DISCUSSION

A. Mode of actions of hMSCs

Compared with rats subjected to MCAO alone, IV injection of hMSCs 1 day after stroke significantly improved functional outcome according to the adhesive-remove test, mNSS, and rotarod test. Controversy surrounds the question of whether true transdifferentation (Cogle et al., 2004; Tondreau et al., 2004), spontaneous cell fusion (Terada et al., 2002; Ying et al., 2002), or trophic supports (Chen et al. (a), 2001) are the primary cause for the improvement in the functional outcome after application of MSCs in animal model for ischemic stroke. Our immunohistochemisty results indicate that endogeneous neuronal progenitor cells (BrdU positive), but not hMSCs (NuMA positive), expressed neuronal or glial phenotype. Our data suggest that, rather than transdifferentiation, upregulation of endogenous recovery mechanism either at the peri-infarct area (neurogenesis) or at area that are remote from the infarct (neuronal plasticity) were important role of hMSCs in the functional recovery after ischemic stroke. This benefit may reflect production of growth factors, including neurotrophins that may promote repair of damaged parenchymal cells, reduce apoptosis in the ischemic boundary zone, and enhance proliferation and differentiation of endogenous neural stem and progenitor cells in the SVZ after stroke in rats (Li et al., 2002). Supporting this hypothesis, a recently study reported that hMSCs have the capacity to secrete BDNF, NGF, VEGF and HGF in culture and that hMSCs are sensitive to the normal and ischemic injured brain environment and

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respond through an increased production of these neurotrophins and angiogenic factors (Chen et al., 2002). The ability of MSCs to secrete multiple growth factors and cytokines is suggestive of their critical trophic roles in a plethora of cellular and physiological functions (Majumdar et al., 1998 and 2000; Wieczorek et al., 2003).

B. Passage of hMSCs as a Factor Affecting the Efficacy of Stem Cell Therapy

1. Passage of hMSCs and neurogenesis

Our data demonstrate that hMSC + P2 hMSC treatment promotes neurogenesis in the SVZ and IBZ and that such effect was more pronounced in the earlier passage MSCs (P2) than in the later passage (P6). Although there have been several studies concerning the growth pattern of MSCs (Zhang et al., 2005; Liu et al., 2003), Zhang et al. recently reported that the population doubling time of P4 rat MSCs was prolonged to nearly 3-fold compared to that on P1 and that, in contrast to the rapid proliferation phase in P1 rat MSCs, P4 rat MSCs entered a growth arrest phase (Zhang et al., 2005). The similar growth pattern of rat MSC, such as rapid proliferation at P1, slow growth at P3, and growth arrest at P4, were observed by Liu et al (Liu et al., 2003). These results suggest that the growth properties of different passage hMSCs might be one of the factors determining the neurogenesis in the ischemic rat brain, But there was no report comparing the effects of MSCs on neurogenesis after stroke depending on their passage.

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Our hypothesis was that the characteristics of hMSCs (such as passage numbers) may be important in consideration of hMSCs for enhancement of the ischemia-induced neurogenesis. Our data showed that neurogenesis was enhanced by the application of hMSCs and that such effects were more prominent in earlier passage of hMSCs than in the later passage. Thus it is conceivable that in the clinical application of hMSCs it might be better to apply a smaller dose of early passage of hMSCs rather than delay to achieve more numbers of hMSCs of late passage with further ex vivo cultivation.

However, further studies comparing smaller dose of early passages and the larger dose of late passages because it was reported that animals with ischemia-induced brain damage infused with a high dose of MSCs (3×10⁶) recovered better than did control animals infused with a low dose of MSCs (1×10⁶) (Chen et al.(a), 2001). In this present study, no significant difference was noted in the behavioral recovery of animals that P2 and P6 hMSC injected MCAo rats. Further studies are needed to decide the ideal passage and numbers of hMSCs.

2. Passage of hMSCs and chemokine expression

The chemokine SDF-1 (also known as CXCL12) and its receptor CXCR4 have been implicated in homing of stem cells to the bone marrow and the homing of bone marrow-derived cells to sites of injury. It was reported that SDF-1 protein expression was as detected by anti-SDF-1 is clearly upregulated in the infarcted hemisphere within 24 hours and it is maintained through at least 30 days post-occlusion (Hill et

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al., 2004). Thus, with respect to the recipient brain condition, earlier application of hMSCs may be important because there exist the possibility that the homing effects of SDF-1 to injured area diminish with time (Abbott et al., 2004).

Our results of the prominent neurogenetic potentials of earlier passage of MSCs compared to that of the later passage MSCs are unlikely caused by the difference in the chemokine levels; our immunohistochemical study of SDF-1 showed that the chemokine levels of the rat were not different between the P2- and P6 MSCs received rat.

C. Conclusions

In conclusion, we demonstrate that MSCs derived from adult bone marrow can enhance neurogenesis in the ischemia rat brain and this potential is restricted by proliferation ability and cell passages.

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Table 1. Modified neurologic severity score test.

Motor tests Points

• Raising the rat by the tail 1=Flexion of forelimb

1=Flexion of hindlimb

1=Head moved >10 degree vertical axis within 30s

3

1=proprioceptive(deep sensation, pushing paw against the table edge to stimulate limb muscles)

2

• Beam balance test (normal=1;maximum=6) 0= balance with steady posture

1= balance with steady posture grasps side of beam 2= hugs the beam & 1 limb falls down from the beam 3= 2 limbs fall down from the beam or spins on the beam 4= attempts to balance on the beam but falls off>40s 5= attempts to balance on the beam but falls off>20s 6=falls off: no attempt to balance or hang on the beam<20

6

• Reflexes and abnormal movements 1=pinna reflex(a head shake when touching the auditory meatus)

1=corneal reflex(an eye blink when lightly ouching the cornea with cotton) 1=startle reflex(a motor response to a brief noise from snapping a clipboard paper)

1=seizures, myoclonus, myodystrophy

4

Maximum points 18 One point is awarded for the inability to perform the tasks or for the lack of a tested reflex.13-18 =sever injury; 7-12 = moderate injury; 1-6 = mild injury.

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Figure 1. TTC stained coronal brain sections. The images of brain section were captured by scanner. Brain slides (2mm Thick) stained by the TTC solution 1 day after MCAo in adult rat. The lesion of cerebral infarct was visualized as the white arer in contrast to the normal red-stained area. The development of the MCAo was limited in the MCA territory.

Figure 2. hMSC labeled in 14d after ischemia rat. Marked increase in NuMA-positive MSCs (B) and PKH2-labeled hMSC (C) in the ischemic border zone. Scar bar 20 ㎛ in B and 100 ㎛

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Figure 3. Fifteen days after post-MCAo brain section labeled for SDF-1 in the border zone. SDF-1 labeled in the ischemia border zone (A and C) than in the contralateral zone (B). Scar bar 100㎛. B. scar bar 20㎛

Figure 4. Results of behave functional tests.

(A) Adhesive-remove test ; (B) Modified neurologic severity score [mNSS] test; and (C) Rotarod test before and after middle cerebral artery occlusion (MCAo).

Group 1: MCAo alone(n=4); group 2: intravenous infusion P2 hMSC; or group 3:

intravenous infusion P6 hMSC.

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Figure 5. Neurogenesis.

BrdU immunostaining in the SVZ of the ipsilateral hemisphere at the 14d after each group (Left); (A) Sham operation, (B) tMCAO + PBS, (C) tMCAO + MSC P2, and (D) tMCAO + MSC P6.

BrdU-labeled cells (Right); In the SVZ and penumbra zone, the numbers of labeled cells were largest in P2 hMSC.

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Figure 6. Phenotype of BrdU-labeled cells in border zone at 14d after MCAO.

(Upper lane) BrdU labeled cells were merged with NF-H( C ), NeuN (F),DCX (I) and GFAP (L). (Lower lane) No NuMA positive cells expressed GFAP (O).

Section were stained for NF-H, NeuN, DCX and GFAP(A,D,G,J) and BrdU (B,E,H,K); NuMA( N) and GFAP(M) were examined by confocal microscopy.

A-O Scale bar 20㎛.

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