In a rodent middle cerebral artery occlusion (MCAO) model, postischemic inflammation events, such as microglial activation, neutrophil infiltration, and various pro-inflammatory cytokines, play a pivotal role in edema formation, infarct progression, and hemorrhagic transformation in the acute phase [11, 12]. Accordingly, in knockout mice or after pharmacological suppression of these inflammatory mediators, the extent of cell death and tissue damage after ischemia is decreased [13].

Bone marrow stromal cells and induced pluripotent stem cells require a period of cell culture before transplantation, whereas MNCs can be collected autologously just prior to administration. This should provide strong advantages in clinical use compared with other cell sources. Systemic transplantation of MNCs potently reduces neutrophil infiltration, microglia/macrophage activation, and inducible nitric oxide synthase (iNOS) expression in the ischemic brain and reduces serum interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) levels while increasing IL-10 levels after stroke [14–17]. The peripheral immune response, mediated by the spleen, is an important contributor to inflammation and enhances neurodegeneration after stroke in animals [18, 19]. Moreover, in humans, changes in spleen volume could be associated with the release of cellular components into the blood stream, which in turn may contribute to the postischemic inflammatory cascade [20]. A previous report demonstrated that systemic injection of NSCs during acute-stage hemorrhagic stroke decreases TNF-α and IL-6 mRNA levels and nuclear factor (NF)-kB protein expression in the spleen. In addition, a number of NSCs exhibit cell-to-cell contact with CD11b⁺ spleen macrophages. Similarly, intravenous transplantation of MNCs results in accumulation of cells in the spleen, particularly in the marginal zone between the red and white pulp, partly internalized by marginal zone macrophages positive for ionized calcium-binding adapter molecule-1. Furthermore, gene expression analysis of spleen tissue has revealed significant increases in monocyte chemotactic protein-1 (MCP-1) and IL-10 [8, 21–23]. Thus, the spleen may be an important target for the development of stem cell therapies in acute stroke.

Because earlier cell transplantation may be more efficacious against spleen-induced inflammatory responses after stroke, MNCs should have advantages over other cell types. Recently, Bing et al. reported that ischemic stroke itself modulates the cytokine profile (IL-10, IL-6, MCP-1, vascular endothelial growth factor [VEGF], and TNF-α) of MNCs within the bone marrow, which may result from changes in specific cell subpopulations within MNCs. These results indicate that the application of autologous MNCs in patients with stroke may be more effective than pre-stroke or allogenic MNCs from healthy humans.

One of the most important facets of early neurovascular damage is manifested as perturbations in blood-brain barrier (BBB) function. BBB disruption leads to vasogenic edema and hemorrhagic transformation, which eventually exacerbates short and long-term disability. Protecting brain endothelial cells is critical for maintaining BBB function. MNCs suppress von Willebrand factor expression, a marker of endothelial injury, in the acute phase of ischemic stroke [16]. Moreover, some reports have shown that cell therapy protects against endothelial injury in a stroke model [24, 25]. Recently, we reported that MNCs confer protective effects against the injury of neurovascular units through inhibition of intracerebral hemorrhage-mediated upregulation of high-mobility group protein box-1, S100β, matrix metalloproteinase 9, and aquaporin 4. Thus, MNCs have protective effects against endothelial damage following stroke.

Interestingly, a recent report showed that MNCs reduce atherosclerotic plaque size and increase the collagen content of plaques through reduction of pro-inflammatory cytokines (IL-1β and TNF-α), matrix metalloproteinase 9 activity, and cleaved caspase-3 expression and upregulation of eNOS, antioxidant enzymes (glutathione peroxidase 1 and superoxide dismutase-1), insulin-like growth factor-1, and its receptor in a rabbit model of atherosclerosis [26]. Taken together, these findings suggest that MNCs modulate brain and systemic inflammation, regulate the immune response, and protect against endovascular injury during the acute phase of ischemic brain injury.

A systematic search of PubMed identified 20 preclinical studies of MNCs in the setting of experimental stroke. MNCs can be safely harvested from rodents and reinfused by intravenous or intra-arterial injection in a rodent stroke model. Moreover, the favorable effects of MNCs on functional recovery were robust across rodents, route of delivery (intravenous or intra-arterial), type of MNCs (allogenic or autologous), time of administration in relation to stroke (from 90 min to 3 days after stroke onset), and MNC dosage (from 1 × 10⁶ to 3 × 10⁷ cells). These findings indicate that, in animal models of stroke, MNCs exert therapeutic effects over a wide dose range, may be administered as late as 3 days after cerebral ischemia, and may be beneficial regardless of cell source or route of delivery.

However, most studies demonstrating the positive effects of MNC treatment in stroke have used healthy young animals. The Stroke Therapy Academia Industry Roundtable (STAIR) Committee has recommended that after initial studies demonstrate positive effects in healthy young animals, additional studies in aged animals and/or animals with comorbidities, such as hypertension and diabetes, should be performed if that is the intended population for clinical trials [46]. The aged brain has faster astroglial scar formation, increased microglial reactivity, and greater pro-inflammatory cytokine release, which impairs axonal growth and neuronal tissue recovery [47, 48]. Clinical and experimental results have indicated that hypertension and hyperglycemia are related to increase infarct volume and worse functional outcomes after stroke [49–52]. Furthermore, studies haves shown that gender may be also a relevant factor affecting injury, outcomes, and the effect of therapies, including thrombolysis [53]. Therefore, the inclusion of older animals, comparisons between genders, and comorbidities should be considered during experimental design in order to assess the actual potential efficacy of therapies for stroke. Several studies examining the treatment of brain ischemia with MNCs have included these factors. For example, intravenous transplantation with MNCs (3 × 10⁷ cells) at 24 h after stroke has been shown to result in sensorimotor recovery of thermocoagulation-induced cortical ischemia in middle-aged male and female rats [54]. In another study, intra-arterial transplantation of autologous MNCs (4 × 10⁶ cells) at 24 h after ischemia was shown to enhance recovery in focal ischemia in middle-aged rats [14]. Intravenous transplantation with MNCs (1 × 10⁷ cells/kg) at 24 h after intracerebral hemorrhage (ICH) was shown to improve spatial learning, alleviate memory impairment, and reduce brain edema and atrophy. On the other hand, intravenous transplantation of MNCs (8 × 10⁶ cells/kg) at 24 h after ischemia could not reduce functional deficits or ischemic lesion volume in aged hypertensive rats, regardless of the donor’s age [55]. Because MNCs offer the particular advantage of acute and autologous transplantability, age, gender, and comorbidities may influence both the patients’ susceptibility to and the functionality of the MNC graft. More studies are needed to identify the impact of these factors when studying the efficacy of MNC transplantation for stroke.

Savitz et al. reported that intravenous infusion of autologous MNCs within 24 and 72 h after stroke may be effective compared with that of control stroke patients matched for age and National Institutes of Health Stroke Scale score [9]. Taguchi et al. reported that intravenous infusion of autologous MNCs within 7–10 days of stroke onset is a safe and feasible therapy, leading to improved functional recovery and increased cerebral blood flow and metabolism in patients with severe ischemic stroke [10]. On the other hand, a phase II, multicenter, randomized clinical trial demonstrated that intravenous infusion of autologous MNCs at median of 18.5 days after stroke onset is safe, but failed to show beneficial effects on clinical outcomes [56]. These clinical results suggest that earlier transplantation of MNCs from stroke onset may have greater effects on outcomes, consistent with previous experimental results [6, 16]. However, patients can deteriorate within the first few days after stroke [51, 57]. Therefore, strategies to expand the therapeutic time window for clinical application of MNCs are also important. Previous studies have shown that the effects of cell therapy may depend on the number of engrafted cells in the injured brain [24, 58]. Most grafted cells are thought to die within a few days of systemic administration [59]. Thus, it is imperative to identify therapies that can protect donor MNCs from this hostile microenvironment after stroke. We speculate that MNCs combined with appropriate pharmacological therapy, hypothermia, and/or rehabilitation may create a suitable microenvironment for transplanted MNCs and local cellular repair in the ischemic brain [60–62].

Understanding the influence of age, gender, comorbidities, and other variables on the therapeutic effects of MNCs after stroke will be important for identifying the characteristics of patients most likely to benefit from MNCs and for improving our understanding of the fundamental limitations of MNCs in the treatment of stroke. Furthermore, we must clarify the appropriate partners for cell-based therapies against stroke.

In conclusion, experimental studies have strongly suggested the therapeutic potential of MNC transplantation against various types of brain damage, including models of focal cerebral ischemia, transient global cerebral ischemia, chronic cerebral ischemia, and ICH. Currently, several clinical trials are examining the efficacy of using MNCs in the treatment of stroke. It is necessary to consider the failures of neuroprotective agents for acute stroke that have occurred within the past 30 years. Further translational studies are needed to establish optimal protocols in the clinical setting.