V. CONCLUSION
10. Statistical Analysis
Statistical differences between groups were evaluated using one way analysis of variance. P values <0.05 were deemed statistically significant.
III. RESULTS
1. Characteristics of hMSC
To determine possible differences in viability, depending on the passage of the hMSCs, cell viability of P2, P6, and P15 hMSCs was measured by observing the amount of MTT reduction; no significant difference was detected between P2 and P6 hMSCs (Fig. 1A).
Similar results were obtained when cell viability was assessed with the trypan blue dye exclusion assay; both P2 and P6 showed over 95% viability.
Flow cytometric analysis (Fig. 1B) indicated that both P2 and P6 cells had high levels of expression of stem cell markers, CD105 (median 90.3%, range 83.1–96.4% for P2; median 91.7%, range 77.1–99.7% for P6) and CD73 (median, 98.0%, range 97.4–98.8% for P2;
median 94.3%, range 83.8–99.9% for P6). No significant difference was found between P2 and P6 MSCs. These cells did not express CD34 or CD45 antigen (data not shown).
Fig. 1. Characteristics of MSCs. (A) Viability of P2, P6, and P15 hMSCs. Cells were cultured and MTT reduction was serially monitored by optical density at 540 nm. The ordinate represents % of cell survival as measured by MTT assay. The day 1 cell viability values obtained was normalized to 100%. Numbers presented are averages ± standard errors.
Some error bars were too small to appear on the graph. (B) Flow cytometric analysis of MSC surface markers, CD105 (upper lane) and CD73 (lower lane), of P2 and P6 hMSCs.
2. Neurologic functional testing
At day 14 after tMCAo, rats that received P2 or P6 hMSCs showed functional recovery compared to the tMCAo-only group (Fig. 2A, B). However, the degree of improvement differed, depending on the passage of the hMSCs received. On the adhesive-removal test, rats that received P2 hMSCs showed a higher level of functional improvement than rats receiving P6 hMSCs (P<0.05). In the same manner, neurological deficits at 14 days after stroke were less severe in the rats that received P2 hMSCs than in those receiving P6 hMSCs (P<0.05) or rats in the tMCAo-only group on the mNSS test (P<0.001).
3. Ischemic lesion size measurement by MRI and TTC analysis
An estimate of lesion size was obtained using both MRI (24 h and 14 day) and TTC staining (14 days after tMCAo) (Fig. 2C). The T2-WI lesion volume at 24 h after tMCAo was similar among the groups. Although a trend was observed in the hMSC-treated group for lesion volumes to be smaller at 14 days after tMCAo compared to those in the tMCA-only group, the difference was not statistically significant (P>0.05). The T2-WI lesion volume was 217 ± 18 mm3 in the tMCAo-only group, 179 ± 20 mm3 in the P6 hMSC-treated group, and 184 ± 19 mm3 in the P2 hMSC-treated group, while the TTC infarct volume in the three groups was 143 ± 16, 122 ± 14, and 129 ± 13 mm3, respectively.
Fig. 2. Results of behavioral tests and infarct volume measurements. Adhesive-removal test (A) and modified neurological severity score (mNSS) test (B), before and after transient middle cerebral artery occlusion (tMCAo). Open circle – tMCAo-only group, filled circle – P6 hMSC-treated group, and open triangle – P2 hMSC-treated group. (C) and (D) Evaluation of ischemic lesion volume with T2-weighted image and TTC staining in rats of (a) the tMCAo-only group, (b) P6 hMSC-treated group, and (c) P2 hMSC-treated group. T2 -weighted image obtained at 24 h (column 1) and 14 days (column 2) after tMCAo. The 24 h images were obtained just before intravenous cell or vehicle injection. Brain slices were stained with TTC to visualize lesions 14 days after tMCAo (column 3). The coronal forebrain sections were obtained at the level of the caudato–putamen complex. (Scale bar =
500 μm). (D) Meserment infarct size of T2-weighted image and TTC staining image at 1day and 14days after tMCAo.
**P<0.01, the P6 hMSC-treated group versus the tMCAo-only group.
***P<0.001, the P2 hMSC-treated group versus the tMCAo-only group.
# P<0.05, the P2 hMSC-treated group versus the P6 hMSC-treated group.
4. Cell proliferation in the SVZ of the ischemia brain
In the sham group, a few BrdU-positive cells were concentrated in the SVZ (Fig. 3A), while in the tMCAo groups, BrdU-labeled cells were more widely dispersed throughout the SVZ (Fig. 3B). In the P2 and P6 hMSC-treated groups, increased numbers of more widely distributed BrdU-positive cells were observed, involving the enlarged SVZ, corpus callosum, and striatum, mainly at the IBZ (Fig. 3C, D).
Unbiased stereological analysis was used to quantify cell proliferation in the SVZ and IBZ at 14 days (Fig. 3E, F). BrdU-labeled cells in the SVZ were counted in a clearly defined region by outlining the dorsolateral SVZ and IBZ. Both ischemia and hMSC stimulated cell proliferation in the SVZ and IBZ on the ischemic side (P<0.001 in both cases). The most BrdU-positive cells were in the hMSC-treated group and the fewest in the sham operation group; the number in the tMCAo-only group was in between. The numbers of BrdU-labeled cells increased significantly in the hMSC-treated groups over the sham operation group or the PBS-treated tMCAo group (P<0.01 in all cases). Of the hMSC-treated groups, BrdU-labeled cells in the SVZ increased significantly in the P2 hMSC-treated group compared to the P6 hMSC-treated group (P<0.01). A similar trend was observed in the IBZ (Fig. 3F).
Fig. 3. Comparison of neurogenesis between the groups. BrdU immunostaining in the subventricular zone of the ipsilateral hemisphere at day 14 in each group: (A) sham operation, (B) tMCAo + PBS, (C) tMCAo + P6 MSC, and (D) tMCAo + P2 MSC. In the inner box, only secondary antibody without BrdU was used as background control (negative
5. Identification and characterization of donor and newly developed cells.
Labeling with NuMA-positive cells (~3–5% of 1 × 106 hMSCs) survived and were observed in multiple areas of the ipsilateral hemisphere, including the cortex and striatum. Most NuMA-labeled hMSC (~50–70% of the total) were located in the ischemic boundary zone.
hMSC themselves were unlikely to proliferate after the time of infusion because no NuMA-labeled hMSC was BrdU-positive (Fig. 4). Few NuMA-positive cells were double-NuMA-labeled with the migrating neuronal marker DCX (~5% of the total; Fig. 4C), and no NuMA-positive cells double-labeled with GFAP (Fig. 4A) or NF-L were detected (Fig. 4B).
Fig. 4. Phenotype of NuMA-labeled cells in the boundary zone 14 days after tMCAo.
BrdU- and NuMA-positive cells double-labeled in the subventricular and boundary zone. No NuMA-positive cells merged with BrdU-positive cells. No NuMA-positive cells expressed GFAP or NF-L (C). Some NuMA-positive cells expressed DCX (D). Scale bar: 100 µm (A, B); 20 µm (C, D).
In contrast, some BrdU-positive cells in the ipsilateral striatum and cortex were reactive for the neural or glial markers used. A significant proportion of BrdU-labeled cells expressed DCX, GFAP, NeuN, and NF-L (Fig. 5). As shown in the Table 1, the proportions of DCX-, GFAP-, NeuN-, and NF-L-expressing BrdU-positive cells were higher in the hMSC-treated groups than in the other groups. Most of the BrdU-positive cells in groups P2 and P6 exhibited neuronal or glial phenotypes. However, no significant difference was observed in the proportion of BrdU-positive cells expressing neuronal or glial phenotypes between groups P2 and P6 (P>0.05 in all cases).
Table 1. Quantitative analysis of BrdU-labeled cells and their phenotypes.
Groups, Mean ± SD
Per section Sham MCAo+PBS MCAo+P6 hMSC MCAo+P2 hMSC
Total BrdU+ cells 56 ± 12 249 ± 14 855 ± 14 947 ± 18 BrdU+ DCX+ cells 0.2 ± 0.1 (0.5) 32.8 ± 1.8 (13.2) 384.7 ± 6.3 (45) 435.6 ± 8.28 (46) BrdU+ GFAP+ cells 0.3 ± 0.1 (0.6) 34.8 ± 2 (14.7) 205.2 ± 3.36 (24) 208.3 ± 3.96 (22) BrdU+ NeuN+ cells 0.1 ± 0.1 (0.3) 19.92 ± 1.2 (8) 102.6 ± 1.68 (12) 142 ± 2.7 (15) BrdU+ NF-L+ cells ― 0.7 ± 0.1 (0.3) 68.4 ± 1.12 (8) 94.7 ± 1.8 (10) Numbers in the parenthesis represent percentages (Numbers of cells / total BrdU+ cells).
6. CXCR4 and chemokine expression
To investigate whether application of hMSC promoted cell migration in the ischemic brain, I assessed SDF-1 levels in the brain (Fig. 6A). Tissue levels of SDF-1 were higher in the ischemic rat brains than in control brains (P<0.05). However, tissue levels of SDF-1 (Fig.
6A) and SDF-1 expression on immunostaining (data not shown) were not different between the tMCAo and hMSC-treated groups. In addition, flow cytometric analysis showed that both P2 and P6 hMSC stained for the CXCR4 antigens (Fig. 6B). However, surface expression of CXCR4 was less than 1% in both P2 and P6 hMSC, and no significant difference was found between them.
Fig. 6. Chemokine expression. (A) SDF-1 chemokine expression in the ischemic brain 14 days after tMCAo. (a) Sham group, (b) tMCAo-only, (c) tMCAo + P6 MSCs, and (d) tMCAo + P2 MSCs. No significant difference was found between the tMCAo-only group
7. Growth factor level
Using a sandwich ELISA, I examined tissue levels of VEGF, HGF, NGF, GDNF, BDNF, and bFGF in ischemic brain tissue 14 days after tMCAo. As shown in Fig. 7, the levels of trophic factors were significantly higher in the brain tissue of rats that received intravenous application of hMSCs than in those of the tMCAo group (P<0.05 in all cases). The brain levels of VEGF, HGF, NGF, and GDNF were significantly higher in the brain tissue of rats that received intravenous application of P2 hMSCs than in those of the P6-hMSC group (P<0.05 in all cases). The level of BDNF was similar between the P2 and P6 hMSCs group, whereas the level of bFGF was higher in the P6 hMSC group than in the P2 hMSC group.
Fig. 7. Brain levels of trophic factors at 14 days after tMCAo. (A) vascular endothelial growth factor (VEGF), (B) hepatocyte growth factor (HGF), (C) nerve growth factor (NGF), (D) glial cell line-derived neurotrophic factor (GDNF), (E) brain-derived neurotrophic factor (BDNF), and (F) basic fibroblast growth factor (bFGF). *P<0.05, **P<0.01, ***P<0.001.
IV. DISCUSSION
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.
V. CONCLUSION
I demonstrated that hMSCs can enhance neurogenesis in the ischemic rat brain and that this potential depends on their passage. Given that the neurogenic potential and trophic support of hMSCs differ depending on their passage, these aspects should be considered when using large-scale expansion of MSCs.
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