My results are consistent with previous reports that functional recovery occurs after treatment with hMSCs. However, the mechanisms underlying the beneficial effects of these therapies have not been fully investigated. MSCs promote endogenous plasticity, angiogenesis, and neurogenesis (Chen et al., 2003; Zhang et al., 2003; Zhang et al., 2001).
The functional benefit of these cells is derived from either their ability to differentiate or integrate into cerebral tissue and act as stem or progenitor cells (Chen et al., 2001a; Chen et al., 2001b; Cogle et al., 2004; Deng et al., 2005; Pisati et al., 2007), spontaneous cell fusion (Terada et al., 2002; Ying et al., 2002), or from their ability to cause endogenous restorative activity by inducing production of neurotrophic factors (Chen et al., 2001a; Savitz et al., 2002; Wang et al., 2007; Zhao et al., 2006). My immunohistochemistry results indicate that most of the endogenous neuronal progenitor cells (BrdU-positive), but no hMSCs (NuMA-positive), expressed neuronal or glial phenotypes. Our data suggest that rather than transdifferentiation, up-regulation of the endogenous recovery mechanism at the peri-infarct area (neurogenesis) is an important role of hMSCs in functional recovery after ischemic stroke. This benefit might reflect the production of growth factors, including neurotrophins, which might promote the repair of damaged parenchymal cells, reduce apoptosis in the IBZ,
good agreement with a previous report that hMSCs have the capacity to secrete trophic factors in culture in response to ex vivo treatment with ischemic brain extract (Chen et al., 2002). The ability of MSCs to secrete multiple growth factors and cytokines suggests their important trophic roles in a plethora of cellular and physiological functions (Majumdar et al., 2000; Majumdar et al., 1998; Wieczorek et al., 2003).
Given that application of MSCs, trophic factors, or both, has been reported to be associated with reduced infarct size, the behavioral recovery observed in the present study may result solely from the neuroprotective effects of hMSCs against ischemic brain injury.
Recent experimental studies suggest that gene transduction into MSCs can enhance their therapeutic potential (Kurozumi et al., 2005; Lichtenwalner et al., 2006). Various trophic factor gene-modified MSCs were reported to be associated with improved behavioral recovery in a stroke model, including GDNF (Horita et al., 2006), HGF (Zhao et al., 2006), BDNF (Noumura et al., 2005), and placental growth factor (a VEGF family member) (Liu et al., 2006). Application of VEGF over-expressing MSCs has also been reported to reduce infarct size in a myocardial infarction model (Matsumoto et al., 2005).
In the present study, although the hMSC-treated groups showed improved functional recovery compared to the control group, no significant difference in infarct size was observed between the hMSC-treated and control group. Possible explanations include the following. First, I used naive MSCs, and thus the degree of expression of trophic factor proteins in my hMSC groups may be lower than those of gene-modified MSCs. Second, in the present study, the time of intravenous application of hMSCs was 24 h after tMCAo, while it was ~2–6 h in most reports using gene-modified hMSCs. A therapeutic window for
intravenous administration of bone marrow in reducing lesion size after cerebral ischemia has been reported (Iihoshi et al., 2004). Transplanted MSCs in the tMCAo model 24 h after ischemia occurred had improved functional recovery, but did not significantly decrease the infarct area (Chen et al., 2001; Li et al., 2002).
My results suggest that the difference in functional recovery between the groups may have resulted from the difference in the levels of trophic factors in brain tissue and also in the degree of neurogenesis. In most previous studies, only neuroprotective effects, in terms of infarct size, were analyzed (Horita et al., 2006; Liu et al., 2006; Nomura et al., 2005; Zhao et al., 2006); other aspects of therapeutic benefit, such as neurogenesis, have seldom been reported. Given the relatively few cells showing brain cell phenotype and the absence of marked reduction in infarct size, neither neurogenesis nor neuroprotection may entirely account for the behavioral recovery of the hMSC-treated rats.
My results showed that ischemia-induced neurogenesis was enhanced by the application of hMSCs. Further studies are needed to evaluate whether the increased BrdU-positive cells are the result of increased proliferation, enhanced survival in a toxic environment, or both.
Intraventricular infusion of VEGF increased neurogenesis, but it is unclear whether this effect resulted from increased proliferation, survival, or both (Lichtenwalner et al., 2006).
The involvement of caspase-mediated apoptotic death in the loss of stroke-generated striatal
provide various cytokines and trophic factors, which may be important in repair mechanisms after stroke (Prockop et al., 1997). Cell transplantation may provoke diverse mechanisms involved in the structural modifications of dendrites and synapses associated with function plasticity after ischemic injury (Dunnett et al., 1994; Iihoshi et al., 2004). Moreover, application of bone marrow stromal cells has recently been reported to reduce scar thickness and increase the number of oligodendrocyte precursor cells (Shen et al., 2007). It was suggested that functional recovery may be enhanced by the interaction between MSCs and the host brain in an anatomically distributed, tissue-sensitive, and temporally ongoing way (Zhao et al., 2006).
In the present study, the therapeutic benefit of MSC transplantation was greatly influenced by the passage of the hMSCs; the application of earlier-passage hMSCs showed more therapeutic benefit than the later-passage cells. Concerning the characteristics and potentiality of MSCs, my results about the effect of culture expansion on stem cell potentiality in neurogenesis and trophic support may have important implications in both clinical trials involving cell therapy and basic research. My data demonstrate that intravenous hMSC treatment promotes neurogenesis in the SVZ and IBZ and that this effect is more pronounced with earlier-passage than with later-passage MSCs. Recently, it was reported that the ability to differentiate into neurons, osteocytes, and adipocyte was more effective in P2 rat MSCs than in P10 cells (Yeon et al., 2006), and several studies have examined the growth pattern of MSCs (Liu et al., 2003; Zhang et al., 2005). However, to my knowledge, no report has compared the effects of MSCs on neurogenesis after stroke, depending on their passage.
Various trophic factors influence neurogenesis (proliferation, survival, and differentiation) in the mature brain (LIchtenwalner et al., 2006), and the capacity to release trophic factors has been suggested as key to the beneficial effect of MSCs in cerebral ischemia (Chen et al., 2002). The application of gene-modified hMSCs is an attractive modality to increase trophic support by hMSCs. However, this method is not feasible in clinical practice.
I hypothesized that characteristics of the hMSCs (such as passage number) would be important when considering hMSCs for enhancing ischemia-induced neurogenesis and trophic support. To My knowledge, differences in the brain levels of trophic factors after application of different-passage MSCs has not been previously reported. My data showed that neurogenesis was enhanced by the application of hMSCs and that the effects were more prominent with earlier-passage hMSCs. Thus, it is conceivable that in the clinical application of hMSCs, a better result might be achieved by applying a smaller dose of earlier-passage hMSCs, rather than waiting to achieve a higher number of later-passage hMSCs by further ex vivo culturing.
In the present study, I examined possible reasons for the different capacities of earlier and later-passage hMSCs. Cell viability, the expression levels of chemokine SDF-1 (also known as CXCL12) and its receptor, and stem cell marker levels were not different between
analysis: when I treated cultured hMSCs ex vivo with ischemic brain extract, the levels of trophic factors secreted by P2 hMSCs were higher than by P6 hMSCs (Choi et al., 2006).
Levels of HGF, VEGF, and NGF markedly increased in cultured P2 hMSCs versus P6 hMSCs after ex vivo treatment with ischemic brain extract, whereas the BDNF level significantly increased in the latter versus the former (Choi et al., 2006).
Both our in vivo and in vitro data suggest that P2 and P6 hMSCs differ in their capacity for trophic support, resulting in superior efficacy of P2 hMSCs in neurogenesis compared to P6 hMSCs. Several trophic factors have been reported to improve adult neurogenesis in ischemic-injured brain, including VEGF, epidermal growth factor, heparin-binding epidermal growth factor, insulin-like growth factor, BDNF, GDNF, and bFGF (Emsley et al., 2005; Kobayashi et al., 2006; Teramoto et al., 2003; Wang et al., 2007; yasuhara et al., 2004). Among these, the increased tissue level of VEGF in ischemic brain tissue was reported to be associated with neurogenesis (Emsley et al., 2005; Kobayashi et al., 2006;
Teramoto et al., 2003; Wang et al., 2007; yasuhara et al., 2004), as was observed in rats that received the earlier-passage hMSCs in the present study. The roles of GDNF, NGF, and HGF in neurogenesis after ischemic injury remain unclear. Further studies using antagonists of these trophic factors are needed.
This study has some limitations. First, I compared only the differential effect of P2 and P6 hMSCs on neurogenesis; hMSCs of later passages were not evaluated. However, my electron microscopy study showed that unlike later-passage hMSCs (such as P15) that show dieing cells, P2 and P6 hMSCs had similar morphologic features and the viability of P6 hMSCs was not different from that of P2 hMSCs. Second, the presence and degree of
apoptotic cell death and angiogenesis was not evaluated in the present study. It has been suggested that the beneficial effects of MSCs may not result from neurogenesis;
neuroprotective and angiogenetic effects may actually be primarily responsible (Chen et al., 2001a). The differences in the number of BrdU-positive cells between the groups may have been caused by differences in the protective effects of neurotrophic factors against apoptotic death of BrdU-positive cells as well as by enhanced neurogenesis per se. In addition, there were no differences in the in vitro survival between P2 and P6 in the present study.
However, in vivo survival rates of early and late passage hMSCs were not evaluated in this study; which could be related to the differential capacity in neurogenesis and trophic supports between P2 and P6 hMSCs. Further studies are needed concerning the presence and degree of apoptotic cell death of hMSCs and BrdU-positive cells. Third, MSCs can give rise to microglia, which in turn may play a major role in the response to ischemia. However, in the present study, I focused on the effect of the passage of the MSCs on neurogenesis and trophic support, and the effects of hMSCs on microglia were not examined. Further studies concerning the immune-modulating effects of hMSCs are needed. Lastly, the numbers of trophic factors involved in neurogenesis are increasing, but the tissue levels of all these trophic factors could not be tested in the present study. Recently, it was reported that VEGF plasmid treatment after stroke enhanced striatal neurogenesis in adult rat brains (Tse et al.,
animals infused with a lower dose of MSCs (1 × 106) (Chen et al., 2001a). Additional studies are needed to determine the ideal passage and numbers of hMSCs.