Alzheimer’s disease (AD) is characterized by the accumulation of amyloid plaques and neurofibrillary tangles accompanied by cognitive dysfunction. The aim of the present study was to elucidate preventive and therapeutic potential of stem cells for AD. Among stem cells, autologous human adipose-derived stem cells (hASCs) elicit no immune rejection responses, tumorigenesis, or ethical problems. We found that intravenously transplanted hASCs passed through the BBB and migrated into the brain. The learning, memory and pathology in an AD mouse model (Tg2576) mice greatly improved for at least 4 months after intravenous injection of hASC. The number of amyloid plaques and Aβ levels decreased significantly in the brains of hASC-injected Tg mice compared to those of Tg-sham mice. Here, we first report that intravenously or intracerebrally transplanted hASCs significantly rescues memory deficit and neuropathology, in the brains of Tg mice by up-regulating IL-10 and VEGF and be a possible use for the prevention and treatment of AD.

Competing interests: Jeong Chan Ra and Hyeong Geun Park are employees and shareholders of RNL BIO Limited, which holds patents on some development technologies in this manuscript. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Funding: This research was supported by the Conversing Research Center Program through the National Research Foundation of Korea (NRF) (2011K000678), Mid-career Researcher Program through NRF grant funded by the MEST (20110027566), and a grant (2011K000270) from Brain Research Center of the 21st Century Frontier Research Program, Science and Technology, the Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © Kim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we first confirmed that intravenously injected stem cells could enter the brain through BBB and hASCs could have beneficial effects in Tg2576, AD model mice by injecting hASCs in two ways: intra-venous and intra-cerebral injection. Intracerebral injection is intended to examine the therapeutic potential of hASCs in the early stage of the disease while intravenous injection is more related to preventing or delaying the onset of disease. With both injection methods, hASCs showed therapeutic or preventive potentials rescuing cognitive impairments and reducing Aβ pathology and especially, very simply, a convenient and safe intravenous injection of hASCs might be very useful for both the prevention and treatment of AD.

Among stem cells, adipose-derived stem cells (ASCs), mesenchymal stem cells isolated from adipose tissue, are well known for their pluripotency and ability to differentiate into mesenchymal and non-mesenchymal lineages [9] . ASCs are readily accessible and show high proliferation rates in vitro with lower senescence ratios than BM-MSCs [10] . Considering clinical applications, ASCs are the most suitable source of stem cells due to the possibility of to intravenous transplantation of autologous ASCs with no immune rejections, ethical problems or tumorigenesis [11] and intravenous injection is the most convenient, simple and safest method. Therapeutic potential of intracerebral injection of human ASCs (hASCs) in neurodegenerative diseases was previously reported in Huntington’s disease (HD) and ischemia mouse models [12] , [13] . However the pathogenesis of AD is very different from those of stroke and HD. Therefore these findings were not indicative if they would be beneficial in AD.

Therapeutic potentials of stem cells in several brain disorders are enticing researchers to apply stem cell-based therapies [5] – [7] . Neural stem cells have been shown to rescue memory impairment in AD model mice by releasing brain-derived neurotrophic factor (BDNF) [2] . Also, Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs) alleviated Aβ deposition and memory deficits in AD model mice by modulating immune response [8] . However, it would almost be impossible to perform intravenous transplantation of neural stem cells and BM-MSCs.

AD is the most prevalent neurodegenerative disorder in The United States affecting approximately 5.3 million Americans [1] . AD is characterized by progressive loss in memory and as well as a decline in the ability to learn that is associated with neuronal death. Well known hallmarks of AD are neuritic plaques and neurofibrillary tangles [2] , [3] and extensive inflammation [4] . Currently, no treatment has been developed to fully cure or prevent the progression of dementia that is associated with AD.

With the data obtained so far, we can conclude that the intravenously transplanted hASCs survive, migrate into the brain and alleviate pathology by reducing the number of amyloid plaques and memory impairment of Tg2576 mice by up-regulating IL-10 and VEGF and elevating endogenous neurogenesis and synaptic and dendritic stability.

Immunohistochemical analysis of MAP-2 showed differences between Tg-Sham and Tg-hASC group after (i.v.) and (i.c.) injection of hASC. MAP-2 stained dendrites of pyramidal cells were formed to be elongated and densely distributed in the brains of the Tg-hASC mice while MAP-2 dendrites were found to be shortened in the brains of Tg-sham mice.

Long-term changes in synaptic interaction are supposed to involve alterations in dendrite morphology [18] . Postsynaptic neuronal dendrites undergo functional and morphological changes in response to pathologically excessive synaptic activation [19] . 4 months after a single hASC injection or the final (13 th ) intravenous injection, MAP2-stained dendrites of pyramidal cells were shortened in the brains of Tg2576 mice, whereas dendrites were elongated and densely distributed in the Tg-hASC group ( Fig. 10 ).

To investigate the effect of transplantation of the hASC on synaptic stability, we checked PSD-95 and synaptophysin levels in the brains of all groups. PSD-95 and synaptophysin are important factors that contribute to synaptic formation and have been proposed as a molecular scaffold for receptors and the cytoskeleton at synapses [17] . At 3 weeks and 6 weeks after the transplantation, the PSD-95 and synaptophysin were increased in the brains of hASC transplanted Tg2576 mice ( Fig. S3 ).

To confirm the effects of hASCs on endogenous neurogenesis, we also examined neurogenesis related molecules such as PSA-NCAM. In our study, the PSA-NCAM was increased in the brains of the Tg-hASC group at both 3 and 6 weeks after the transplantation compared to the Tg-sham group ( Fig. 9b ). Brain sections of mice sacrificed 4 months after transplantation were labeled with anti-doublecortin (DCX) antibody and counted the DCX-positive cells in the dentate gyrus of the hippocampus ( Fig. 9c ). Quantitative analysis showed that endogenous neurogenesis was increased by 1.7-fold in the Tg-hASC group compared to the Tg-sham group (from 49.89±8.26 to 85.41±10.77, p<0.05; Fig. 9c ).

(a) At 3 weeks after (i.c.) injection, the CSF-hASCs still stay at the hippocampal region with mouse Nestin and BrdU positive cells produced around them. (b) At 3 and 6 weeks after (i.c.) injection, PSA-NCAM level was increased in Tg-hASC group. One representative of three separate experiments is shown. (c-d) Immunohistochemical analysis showed significantly increased number of DCX positive cells. (c) Tissues were immunostained with anti-DCX (red) antibody and counterstained with DAPI (blue). Scale bar = 50µm. (d) Quantitative data of DCX positive cells are represented as mean ± SEM of three independent experiments (n = 8). Asterisk *, P<0.05 by one-way ANOVA.

Adult hippocampal neurogenesis was affected by neurotrophic or growth factors [15] . We hypothesized that hASC transplantation would induce endogenous neurogenesis through neuroprotective factors. Brain sections from mice sacrificed 3 weeks after transplantation were labeled with anti-mouse Nestin and anti-BrdU antibodies. Increased cells positive for Nestin, a primitive neurofilament protein were found around the engrafted hASCs [16] ( Fig. 9a ). There were some cells positive for both anti-mouse Nestin and BrdU antibodies.

Primary neurons were grown in 10 µM of oligomeric Aβ 42 with or without blocking IL-10 and IL-10 receptor interaction. After 48 h oligomeric Aβ 42 treatment, LDH assay was performed. (a, b) The results shown are expressed as mean ± SEM from three independent experiments (n = 16). Asterisk *, P<0.05, **, P<0.01, ***, P<0.001; by One-Way ANOVA; Tukey’s HSD Post Hoc test.

Next, by lactate dehydrogenase (LDH) release assay, we evaluated the viabilities of mouse primary cortical neurons treated with oligomeric Aβ42. After 48 h posttreatment of Aβ42, co-culture with hASC showed significantly decreased LDH release (8.6±1.0%) versus the control (13.4±1.6%), but LDH release was increased by co-culture of BV2 (17.1±0.1%) ( Fig. 8a ). However, LDH release was significantly reduced to the level of the control in co-culture of both ASC and BV2 (14.4±0.1%) ( Fig. 8a ). Treatment with blocking antibodies for either IL-10 or IL-10 receptor significantly increased the neuronal cell death induced by oligomeric Aβ42 (from 14.4±0.1 to 25.9±1.3, p<0.001; Fig. 8b ).

(a–c) primary mouse neurons were grown in coated 24-well culture dishes to near confluence 80% in neurobasal media containing B27 for 7 days. They were then added to 10 µM of oligomeric Aβ 42 peptides and co-cultured with hASCs and/or BV2 cells. Blocking of IL-10 receptor interaction was performed for 48 h and then LDH and TUNEL assay were performed. A neutralizing IL-10 or IL-10 receptor antibody (5 µg/ml, respectively) was used in the indicated groups. (a) Phase contrast and TUNEL staining of primary neurons treated with 10 µM of oligomeric Aβ 42 peptides. The TUNEL-positive cells are stained red. Scale bar, 50 µm. (b, c) Data represent mean ± SEM of three independent experiments (n = 30). Asterisk *, P<0.05, **, P<0.01, ***, P<0.001; by One-Way ANOVA; Tukey’s HSD Post Hoc test.

We found that Aβ 42 -treated neurons displayed apoptotic nuclei and co-culture with hASC or hASC/BV2 reduced the number of apoptotic nuclei ( Fig. 7a ). After 48 h posttreatment of oligomeric Aβ42, the apoptotic index of cortical neurons was 55.5±6.7%, and apoptotic indexes of co-culture with hASC only, BV2 only, both hASC and BV2 were 22.7±3.2%, 65.7±7.2% and 37.96.3%, respectively, compared to the total number of cells ( Fig. 7b ). Neutralizing antibodies against IL-10 abrogated the neuroprotective effect of the hASCs (from 37.9±6.3 to 93.4±1.2, p<0.001; Fig. 7c ).

Next, we confirmed the level of IL-1β in the culture system. The levels of IL-1β were significantly increased in the primary cortical neurons co-cultured with BV2 in the presence of 10 µM oligomeric Aβ peptides (from 9.7±1.0 to 20.8±0.6 pg/ml, p<0.05; Fig. 6c ), but IL-1β level was recovered when primary cortical neurons were co-cultured with hASC/BV2 ( Fig. 6c ). Treatment with blocking antibodies for either IL-10 or IL-10 receptor, the level of IL-1β significantly increased (from 11.3±1.9 to 19.6±1.0 pg/ml, p<0.01; Figure 6d ). However, the level of IL-1β was significantly reduced in neurons treated with only one antibody of specific human or mouse IL-10 antibodies, compared with those in neurons treated with two kinds of antibody (hIL-10, 14.0±1.1 pg/ml; mIL-10, 13.5±0.5 pg/ml, p<0.05; Fig. 6d ).

(a) Primary mouse neurons were grown in coated 24-well culture dishes to near confluence 80% in neurobasal media containing B27 for 7 days. They were then added to 10 µM of oligomeric Aβ 42 peptides and co-cultured with hASCs and/or BV2 cells. Blocking of IL-10 and IL-10 receptor interaction was performed for 48 h. A neutralizing IL-10 or IL-10 receptor antibody (5 µg/ml, respectively) was used in the indicated groups and IL-10 or IL-1β ELISA was performed. (a–b) The concentration of IL-10 in hASC/BV2 co-culture system was measured with ELISA. (c–d) The concentration of IL-1β in hASC/BV2 co-culture system was measured with ELISA. Data represent mean ± SEM of three independent experiments (n = 30). Asterisk *, P<0.05, **, P<0.01, ***, P<0.001; by One-Way ANOVA: Tukey’s HSD Post Hoc test.

To examine which cells are responsible for the increase of IL-10 in brain tissue after hASC treatment, mouse primary neurons were co-cultured with hASC and/or BV2 cells in the presence of 10 µM oligomeric Aβ peptides, with or without IL-10 or IL-10 receptor neutralizing antibodies and was analyzed by an ELISA assay. We found that concentration of IL-10 was increased in the co-culture of hASC, BV2 or hASC/BV2 group (14.9±0.8; 15.3±0.4; 23.0±2.5 pg/ml, respectively). Especially, IL-10 was significantly increased in the co-culture of BV2 or hASC/BV2, compared to the sham group ( Fig. 6a ). We also investigated whether IL-10 release was blocked by treatment with antibodies for either IL-10 or IL-10 receptor. As shown in Figure 6b , IL-10 induced by co-cultured with both ASC and BV2 was significantly reduced by treatment with each antibody (13.6±1.3 pg/ml). In addition, both specific mouse IL-10 antibody (mIL-10) and specific human IL-10 (hIL-10) antibody suppressed IL-10 release induced by co-culture with hASC/BV2 ( Fig. 6b ). These findings suggested that hASC drove BV2 to produce IL-10 as well as hASC secretion of IL-10 itself. Moreover, hASC-medicated IL-10 production induced that neuroprotection to primary cortical neurons by a paracrine effect.

Next, we examined the levels of several neurotrophic factors at early and late stages after the transplantation. VEGF level also increased in the Tg-hASC group compared to the Tg-sham group at 6 weeks (from 1.0±0.02 to 1.07±0.02, p<0.01), 4 months (from 1.0±0.02 to 1.23±0.11, p<0.05) after intracerebral injection, 4 months after the final (13 th ) intravenous injection (from 1.0±0.02 to 1.22±0.03,dda p<0.05; Fig. 5e ). GDNF, NT3 and NeuroD1 were significantly increased by both 3 weeks and 6 weeks after the transplantation, especially NT3 levels increased until 4 months after the injection ( Fig. S3 ). However, there was no change in BDNF between Tg-sham and Tg-hASC groups ( Fig. S3 ).

ELISA for mouse brain lysates obtained (a) 6 weeks and (b) 4 months after the (i.c.) injection, and (c) 4 months after the 13 th (i.v.) injection revealed significant change in IL-10 level (n = 5). (d) Western blot of mouse brain lysates obtained from 6 weeks and 4 months after single intracerebral injection and 4 months after the 13 th (i.v.) injection revealed a significant increase in VEGF. (e) Quantitative data of VEGF level was obtained using western blot analysis (n = 5). All data are represented as mean ± SEM of three independent experiments. Asterisk *, P<0.05, **, P<0.01 by one-way ANOVA.

4 months after the transplantation, we found microglia were co-localized with Aβ deposition in the brains of Tg2576 mice ( Fig. S2 ). To investigate whether transplanted hASCs mediated immune and inflammatory reaction, we quantified IL-10 and IL-1β levels by sandwich ELISA. There were significant increases in the levels of IL-10 at 6 weeks (from 14.03±3.02 to 32.16±0.58, p<0.05; Fig. 5a ), 4 months (from 19.87±1.81 to 27.33±2.41, p<0.05; Fig. 5b ) after intracerebral injection, 4 months after the final (13 th ) intravenous injection (from 25.47±1.78 to 31.06±2.45, p<0.05; Fig. 5c ), while there was no change in IL-1β level ( Fig. S2 ).

(a) Western blot analysis was performed with lysates from the cortical region of the brains in each group using 6E10 and GAPDH antibodies 4 months after injection. (b, c) Aβ and CT expressions were normalized with those of APP and GAPDH for quantification (n = 5). (d) Neprilysin level was significantly increased in Tg-hASC group (n = 4). All data are represented as mean ± SEM. Asterisk *, P<0.05 by one-way ANOVA.

We examined protein levels of Amyloid Precursor Protein (APP), APP C-terminal fragment (APP-CT) and Aβ using 6E10 antibody 4 months after injection based on Congo red staining data ( Fig. 3 ) obtained. In both injection groups, the levels of Aβ and APP-CT were dramatically reduced in the cortical region of Tg-hASC group compared to the Tg-sham group ( Fig. 4a ). In intracerebral injection group, the levels of Aβ and APP-CT in Tg-hASC were significantly reduced (Aβ, from 1.0±0.039 to 0.55±0.018, P<0.05, Fig. 4b ; APP-CT, from 1.0±0.033 to 0.63±0.029, P<0.05, Fig. 4c ). In intravenous hASC injection group, Aβ and APP-CT levels in Tg-hASC mice were also significantly reduced (Aβ, from 1.0±0.039 to 0.82±0.018, P<0.05, Fig. 4b ; APP-CT, from 1.0±0.033 to 0.78±0.029, P<0.05, Fig. 4c ).

Intravenously transplanted hASCs reduced the number of amyloid plaques in the 14-month-old Tg2576 mice brains ( Fig. 3b ). In the Tg-hASC group, there was a significant reduction in the cortex (from 6.67±1.21 to 3.17±0.43, p<0.01) and notable difference in the hippocampus (from 2.3127±0.71 to 1.38±0.28, p<0.14) compared to the Tg-sham group ( Fig. 3b ). There was a significant difference between intracerebrally transplanted hASC and sham groups of 15-month-old Tg2576 mice in both cortex (sham; 16.75±5.30, hASC; 9.11±2.78, p<0.05) and hippocampus (sham; 5.34±2.312, hASC; 2.29±1.082, p<0.05) ( Fig. 3c ).

(a) Congo red staining for the detection of amyloid plaques was carried out in the hippocampus of each group 4 months after (i.v.) injection. (b) 4 months after the 13 th (i.v.) injection, the number of plaques was counted in the hippocampal region of the Tg-hASC and the Tg-sham group. (c) At 4 months after hASC (i.c.) injection, the number of plaques was counted in the hippocampal region of Tg-hASC and Tg-sham groups. All data are represented as mean ± SEM (n = 9∼15 per group). Asterisk *, P<0.05, **, P<0.01 by one-way ANOVA.

To investigate whether hASC transplantation could alleviate toxic amyloid plaque formation, we performed Congo red staining on postmortem brains 4 months after injection with WT-sham, WT-hASC, Tg-sham and Tg-hASC mice. While Tg2576 mice showed amyloid plaque formation in almost all regions of the brain, there was no plaque observed in age-matched WT group mice ( Fig. 3a ).

(a) A Morris water maze task was performed 3 months after final intravenous hASC injection. The path shapes of the movement of the mice during the training period were obtained. (b) The task was conducted for 7 consecutive days 4 months after the last intravenous injection. A significant difference was observed between the Tg-hASC group and the Tg-sham group from the 5 th day of the Morris water maze task. (c) The probe test was carried out 48 h after the final trial. The Tg-hASC group showed memory improvement compared to the Tg-sham group in zone 4 where the platform had been hidden (n = 11∼20 per group) (d) A Morris water maze task was performed 3 months after hASC i.c. injection. The task was conducted for 6 consecutive days. A significant difference was observed between the Tg-hASC group and the Tg-sham group on the 5 th day of the Morris water maze task. (e) The probe test was carried out 48 h after the final trial. The Tg-hASC group showed memory improvement compared to the Tg-sham group in zone 4 where the platform had been hidden (n = 10∼15 per group), All data are represented as mean ± SEM. Asterisk *, P<0.05, **, P<0.01 by one-way ANOVA.

To determine whether engrafted hASC transplantation improved cognitive deficits, we performed the Morris Water Maze 3 months after the final (13 th ) intravenous injection or single intracerebral hASC injection. With trainings repeating daily, WT-sham, WT-hASC and Tg-hASC groups found the hidden platform with less movement while the Tg-sham group kept wandering with no regular pattern ( Fig. 2a ). Analysis of the escape latency of each group showed significant difference between the Tg-hASC and Tg-sham groups ( Fig. 2b and d ). We found no noticeable difference between WT-sham and WT-hASC groups ( Fig. 2b and d ). 48 hours after the final trial, we performed the probe test without the platform and checked the duration of time spent in the zone 4 where the platform was previously hidden. The Tg-hASC group spent significantly more time in zone 4 than in other 3 zones (zones 1–3) ( Fig. 2c and e ), as WT groups did. However, in the case of Tg-sham, no significant difference between times spent in each zone was observed ( Fig. 2c and e ). These data show that both intravenous and intracerebral hASC transplantation improved spatial learning inTg2576 mice.

We checked whether intravenously transplanted hASCs passed through the BBB and migrated into the brain, we injected hASCs labeled with fluorescence magnetic nanoparticles into the tail vein of mice and monitored hASCs at 0, 1, 3 and 10 days after i.v. injection of labeled hASCs in live mice ( Fig. S1a ). One day after injection of labeled cells, the fluorescence signal was mostly detected in the liver, however some were detected in the brain of Tg2576 ( Fig. S1a ). On the 3 rd day after injection, prominent fluorescence signals from hASCs were detected in the brains, and we found that the cells remained in the brain up to 10 days ( Fig. S1a ). Fluorescence signals from the organs extracted 3 days after cell transplantation show that the cells had spread throughout the entire organs including the brain ( Fig. S1b ). After dissecting the brain into 5 distinct regions (olfactory bulb, hippocampus, cerebellum, brainstem, midbrain and cortex), we found fluorescent nanoparticle signals in all brain regions except the olfactory bulb ( Fig. S1c ). Our data clearly shows that the intravenously transplanted hASCs survive and migrate into the brain.

The hASCs were intravenously transplanted into Tg2576 and WT mice biweekly a total of 13 times from 3 months of age ( Fig. 1a ) or bilaterally transplanted into the dentate gyrus (DG) of the hippocampus of the 11-month-old Tg2576 and age-matched wild type (WT) mice ( Fig. 1b ).

Discussion

There are two hallmark factors in the AD brain: amyloid plaques formed by a small peptide called Aβ and neurofibrillary tangles formed by the hyperphosphorylated microtubule-binding tau [20]. Previously, it was reported that the accumulation of Aβ plays a major role in the progression of the disease including memory impairment [21], [22]. Once progressed, pathological development is inevitable, and although there are various reports regarding a cure for AD, there is no perfect treatment that can fully diminish Aβ plaques and prevent the disease [23].

Recently, treatment of disease using stem cells has been in the spotlight due to the advantages stem cells possess. For regenerative clinical applications, stem cells should meet following criteria [24]: 1. they can be extracted in abundant quantities; 2. they can be harvested by a minimally invasive procedure; 3. they can be differentiated in to multiple cell lineages; and 4. they can be safely and effectively transplanted into either an autologous or allogenic host without immune rejection. MSCs meet the above criteria and have been shown to reduce neuronal damage and support nerve regeneration in nerve injury models [25], [26]. Among MSCs, ASCs, which originate from adipose tissue, are the most suitable for clinical application. Adipose tissue is known to contain cells that have a high proliferation capacity in vitro and have the ability to undergo differentiation into multiple cell lineages [27]. As adipose tissue is easily accessible, autologous ASCs transplantation like other MSCs transplantation can be performed safely without immune rejection or tumorigenesis. It has been reported that ASCs also secrete growth factors including VEGF, GDNF, NT-3, NGF and bFGF, and are thought to participate in immunization by regulating cytokine release [28], [29].

In our experiment, we showed preventive or therapeutic potential of hASCs on Tg2576 mice using two injection methods: intravenous, the simplest, convenient and safest method, and intracerebral injection. Intravenous injection is more related to preventing or delaying the onset of disease, while intracerebral injection is intended to examine the therapeutic potential of hASCs when the disease has already progressed.

Since disruption in the blood-brain barrier (BBB) was observed in the case of AD [30], after peripheral injection the amount of ASCs migrated into the brain might be increased compared with the control. We checked the organ distributions of intravenous injected hASCs using hASC labeled fluorescence magnetic nanoparticles for in vivo live tracking of hASCs. The present data demonstrated that fluorescent nanoparticle signals in all brain regions except the olfactory bulb (Fig. S1). Our data clearly shows that the intravenously transplanted hASCs migrate into the brain.

At first, to assess the functional effects of hASC transplantation into the AD model mouse, behavior test and pathological analysis was performed with intravenously or intracerebrally transplanted Tg or WT mice. The Morris water maze test demonstrated that spatial learning ability in the intravenously or intracerebrally transplanted Tg-hASC group was improved and this result was confirmed by the probe test. The number of amyloid plaques in Tg-hASC mice brain sections was significantly reduced compared to that of the Tg-sham group 4 months after injection. Moreover, protein levels of Aβ and APP-CT were significantly decreased in the Tg-hASC group mice brains through the induction of neprilysin till at least 4 months after injection. Other investigators checked that memory deficits and neuropathology were only reduced 2 weeks – 2 months after transplantations of stem cells [2], [8], [31].

In this study, we examined the levels of several neurotrophic or growth factors such as VEGF, GDNF, NT3, BDNF and neuroD1 (Fig. S4). Among them, VEGF, GDNF and NT3 were significantly increased by transplantation of the hASC in Tg2576 mice compared to Tg-sham group lasting for several months (Fig. S4). IL-10, anti-inflammatory cytokine was also increased in Tg-ASC mice brains for at least 4 months, while IL-1β did not change (Fig. 5). At 3 and 6 weeks post-injection, there were increased Nestin and BrdU positive cells particularly around the engrafted hASCs compared to other areas. As in previous reports, some of the injected stem cells might have sensed a damaged area, migrated, differentiated into neuronal lineages and replaced the functions of dead neurons [32]. Recent studies demonstrate an alternative mechanism referred to as the “bystander effect” of stem cell treatment [33].

Transplanted human BM-MSCs in the brains of cerebral ischemia rats induced behavioral recovery by elevating BDNF, NT-3 and VEGF levels [34], but our results showed that ASCs didn’t significantly affect the level of BDNF (Fig. S4). In addition, J. K. Lee et al. suggested that intracerebral transplantation of BM-MSCs promoted activation of microglia that secreted neurotrophic agents and resulted in cognitive improvements and reduction of Aβ pathology [8]. MSCs transplanted into the brains of cerebral infarction model rats have been reported to affect cytokine release by up-regulating IL-10 and down-regulating TNF-α [35]. Recent reports suggest that proinflammatory cytokines have a negative effect on neurogenesis, whereas anti-inflammatory cytokines exert an opposite effect [36], [37]. Growth and neurotrophic factors including VEGF has also been reported to exert neuroprotective and neurogenic effects [38].

Neuroprotective capacity of hASCs could be attributed to the soluble mediators, which was confirmed by transwell experiments. Mouse primary neurons were co-cultured with hASC or BV2 cells in the absence or presence of 10 µM oligomeric Aβ peptides, with or without IL-10 or IL-10 receptor neutralizing antibodies. BV2 activated in the presence of hASCs produced a significantly higher amount of IL-10; additionally, a blockade of IL-10/IL-10R interaction by antibodies abrogated the neuroprotective capacity of hASC culture supernatants. Therefore, we concluded that both ASC and BV2 cells affected the cell survival against Aβ by increasing IL-10. And also, we checked the secreted level of IL-1β from BV2 cells in each group and found that the level of IL-1β was decreased by co-culture with hASC. But, treatment of anti-IL-10 and anti-IL-10 receptor antibodies increased the reduced level of IL-1β again. The secreted level of IL-1β might be controlled by the secreted IL-10 from hASC. These immunomodulatory effects of hASCs might be supported by recent study, which MSCs inhibit the proliferation of either syngeneic or allogeneic T cells by inducing IL-10 [39].

Our immunohistochemical analyses suggest that transplanted hASCs increased proliferation of endogenous stem cells. We examined neurogenesis related molecules such as PSA-NCAM, which is highly expressed in the population of newly generated granule cell precursors and is closely related to neurogenesis and brain plasticity. PSA-NCAM level was also increased in the Tg-hASCs group compared to the Tg-sham group. Four months after hASC transplantation, we also found an increase of DCX positive cells, which provides additional molecular evidence for increasing the endogenous neurogenesis. These data show that the hASC transplantation is involved with the increase of endogenous neurogenesis through increasing neurotropic factors. Therefore, we suggest that intravenously or intracerebrally transplanted hASCs benefit the brain by inducing proliferation of endogenous early-stage neurons and surrounding cells in the hippocampus region. In addition, MAP2 levels were enhanced by hASC transplantation along with PSD-95, suggesting the increase of dendrite and synaptic stability.

Our previous study demonstrated that ASCs have no side effects such as tumorigenicity, chromosomal abnormalities, or immune rejection [11], confirming the systemic transplantation of hASCs in animals and humans to be safe [11]. In this study, there was also no sign of distortion or tumor formation and no noticeable anti-graft immunoreactivity.

From these data, we conclude that intracerebrally or intravenously injected hASCs dramatically improved learning and memory ability and neuropathology of Tg2576 mice by diminishing the formation of amyloid plaques, decreasing Aβ and CT levels and up-regulating IL-10, VEGF and elevating endogenous neurogenesis and synaptic and dendritic stability.

Although it is yet unclear how hASCs up-regulated IL-10 and growth factors such as VEGF and GDNF, our findings that intravenously transplanted hASCs prevent the onset and progression of the disease clearly provide an important preclinical platform for the development of prevention and therapy for AD patients.