Table 2.

Studies of cell-based therapies with immunomodulatory properties in animal models of Alzheimer’s disease (AD)

Study	Details
M2 macrophages
Reference	Zhu et al. (2016)
Number of animals, gender, ages, and treatment	Adult male F344 rats, 8–9 weeks of age, were randomly divided into groups: model group (intracerebroventricular injection (i.c.v.) amyloid-β (Aβ42) + intravenous injection (i.v.) phosphate buffer saline (PBS), n = 15) and M2-transplantation group (i.c.v. Aβ42 + i.v. M2 macrophages, n = 15). Aβ42 peptide was dissolved in sterile water at a concentration of 6 mg/mL and diluted to 2 mg/mL with 0.01 M PBS. The peptide was incubated at 37°C for 4 days to aggregate it before injection. Rats were anesthetized, placed in the stereotaxic apparatus, and a hole driled in the skull. Then 5 µL of aggregated Aβ42 suspension was administered by i.c.v. injection. Bone marrow cells were collected from the tibial and femoral shafts of male F344 rats. Macrophages were isolated from the bone marrow cell suspensions, cultured and differentiated in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum and 10 ng/mL recombinant macrophage colony-stimulating factor for 6 days. On 7th day, macrophages were skewed to M2 phenotype by adding 15 ng/mL recombinant interleukin (IL)-4 for 48 hours. On 9th day, M2 macrophages were labeled with DiI and then detached with 0.25% trypsin for 10 minutes. At 5 days after stereotactic surgery, 1 × 106 M2 macrophages/rat were injected via tail vein. Morris water maze test was carried out from day 12 to day 14 after Aβ42 injection. Spontaneous locomotor activity was tested by Y maze on day 16. For immunohistochemistry, rats in each group (n = 5) were anesthetized, and perfused with 0.1 M PBS followed by 4% paraformaldehyde. Brains were removed and postfixed in 4% paraformaldehyde. The remaining rats were euthanized and the cortex isolated and homogenized for analysis of cytokine levels.
Comparison	Rats received i.c.v. PBS + i.v. PBS as control sham group (n = 15)
Functional outcomes	M2 macrophages were labeled with DiI to distinguish transplanted cells from endogenous M2 macrophages/microglia. In Morris water maze, escape latency was prolonged in the model group compared with the sham group, indicating spatial memory deficits. In the Y maze, the proportion of AD model rats that entered three consecutive different arms was significantly lower than that of the sham control rats. Compared to model group rats, M2-transplantation rats showed an increase in both tests. Immunohistochemistry analysis of the density of total neurons (NeuN+ cells) in cortex and cholinergic neurons (ChAT+ cells) in nucleus basalis of Maynert were significantly decreased in the model group compared to sham group. In the M2-transplantation group, the densities were restored to the levels of the sham group. These findings demonstrated that M2 macrophage transplantation significantly improved learning and memory in the AD model rats and exerted neuroprotective effects. Immunohistochemistry analysis showed that the densities of inducible nitric oxide synthase-positive cells (M1 macrophages/microglia) and CD206+ cells in the cortex of rats in the model group were significantly increased and decreased, respectively, compared to sham group. In the M2-transplantation group, these changes were reversed. immunohistochemistry showed no DiI+ cells in the brain. These findings demonstrated that transplanted M2 macrophages were not trafficked to the brain, but were able to induce an increase in the numbers of endogenous M2 microglia. Expression levels of IL-4, IL-5 and β-nerve growth factor in the cortex of rats in the model group were decreased compared to the sham group, indicating that administration of Aβ42 led to the supression of anti-inflammatory factors and triggered an inflammatory response and neuroinflammatory injury. All of these changes were reversed by M2 macrophage transplantation. Western blots showed interferon regulatory factor (IRF)5 levels in cortex were significantly raised, while levels of IRF4 were significantly lowered, in model rats compared to sham rats. M2 macrophage transplantion rats had significantly lower levels of IRF5 and significantly higher levels of IRF4 compared to model rats. These results indicated that transplantation promoted M2 phenotype polarization. In vitro studies with primary microglia isolated from cortex of neonatal rats at postnatal day 1 suggested that Aβ42 induced a neuroinflammatory response that stimulated microglia to polarize into the M1 phenotype. In contrast, nerve growth factor induced polarizartion of microglia toward M2 and rebalanced the ratio of M1 and M2 phenotype cells.
Conclusion	M2 macrophage transplantation attenuated neuroinflammation, reversed Aβ42-induced changes in IRF4 and IRF5, drove endogenous microglial polarization toward M2 phenotype, and ameliorated cognitive impairment.
CD4+CD25+Foxp3+ Treg cells
Reference	Baek et al. (2016)
Number of animals, gender, ages, and treatment	Adult male 3xFAD Tg AD mice with PS1M146V, APPKM670/671NL, tauP301L transgenes, 4 months of age, were used. CD4+CD25+ T cells and CD4+CD25– T cells were isolated from the spleens obtained from male 6-week-old C57BL6 mice. Either 1 × 106 Treg or Teff cells were adoptively transferred by i.v. injection (tail vein) of 3xTg AD mice. Spatial learning and memory in mice was tested using Morris water maze. After the behavioral test, mice were transcardially perfused with saline containing 0.5% sodium nitrate and heparin (10 U/mL) and then fixed with 4% paraformaldehyde in 0.1 M PBS. Each brain was removed and post-fixed overnight at 4°C, and stored in 30% sucrose for sectioning and immunohistochemistry. Single-cell suspension of splenocytes was cultured in RPMI-1640 with 10% fetal bovine serum and antibiotics. Cultures were activated in the presence of plate-bound anti-CD3 and soluble anti-CD28 antibody. Cytokines were assayed in the supernatants.
Comparison	Male 3xTg AD mice as control. Also wild type (WT) mice.
Functional outcomes	Treg treatment of 3xTg AD mice improved cognitive impairments as indicated by a decreased escape latency response and increased platform entries in the spatial probe test compared with the 3xTg group. The expression of IL-2, IL-6, interferon-γ and IL-17A in supernatants of splenocyte cultures was significantly increased for 3xTg AD mice compared with WT group. Administration of CD4+CD25+ T cells significantly decreased these cytokine levels compared with the 3xTg group. Administration of CD4+CD25– T cells significantly reduced interferon-γ levels but was without effect on the other cytokines compared to 3xTg group. In addition, IL-10 levels were significantly increased in Treg transferred to 3xTg AD mice compared with the 3xTg group, but not with Teff transfer. Treatment with CD4+CD25+ T cells of 3xTg AD mice significantly decreased Aβ burdens in both cortex and hippocampus compared with the 3xTg group. In contrast to treatment with CD4+CD25+ T cells, treatment with CD4+CD25– T cells had no effect on Aβ in hippocampus or cortex. Treg treatment reduced the number of Iba-1+ microglia in hippocampal CA1 region of 3xTg AD mice, whereas Teff treatment had no effect on Iba-1 expression, compared with the 3xTg group.
Conclusion	Transplantation of purified CD4+CD25+ T cells into 3xTg AD mice improved cognitive functions and reduced Aβ deposition.
Th1 T cells stimulated with anti-CD3
Reference	Fisher et al. (2014)
Number of animals, gender, ages, and treatment	Adult amyloid precursor protein (APP)/presinilin 1 (PS1) transgenic (Tg) mice, 5 months and 12–15 months of age, were used. Aβ-specific T cell line was generated by immunizing mice 2 months of age by footpad injection of Aβ42 (100 µg) emulsified in complete Freund’s adjuvant H37Ra. At 10 days later, popliteal, ingunal, and iliac lymph nodes were extracted and cells seeded (5 × 106 cells/mL) in Biotarget medium supplemented with 10 µg/mL Aβ42. Every other day thereafter, human recombinant IL-2 (rIL-2) (10 U/mL) in complete Dulbecco’s modified Eagle medium was added. Following 1 week and every 2 weeks later, the T cell cultures were restimulated with irradiated (6000 rad) splenocytes and reseeded (2 × 105 T cells/mL, 5 × 106 irradiated splenocytes/mL) CD4+ T cells were restimulated with 1 µg/mL anti-CD3 for 48 hours. Cells were then harvested and resuspended in PBS at a concentration of 5 × 104 cells/µL. For Th1 cell subpopulation generation, anti-IL-4 (20 µg/mL) and mouse IL-12 (20 ng/mL) were supplemented in the first three stimulations during seeding and then 2 days later. OVA-specific T cell line was also generated from spleens from OT-II OVA (ovalbumin) TCR Tg mice cultured with OVA 323–339 peptide (OVA323–339) (10 µg/mL). Cells (2.5 × 105) were slowly injected over 5 minutes into each of the lateral ventricles of the brain of APP/PS1 Tg mice, 5 months of age, with a stereotactic device (n = 3–5).
Comparison	WT mice were i.c.v. injected with Aβ-specific Th1 T cells (n = 3–5)
Functional outcomes	Significantly more CD4 T cells were observed at 28 days post-injection in the hippocampus, thalamus and cortex of 5 month-old APP/PS1 Tg mice injected i.c.v. with Aβ-specific Th1 T cells (Aβ -> AD mice) as compared with WT mice injected i.c.v. with Aβ-specific Th1 T cells (Aβ -> WT mice) and with APP/PS1 Tg mice injected i.c.v. with OVA323–339-specific T cells (OVA-> AD mice). Immunohistochemistry of the hippocampus showed that whereas the cells were randomly distributed in Aβ -> WT mice, they were clustered around Aβ plaques in Aβ -> AD mice at 28 days. In Aβ -> AD mice compared to OVA-> AD mice, major histocompatibility complex II was increased and was colocalized with T cells at the sites of Aβ plaques. These findings suggested that the accumulation of Aβ in the brain of APP/PS1 Tg mice promoted the targeting of T cells specifically to their Aβ antigens and thereby increasing major histocompatibility complex II expression, which presumably facilitated longer retention of these cells in the brain. Aβ-specific Th1 cells or PBS were injected i.c.v. to APP/PS1deltaE9 Tg mice (Aβ -> AD and PBS ->AD, respectively) 12–15 months of age. Brain sections were immunolabeled with anti-Aβ at 28 days post-injection and the areas occupied by Aβ plaques were analyzed. Compared with PBS ->AD mice, a 56% and 30% reduction in plaque burden was found in the hippocampus and cortex, respectively, of Aβ-> AD mice. By real time polymerase chain reaction analysis, major histocompatibility complex II, interferon-γ and tumor necrosis factor (TNF)-α in the brains of Aβ -> AD mice remained significantly upregulated as compared with PBS ->AD mice at 28 days post-injection (~3-fold), but to a markedly lower extent than at 5 days post-injection. Similarly, signal regulatory protein-1β, which was shown to increase Aβ uptake by microglia, was induced by Th1 cells (but not Th2 or Th17 cells) at 5 days post-injection and to a lesser extent at 28 days post-injection. Moreover, of all of the chemokines induced at 5 days post-injection, only chemokine (C-X-C motif) ligand 9 remained significantly upregulated. These results suggested a very low grade inflammatory reaction induced by Aβ-plaque-associated Th1 T cells sufficient to enhance the clearance of Aβ by microglia. Using the terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling staining, the number of apoptotic cells was very low and similar in the brain of Aβ -> AD mice and of PBS ->AD mice, whereas the positive control (sections from a mouse brain following vascular injury) showed markedly increased apoptosis. The amount of proliferating and differentiating neuronal progenitors in the hippocampus was measured in brain sections from 6-month-old WT mice and from 10- to 12-month-old APP/PS1deltaE9 Tg mice, each left untreated or i.c.v. injected with Aβ-specific Th1 T cells or with PBS. Sections were immunolabeled with anti-doublecortin, a microtubule-associated protein expressed by neuronal precursor cells. The total number of doublecortin-positive cells was similar in all groups. However, while the number of neuronal progenitors in subgranular layer of dentate gyrus was also similar in all 6-month-old WT groups, it was significantly increased in 10- to 12-month-old APP/PS1deltaE9 Tg mice i.c.v. injected with Aβ-specific Th1 T cells. These findings suggested that i.c.v. injected Aβ-specific Th1 T cells induced neither neuronal loss nor chronic neuroinflammation, and that in APP/PS1deltaE9 Tg mice they induced a temporary increase in dentate gyrus proliferating neuronal progenitors.
Conclusion	Aβ-specific Th1 T cells when injected into APP/PS1 Tg mice target Aβ plaques, increase Aβ uptake, and promote neurogenesis with no evidence of neuronal loss.
Apolipoprotein (APOE) 3 bone marrow cells (BMCs), APOE4 BMCs
Reference	Yang et al. (2013)
Number of animals, gender, ages, and treatment	Adult APPswe/PS1deltaE9 mice, 5 months of age, received total body 10.5 Gy single dose irradiation at approximately 2 Gy/min from a cesium-137 source. BMCs were isolated from 8-week-old male APOE3/3;GFP (green fluorescent protein) or APOE4/4;GFP transgenic mice by flushing femurs and tibias with RPMI media with 10% fetal bovine serum. The samples were combined, passed through a 25-G needle filtered through a 70-µm nylon mesh and centrifuged. Erythrocytes were lysed in ammonium chloride potassium buffer, and the remaining leukocytes resuspended in sterile PBS at a concentration of approximately 5 × 106 viable nucleated cells/200 µL. Irradiated APPswe/PS1deltaE9 mice received APOE3/3;GFP (n = 11) or APOE4/4;GFP (n = 8) BMCs via retro-orbital venous plexus injections 1 day after total body irradiation. Chimeric mice underwent behavioral testing at 8 months after transplantation and were then euthanized for tissue analysis. Blood was collected by cardiac puncture for complete blood counts and flow cytometry and mice were transcardially perfused with ice-cold PBS. Brains were removed for analysis.
Comparison	–
Functional outcomes	White bood cell, red blood cell, and platelet counts did not differ between groups. Multilineage differentiation of hematopoietc stem cells was within the normal range, with no significant differences between groups. There was no differential influence of APOE on the proportions of T and B lymphocytes and neutrophils. Although differential blood counts showed no differences in total monocytes, flow cytometry of peripheral blood indicated APOE4/4 BMC transplantation (BMT) gave rise to fewer CD11b+ monocytes/macrophages than did APOE3/3 BMT, suggesting effects of APOE on monocyte molecular phenotype in the periphery. Mononuclear cells were isolated for flow cytometry from cerebral cortex and were then probed for microglia, which unlike peripheral monocytes are CD11b+ and CD45low cells. Although almost half of the CD11b+CD45low cells were BMT derived (GFP+) in APOE3/3 recipients, less than a third of microglia in APOE4/4 recipients were derived from BMT. Using immunofluorescence histology, hippocampus and cerebral cortex from the contralateral hemisphere were analysed to further quantitate APOE genotype effects on BMT-derived monocyte/microglia engulfment. BMT-derived cells were identified by strong GFP autofluorescence in both groups, and on the basis of Iba-1 immunopositivity were almost uniformly microglia. Stereological analysis showed significantly increased donor-derived microglia in APOE3/3 compared to APOE4/4 recipients in cerebral cortex and in hippocampus. Overall, cerebral cortical and hippocampal microglia densities were not significantly different between the two groups, and there was no significant APOE effect on total microglia density between BMC recipients. APOE3/3 transplantation resulted in 45% and 40% greater APOE protein levels in the cerebral cortex and hippocampus, respectively, than did APOE4/4 transplantation. In the behavior tests, APOE3/3 mice displayed habitation to a novel environment as shown by a progressive significant decrease in total distance traveled over successive days, whereas APOE4/4 mice showed no significant decrease in distance traveled over successive days. There was no significant difference in baseline locomotor function between the two groups, nor any significant differences in the acquisition phase of the Barnes maze test; however, reversal learning was significantly preserved in APOE3/3 recipients compared with APOE4/4 recipients. APOE3/3 mice exhibited decreased distance traveled, shorter escape latency, and fewer errors than APOE4/4 mice. The APOE4/4 recipients only used a spatial or serial search strategy 16% of the time, whereas APOE3/3 recipients used one of these strategies 50% of the time. These findings demonstrated better spatial working memory in APPswe/PS1deltaE9 recipients of APOE3/3 compared to APOE4/4 BMT. Using immunohistochemistry of sections stained with a pan-Aβ antibody, total area occupied by Aβ plaques and plaque frequency were significantly decreased in the hippocampus of APOE3/3 mice compared to APOE4/4 mice. APOE genotype effects were less apparent in the cerebral cortex, where plaque frequency was lower in APOE3/3 recipients, but there was no significant effect of donor APOE genotype on total area occupied by plaques. Average plaque size was not affected by donor genotype in either cortex or hippocampus. Qualitatively more bone marrow-derived cells were found in association with Aβ plaques in the hippocampi of APOE3/3 recipients compared with APOE4/4 recipients. In both groups, GFP+ cells around plaques had a less ramified morphology, with blunted processes extending around and into the immunopositive amyloid core. There were no significant differences in Tris-HCl buffer-soluble Aβ40 or Aβ42 between the two groups in cerebral cortex or hippocampus. However, APOE3/3 recipients had less guanidine-soluble Aβ40 in cerebral cortex and hippocampus compared to APOE4/4 recipients. There was no significant effect of donor APOE genotype on levels of guanidine-soluble Aβ42 in cerebral cortex or hippocampus. Using real time polymerase chain reaction, the levels of TNF-α and macrophage migration inhibitory factor in cerebral cortex were significantly increased in APOE4/4 compared to APOE3/3 recipients, and levels of IL-10 were significantly lower in the APOE4/4 compared to APOE3/3 recipients. Donor APOE genotype did not promote differences in cerebral cortex expression levels of IL-6, IL-4, chemokine (C-C motif) ligand 2, chemokine (C-X3-C motif) ligand 1, and chemokine (C-C motif) ligand 8. Also there was no effect of donor APOE genotype on major histocompatibility complex II microglia expression. Overall, these findings indicated that APOE4/4 transplantation resulted in a more proinflammatory state in cerebral cortex and hippocampus than did APOE3/3 transplantation.
Conclusion	Bone marrow-derived APOE3 expressing cells were superior to those expressing APOE4 in their ability to mitigate the behavioral and neuropathological changes in APP/PS1 mouse model of AD.
Amniotic stem cells (AMSCs)
Reference	Kim et al. (2013)
Number of animals, gender, ages, and treatment	Tg2576 mice were used to evaluate the effect of AMSC transplantation. Adult female APPswe mice. 15–16 months of age, were used for the behavioral studies and for pathological analysis at 12 weeks after transplantation (n = 8/group). Also adult male APPswe mice. 12–13 months of age, were used for additional pathological analysis at 1 week after transplantation (n = 8/group). To evaluate the immunomodulatory effects of AMSCs on AD pathology, 3xTg AD mice, 6–7 months of age, were used (3 female mice at each time point). Normal human placentae (≥ 37 gestational weeks) were obtained after Cesarean section. Each placenta was carefully dissected and the tissue washed in PBS and then mechanically minced and digested with 0.5% collagenase IV for 30 minutes at 37°C. Harvested cells were cultured in α-MEM with 10% fetal bovine serum, antibiotics, 25 ng/mL fibroblast growth factor 4 and 1 µg/mL heparin. Cells were cultured in complete medium containing 25 ng/mL fibroblast growth factor 4 and 1 µg/mL heparin for 6 days at 37°C. For i.v., 200 µL of cell suspension (approximately 2 × 106 cells) was injected into the tail vein (AMSC-injected group). All behavioral tests were performed 6 weeks after AMSC transplantation. For immunohistological analysis, mice were euthanized at 1 week (n = 8/group) and 12 weeks (n = 8/group) after injection.
Comparison	WT littermates (n = 10) were used as normal control group. Also female 3xTg AD mice, 6–7 months of age, injected i.v. with PBS (PBS-injected group)
Functional outcomes	The water maze test (WMT) was performed 6 weeks after transplantation. In trial block 1, the normal control mice tended to find the hidden platform more quickly than the PBS- and AMSC-injected groups, while in trial block 2 the mean escape latencies of the 3 groups were not significantly different. The latency in the normal control group was significantly faster in trial block 3 than the PBS- and AMSC-injected groups. From trial block 3 to trial block 5, the AMSC-injected group showed a marked change in mean escape latency, indicating that impaired memory function was reversed by stem cell injection. Although there was no significant difference between the AMSC-injected group and the normal control group in trial blocks 4 and 5, there was a significant difference between the AMSC-injected group and the PBS-injected group for trial blocks 4 and 5. A probe test was performed on day 5, 24 hours after the last training trial. This involved removing the platform and recording the length of time each nouse spent within the zone previously occupied by the platform over a period of 60 seconds. The time spent in the zone was significantly less for the PBS-injected group than for the normal group and the AMSC-injected group. There was no difference between the normal group and the AMSC-injected group in terms of swimming velocity throughout the first four trial blocks, but there was a significant difference between the normal group and the PBS-injected group. There was no difference between the WT mice (normal control group), APPswe mice injected with PBS (PBS-injected group) or APPswe mice injected with AMSCs (AMSC-injected group) in terms of locomotor tests, the elevated plus maze test, light/dark transition tests, or open field test tests. Analysis of sections stained eith cresyl violet and thioflavin S showed that the number of Aβ plaques in the cortex and hippocampus was significantly lower in the AMSC-injected group (treated for 12 weeks with AMSCs) than in the PBS-injected group. The numbers of small (< 50 µm in diameter) and intermediate (50–100 µm in diameter) plaques were significantly lower in AMSC-injected group than in the PBS-injected group, whereas the number of large plaques (> 100 µm in diameter) was not different between the groups. The AMSC-injected group tended to show a positive correlation between mean escape latency in the WMT and the number of amyloid plaques (P < 0.06), but no such correlation was found in the PBS-injected group. At 1 weeks post-injection, the number of Iba-1+ microglia was significantly greater in the AMSC-injected group compared with the PBS-injected group, whereas the number of Iba-1+ microglia was not different beween the two groups at 12 weeks post-injection. The area of Iba-1+ microglia was significantly higher in the AMSC-injected group than in the PBS-injected group at 1 week post-injection, but significantly lower at 12 weeks post-injection. These findings suggested that AMSCs were able to recruit microglial cells during the initial acute stage after transplantation in the AD mouse model, and after the initial stage AMSCs maintained a lower number of resident microglial cells despite the proinflammatory environment. This supports an immunosuppresive role for AMSCs. At 1 week post-injection, the levels of mRNA expression of the proinflammatory cytokines IL-1 and TNF-α in the brain were significantly lower in the AMSC-injected group than in the PBS-injected group, whereas the levels for the anti-inflamatory cytokines IL-10 and transforming growth factor-β were significantly higher in the AMSC-injected group than in the PBS-injected group. At 12 weeks post-injection, the levels of mRNA expression of IL-1 and TNF-α in the brain were not significantly different between the two groups, whereas the levels for IL-10 and transforming growth factor-β were significantly higher in the AMSC-injected group than in the PBS-injected group. At 1 week post-injection, the levels of Aβ-degrading enzymes, including insulin-degrading enzyme and matrix metalloprotein-9, were significantly higher in the AMSC-injected group than in the PBS-injected group.
Conclusion	At 6 weeks after treatment with AMSCs, AD mice showed improved spatial learning which was significantly correlated with fewer Aβ plaques in the brain. The level of proinflammatory cytokines IL-1 and TNF-α was lower and that of anti-inflammatory cytokines IL-10 and transforming growth factor-β was higher in AMSC-injected mice than in PBS-injected mice.