Effects of medium of hBM-MSCs on apoptosis induced SH-SY5Ys

To determined whether addition of medium of hBM-MSCs has neuroprotective effects against apoptotic cell death, the media of SH-SY5Ys were replaced by the media of hBM-MSCs. The media of hBM-MSCs did not show any protective effect against apoptosis. To eliminate the possibility of the depletion of nutritional supports due to 24 hours culture of hBM-MSCs, hBM-MSCs were diluted with fresh DMEM with 10%

FBS in 1:1 ratio. As shown in Fig. 3, apoptotic cell death induced SH-SY5Ys were not protected with medium of hBM-MSCs. These results demonstrated that hBM-MSCs medium alone is not enough to protect cells from apoptotic cell death.

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3. Neuroprotective effect of hBM-MSCs on apoptosis induced SH-SY5Ys

To determine the effect of hBM-MSCs on apoptosis induced SH-SY5Ys, apoptosis induced SH-SY5Ys were cocultured with hMSCs using transwell inserts for 5days.

Number of viable cells was counted and compared with control that was cocultured with SH-SY5Ys using same transwell inserts from before coculture until 5 days after. For initial two to three days of coculture, number of Trypan blue excluded viable cells did not changed (Fig 4). However, number of SH-SY5Ys increased significantly at day 3 (p<0.05) and day 5 (p<0.01). When apoptosis induced SH-SY5Ys were cocultured with hBM-MSCs using transwell inserts for 5days, number of cells did not changed for 3 days (Fig. 4). Under phase contrast microscope, cells exhibited morphological characteristics of apoptosis, including extreme nuclear shrinkage and chromatin concentration, as well as apoptotic bodies were observed before coculture with hBM-MSCs and were decreased in number progressively.

4. Flow cytometric measurement of cell death using annexin-v/PI

To observe the influence of hBM-MSCs on the apoptosis induced SH-SY5Ys overtime, repeated flow cytometric assays was performed. Cultured and STS treated SH-SY5Ys with or without hBM-MSCs were analyzed. It was demonstrated that amount of Annexin V positive cells was same in pattern with data from trypan blue exclusion cell counting. These results indicate that hBM-MSCs protect SH-SY5Ys from apoptotic cell death. As shown in Fig. 5, cell proportion of live cells stained negative for both annexin-V and PI (PS^-/PI^-), in the lower left quadrant of each diagram was higher in hBM-MSCs

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co-cultured group(38.1and 33.3%) than cells without hBM-MSCs (69.3 and 84.6%) at 3 days and 5 days. Annexin-V positive and PI negative (PS⁺/PI^-) cell population in early stages of apoptosis but not in phase of necrosis (lower right quadrant) were increased significantly in Cultured SH-SY5Ys without hBM-MSCs. Cell population according to the response to annexin-V and PI were quantified and compared between cells with hBM-MSCs treated or not (Fig 5-B).

5. Western blot analysis

To investigate role of Bcl-2 family in recovery of apoptosis induced cell death, western blot analysis of bcl-family including bcl-2 (apoptosis suppressor) and bax (apoptosis promoter) were performed in cultured and STS treated SH-SY5Ys with or without hBM-MSCs. According to our data, expression of bax marked at day 1, increased progressively and peaked at day 5 in SH-SY5Ys without hBM-MSCs but, decreased progressively and significantly cells with hBM-MSCs (Fig 6). However, expression of bcl-2 increased progressively and reached normal level at five days after addition of hBM-MSCs (Fig 7).

6. Caspase-3 activity assay

To determine further apoptosis related cell death, activation of cysteine proteases in cultured and STS treated SH-SY5Ys with or without hBM-MSCs was analyzed. We examined the activation of caspase-3 proteases directly in cell lysates by analyzing the hydrolysis of specific colorimetric substrates. There was significant decrease of

caspase-- １４ -

3 activity in STS treated SH-SY5Ys with hBM-MSCs than cells without hBM-MSCs overtime (Fig 8). Significant decrease in caspase-3 observed at 2,3 and 5day following coculture with hMSC.

- １５ - Fig. 1. Experimental design

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Fig.2. The ability of STS to act as an effective inducer of apoptosis upon the SH-SY5Y cells was initially characterized by analyzing its effect in a range from 0.25mM to 2mM.

The cell death phenomenon appeared to be concentration, time-dependent in SH-SY5Y cells. Loss of cell viability was monitored by MTS assy

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Fig. 3. Medium of hMSCs did not reduce apoptosis in apoptosis induced SH-SY5Ys.

Apoptosis indeced SH-SY5Ys were incubated with medium of hMSC in different concentrations upto 7days. Apoptotic ell death was quantified by using MTS assay.

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Fig. 4. The effect of hMSCs on apoptosis induced SH-SY5Ys.

Cell counting with trypan blue showed the number of SH-SY5Ys cocultured with hMSCs was dramatically increased than that without hMSCs at 3 day and 5 day (A).

The morphological changes of SH-SY5Ys cocultured with hMSCs observed at 2day but not without hMSCs. (n=4/group, *P < 0.005, **P ≤ 0.001 ) Values are means ± SD.

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Fig. 5. Flow cytometry analysis of apoptosis induced SH-SY5Ys cocultured with hMSCs or not. Folw cytogram showing cell death as assessed by flow cytometric analysis using annexin V/PI (A). Histogram showing cell death as calculated flow cytometric analysis (B).

Annexin V⁺ cells were reduced in SH-SY5Ys cocultured with hMSCs at 2days. (n=4/group, *P

< 0.005, **P ≤ 0.001 ) Values are means ± SD.

- ２０ - Fig 6. Pro-apoptotic protein Bax Western blot

Bax translocation was induced in SH-SY5Y. However hMSC inhibit translocation of Bax.

Western blot analysis was performed to determine the expression levels of Bax(21kDa). Actin-b was used as the loading control for cytosolic proteins. Sighificant difference is between the apoptotic SH-SY5Y cocultured with hMSC and without hMSC.

- ２１ - Fig. 7 Anti-apoptotic protein Bcl-2 Western blot

Westrn blot analysis of untreated SH-SY5Ys, apoptotic SH-SY5Ys cocultured with hMSCs and without hMSCs for 1-5days. The proteins were stained using specific antibodies against Bcl-2(30kd). β-actin was used as the loading control

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Fig.8. Caspase-3 activities in SH-SY5Y cocultured with or without hMSCs treated with staurosporine. Activity was quantified by monitoring the hydrolysis of the corresponding caspase-3 specific colorimetric substrate. No change of caspase-3 activity is found in normal SH-SY5Y. A significant decrease in caspase-3 occurs at 2day, 3day and 5day. Activity of caspase-3 decrease at day in SH-SY5Y cocultured with hMSCs, whereas caspase-3 activity peaks at 3day in SH-SY5Y without hMSCs (n=3/group, *P <

0.005, **P ≤ 0.001 ). Values are means ± SD.

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Ⅳ Ⅳ Ⅳ

Ⅳ. Discussion

Apoptosis is a mechanism of programmed cell death involved in the homeostasis of nervous tissues and it’s dysregulation has been associated with the pathology of neurodegenerative diseases, stroke, and neurotrauma. Therefore, therapeutic strategies to prevent neuronal cell apoptosis is critical in Neurologic diseases (Siren, Fratelli et al.

2001; Pregi, Vittori et al. 2006) Recent experimental studies raised the possibility of using hMSCs in neurological disease therapy. There is increasing evidence that hMSC promote fuctional recovery in animal models. In this regard, hMSCs are considered to have neuroprotective role.(Bang, Lee et al. 2005) This study investigates that hMSCs have protective effects against apoptosic stress by expression of anti-apoptotic protein Bcl-2.

Staurosporine is able to induce a prominent neuritogenesis in human neuroblastoma cells. Because of this neurotrophin-like effect, staurosporine has been proposed as a potential prototype for future neurothrphic drugs, that could stimulate the process of regeneration and neurite outgrowth in damaged neurons.(Rasouly and Lazarovici 1994) However staurosproine can also be toxic for cells of neural origine at the concentrations and neurite outgrowth occurs early and cell death phenomenon takes place at later times.(Boix, Llecha et al. 1997) SH-SY5Ys have been used extensively in the study of neuronal cell death. Staurosporine-induced cell death in SH-SY5Ys is accompanied by endogenous Bax translocation and concomitant translocation of cytochome c from the

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mitochondrial to the cytosolic fractions.(McGinnis, Gnegy et al. 1999; Lopez and Ferrer 2000) Our results indicate that staurosporine not only triggers the apoptotic phenomenon upon the SH-SY5Ys but also induce cell death in concentration-, and time-dependent manner. We decided that 0.25uM of staurosporine treatment for 24 hours was optimal to observe the effect of hBM-MSCs against the STS-induced apoptotic cell death. Also I obtained the results that treatment with both fresh culture media including 10% FBS and culture media of the hMSCs failed to rescue the apoptotic stress of SH-SY5Ys. Isele N et al reported that the protective effects of hMSCs conditioned medium was further accelerated when hMSCs were incubated with Neurobasal medium of neurons previously exposed to apoptotic stress indication a potential cross talk between stressed neurons and hMSCs. But heating of the conditioned medium abolish the protective effect, suggesting that heat labile factors, i.e. protein, released by hMSCs mediated the observed anti-apoptotic effect in the neurons(Isele, Lee et al. 2006). And we found that the presence of hMSCs reduced cell death, which suggested that hMSCs exert protective roles on cell death. This indicates the release of protective factors which exert their effects across species barriers, which is typical for growth factors and cytokines. In addition, these results offer the perspective to use hMSCs in forcal cerebral ischemia. Li et al. reported that hMSCs treatment of stroke in rats increased astrocytic proliferation and activation in the subventricular zone, and an increased expression of an axonal marker(GAP-43) was observed among reactive astrocytes in the scar boundary zone and SVZ. (Li, Chen et al. 2005) This indicates that the beneficial beneficial functional recovery that hMSCs provide after stroke may be derived from the

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protection of astrocytes from cell death post-ischemia. The increased survival of astorcytes in the injured brain might subsequently increase neuronal cell survival, enhance neuroregeneration, and promote plasticity.(Gao, Li et al. 2005)

Another important aspect is that the neuroprotective effect of hMSCs required coculture with SH-SY5Ys induced cell death. When cultured media of hMSCs was supplied simultaneously with the apoptotic challenge, the neuroprotective effect was not detectable, whereas coculture of apoptosis induced SH-SY5Ys with hMSCs significantly attenuated neuronal apoptosis. This supports the idea that hMSCs mediated neuroprotection depends on expression of survival factors.

To further understand the mechanism underlying hMSCs effects on apoptosis, we examined Bcl-2 family protein. Bax translocates from the cytosol to the mitochondria in neuronal cells undergoing apoptosis. Bax has been previously found in various cell types.(Jurgensmeier, Xie et al. 1998) Bax translocation is accompanied by release of cytochrome c from the mitochondria in both CFNs and SH-SY5Ys. Cytochrom c released from the mitochondria is a cofactor for caspase-3 activity.(McGinnis, Gnegy et al. 1999) Apoptotic signals induced Bax translocation in SH-SY5Ys suggest that Bax translocation is a universal component of apoptosis in neuronal cells. Bax expression is known to be critical for neuronal apoptosis. Anti-apoptotic protein such as Bcl-2 inhibit the mitochondrial permeability transition and cytochrom c release, although effects downstream of cychrome c release have been documented.(Kluck, Bossy-Wetzel et al.

1997; Rosse, Olivier et al. 1998) Cytochrome c plays an active role in the apoptotic cascade by facilitating activation of downstream caspases through processing of

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procaspase-9.(Li, Nijhawan et al. 1997) While it has been suggested that the prima(Susin, Zamzami et al. 1996; Kluck, Bossy-Wetzel et al. 1997)ry site for Bcl-2 regulation of apoptosis is by preventing cytochrome release.(Susin, Zamzami et al.

1996; Kluck, Bossy-Wetzel et al. 1997) (Murphy, Ranganathan et al. 2000)In monitoring Bax expression during coculturing period, Bax expression was increased in time dependent manner. It was peaked at 5days. While Bcl-2 expression was gradually increased. These our results suggest that the mechanism by which Bcl-2 blocks drug-induced apoptosis is by preventing Bax overexpression. These finding suggests that the hMSCs neuroprotective effects on apoptosis induced cells might partially be derived from upregulation of anti-apoptotic protein and downregulation of pro-apoptotic protein.

In summary, out findings strongly suggest that hMSCs secrete protective factors that prevent neuronal apoptosis that prevent neuronal apoptosis through stimulation of expression of endogenous anti-apoptotic protein. These findings suggest that the therapeutic benefit hMSCs provide to injured brain after ischemic infarct may be inpart due to hMSCs stimulation of neuroprotection.

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Ⅴ Ⅴ Ⅴ

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