The question addressed by the present study was whether age influences the therapeutic concept of autologous BM MNC transplantation after acute stroke. The Framingham Study clearly demonstrated the relevance of age and high blood pressure for the lifetime risk of stroke  indicating the need to mimic these risk factors in preclinical stroke studies. Since BM MNCs offer the particular advantage of acute and autologous transplantability, age might influence both the patients’ susceptibility to and the functionality of the BM MNC graft.
By comparing the neuroprotective properties of human BM MNCs from young (24 ± 4 years) with aged (68 ± 1 years) donors in a cell culture model of cerebral hypoxia we found that young BM MNCs significantly suppress hippocampal cell death after oxygen glucose deprivation (OGD). This finding is in accordance with another recent in vitro study that revealed an attenuated amount of apoptotic neurons after hypoxic injury and cotreatment with BM MNC-derived supernatants . The authors discussed the release of trophic factors and the modulation of microglia as contributing factors to the observed neuroprotective effects.
In our hands, aged BM MNCs did not show neuroprotective capabilities in the hippocampal OGD model. A BM MNC subpopulation, the bone marrow derived mesenchymal stromal cells (BMSC) are supposed to account for antiapoptotic effects after ischemic damage [18, 19]. However, several studies revealed a decline in BMSC numbers and fitness with age. Thus it is tempting to speculate that senescent BM MNCs lack neuroprotective effects, as shown in the present study, due to an age-related drop in BMSC frequency and functionality .
In a second step, we evaluated the therapeutic efficacy of intravenously transplanted young and aged BM MNCs in aged hypertensive rats (SHR). Both BM MNCs from juvenile and elderly donors failed to decrease the lesion volume after experimental brain ischemia. Beyond that, functional recovery was not improved over a period of 56 days. These findings are, at least partly, in clear contrast to recent stroke studies demonstrating significant benefits of BM MNC transplantation in young and middle-aged (12 months old) healthy animals [5, 6, 8]. The differing results might be explainable by age and comorbid status of the laboratory animals used in this study. Aged individuals show a dysregulated cellular and genetic response to cerebral ischemia that finally determines a poor neurofunctional recovery. More precisely, augmented apoptosis and massive microglia activation that induces an enhanced inflammatory response account for accelerated infarct development in the aged brain [21, 22]. Likewise, stroke in senescent rats is associated with a greater degree of oxidative cellular injury . Another contributing factor might be the exacerbated astrocytic reaction in geriatric stroke rats that, in turn, impairs neurite outgrowth . One of the cellular mechanisms of neuronal plasticity after stroke is axonal sprouting. Interestingly, aging delays the expression of growth-promoting genes during the sprouting response while growth-inhibitory genes are induced at earlier stages than in its younger counterparts . Of note, enhancement of endogenous plasticity mechanisms such as axonal sprouting and synaptogenesis, amongst others, is considered to be a critical mode of action for cell-based therapies . Thus, the temporal mismatch of growth-associated gene expression in the aging brain may interfere with the plasticity-related effects of cell therapy. Most importantly, the age-related shift of pathophysiological sequences in stroke might require adjustments of the therapeutic setting such as a prolonged treatment period or higher dosages of BM MNCs.
Moreover, previous studies revealed that the final infarct volume in SH rats develops rapidly within 1 h after the onset of permanent ischemia . Thus, the lack of secondary infarct expansion in SH rats might additionally account for the absence of neuroprotective effects in this study. Unfortunately, our study is limited by the exclusive use of aged spontaneously hypertensive rats without controlling the age factor. Thus, the discrimination between the influence of hypertension, including its substantial impact on the cerebral microvessel system , and age is not possible. On the other hand, the combination of age, significant cardiovascular comorbidities, and a relatively advanced ischemic lesion development (sub-acute stage) describes the population of stroke patients which would primarily be eligible for BM MNC treatment in upcoming clinical trials.
Cell storage and processing may further influence the therapeutic potential of BM MNC. The cells used in our study had been cryopreserved prior to use, while the majority of positive therapeutic results were obtained in studies using freshly prepared BM MNC. Cryopreservation does not seem to affect the hematopoietic potential . However, it is unclear whether freezing and/or thawing alters potential neuroprotective capabilities, which probably depend on completely different physiological processes. In fact, it has been shown that cryopreservation affects BM MNC physiology. Even single freezing-thawing cycles can enhance intrinsic proteolytic activity leading to the cleavage of apoptosis-related proteins , even though the impact of this alteration (and potentially others) on neuroprotection is unknown. Thus, thorough research is needed to elucidate this impact.
Another limitation of our study is the transplantation of human BM MNCs to rodents without an immunosuppressive treatment. The absence of beneficial effects in aged SHRs might be simply a consequence of xenograft rejection. However, recent animal stroke studies described significantly improved recovery by BM MNCs, albeit the transplanted cells died immediately after injection [5, 31]. It is not conclusively elucidated so far, if and how long transplanted cells must survive to facilitate beneficial effects. In line with this, Thum and colleagues introduced the “dying stem cell hypothesis”. They suppose that apoptotic, rather than viable cells are responsible for the functional restoration after stem cell transplantation in ischemic injury via modulating the local immune response . A further decisive reason why we desisted from immunosuppression is that the commonly applied agents may distort the value of this study by their neuroprotective properties  and by impacting the immunological processes that ultimately determine progress and outcome after stroke .
Although not investigated in the present study, several further mechanisms by which BM MNCs might improve recovery after ischemic stroke are age-dependent. It has been shown that young BM MNCs promote neovascularization in models of limb ischemia  myocardial infarction  and ischemic white matter damage . However, aging impairs the angiogenic capacity of BM MNCs , a finding that is in agreement with an impaired VEGF production and migratory response to VEGF in aged BM MNCs . Furthermore, it has been shown that vasculogenesis after BM MNC transplantation is dependent on the CD34+ cell fraction  and that CD34+ cells enhance post-ischemic neurogenesis . Our finding that the frequency of hematopoietic progenitor cells is significantly reduced in senescent BM MNCs might therefore compromise the therapeutic efficacy of self-donated BM MNCs in aged patients.