Mesenchymal stem cells increase hippocampal neurogenesis and ...

Molecular Psychiatry (2010) 15, 1164–1175 & 2010 Macmillan Publishers Limited All rights reserved 1359-4184/10 www.nature.com/mp

ORIGINAL ARTICLE

Mesenchymal stem cells increase hippocampal neurogenesis and counteract depressive-like behavior M Tfilin1,2, E Sudai2, A Merenlender2, I Gispan2, G Yadid2 and G Turgeman1 1 Department of Molecular Biology, Ariel University Center of Samaria, Ariel, Israel and 2Neuropharmacology Section, the Mina and Everard Goodman Faculty of Life Sciences and the Leslie and Susan Gonda (Goldschmied) Multidisciplinary Brain Research Center, Bar-Ilan University, Ramat-Gan, Israel

Adult bone marrow-derived mesenchymal stem cells (MSCs) are regarded as potential candidates for treatment of neurodegenerative disorders, because of their ability to promote neurogenesis. MSCs promote neurogenesis by differentiating into neural lineages as well as by expressing neurotrophic factors that enhance the survival and differentiation of neural progenitor cells. Depression has been associated with impaired neurogenesis in the hippocampus and dentate gyrus. Therefore, the aim of this study was to analyze the therapeutic potential of MSCs in the Flinders sensitive line (FSL), a rat animal model for depression. Rats received an intracerebroventricular injection of culture-expanded and 1,10 dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate (DiI)-labeled bone marrow-derived MSCs (105 cells). MSC-transplanted FSL rats showed significant improvement in their behavioral performance, as measured by the forced swim test and the dominant–submissive relationship (DSR) paradigm. After transplantation, MSCs migrated mainly to the ipsilateral dentate gyrus, CA1 and CA3 regions of the hippocampus, and to a lesser extent to the thalamus, hypothalamus, cortex and contralateral hippocampus. Neurogenesis was increased in the ipsilateral dentate gyrus and hippocampus of engrafted rats (granular cell layer) and was correlated with MSC engraftment and behavioral performance. We therefore postulate that MSCs may serve as a novel modality for treating depressive disorders. Molecular Psychiatry (2010) 15, 1164–1175; doi:10.1038/mp.2009.110; published online 27 October 2009 Keywords: mesenchymal stem cells; depression; dentate gyrus; neurogenesis; flinders sensitive line

Introduction Major depressive disorders present a challenge for clinicians, as studies indicate that approximately 50% of these patients fail to respond to treatment with selective serotonin reuptake inhibitors, and approximately 65% fail to achieve remission.1 Moreover, many patients who achieve remission still suffer from residual symptoms of depression.2 These findings call for the development of different treatment modalities that will potentially overcome current treatment limitations.3 In this study we explored a novel stem cell therapy approach for treating major depressive disorders that is based on mesenchymal stem cell transplantation. Bone marrow-derived mesenchymal stem cells (MSCs) are easily isolated and expanded from bone marrow and even from adipose tissues. MSCs are pluripotent adult stem cells that differentiate into various mesenchymal lineages (for example, bone, cartilage and adipose cells).4 However, MSCs also Correspondence: Dr G Turgeman, Department of Molecular Biology, Ariel University Center of Samaria, Ariel 40700, Israel. E-mail: [email protected] Received 27 March 2009; revised 21 August 2009; accepted 18 September 2009; published online 27 October 2009

show differentiation plasticity into other non-skeletal cell types, including neural cells (neurons and glial cells).5 After transplantation, MSCs can engraft into brain tissue and differentiate in situ into neurons and glial cells.6 In addition to their neural differentiation ability, MSCs are known for their ability to promote the neurogenesis of primary neural progenitors and survival of neural cells by expressing neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), b-nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1).7 Moreover, transplantation of MSCs in the central nervous systems prevents apoptosis and promotes neurogenesis (proliferation and differentiation) of ‘host’ neural cells and stem cells in the engrafted site.7–9 MSCs were therefore suggested as candidates for treating a variety of neurodegenerative diseases, in particular Parkinson’s disease, multiple sclerosis, cerebral hemorrhage and brain cancer.10–13 Neurogenesis is a continuous process of the formation of new neurons, which occurs in the dentate gyrus in the hippocampus throughout life. Growing evidence has recently linked impaired neurogenesis in the hippocampus with affective disorders, including depression.14,15 In humans, morphological atrophic changes in the hippocampus were also observed.16 Impaired neurogenesis was reflected in

MSCs increase hippocampal neurogenesis M Tfilin et al

decreased survival, proliferation and differentiation of neural lineages in the dentate gyrus as observed in animal models.14,17 Molecular analysis has further correlated these observations with decreased expression of neurotrophic factors, such as BDNF and fibroblast growth factor-2 (FGF-2).18,19 Although doubts have been raised regarding the actual role of hippocampal neurogenesis in the etiology of major depression, its role in mediating hippocampal aspects of depression is recognized.20 Furthermore, some antidepressants were found to elicit hippocampal neurogenesis, thus indicating its importance in mediating the therapeutic effect of antidepressants.14,21 Similarly, injection of human MSCs into the hippocampus of normal mice resulted in a significant increase in neurogenesis, originating primarily from host neural stem cells.8 Moreover, the engraftment of neural stem cells or MSCs in the hippocampus and dentate gyrus resulted in increased neurogenesis and induced an improved behavioral phenotype in prenatal heroin exposure and in apolipoprotein E knockout mice, respectively.22,23 In this study, we therefore analyzed the possible therapeutic potential of MSCs in depression. Bone marrow-derived MSCs have been shown to engraft in the hippocampus and dentate gyrus of Flinders Sensitive Line (FSL) rats, a rodent model for depression. FSL rats show normal cognitive behavior, but reduced appetite, psychomotor behavior, sleep and immune abnormalities similar to depressed individuals.17,24,25 FSL rats have been successfully used as a screening model for various antidepressants over the years.25 In this study we show that engraftment of MSCs into the hippocampus and dentate gyrus of FSL rats resulted in enhanced neurogenesis and affected behavioral changes, reversing the depressive-like phenotype normally presented by these animals.

Materials and methods Animals Male FSL rats (230–260 g) were obtained from D Overstreet, North Carolina, bred at Bar-Ilan University and housed two rats per cage under conditions of constant temperature (22 1C) and humidity (50%) in a 12:12 h light:dark cycle with lights off at 0600 h. Food and water were provided ad libitum. All animal studies were approved by the animal care and use committee of the University and were performed in accordance with the guidelines of the National Institute of Health. Bone marrow-derived mesenchymal stem cell (MSC) isolation and expansion Mesenchymal stem cells were isolated from the bone marrow of male FSL rats as previously described.26 In brief, after the sacrifice of male FSL rats, the tibias and femurs were removed and cleaned of connective tissue. Marrow was flushed out of the cut bones after removal of the epiphysis and suspended in Dulbecco’s modified Eagle’s medium (DMEM; Biological

Industries, Beit Haemek, Israel) supplemented with 10% fetal bovine serum (Biological Industries), 100 units ml1 of penicillin (Biological Industries), 100 mg ml1 of streptomycin (Biological Industries) and 2 mM of L-glutamine (Biological Industries). Marrow cells were separated and suspended by repeated passage through 19, 20, 21, 23 and 25 G syringe needles. Suspended marrow cells (108) were plated in 100 mm2 dishes and cultured under conditions of 37 1C and 5% carbon dioxide. The nonadherent cells were removed at 24 and 48 h after plating. MSCs were expanded in culture for 3–8 passages. The medium was changed twice weekly.

1165

In vitro mesenchymal differentiation of mesenchymal stem cells (MSCs) Mesenchymal stem cells were plated in 60 mm2 cell culture plates (2 105 cells per plate) supplemented with appropriate differentiation media for the induction of MSC differentiation into different mesenchymal cell phenotypes. For osteogenic differentiation, MSCs were cultured in modified Eagle’s medium-a (Biological Industries) supplemented with 10% fetal bovine serum (Biological Industries), 50 mg ml1 of ascorbic acid (Sigma, Rehovot, Israel ), 10 mM of bglycerol 2 phosphate (Sigma) and 107 M of dexamethasone (Sigma) for 4 weeks. MSC cultures were stained for mineralization with a 40-mM alizarin red (Sigma) solution (pH 4.1) to confirm osteogenic differentiation. For induction of adipogenic differentiation, MSCs were cultured in DMEM high glucose (Biological Industries) supplemented with 10% fetal bovine serum (Biological Industries), 0.5 mM of 1-methyl-3-isobutylxanthine (Sigma), 107 M of dexamethasone (Sigma) and 10 mM of insulin (Sigma) for 4 weeks. Adipocytes were detected by oil red-O (Sigma) staining (0.5%), which stained the intracellular lipid droplets. Chondrogenic differentiation was induced in high glucose (4 g l1) DMEM (Biological Industries) supplemented with 3% fetal bovine serum (Biological Industries), 10 ng ml1 of transforming growth factor-b 1 (Biological Industries), 107 M of dexamethasone (Sigma) and 50 nM of ascorbic acid (Sigma) for 4 weeks. An Alcian blue (Sigma) solution (10 mg ml1, pH 1) was applied for the detection of chondrogeneic cells. Assessment of the neurogenic potential of mesenchymal stem cells (MSCs) The mRNA expression of BDNF, FGF-2 and IGF-1 in bone marrow-derived MSCs was assessed using realtime PCR (see below) to analyze the potential of these cells to support neurogenesis. Furthermore, a bioassay was constructed to assess the potential of MSCsecreted factors to support neurosphere development in a rat neonatal cortical cell culture. In brief, MSCs were incubated with conditioning medium (serumfree high glucose DMEM) for 24 h. Harvested conditioned medium was filtered using a 0.2-mm sterile filter supplemented with 1% B27 supplement (Invitrogen, Carlsbad, CA, USA) and was used to Molecular Psychiatry


1166

culture rat neonatal cortical cells in 24-well plates (104 cells per well). A rat neonatal cortical cell suspension was obtained after incubation of dissected cortices obtained from the sacrificed neonatal Sprague-Dawley rats (Harlan, Jerusalem, Israel) with 0.25% trypsin (Biological Industries) at 37 1C for 10 min. Rat neonatal cortical cells that were cultured in MSC-conditioned medium were assayed after 5 days of culture for the number of neurospheres that developed in the culture and were counted under a microscope. Neurospheres were further analyzed for the presence of differentiating neuronal and glial cells using immunostaining for nestin, glial fibrillary acidic protein (GFAP) and doublecortin (DCX; see below). Rat neonatal cortical cells were also cultured in NIH3T3 fibroblast-conditioned medium and with non-conditioned medium consisting of serum-free DMEM (high glucose) supplemented with 1% B27 as controls. Mesenchymal stem cell (MSC) fluorescence labeling Suspended MSCs were labeled with 1,10 -dioctadecyl3,3,30 ,30 -tetramethylindocarbocyanine perchlorate (DiI, Sigma) fluorescent dye as described previously,27 to trace the migration of the cells in the brain. In brief, trypsinized MSCs were suspended in phosphate buffered saline (106 cells ml1) in the presence of DiI at a final concentration of 1 mg ml1 and incubated for 10 min at 37 1C followed by 5 min at 4 1C and finally washed thrice with PBS. Mesenchymal stem cell (MSC) transplantation into Flinders sensitive line (FSL) rats Approximately 105 DiI-labeled rat MSCs in a 10-ml volume were stereotactically injected into the left lateral ventricle of anesthetized FSL rats (coordinates referring to bregma A = 0.8, L = þ 1.5 and V = 4.2). Control animals were injected with vehicle only. The rats were tested for behavioral changes at 2–3 weeks after injection. Some animals were then sacrificed by decapitation and their brains were removed for RNA transcription analysis, whereas other animals underwent intracardial perfusion for immunofluorescence analysis. Behavioral assays Several behavioral tests were applied to evaluate the depressive-like behavior in FSL rats after MSC transplantation as detailed below. Modified forced swim test. Flinders sensitive line rats were placed individually in plastic cylinders (height 40 cm, diameter 18 cm) filled with water in accordance with the animal’s weight and pre-warmed to 23±1 1C. The animals were left in the cylinder for 5 min. The total immobility duration of the rats was measured.28 Dominant–submissive relationship (DSR) paradigm. The DSR paradigm uses a single apparatus with two chambers connected by a tunnel that has a feeder with sweetened milk at the midpoint.29,30 FSL rats were randomly paired. In each pair, the animals were placed in the opposite chambers of the DSR apparatus

Molecular Psychiatry

and were allowed to compete for the milk for a 5-min period after 30 s of acclimation. The test was repeated daily for 10 days before the MSC transplantation. The drinking duration was measured for each animal in each test. In each pair the animal that had a lower drinking duration was injected with MSCs to the lateral ventricle, whereas its counterpart was injected with the vehicle. At 10 days after the operation, the same pairs were tested again in the DSR paradigm for an additional 7 days. Open field. The open field apparatus consisted of a black plastic box measuring 90 90 30 cm (width length height). The rats were placed near the midpoint of the open field apparatus for a period of 5 min and the total distance they walked was measured. Real-time PCR Total RNA was extracted from MSC cultures or dissected hippocampi of FSL rats using the Versagene RNA isolation kit (Gentra Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol for the quantitative detection of mRNA levels of various genes. RNA was quantified according to absorbance at 260 nm measured using spectrophotometry. The quality of the RNA was confirmed by running 0.5 mg of the RNA in 1% agarose gel electrophoresis for 30 min. Complementary DNA (cDNA) was transcribed from 1 mg RNA using the Stratascript 5 reverse transcription kit (Stratagene, La Jolla, CA, USA) and was used as a template for real-time PCR analysis. cDNA samples were analyzed for the expression of the following rat genes: BDNF, FGF-2, IGF-1, DCX, microtubule-associated protein 2 (MAP-2), GFAP, stromal cell-derived factor 1 (SDF-1), CXC chemokine receptor 4 (CXCR4) and hypoxanthine-guanine phosphoribosyl transferase (HPRT) as a reference gene by real-time PCR using the Stratagene MX3000P realtime PCR machine. Primers were synthesized commercially (Sigma) and are listed in Table 1. Primers were designed to include exon–exon junctions, thereby yielding no genomic product, or on the following exons, thereby yielding a genomic PCR product of at least 10 times larger than the cDNA product, to distinguish between the cDNA product and possible genomic products. Amplification was performed using DyNAmo SYBR Green qPCR Kit (Finnzymes, Espoo, Finland) under the following conditions: 30 s denaturation at 95 1C, followed by 1 min annealing at 60 1C and then followed by 1 min extension at 72 1C for a total of 35 cycles. In addition to cDNA samples, RNA samples that were not reverse transcribed were used as controls. PCR product specificity was confirmed using melting curve analysis and agarose gel electrophoresis. Immunofluorescence Immunoassays for the detection of the proteins nestin, DCX, GFAP, proliferating cell nuclear antigen (PCNA) and BDNF were performed on cell cultures and on frozen sections of FSL rat brains. All primary


Table 1

1167

Real time PCR primers list

Primer name

Rat HPRT Rat MAP-2 Rat FGF-2 Rat GFAP Rat PCNA Rat CXCR4 Rat SDF-1 Rat doublecortin Rat BDNF

Sequence

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

50 -AGGCCAGACTTTGTTGGATT-30 50 -GCTTTTCCACTTTCGCTGAT-30 50 -CAAACGTCATTACTTTACAACTTGA-30 50 -CAGCTGCCTCTGTGAGTGAG-30 50 -AGCGGCTCTACTGCAAGAAC-30 50 -GCCGTCCATCTTCCTTCATA-30 50 -GGTGGAGAGGGACAATCTCA-30 50 -CCAGCTGCTCCTGGAGTTCT-30 50 -AGGACGGGGTGAAGTTTTCT-30 50 -CAGTGGAGTGGCTTTTGTGA-30 50 -GTGCAGCAGGTAGCAGTGAC-30 50 -CCAGGATTACCAACCCATTG-30 50 -TTTCACTCTCCGTCCACCTC-30 50 -ATCTGAAGGGCACAGTTTGG-30 50 -GGGGATTGTGTACGCTGTTT-30 50 -CGACCAGTTGGGATTGACAT-30 50 -GCGGCAGATAAAAAGACTGC-30 50 -GCCAGCCAATTCTCTTTTTG-30

cDNA product size (bp)

Genomic product size (bp)

119

None

122

21 259

183

42 506

233

2008

173

1621

250

2447

251

3075

244

7649

238

None

Abbreviations: BDNF, brain-derived neurotrophic factor; CXCR4, CXC chemokine receptor 4; FGF-2, fibroblast growth factor-2; GFAP, glial fibrillary acidic protein; HPRT, hypoxanthine-guanine phosphoribosyl transferase; MAP-2, microtubuleassociated protein 2; PCNA, proliferating cell nuclear antigen; SDF-1, stromal cell-derived factor 1.

antibodies were purchased from Abcam Inc. (Cambridge, MA, USA) and secondary antibodies were purchased from KPL Inc. (Gaithersburg, MD, USA). Specifically, FSL rats were anesthetized and perfused transcardially with 10 U ml1 of heparin followed by PBS (pH 7.4) and finally by 4% paraformaldehyde (Sigma) in 0.1 M of phosphate buffer (pH 7.4). Brains were removed, post fixed overnight and equilibrated in phosphate buffered 30% sucrose. Free-floating, 20– 40 mm thick coronal hippocampal sections were prepared in a cryostat and stored in 0.1% sodium azide (Sigma) at 4 1C before immunofluorescence. Frozen tissue sections and cultured cells were washed with PBS, incubated in 0.1% Triton X-100 (Sigma) for 5 min and then blocked for 45 min with blocking solution (0.1% Triton X-100 and 5% bovine serum albumen in PBS). Samples were then incubated with the following primary antibodies for 1 h at room temperature: rabbit polyclonal anti-BDNF (6.6 ng ml1), mouse monoclonal anti-PCNA (1.4 mg ml1), mouse monoclonal anti-nestin (56 mg ml1), rabbit polyclonal anti-DCX (1 mg ml1) and rabbit polyclonal anti-GFAP (1:100 dilution). Incubation with the appropriate secondary antibody (fluorescein isothiocyanate goat anti-rabbit and goat anti-mouse) at a 1:100 dilution was followed for 1 h at room temperature. Between incubations, samples were washed thrice with PBS. Sample assay results were visualized using a fluorescence microscope (TE2000-U, Nikon, Tokyo, Japan). Statistics All graphs and statistical analyses were prepared using GraphPad Prism 4 software (La Jolla, CA, USA). Statistical tests applied for statistical significance

were a two-tailed Student’s t-test and analysis of variance. The Spearman’s test was applied for calculating correlations. Data are presented as mean±s.e.

Results Rat bone marrow mesenchymal stem cells (MSCs) express neurotrophic factors and promote neurogenesis in vitro Rat bone marrow MSCs were isolated and expanded in culture for five passages. During in vitro expansion the MSCs maintained their pluripotency as shown by their differentiation along the osteogenic, chondrogenic and adipogenic lineages upon induction with the appropriate induction media (data not shown). Interestingly, we observed that expanded non-differentiated MSCs expressed varying mRNA levels of several neurotrophic factors, including IGF-1, BDNF and FGF-2 (Figure 1a). We assessed the ability of conditioned medium collected from MSC cultures to support in vitro neurogenesis of rat neonatal primary cortical neuroprogenitors to evaluate the paracrine influence of factors secreted by MSCs on neurogenesis. MSC-conditioned medium collected for 24 h and consisting of serum-free DMEM was able to support the growth and development of neurospheres originating from neuroprogenitors present in the neonatal rat cortical cell culture. The neurospheres that developed in vitro consisted of proliferating neuroprogenitors differentiating into both glial and neuronal lineages as shown by the presence of cells expressing nestin, DCX and GFAP inside the neurospheres (Figure 1b). Such neurospheres can otherwise be obtained from cultures of rat neonatal cortical cells Molecular Psychiatry


1168

Figure 1 In vitro neurogenic potential and in vivo engraftment of mesenchymal stem cells (MSCs). (a) Cultured rat bone marrow-derived MSCs express basal mRNA levels of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1(IGF-1) and fibroblast growth factor-2 (FGF-2), as detected using real-time PCR. Results are presented as relative expression compared with hypoxanthine-guanine phosphoribosyl transferase (HPRT). The bars represent mean±s.e. (b) Conditioned medium from MSC cultures can support the in vitro growth and development of neurospheres originating from neuroprogenitors present in a culture of rat neonatal cortical cells. Active neurogenesis is occurring inside the neurospheres as reflected by the detection of cells positive for nestin, glial fibrillary acidic protein (GFAP) and doublecortin (DCX) using immunofluorescence (magnification 200). (c) MSCs engraft into Flinders sensitive line (FSL) rat brains after injection into the lateral ventricle. 1,10 -dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate (DiI)-labeled MSCs injected into the lateral ventricle were found to engraft (indicated by arrows) mainly into the hippocampus CA3, CA1 regions (CA) and dentate gyrus (DG), magnification 200. (d) Clusters of engrafted MSCs were also found in the thalamus adjacent to blood vessels and aligned in rows. Some engrafted cells (DiI-positive) also expressed DCX as detected using immunofluorescence (magnification 200).



1169

only in the presence of neurotrophic factors, such as FGF-2 and EGF.31,32 In contradistinction, non-conditioned medium and conditioned medium collected from NIH3T3 fibroblasts failed to support the growth and development of neurospheres compared with MSC-conditioned media (3±1 and 103.5±19.1, respectively, P < 0.05, Student’s t-test). It should be noted that under these culture conditions MSCs did not differentiate into any neural lineage and were found to be negative for the expression of neural markers such as GFAP, DCX and microtubule-associated protein 2 (data not shown). After injection into the lateral ventricle, mesenchymal stem cells (MSCs) migrate and engraft mainly into the hippocampus and dentate gyrus of Flinders sensitive line (FSL) rats To study the engraftment patterns of MSCs in depressed animals, 105 DiI-labeled MSCs were injected into the left lateral ventricle of FSL rats. At 2 weeks after transplantation, labeled cells were observed to engraft mainly into the ipsilateral dentate gyrus mainly in the granular cell layer, dentate hilus and the CA1–CA3 regions of the hippocampus, adjacent to the pyramidal cell layer (Figure 1c). Engrafted cells were found to a much lesser extent in the contralateral dentate gyrus, hippocampus and ipsilateral hypothalamus. Cells engrafted to the hypothalamus are often observed aligned with or along blood vessels appearing to be migrating. Some engrafted MSCs expressed DCX, a marker for migrating neurons (Figure 1d). Mesenchymal stem cell (MSC) injection into the lateral ventricle of Flinders sensitive line (FSL) rats reverses their depressive-like behavior At 2 weeks after injection of MSCs into the lateral ventricle, injected rats showed a significant reduction in their immobility duration in the forced swim test compared with vehicle (control)-injected FSL rats (Figure 2a). In the DSR paradigm, randomly paired FSL rats failed to show significant dominant–submissive characteristics. Among the randomly selected pairs of rats in each pair, the rat with the lower drinking duration was injected with MSCs into the lateral ventricle and its counterpart was injected with vehicle (control). At 17 days after injection, FSL pairs showed significant dominant–submissive relations, in which MSC-injected animals showed dominant behavior over their counterparts (Figure 2b). The effect of MSC injection on the locomotion of FSL rats at 2 weeks after injection was assessed in an open field test (n = 5). A nonsignificant increase was observed in MSC-injected animals (1145±298 cm) when compared with controls (890±136 cm). Moreover, no significant correlation was observed between the results of the open field and those of the swim test (r = 0.52, P = 0.13) and of the DSR paradigm (r = 0.59, P = 0.12).

Figure 2 Mesenchymal stem cell (MSC) injection into the lateral ventricle reverses depressive-like phenotype. Flinders sensitive line (FSL) rats injected with 105 MSCs were analyzed for behavioral changes at 2 weeks after injection by the forced swim test (n = 8) and the dominant–submissive relationship (DSR) paradigm (n = 5). Results are presented in graphs as mean±s.e. *Statistically significant difference (P < 0.05 analysis of variance (ANOVA)) between MSCtreated FSL rats and control animals (vehicle). (a) Results of the forced swim test show a significant reduction in immobility duration in MSC-injected animals compared with control animals injected with vehicle. (b) In the DSR paradigm, FSL rat pairs failed to show a significant dominant–submissive relation. In each pair, the lower scoring animal was injected with 105 MSCs, whereas its counterpart was injected with vehicle. At 17 days after injection, significant relations were established favoring the MSC-injected animals as dominant.

Mesenchymal stem cells (MSCs) enhance neurogenesis in the hippocampus and dentate gyrus after engraftment As most of the injected MSCs were engrafted into the ipsilateral hippocampus of FSL rats, our next aim was to test the effect of this engraftment on hippocampal neurogenesis. Neurogenesis was therefore analyzed using immunofluorescence staining of dorsal hippocampus sections for the detection of the proliferation markers PCNA, DCX, GFAP and BDNF. Immunostaining revealed more PCNA-positive nuclei in the Molecular Psychiatry


1170

Figure 3 Mesenchymal stem cell (MSC) engraftment enhances hippocampal neurogenesis. At 2 weeks after transplantation of MSCs, frozen sections of the hippocampus and dentate gyrus were analyzed for MSC engraftment (detected using 1,10 dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate (DiI) labeling) and neurogenesis. Proliferating cells were shown by immunofluorescence detection of proliferating cell nuclear antigen (PCNA). Results show an increased number of PCNA-positive cells in the ipsilateral hippocampus (indicated by arrows) compared with the contralateral hippocampus (a) magnification 200. Insert shows a positive nucleus at 400 magnification. (b) Immunofluorescence detection of doublecortin (DCX)-positive cells revealed abundant DCX-positive cells in the granular cell layer of the ipsilateral dentate gyrus compared with the contralateral dentate gyrus (magnification 200). Similarly, higher numbers of brain-derived neurotrophic factor (BDNF)-expressing cells were detected in the subgranular zone and higher numbers of glial fibrillary acidic protein (GFAP)-expressing cells were detected in the dentate hilus of the ipsilateral dentate gyrus compared with the contralateral (c, d, respectively, magnification 200). Expression of these markers seems to correspond to the distribution of engrafted MSCs (DiI). Inserts (b– d) show engrafted MSCs positive for both DiI labeling and for each marker (magnification 400). In control animals neurogenesis resembled that of the contralateral hippocampus and dentate gyrus of MSC-injected animals.

ipsilateral hippocampus (stratum radiatum) than in the contralateral hippocampus or in control vehicleinjected animals (Figure 3a). Although PCNA-positive nuclei were not observed in the dentate gyrus at 2 weeks after treatment, an increase was observed in DCX-expressing cells in the granular cell layer of the ipsilateral dentate gyrus compared with the contralateral dentate gyrus and control animals (Figure 3b). Similarly, an increase in BDNF-expressing cells was observed in the subgranular zone in the dentate gyrus and an increase in GFAP-expressing cells was observed in the dentate hilus (Figures 3c and d). It is important to note that although some engrafted MSCs were also found to express the neural markers Molecular Psychiatry

DCX and GFAP (Figures 3b–d), the majority of engrafted DiI-labeled cells did not. Quantitative real-time PCR analysis performed on total hippocampal RNA collected from treated animals revealed a significant (P < 0.05) decrease in mRNA expression of GFAP, stromal cell-derived factor 1 and CXCR4 in the ipsilateral hippocampus of MSC-treated animals compared with controls (Figures 4a and b). A smaller decrease was also observed in IGF-1 and PCNA mRNAs, whereas microtubule-associated protein 2 and BDNF remained unchanged (Figures 4a and b). A positive correlation was observed between GFAP and PCNA mRNA expression (r = 0.93, P < 0.001). In contradistinction,


Figure 4 Differential gene expression in hippocampi of mesenchymal stem cell (MSC)-injected Flinders sensitive line (FSL) rats and controls. Total RNA was extracted from the ipsilateral hippocampi of MSC-injected FSL rats and control animals (vehicle) at 2 weeks after injection. Quantitative real-time PCR was performed on complementary DNA (cDNA) samples prepared from these RNA sample for the detection of the following genes: glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2), proliferating cell nuclear antigen (PCNA), brainderived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF-2), CXCR4, stromal cell-derived factor 1 (SDF-1) and doublecortin (DCX). Results are presented as mean±s.e. for the relative gene expression compared with hypoxanthine-guanine phosphoribosyl transferase (HPRT; n = 4–5). *Significant difference (P < 0.05 analysis of variance (ANOVA)) between MSC-treated FSL rats compared with controls (vehicle injected).

a significant increase in the expression of FGF-2 and DCX mRNAs was observed in MSC-treated animals as compared with controls (Figure 4b). Moreover, a significant correlation was found between FGF-2 mRNA levels in the ipsilateral hippocampus and the scores obtained by the animals in both the forced swim test (r = 0.82, P < 0.005) and the DSR test (r = 0.89, P < 0.05).

Discussion Bone marrow-derived MSCs are known to secrete neurotrophic factors and promote the survival, proliferation and differentiation of neural cells

in vitro.33,34 It has also been suggested to be the main mechanism by which MSCs can mediate recovery of neural damage.35 Our in vitro data confirm this notion, showing the expression of several neurotrophic factors. Moreover, we showed that conditioned medium from cultured MSCs can support the growth and differentiation of neonatal rat neuroprogenitors and form differentiating neurospheres in vitro (Figure 1). Similar support of neurosphere formation and propagation was reported by co-culturing with MSCs or with MSC-driven conditioned medium.33,36,37 Although our data suggest mainly the involvement of secreted factors in supporting neurogenesis, the involvement of a direct interaction between MSCs and neuronal cells should not be excluded.38 Injection of MSCs into the lateral ventricle of FSL rats resulted in their engraftment mainly to the hippocampus and dentate gyrus, with smaller amounts of cells localized in the hypothalamus at 2 weeks after injection. Specific migration of MSCs in the brain is well documented for lesion sites in the brain,39,40 as well as in gliomas.41–43 However, tracking MSCs in normal brains shows a variety of engraftment sites, ranging from the cerebellum to the forebrain.44,45 Sites in which active neurogenesis occurs in adulthood, such as the hippocampus, subventricular zone and olfactory bulb, are often included in these preferred engraftment sites.6,46–48 In this respect the results of this study are not surprising. However, the non-proportional engraftment of MSCs in the hippocampus and dentate gyrus suggests a possible preference to these sites in ‘depressed’ FSL rats. As most studies relate MSC migration and engraftment with the expression of chemokine receptors and adhesion molecules,45,47 it would be interesting to test their role in the engraftment of MSCs in ‘depressed’ FSL rats in the future. Engraftment of MSCs to the dentate gyrus in this study resulted in altered neurogenesis at this site, as observed using immunofluorescence analysis (Figure 3). Increased DCX-expressing cells in the granular cell layer, BDNF-expressing cells in the subgranular zone and GFAP-expressing cells at the dentate hilus indicate active neurogenesis that takes place in the dentate gyrus. Similar observations indicating increased neurogenesis were reported by Monuz et al.8 after implantation of human MSCs into the dentate gyrus of mice and by Ben Shaanan et al.23 after transplantation of neural progenitors into the hippocampus of prenatally heroin-exposed mice. Both studies also reported that neurogenesis was maintained mainly by endogenous cells, with a minor contribution of engrafted stem cells to the newly formed neurons and glial cells. Our data confirm these observations, as the majority of the engrafted MSCs failed to express markers typical of the newly formed cells (that is, GFAP and DCX). Similar observations outside the hippocampus in stroke and even in spinal injury have shown that MSCs mediate neurogenesis mainly by influencing endogenous neuroprogenitors.9,49 These observations imply that

1171



1172

MSCs mediate their therapeutic effect in the nervous system mainly by interacting with local neuroprogenitors, rather than by differentiating into neural progeny. PCNA-expressing cells were not detected in the dentate gyrus at 2 weeks after transplantation. Moreover, PCNA mRNA expression in the total hippocampus was reduced in MSC-treated animals compared with controls (Figure 4a). However, this observation does not exclude the possibility that PCNA mRNA expression was upregulated at earlier time points and reduced thereafter. Indeed, Monuz et al.8 showed an increase in hippocamapal mRNA expression of PCNA at 2 and 3 days, which diminished at 7 days after implantation of human MSCs into the dentate gyrus of mice. The reduction in PCNA mRNA after 2 weeks suggests reduced proliferation in the treated hippocampi rather than merely a lack of increase in proliferating cells. This observation confirms previous studies that reported increased 5-bromodeoxyuridinepositive cells in the hippocampus of ‘depressed’ FSL rats compared with controls.17 In this animal model, the number of 5-bromodeoxyuridine-positive cells in the hippocampus was reduced after treatment of FSL rats with the antidepressant ecitalopram.50 However, in this study we also found that the reduction in PCNA mRNA was significantly correlated with a reduction in GFAP mRNA expression, indicating that the reduction in PCNA expression is probably because of a reduction in GFAP-expressing cells and not in neuronal cells. Similarly, the observed reduction in the chemokine stromal cell-derived factor 1 and its receptor CXCR4 (Figure 4b) can be explained by the reduction in GFAPexpressing glial cells, as they too express these chemokines.51,52 In contradistinction to this reduction in GFAP expression, DCX mRNA expression was significantly increased in MSC-treated hippocampi compared with controls (Figure 4b), indicating an increased number of newly formed neurons in the hippocampus. Similarly, the mRNA of FGF-2, a potent modulator of hippocampal neurogenesis,53,54 increased significantly in MSCtreated hippocampi compared with controls (Figure 4b). In contradistinction to FGF-2, IGF-1 mRNA expression was slightly reduced in MSC-treated hippocampi (Figure 4b). Such a reduction in IGF-1 mRNA expression in the neocortex was previously correlated with dominant behavior in a social dominant– submissive gene expression study.55 Similarly, MSCtreated FSL rats showed dominant behavior over control FSL rats after treatment (Figure 2c). These results indicate that MSC engraftment does not merely enhance neurogenesis in the dentate gyrus, but also alters total hippocampal neurogenesis by promoting the formation of new neurons over GFAP-expressing glial cells. Such differential neurogenesis has been reported previously for the effect of WNT and bone morphogenetic protein (BMP) signaling cascades.56 WNT signaling activating factors and bone morphogenetic protein inhibitors promote the production of new neurons in the dentate gyrus


and hippocampus,57,58 whereas bone morphogenetic protein promotes glial differentiation.59. It is therefore possible that preferential activation of WNT signaling or alternatively blocking of bone morphogenetic protein signaling are involved in the observed alteration in neurogenesis in this study. In this study, MSC injection into the lateral ventricle resulted in a dramatic change in the ‘depressive’ behavior of FSL rats when compared with controls at 14–17 days after injection (Figure 2). The forced swim test, a conventional assay for assessment of FSL rat depressive-like behavior,25,60 showed a significant reduction in immobility time after MSC treatment. Similarly, it was previously shown that monoaminergic grafts originating from embryonic stem cells decreased the immobility duration in forced swim tests after engraftment to the prefrontal cortex of Sprague-Dawley rats61. We also applied the dominant–submissive social behavior paradigm, in which the relative behavioral tendency toward submissiveness or dominance reflects the behavioral status on the axis between depression and mania.29,62 FSL rats were recently shown in this model to have a submissive phenotype that was abolished after electrical stimulation of the ventral tegmental area.30 Our results further show the establishment of dominant behavior in MSC-injected rats compared with submissive behavior observed in their vehicle-injected counterparts (Figure 2b). Both neuronal stem cells and bone marrow MSCs have previously been shown to improve cognitive impairment inflicted by cerebral ischemia.63–66 This effect was directly correlated with the engraftment of stem cells to the injury sites and with the induction of neurogenesis.66 More specifically, stem cell engraftment to the hippocampus has resulted in increased neurogenesis in the dentate gyrus and improvement of cognitive impairment in prenatally heroin-exposed mice and in models of Alzheimer’s disease.22,23,67,68 Nevertheless, this study shows the effect of stem cells on depressive-like behavior for the first time. The observed alterations in hippocampal neurogenesis resulting from MSC transplantation into the brain of FSL rats strongly suggest neurogenesis as the underlying mechanism for the observed changes in the depressive-like behavior shown by these animals. Nonetheless, fundamental support for this mechanism came from the finding that FGF-2 mRNA expression in the hippocampi of the FSL rats was significantly correlated with the behavioral tests scores in both the forced swim test and the DSR paradigm. FGF-2 is a potent neurogenic factor promoting early neurogenesis, supporting proliferation of neural stem cells and progenitors and enhancing the survival of mature neurons.69–72 Moreover, a reduction in FGF-2 expression and an increase in FGF receptor expression in the frontal cortex and hippocampus were observed in post-mortem human brain samples of patients suffering from major depression.73,74 Similarly, animal models further support the role of the FGF system in depression


and its potential involvement in antidepressant treatment.75,76 Intraventricular administration of FGF-2 was also shown to have an antidepressive-like effect in rats.77 However, although the theory identifying hippocampal neurogenesis as a cause of depression has long been propagated,78 doubts have recently been raised concerning its soundness.20,79 Nevertheless, regardless of the doubted role of hippocampal neurogenesis in the etiology of depression, there is wide recognition of its role in mediating the therapeutic effect of antidepressants.20,79–81 Our data also strongly suggest that the improvement in the depressive-like behavior of FSL rats implanted with MSCs is mediated by increased neurogenesis in the dentate gyrus and hippocampus. Finally, we conclude that MSC transplantation into the central nervous system can modulate neurogenesis in the hippocampus and reduce depressive-like behavior. Future studies should further analyze the long-term effect of MSC transplantation on behavior and neurogenesis and should explore the possible influence of MSCs modes of administration (that is, bilateral, repeated and systemic injections) in various animal models of depression. Taking into consideration the growing need for novel treatments for depression to address the high rates of resistance to current major depressive disorder treatment and the longitudinal residual symptoms in many post-treated patients, we propose stem cell-based therapy. This approach is directed toward attaining increased hippocampal neurogenesis as a potential novel strategy. As MSCs are relatively easy to isolate and expand in culture from various tissues (that is, bone marrow and adipose tissue), they may serve as preferable candidates for stem cell therapeutic modalities for depressive disorders.

Conflict of interest The authors declare no conflict of interest.

Acknowledgments We thank Dr Albert Pinhasov, Department of Molecular Biology, Ariel University Center of Samaria for his helpful comments and Mrs Esther Furman for editing this paper.

References 1 Trivedi MH, Rush AJ, Wisniewski SR, Nierenberg AA, Warden D, Ritz L et al. Evaluation of outcomes with citalopram for depression using measurement-based care in STAR*D: implications for clinical practice. Am J Psychiatry 2006; 163: 28–40. 2 Nierenberg AA, Keefe BR, Leslie VC, Alpert JE, Pava JA, Worthington III JJ et al. Residual symptoms in depressed patients who respond acutely to fluoxetine. J Clin Psychiatry 1999; 60: 221–225. 3 Trivedi MH, Hollander E, Nutt D, Blier P. Clinical evidence and potential neurobiological underpinnings of unresolved symptoms of depression. J Clin Psychiatry 2008; 69: 246–258. 4 Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997; 276: 71–74.

5 Grove JE, Bruscia E, Krause DS. Plasticity of bone marrow-derived stem cells. Stem Cells 2004; 22: 487–500. 6 Zhang H, Huang Z, Xu Y, Zhang S. Differentiation and neurological benefit of the mesenchymal stem cells transplanted into the rat brain following intracerebral hemorrhage. Neurol Res 2006; 28: 104–112. 7 Crigler L, Robey RC, Asawachaicharn A, Gaupp D, Phinney DG. Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis. Exp Neurol 2006; 198: 54–64. 8 Munoz JR, Stoutenger BR, Robinson AP, Spees JL, Prockop DJ. Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice. Proc Natl Acad Sci 2005; 102: 18171–18176. 9 Yoo SW, Kim SS, Lee SY, Lee HS, Kim HS, Lee YD et al. Mesenchymal stem cells promote proliferation of endogenous neural stem cells and survival of newborn cells in a rat stroke model. Exp Mol Med 2008; 40: 387–397. 10 Hamada H, Kobune M, Nakamura K, Kawano Y, Kato K, Honmou O et al. Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy. Cancer Sci 2005; 96: 149–156. 11 Dezawa M. Systematic neuronal and muscle induction systems in bone marrow stromal cells: the potential for tissue reconstruction in neurodegenerative and muscle degenerative diseases. Med Mol Morphol 2008; 41: 14–19. 12 Karussis D, Kassis I, Kurkalli BG, Slavin S. Immunomodulation and neuroprotection with mesenchymal bone marrow stem cells (MSCs): a proposed treatment for multiple sclerosis and other neuroimmunological/neurodegenerative diseases. J Neurol Sci 2008; 265: 131–135. 13 Kan I, Melamed E, Offen D. Autotransplantation of bone marrowderived stem cells as a therapy for neurodegenerative diseases. Handb Exp Pharmacol 2007; 180: 219–242. 14 Dranovsky A, Hen R. Hippocampal neurogenesis: regulation by stress and antidepressants. Biol Psychiatry 2006; 59: 1136–1143. 15 Thomas RM, Hotsenpiller G, Peterson DA. Acute psychosocial stress reduces cell survival in adult hippocampal neurogenesis without altering proliferation. J Neurosci 2007; 27: 2734–2743. 16 Fossati P, Radtchenko A, Boyer P. Neuroplasticity: from MRI to depressive symptoms. Eur Neuropsychopharmacol 2004; 14(Suppl 5): S503–S510. 17 Husum SA. Exacerbated loss of cell survival, neuropeptide Y-immunoreactive (IR) cells, and serotonin-IR fiber lengths in the dorsal hippocampus of the aged flinders sensitive line ‘depressed’ rat: implications for the pathophysiology of depression? J Neurosci Res 2006; 84: 1292–1302. 18 Gronli J, Bramham C, Murison R, Kanhema T, Fiske E, Bjorvatn B et al. Chronic mild stress inhibits BDNF protein expression and CREB activation in the dentate gyrus but not in the hippocampus proper. Pharmacol Biochem Behav 2006; 85: 842–849. 19 Gaughran F, Payne J, Sedgwick PM, Cotter D, Berry M. Hippocampal FGF-2 and FGFR1 mRNA expression in major depression, schizophrenia and bipolar disorder. Brain Res Bull 2006; 70: 221–227. 20 Kempermann G, Krebs J, Fabel K. The contribution of failing adult hippocampal neurogenesis to psychiatric disorders. Curr Opin Psychiatry 2008; 21: 290–295. 21 Malberg JE, Schechter LE. Increasing hippocampal neurogenesis: a novel mechanism for antidepressant drugs. Curr Pharm Des 2005; 11: 145–155. 22 Peister A, Zeitouni S, Pfankuch T, Reger RL, Prockop DJ, Raber J. Novel object recognition in Apoe/ mice improved by neonatal implantation of wild-type multipotential stromal cells. Exp Neurol 2006; 201: 266–269. 23 Ben Shaanan TL, Ben Hur T, Yanai J. Transplantation of neural progenitors enhances production of endogenous cells in the impaired brain. Mol Psychiatry 2007; 13: 222–231. 24 Yadid G, Friedman A. Dynamics of the dopaminergic system as a key component to the understanding of depression. In: Di Giovanni G, Di Matteo V, Esposito E (eds). Progress in Brain Research Serotonin-Dopamine Interaction: Experimental Evidence and Therapeutic Relevance, Vol. 172 edn. Elsevier: Amsterdam, 2008 pp. 265–286.

1173



1174

25 Overstreet DH, Friedman E, Mathe AA, Yadid G. The flinders sensitive line rat: a selectively bred putative animal model of depression. Neurosci Biobehav Rev 2005; 29: 739–759. 26 Zangi L, Rivkin R, Kassis I, Levdansky L, Marx G, Gorodetsky R. High-yield isolation, expansion, and differentiation of rat bone marrow derived mesenchymal stem cells with fibrin microbeads. Tissue Eng 2006; 12: 2343–2354. 27 Dai W, Hale SL, Martin BJ, Kuang JQ, Dow JS, Wold LE et al. Allogeneic mesenchymal stem cell transplantation in postinfarcted rat myocardium: short- and long-term effects. Circulation 2005; 112: 214–223. 28 Genud R, Merenlender A, Gispan-Herman I, Maayan R, Weizman A, Yadid G. DHEA lessens depressive-like behavior via GABAergic modulation of the mesolimbic system. Neuropsychopharmacology 2008; 34: 577–584. 29 Malatynska E, Knapp RJ. Dominant-submissive behavior as models of mania and depression. Neurosci Biobehav Rev 2005; 29: 715–737. 30 Friedman A, Frankel M, Flaumenhaft Y, Merenlender A, Pinhasov A, Feder Y et al. Programmed acute electrical stimulation of ventral tegmental area alleviates depressive-like behavior. Neuropsychopharmacology 2009; 34: 1057–1066. 31 Shetty AK, Rao MS, Hattiangady B. Behavior of hippocampal stem/progenitor cells following grafting into the injured aged hippocampus. J Neurosci Res 2008; 86: 3062–3074. 32 Sinead MG. Differentiation of oligodendrocytes in neurospheres derived from embryonic rat brain using growth and differentiation factors. J Neurosci Res 2007; 85: 1912–1920. 33 Jin GZ, Cho SJ, Choi EG, Lee YS, Yu XF, Choi KS et al. Rat mesenchymal stem cells increase tyrosine hydroxylase expression and dopamine content in ventral mesencephalic cells in vitro. Cell Biol Int 2008; 32: 1433–1438. 34 Koh SH, Noh MY, Cho GW, Kim KS, Kim SH. Erythropoietin increases the motility of human bone marrow multipotent stromal cells (hBM-MSCs) and enhances the production of neurotrophic factors from hBM-MSCs. Stem Cells Dev 2009; 18: 411–421. 35 Hardy SA, Maltman DJ, Przyborski SA. Mesenchymal stem cells as mediators of neural differentiation. Curr Stem Cell Res Ther 2008; 3: 43–52. 36 Lou SJ, Gu P, Chen F, He C, Wang MW, Lu CL. The effect of bone marrow stromal cells on neuronal differentiation of mesencephalic neural stem cells in Sprague-Dawley rats. Brain Res 2003; 968: 114–121. 37 Pomp O, Brokhman I, Ziegler L, Almog M, Korngreen A, Tavian M et al. PA6-induced human embryonic stem cell-derived neurospheres: a new source of human peripheral sensory neurons and neural crest cells. Brain Res 2008; 1230: 50–60. 38 Scuteri A, Donzelli E, Ravasi M, Tredici G. Adult mesenchymal stem cells support cisplatin-treated dorsal root ganglion survival. Neurosci Lett 2008; 445: 68–72. 39 Hellmann MA, Panet H, Barhum Y, Melamed E, Offen D. Increased survival and migration of engrafted mesenchymal bone marrow stem cells in 6-hydroxydopamine-lesioned rodents. Neurosci Lett 2006; 395: 124–128. 40 Sadan O, Shemesh N, Barzilay R, Bahat-Stromza M, Melamed E, Cohen Y et al. Migration of neurotrophic factors-secreting mesenchymal stem cells toward a quinolinic acid lesion as viewed by magnetic resonance imaging. Stem Cells 2008; 26: 2542–2551. 41 Wu X, Hu J, Zhou L, Mao Y, Yang B, Gao L et al. In vivo tracking of superparamagnetic iron oxide nanoparticle-labeled mesenchymal stem cell tropism to malignant gliomas using magnetic resonance imaging laboratory investigation. J Neurosurg 2008; 108: 320–329. 42 Nakamura K, Ito Y, Kawano Y, Kurozumi K, Kobune M, Tsuda H et al. Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Therapy 2004; 11: 1155–1164. 43 Bexell D, Gunnarsson S, Tormin A, Darabi A, Gisselsson D, Roybon L et al. Bone marrow multipotent mesenchymal stroma cells act as pericyte-like migratory vehicles in experimental gliomas. Mol Ther 2009; 17: 183–190. 44 Phinney DG, Baddoo M, Dutreil M, Gaupp D, Lai WT, Isakova IA. Murine mesenchymal stem cells transplanted to the central nervous system of neonatal versus adult mice exhibit distinct engraftment kinetics and express receptors that guide neuronal cell migration. Stem Cells Dev 2006; 15: 437–447.


45 Isakova IA, Baker K, DuTreil M, Dufour J, Gaupp D, Phinney DG. Age- and dose-related effects on MSC engraftment levels and anatomical distribution in the central nervous systems of nonhuman primates: identification of novel MSC subpopulations that respond to guidance cues in brain. Stem Cells 2007; 25: 3261–3270. 46 Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA 1999; 96: 10711–10716. 47 Lee RH, Hsu SC, Munoz J, Jung JS, Lee NR, Pochampally R et al. A subset of human rapidly self-renewing marrow stromal cells preferentially engraft in mice. Blood 2006; 107: 2153–2161. 48 Qu C, Mahmood A, Lu D, Goussev A, Xiong Y, Chopp M. Treatment of traumatic brain injury in mice with marrow stromal cells. Brain Res 2008; 1208: 234–239. 49 Hardy SA, Maltman DJ, Przyborski SA. Mesenchymal stem cells as mediators of neural differentiation. Curr Stem Cell Res Ther 2008; 3: 43–52. 50 Peterseń A, Wo¨rtwein G, Gruber SH, Mathe´ AA. Escitalopram reduces increased hippocampal cytogenesis in a genetic rat depression model. Neurosci Lett 2008; 436: 305–308. 51 Tran PB, Banisadr G, Ren D, Chenn A, Miller RJ. Chemokine receptor expression by neural progenitor cells in neurogenic regions of mouse brain. J Comp Neurol 2007; 500: 1007–1033. 52 Berger O, Li G, Han SM, Paredes M, Pleasure SJ. Expression of SDF-1 and CXCR4 during reorganization of the postnatal dentate gyrus. Dev Neurosci 2007; 29: 48–58. 53 Rai KS, Hattiangady B, Shetty AK. Enhanced production and dendritic growth of new dentate granule cells in the middle-aged hippocampus following intracerebroventricular FGF-2 infusions. Eur J Neurosci 2007; 26: 1765–1779. 54 Laskowski A, Schmidt W, Dinkel K, Martıńez-Sańchez M, Reymann KG. bFGF and EGF modulate trauma-induced proliferation and neurogenesis in juvenile organotypic hippocampal slice cultures. Brain Res 2005; 1037: 78–89. 55 Kroes RA, Panksepp J, Burgdorf J, Otto NJ, Moskal JR. Modeling depression: social dominance-submission gene expression patterns in rat neocortex. Neuroscience 2006; 137: 37–49. 56 Duan X, Kang E, Liu CY, Ming Gl, Song H. Development of neural stem cell in the adult brain. Curr Opin Neurobiol 2008; 18: 108–115. 57 Lie DC, Colamarino SA, Song HJ, Desire L, Mira H, Consiglio A et al. Wnt signalling regulates adult hippocampal neurogenesis. Nature 2005; 437: 1370–1375. 58 Lim DA, Tramontin AD, Trevejo JM, Herrera DG, Garcıá-Verdugo JM, Alvarez-Buylla A. Noggin antagonizes BMP signaling to create a niche for adult neurogenesis. Neuron 2000; 28: 713–726. 59 Bonaguidi MA, McGuire T, Hu M, Kan L, Samanta J, Kessler JA. LIF and BMP signaling generate separate and discrete types of GFAP-expressing cells. Development 2005; 132: 5503–5514. 60 Bjornebekk A, Mathe AA, Brene S. The antidepressant effect of running is associated with increased hippocampal cell proliferation. Int J Neuropsychopharmacol 2005; 8: 357–368. 61 Cunningham MG, Donalds RA, Carlezon WAJ, Hong S, Kim DS, Kim DW et al. Antidepressant effect of stem cell-derived monoaminergic grafts. [Miscellaneous Article]. NeuroReport 2007; 18: 1663–1667. 62 Malatynska E, Rapp R, Harrawood D, Tunnicliff G. Submissive behavior in mice as a test for antidepressant drug activity. Pharmacol Biochem Behav 2005; 82: 306–313. 63 Mochizuki N, Takagi N, Onozato C, Moriyama Y, Takeo S, Tanonaka K. Delayed injection of neural progenitor cells improved spatial learning dysfunction after cerebral ischemia. Biochem Biophys Res Commun 2008; 368: 151–156. 64 Mochizuki N, Takagi N, Kurokawa K, Onozato C, Moriyama Y, Tanonaka K et al. Injection of neural progenitor cells improved learning and memory dysfunction after cerebral ischemia. Exp Neurol 2008; 211: 194–202. 65 Toda H, Takahashi J, Iwakami N, Kimura T, Hoki S, MozumiKitamura K et al. Grafting neural stem cells improved the impaired spatial recognition in ischemic rats. Neurosci Lett 2001; 316: 9–12. 66 Chopp M, Li Y. Treatment of neural injury with marrow stromal cells. Lancet Neurol 2002; 1: 92–100.

MSCs increase hippocampal neurogenesis M Tfilin et al 67 Qu T, Brannen CL, Kim HM, Sugaya KC. Human neural stem cells improve cognitive function of aged brain. NeuroReport 2001; 12: 1127–1132. 68 Wu S, Sasaki A, Yoshimoto R, Kawahara Y, Manabe T, Kataoka K et al. Neural stem cells improve learning and memory in rats with Alzheimer’s disease. Pathobiology 2008; 75: 186–194. 69 Frinchi M, Bonomo A, Trovato-Salinaro A, Condorelli DF, Fuxe K, Spampinato MG et al. Fibroblast growth factor-2 and its receptor expression in proliferating precursor cells of the subventricular zone in the adult rat brain. Neurosci Lett 2008; 447: 20–25. 70 Maric D, Fiorio Pla A, Chang YH, Barker JL. Self-renewing and differentiating properties of cortical neural stem cells are selectively regulated by basic fibroblast growth factor (FGF) signaling via specific FGF receptors. J Neurosci 2007; 27: 1836–1852. 71 Kalluri HS, Dempsey RJ. Growth factors, stem cells, and stroke. Neurosurg Focus 2008; 24: E14. 72 Reuss B, von Bohlen und Halbach O. Fibroblast growth factors and their receptors in the central nervous system. Cell Tissue Res 2003; 313: 139–157. 73 Gaughran F, Payne J, Sedgwick PM, Cotter D, Berry M. Hippocampal FGF-2 and FGFR1 mRNA expression in major depression, schizophrenia and bipolar disorder. Brain Res Bull 2006; 70: 221–227. 74 Evans SJ, Choudary PV, Neal CR, Li JZ, Vawter MP, Tomita H et al. Dysregulation of the fibroblast growth factor system in major depression. Proc Natl Acad Sci USA 2004; 101: 15506–15511.

75 Aonurm-Helm A, Jurgenson M, Zharkovsky T, Sonn K, Berezin V, Bock E et al. Depression-like behaviour in neural cell adhesion molecule (NCAM)-deficient mice and its reversal by an NCAMderived peptide, FGL. Eur J Neurosci 2008; 28: 1618–1628. 76 Turner CA, Calvo N, Frost DO, Akil H, Watson SJ. The fibroblast growth factor system is downregulated following social defeat. Neurosci Lett 2008; 430: 147–150. 77 Turner CA, Gula EL, Taylor LP, Watson SJ, Akil H. Antidepressantlike effects of intracerebroventricular FGF2 in rats. Brain Res 2008; 1224: 63–68. 78 Jacobs BL, Praag H, Gage FH. Adult brain neurogenesis and psychiatry: a novel theory of depression. Mol Psychiatry 2000; 5: 262–269. 79 Eisch AJ, Cameron HA, Encinas JM, Meltzer LA, Ming GL, Overstreet-Wadiche LS. Adult neurogenesis, mental health, and mental illness: hope or hype? J Neurosci 2008; 28: 11785–11791. 80 Balu DT, Lucki I. Adult hippocampal neurogenesis: regulation, functional implications, and contribution to disease pathology. Neurosci Biobehav Rev 2009; 33: 232–252. 81 Sahay A, Drew MR, Hen R. Dentate gyrus neurogenesis and depression. In: Scharfman HE (ed). Progress in Brain Research). The Dentate Gyrus: A Comprehensive Guide to Structure, Function, and Clinical Implications, 163 edn. Elsevier: Amsterdam, 2007, pp 697–722, 822.

1175
