrus, where unique niche architectures permit continuous neurogenesis [9,10]. In pathological situations, recent studies using experimental models have revealed that endogenous neurogenesis is activated around injured areas where neurogenesis does not occur under normal conditions [11]. Consistent with these findings, histopathological studies in stroke patients have pointed out the presence of neural stem/progenitor cells in the post-stroke human cerebral cortex, and that the peak in endogenous neurogenesis occurs approximately 1-2 weeks after a stroke [12]. These findings indicate the potential for a novel therapeutic strategy using injury-induced neurogenesis for functional recovery in patients with cerebral infarction.

The post brain-injury neurogenic response eventually yields only a very small number of mature neurons, as most of them die after the initial boosting [11]. To achieve functional recovery by endogenous neuroregeneration, appropriate support for their survival is essential and angiogenesis has been proposed as the key element in this [7]. In the adult songbird, testosterone-induced angiogenesis leads to neuronal recruitment into the higher vocal center [13]. In the adult rat, endogenous neurogenesis and neovascularization occur in proximity to one another in the cortex following focal ischemia [14]. Moreover, angiogenesis and neurogenesis have been shown to use the same molecules for their regulation; sphingosine-1-phosphate, for example, plays a critical role in neurogenesis and angiogenesis during embryonic development [15]. This accumulating evidence indicates a close relationship between the vascular system and neurogenesis in the central nervous system, and recent studies have focused on the promotion of neurogenesis in association with angiogenesis [6].

To achieve angiogenesis in ischemic tissue, an approach using bone marrow-derived mononuclear cells, a rich cell source of both hematopoietic stem cells and endothelial stem/progenitor cells, has been proposed. Local transplantation of bone marrow-derived mononuclear cells in experimental models of limb ischemia significantly induces angiogenesis and releases ischemic stress in experimental models [16]. Based on these results, clinical trials were initiated, and a cure for ischemic ulcer, with significant angiogenesis in ischemic limb, has been reported [17]. The potential for transplantation of bone marrow-derived mononuclear cells to myocardial ischemia patients was also investigated and demonstrated a therapeutic effect in experimental models. Clinical trials were initiated in patients with ischemic heart disease and the therapeutic potential for improvement in regional perfusion and heart function has been reported [18].

Based on these experimental and clinical findings, we investigated the effect of intravenous transplantation of bone marrow-derived mononuclear cells [19] and hematopoietic stem cells [7] in an experimental model. As a result, we found the following three effects: (a) cell therapy enhances neovascularization at the border of the ischemic zone; (b) neovascularization is essential for the survival of injury-induced neuronal stem cells, and (c) supporting the survival of endogenous neurogenesis improves functional outcomes [19]. The positive effect of bone marrow-derived mononuclear cells was negated by administration of an anti-angiogenesis reagent [19]. It is noteworthy that survival of transplanted cells was rarely observed, despite significant activation of angiogenesis by cell therapy. These findings indicate that the differentiation of the stem cells into endothelial cells in the ischemic brain is not essential for angiogenesis after stroke and therapeutic angiogenesis could be a novel therapeutic strategy to enhance functional recovery after stroke.

To examine the effects of the mobilization of hematopoietic stem cells from bone marrow by drug administration, granulocyte colony-stimulating factor was given in an experimental stroke model and found to impair functional recovery with brain atrophy and with exaggerated inflammatory response at the border of the ischemic region [20]. This result suggested that the mobilization of bone marrow cells, including both granulocytes and hematopoietic stem cells, by granulocyte colony-stimulating factor might augment the inflammatory response consequent to ischemic tissue damage. We also investigated the effect of intravenous transplantation of bone marrow-derived mesenchymal stem cells in an experimental stroke model but found only a mild or nonsignificant effect on functional recovery (unpublished data), though mesenchymal stem cells have the potential to suppress excessive inflammation [21].

In a preclinical trial, we investigated the appropriate cell numbers and optimal therapeutic time window using a highly reproducible murine stroke model [22] and found that administration of a relatively small number of bone marrow-derived mononuclear cells had a significantly beneficial effect on the regeneration of injured brain tissue [23]. Analysis of the therapeutic time window revealed that administration of bone marrow-derived mononuclear cells at 24 h after stroke had a mild or nonsignificant effect on regeneration following ischemia, but administration of these cells between day 2 and day 14 after the ischemic event had a significantly positive effect. This result may be attributed to the time lag between the onset of stroke and the peak of neurogenesis [12].

Based on these results, we initiated a clinical trial to enhance neurogenesis and functional recovery through activating angiogenesis in patients with cerebral infarction. A schematic representation of this therapy is shown in figure 1. Our clinical trial is an unblinded, uncontrolled phase 1/2a study (ClinicalTrials.gov Identifier: NCT01028794). The major inclusion criteria are patients with cerebral embolism, day 7 after stroke, a National Institutes of Health Stroke Scale (NIHSS) score of more than (or equal to) 10, and an improvement in the NIHSS score of less than (or equal to) 5 since admission. On days 7-10 after stroke, either a 25-ml (low-dose group, n = 6) or a 50-ml (high-dose group, n = 6) aspiration of bone marrow cells was performed. These mononuclear cells were purified by Ficoll-Paque Premium (GE-Healthcare, USA) and administered intravenously on the day of the bone marrow aspiration. The primary outcome measures are improvement of the NIHSS score at 30 days after treatment and frequency of change for the worse on the NIHSS at 30 days after treatment, compared with historical control. Though this clinical study is currently still underway, we have already treated 11 patients (6 in the low-dose and 5 in the high-dose group), and no side effects or safety problems have been observed to date. Results related to the therapeutic effects of the treatment are expected in a year. Similar clinical trials are being carried out in other countries, including the USA, UK, Brazil and Spain, with promising results [24,25]. Though the route of administration (intravenous or intra-arterial) and cell source (bone marrow mononuclear cells or CD34-positive cells) vary, no side effects or safety problems with cell therapy have been reported. The current status of most of these ongoing clinical trials can be searched through http://clinicaltrials.gov/.

Previously, we have shown that patients with cerebrovascular disease have a decreased level of circulating bone marrow-derived immature cells, the latter associated with impaired cerebrovascular function [26] and impaired cognition [27], whereas increased levels of bone marrow-derived immature cells are associated with neovascularization of the ischemic brain [28]. In addition, we have demonstrated that partial rejuvenation of bone marrow stem cells in aged rats improves vascular function and reduces ischemic damage after induction of stroke in stroke-prone spontaneously hypertensive rats [29]. Furthermore, we investigated the effect of bone marrow-derived stem cells on white matter damage in a mouse model of cerebral hypoperfusion and found that administration of bone marrow-derived stem cells has a significant protective effect against white matter damage by enhancing cerebral blood flow via the activation of nitric oxide synthase [30]. These findings clearly indicate that bone marrow-derived stem/immature cells have the potential to improve microvascular circulation and prevent cerebrovascular diseases, and the challenge to find novel strategies using autologous, allogeneic or induced pluripotent stem cell-derived hematopoietic stem cells to regenerate the aged brain is ongoing.

Currently, for patients after stroke, there is no specific recovery-targeted treatment other than physical and cognitive rehabilitation techniques after the period of thrombolysis. However, accumulating evidence indicates significant activation of neurogenesis after stroke, and utilization of the stroke-induced neuronal stem cells, we believe, will become a major therapeutic target for the acceleration of functional recovery. The mechanism that links angiogenesis and neurogenesis cannot be attributed to a single molecule or signaling pathway. It is likely that multiple cytokines, growth factors, and cell adhesion molecules are involved, and the balance between these molecules may determine the fate of injured brain tissue. Careful, step-by-step investigation will lead to more efficient neurogenesis with a longer therapeutic time window. Experimental and clinical research focusing on neuroregeneration is needed to enhance functional recovery in patients after stroke.