Adenosine A1 Receptor-Mediated Endocytosis of AMPA Receptors Contributes to Impairments in Long-Term Potentiation (LTP) in the Middle-Aged Rat Hippocampus

Aging causes multiple changes in the mammalian brain, including changes in synaptic signaling. Previous reports have shown increased extracellular adenosine in the aging brain, and we recently reported that activation of adenosine A1 receptors (A1Rs) induces AMPA receptor (AMPAR) internalization in rat hippocampus. This study investigated whether aging-related changes in the rat hippocampus include altered surface expression of adenosine A1 and A2A receptors, and whether these changes correspond to changes in AMPAR surface expression and altered synaptic plasticity. We found reduced A1R surface expression in middle-aged rat hippocampus, and also reduced GluA1 and GluA2 AMPAR subunit surface expression. Using a chemically-induced LTP (cLTP) experimental protocol, we recorded fEPSPs in young (1 month old) and middle-aged (7–12 month old) rat hippocampal slices. There were significant impairments in cLTP in middle-aged slices, suggesting impaired synaptic plasticity. Since we previously showed that the A1R agonist N⁶-cyclopentyladenosine (CPA) reduced both A1Rs and GluA2/GluA1 AMPARs, we hypothesized that the observed impaired synaptic plasticity in middle-aged brains is regulated by A1R-mediated AMPAR internalization by clathrin-mediated endocytosis. Following cLTP, we found a significant increase in GluA1 and GluA2 surface expression in young rats, which was blunted in middle-aged brains or in young brains pretreated with CPA. Blocking A1Rs with 8-cyclopentyl-1,3-dipropylxanthine or AMPAR endocytosis with either Tat-GluA2-3Y peptide or dynasore (dynamin inhibitor) similarly enhanced AMPAR surface expression following cLTP. These data suggest that age-dependent alteration in adenosine receptor expression contributes to increased AMPAR endocytosis and impaired synaptic plasticity in aged brains.     

Mesenchymal stem cells overexpressing integrin-linked kinase attenuate left ventricular remodeling and improve cardiac function after myocardial infarction

In the present study, we investigated whether mesenchymal stem cells (MSCs) overexpressing integrin-linked kinase (ILK) might regulate ventricular remodeling and cardiac function in a porcine myocardial infarction model. ILK-modified MSCs (ILK-MSCs) (n = 8), MSCs (n = 8) or placebo (n = 8) were injected into peri-infarct myocardium 7 days after ligation of the left anterior descending coronary artery. ILK expression was confirmed by immunofluorescence, real-time PCR, Western blot analysis, and flow cytometry. In vitro assays indicated increased proliferation and reduced apoptosis of MSCs due to overexpression of ILK. Echocardiographic, single-photon emission computed tomography and positron emission tomography analyses demonstrated preserved cardiac function and myocardial perfusion. Reduced fibrosis, increased cardiomyocyte proliferation, and enhanced angiogenesis were observed in the ILK-MSC group. Reduced apoptosis, as demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis, was also noted. In conclusion, ILK promotes MSC proliferation and suppresses apoptosis. ILK-MSC transplantation improves ventricular remodeling and cardiac function in pigs after MI. It is associated with increased angiogenesis, reduced apoptosis, and increased cardiomyocyte proliferation. This may represent a new approach to the treatment of post-infarct remodeling and subsequent heart failure.     

Dynamic Changes of Astrocytes and Adenosine Signaling in Rat Hippocampus in Post-status Epilepticus Model of Epileptogenesis

It is of great importance to explore the development of epileptogenesis, and the adenosine and adenosine kinase (ADK) system seems to play a key role in this process. The aim of this study is to explore the dynamic changes of astrocytes and adenosine signaling during epileptogenesis in rat hippocampus in a post-status epileptogenesis (SE) model. Rat SE models were built and killed for experiments at 1 day (acute phase of epileptogenesis), 5 days (latent phase), 4 weeks (chronic phase), and 8 weeks (late chronic phase of epileptogenesis) after SE induction. Immunofluorescence staining, high-performance liquid chromatography, and Western blotting were performed to assess changes of astrocytes, adenosine, ADK, and ADK receptors (including A1R, A2aR, A2bR, and A3R) in hippocampus. The expression level of glial fibrillary acidic protein significantly increased from latent to late chronic phase. The concentration of adenosine sharply increased in acute phase and gradually decreased in the remaining phases of post-SE, being significantly lower than in the control group in late chronic phase. Protein levels of A1R and A2aR in post-SE models increased in acute phase, whereas A2bR and A3R protein expression decreased in latent phase, chronic phase, and late chronic phase following post-SE epileptogenesis. Protein expression of ADK significantly increased during latent phase, chronic phase, and late chronic phase of post-SE epileptogenesis. In conclusion, the levels of adenosine and protein expression of A1R and A2R significantly increased during acute phase of post-SE. During the remaining phases of post-SE epileptogenesis, there was imbalance among astrocytes, adenosine, adenosine receptors, and ADK. Regulation of the ADK/adenosine system may provide potential treatment strategies for epileptogenesis.     

Molecular mechanisms involved in the adenosine A and A receptor-induced neuronal differentiation in neuroblastoma cells and striatal primary cultures

Adenosine A1 receptors (A1Rs) and adenosine A(2A) receptors (A(2A)Rs) are the major mediators of the neuromodulatory actions of adenosine in the brain. In the striatum A1Rs and A(2A)Rs are mainly co-localized in the GABAergic striatopallidal neurons. In this paper we show that agonist-induced stimulation of A1Rs and A(2A)Rs induces neurite outgrowth processes in the human neuroblastoma cell line SH-SY5Y and also in primary cultures of striatal neuronal precursor cells. The kinetics of adenosine-mediated neuritogenesis was faster than that triggered by retinoic acid. The triggering of the expression of TrkB neurotrophin receptor and the increase of cell number in the G1 phase by the activation of adenosine receptors suggest that adenosine may participate in early steps of neuronal differentiation. Furthermore, protein kinase C (PKC) and extracellular regulated kinase-1/2 (ERK-1/2) are involved in the A1R- and A(2A)R-mediated effects. Inhibition of protein kinase A (PKA) activity results in a total inhibition of neurite outgrowth induced by A(2A)R agonists but not by A1R agonists. PKA activation is therefore necessary for A(2A)R-mediated neuritogenesis. Co-stimulation does not lead to synergistic effects thus indicating that the neuritogenic effects of adenosine are mediated by either A1 or A(2A) receptors depending upon the concentration of the nucleoside. These results are relevant to understand the mechanisms by which adenosine receptors modulate neuronal differentiation and open new perspectives for considering the use of adenosine agonists as therapeutic agents in diseases requiring neuronal repair.     

Mesenchymal stem cell-based HSP70 promoter-driven VEGFA induction by resveratrol promotes angiogenesis in a mouse model

Several studies of stem cell-based gene therapy have indicated that long-lasting regeneration following vessel ischemia may be stimulated through VEGFA gene therapy and/or MSC transplantation for reduction of ischemic injury in limb ischemia and heart failure. The therapeutic potential of MSC transplantation can be further improved by genetically modifying MSCs with genes which enhance angiogenesis following ischemic injury. In the present study, we aimed to develop an approach in MSC-based therapy for repair and mitigation of ischemic injury and regeneration of damaged tissues in ischemic disease. HSP70 promoter-driven VEGFA expression was induced by resveratrol (RSV) in MSCs, and in combination with known RSV biological functions, the protective effects of our approach were investigated by using ex vivo aortic ring coculture system and a 3D scaffolds in vivo model. Results of this investigation demonstrated that HSP promoter-driven VEGFA expression in MSC increased approximately 2-fold over the background VEGFA levels upon HSP70 promoter induction by RSV. Exposure of HUVEC cells to medium containing MSC in which VEGFA had been induced by cis-RSV enhanced tube formation in the treated HUVEC cells. RSV-treated MSC cells differentiated into endothelial-like phenotypes, exhibiting markedly elevated expression of endothelial cell markers. These MSCs also induced aortic ring sprouting, characteristic of neovascular formation from pre-existing vessels, and additionally promoted neovascularization at the MSC transplantation site in a mouse model. These observations support a hypothesis that VEGFA expression induced by cis-RSV acting on the HSP70 promoter in transplanted MSC augments the angiogenic effects of stem cell gene therapy. The use of an inducible system also vastly reduces possible clinical risks associated with constitutive VEGFA expression.     

Aspirin induces apoptosis in mesenchymal stem cells requiring Wnt/β-catenin pathway

Background and Objectives:  Mesenchymal stem cells (MSC) are multipotent progenitor cells that are have found use in regenerative medicine. We have previously observed that aspirin, a widely used anti-inflammatory drug, inhibits MSC proliferation. Here we have aimed to elucidate whether aspirin induces MSC apoptosis and whether this is modulated through the Wnt/β-catenin pathway.
Materials and methods:  Apoptosis of MSCs was assessed using Hoechst 33342 dye and an Annexin V–FITC/PI Apoptosis Kit. Expression of protein and protein phosphorylation were investigated using Western blot analysis. Caspase-3 activity was detected by applying a caspase-3/CPP32 Colorimetric Assay Kit.
Results:  In these MSCs, aspirin induced morphological changes characteristic of apoptosis, cytochrome  c release from mitochondria, and caspase-3 activation. Stimulating the Wnt/β-catenin pathway by both Wnt 3a and GSK-3β inhibitors (LiCl and SB 216763), blocked aspirin-induced apoptosis and protected mitochondrial function, as demonstrated by decreased cytochrome  c release and caspase-3 activity. Aspirin initially caused a time-dependent decrease in COX-2 expression but subsequently, and unexpectedly, elevated the latter. Stimulation of COX-2 expression by aspirin was further enhanced following stimulation of the Wnt/β-catenin pathway. Application of the COX-2 inhibitor NS-398 suppressed elevated COX-2 expression and promoted aspirin-induced apoptosis.
Conclusion:  These results demonstrate that the Wnt/β-catenin pathway is a key modulator of aspirin-induced apoptosis in MSCs by regulation of mitochrondrial/caspase-3 function. More importantly, our findings suggest that aspirin may influence MSC survival under certain conditions; therefore, it should be used with caution when considering regenerative MSC transplantation in patients with concomitant chronic inflammatory diseases such as arthritis.     

Evaluation of persistence and distribution of intra-dermally administered PKH26 labelled goat bone marrow derived mesenchymal stem cells in cutaneous wound healing model

The current study was designed to study the persistence and distribution of caprine bone marrow derived mesenchymal stem cells (cBM-MSCs) when administered intra-dermally in experimentally induced cutaneous wounds in rabbits. MSC’s from goat bone marrow were isolated and their differentiation potential towards adipogenic and osteogenic lineages were assayed in vitro. The isolated cells were phenotypically analysed using flow cytometry for the expression of MSC specific matrix receptors (CD73, CD105 and Stro-1) and absence of hematopoietic lineage markers. Further, these in vitro expanded MSCs were stained with PKH26 lipophilic cell membrane red fluorescent dye and prepared for transplantation into cutaneous wounds created on rabbits. Five, 2 cm linear full thickness skin incisions were created on either side of dorsal midline of New Zealand white rabbits (n = 4). Four wounds in each animal were implanted intra-dermally with PKH26 labelled cBM-MSCs suspended in 500 µl of Phosphate Buffer Saline (PBS). Fifth wound was injected with PBS alone and treated as negative control. The skin samples were collected from respective wounds on 3, 7, 10 and 14 days after the wound creation, and cryosections of 6 µM were made from it. Fluorescent microscopy of these cryosections showed that the PKH26 labelled transplanted cells and their daughter cells demonstrated a diffuse pattern of distribution initially and were later concentrated towards the wound edges and finally appeared to be engrafted with the newly developed skin tissues. The labelled cells were found retained in the wound bed throughout the period of 14 days of experimental study with a gradual decline in their intensity of red fluorescence probably due to the dye dilution as a result of multiple cell division. The retention of transplanted MSCs within the wound bed even after the complete wound healing suggests that in addition to their paracrine actions as already been reported, they may have direct involvement in various stages of intricate wound healing process which needs to be explored further.     

Sodium Butyrate Promotes the Differentiation of Rat Bone Marrow Mesenchymal Stem Cells to Smooth Muscle Cells through Histone Acetylation

Establishing an effective method to improve stem cell differentiation is crucial in stem cell transplantation. Here we aimed to explore whether and how sodium butyrate (NaB) induces rat bone marrow mesenchymal stem cells (MSCs) to differentiate into bladder smooth muscle cells (SMCs). We found that NaB significantly suppressed MSC proliferation and promoted MSCs differentiation into SMCs, as evidenced by the enhanced expression of SMC specific genes in the MSCs. Co-culturing the MSCs with SMCs in a transwell system promoted the differentiation of MSCs into SMCs. NaB again promoted MSC differentiation in this system. Furthermore, NaB enhanced the acetylation of SMC gene-associated H3K9 and H4, and decreased the expression of HDAC2 and down-regulated the recruitment of HDAC2 to the promoter regions of SMC specific genes. Finally, we found that NaB significantly promoted MSC depolarization and increased the intracellular calcium level of MSCs upon carbachol stimulation. These results demonstrated that NaB effectively promotes MSC differentiation into SMCs, possibly by the marked inhibition of HDAC2 expression and disassociation of HDAC2 recruitment to SMC specific genes in MSCs, which further induces high levels of H3K9^ace and H4^ace and the enhanced expression of target genes, and this strategy could potentially be applied in clinical tissue engineering and cell transplantation.     

Is the subarachnoid administration of mesenchymal stromal cells a useful strategy to treat chronic brain damage?

Background aimsTraumatic brain injury (TBI) is a leading cause of mortality and morbidity worldwide. Developing effective protocols for the administration of mesenchymal stromal cells (MSCs) is a promising therapeutic strategy to treat TBI. It is important to develop alternatives to direct parenchymal injection at the injury site because direct injection is an expensive and invasive technique. Subarachnoid transplantation, a minimally invasive and low-risk procedure, may be an important and clinically applicable strategy. The aim of this study was to test the therapeutic effect of subarachnoid administration of MSCs on functional outcome 2 months after an experimental TBI in rats.MethodsTwo months after TBI, 30 female Wistar rats were divided into 3 groups (n = 10 in each group): sham, MSC (received 2 × 10⁶ MSCs) and saline (received only saline) groups. Neurological function, brain and spinal cords samples and cerebrospinal fluid were studied.ResultsNo significant differences were found in neurological evaluation and after histological analysis; differences in the expression of neurotrophins were present but were not statistically significant. MSCs survived in the host tissue, and some expressed neural markers.ConclusionsSimilar to direct parenchymal injections, transplanted MSCs survive, migrate to the injury cavity and differentiate into mature neural cell types for at least 6 months after engraftment. These results open the possibility that MSC administration through subarachnoid administration may be a treatment for the consequences of TBI. The transplantation technique and cell number should be adjusted to obtain functional outcome and neurotrophin production differences.     

Activation of CD74 inhibits migration of human mesenchymal stem cells

Therapeutic administration of mesenchymal stem cells (MSCs) by systemic delivery utilizes the innate ability of the cells to home to damaged tissues, but it can be an inefficient process due to a limited knowledge of cellular cues that regulate migration and homing. Our lab recently discovered that a potent pro-inflammatory cytokine, macrophage migration inhibitory factor (MIF), inhibits MSC migration. Because MIF may act on multiple cellular targets, an activating antibody (CD74Ab) was employed in this study to examine the effect of one MIF receptor, CD74 (major histocompatibility complex class II-associated invariant chain), on MSC motility. CD74 activation inhibits in a dose-dependent manner up to 90% of in vitro migration of MSCs at 40 μg/ml CD74Ab (p?<?0.001), with consistent effects observed among three MSC donor preparations. A blocking peptide from the C-terminus of CD74 eliminates the effect of CD74Ab on MSCs. This suggests that MIF may act on MSCs, at least in part, through CD74. Late-passage MSCs exhibit less chemokinesis than those at passage 2. However, MSCs remain responsive to CD74 activation during ex vivo expansion: MSC migration is inhibited ～2-fold in the presence of 5 µg/ml CD74Ab at passage 9 vs. ～3-fold at passage 2 (p?<?0.001). Consistent with this result, there were no significant differences in CD74 expression at all tested passages or after CD74Ab exposure. Targeting CD74 to regulate migration and homing potentially may be a useful strategy to improve the efficacy of a variety of MSC therapies, including those that require ex vivo expansion.     

Acetylcholine induces mesenchymal stem cell migration via Ca2+ /PKC/ERK1/2 signal pathway

Acetylcholine (ACh) plays an important role in neural and non-neural function, but its role in mesenchymal stem cell (MSC) migration remains to be determined. In the present study, we have found that ACh induces MSC migration via muscarinic acetylcholine receptors (mAChRs). Among several mAChRs, MSCs express mAChR subtype 1 (m1AChR). ACh induces MSC migration via interaction with mAChR1. MEK1/2 inhibitor PD98059 blocks ERK1/2 phosphorylation while partially inhibiting the ACh-induced MSC migration. InsP3Rs inhibitor 2-APB that inhibits MAPK/ERK phosphorylation completely blocks ACh-mediated MSC migration. Interestingly, intracellular Ca(2+) ATPase-specific inhibitor thapsigargin also completely blocks ACh-induced MSC migration through the depletion of intracellular Ca(2+) storage. PKCα or PKCβ inhibitor or their siRNAs only partially inhibit ACh-induced MSC migration, but PKC-ζ siRNA completely inhibits ACh-induced MSC migration via blocking ERK1/2 phosphorylation. These results indicate that ACh induces MSC migration via Ca(2+), PKC, and ERK1/2 signal pathways.     

Down-Regulation of AMPA Receptor Subunit GluR2 in Amygdaloid Kindling

Abstract: Alterations in glutamatergic transmission are postulated to be important in kindling and epilepsy. The levels of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits (GluR1, 2, and 4) were compared in amygdalakindled and sham-operated animals using subunit-specific antibodies and quantitative western blotting. Four limbic regions were examined: limbic forebrain, piriform cortex/amygdala, hippocampus, and entorhinal cortex. When subunit levels were examined 24 h after the last stage 5 seizure, levels of GluR2 were found to be selectively reduced in limbic forebrain (30%) and piriform cortex/amygdala (25%), with no changes in other regions examined. In addition, no changes in the other subunits were observed in any region. The decrease in GluR2 that was observed in kindled animals at 24 h was no longer present at 1 week and 1 month after the last stage 5 seizure. Because the GluR2 subunit uniquely determines the calcium permeability of these receptors and because the piriform cortex has been implicated as a source of excitatory drive for limbic seizures, reduced GluR2 expression may be important in increasing neuronal excitability in kindling-induced epilepsy, or may reflect a compensatory mechanism resulting from kindling.     

Leukemia inhibitory factor: Role in human mesenchymal stem cells mediated immunosuppression

The interactions between mesenchymal stem cells (MSCs) and immune system are currently being explored. Leukemia inhibitory factor (LIF) is linked to regulatory transplantation tolerance. Our aim was to study the expression of LIF on human MSCs at both gene and protein level in mixed lymphocyte reaction (MSC/MLR), and its implication in MSC immunosuppressive effect. There was a 7-fold increase (611pg/ml) in LIF in MSC/MLR as compared to MSCs alone. Using LIF neutralizing antibody, a significant restoration of up to 91% of CD3+ lymphocyte proliferation in MSC/MLR was observed (p=0.021). LIF was implicated in the generation of regulatory lymphocytes, as demonstrated by decrease of Foxp3+ regulatory cells after using LIF neutralizing antibody in MSC/MLR (p=0.06) by flow cytometry. A positive correlation between LIF and human leukocyte antigen (HLA-G) gene expression by MSCs was found (R(2)=0.74). Our findings provide evidence supporting the immunomodulatory effect of MSCs.     

Expression of adenosine A2b receptor in rat type II and III taste cells

We previously demonstrated that equilibrative nucleoside transporter 1 was expressed in taste cells, suggesting the existence of an adenosine signaling system, but whether or not the expression of an adenosine receptor occurs in rat taste buds remains unknown. Therefore, we examined the expression profiles of adenosine receptors and evaluated their functionality in rat circumvallate papillae. Among adenosine receptors, the mRNA for an adenosine A2b receptor (A2bR) was expressed by the rat circumvallate papillae, and its expression level was significantly greater in the circumvallate papillae than in the non-taste lingual epithelium. A2bR-immunoreactivity was detected primarily in type II taste cells, and partial, but significant expression was also observed in type III ones, but there was no immunoreactivity in type I ones. The cAMP generation in isolated epithelium containing taste buds treated with 500 μM adenosine or 10 μM BAY60-6583 was significantly increased compared to in the controls. These findings suggest that adenosine plays a role in signaling transmission via A2bR between taste cells in rats.     

Differential Migration of Mesenchymal Stem Cells to Ischemic Regions after Middle Cerebral Artery Occlusion in Rats

To evaluate the optimal timing of mesenchymal stem cell (MSC) transplantation following stroke, rats were transplanted with MSCs at 1 (D1), 4 (D4), and 7 days (D7) after middle cerebral artery occlusion (MCAo). Rats in the D1 group showed a better functional recovery than those in the D4 or D7 groups after MCAo. MSCs preferentially migrated to the cortex in the D1 group, while the MSCs in the D4 or D7 groups preferentially migrated to the striatum. Interestingly, the level of monocyte chemotactic protein-1 (MCP-1) in the cortex was highest at 1 day after MCAo, while the level of stromal cell-derived factor-1 (SDF-1) in the striatum was lowest at 1 day after MCAo and then increased over time. The pattern of MCP-1 and SDF-1 level changes according to the time after MCAo was consistent with in vivo and in vitro migration patterns of MSCs. The results suggest that an earlier MSC transplantation is associated with a better functional recovery after stroke, which could be explained by the preferential migration of MSCs to the cortex in the early transplantation group. The time-dependent differential expression of MCP-1 and SDF-1 between ischemic regions seemed to mediate the differential migration of MSCs. Highest level of MCP-1 at one day of stroke may induce preferential migration of MSCs to the cortex, then better functional improvement.     

Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells treated by GSK-3 inhibitors

A study of the cartilage differentiation of mesenchymal stem cells (MSCs) would be of particular interest since one strategy for cell-based treatment of cartilage defects emphasizes the use of cells that are in a differentiated state. The present study has attempted to evaluate the effects of two well-known glycogen synthase kinase-3 inhibitors, including lithium chloride (LiCl) and SB216763 on a human marrow-derived MSC (hMSC) chondrogenic culture. Passaged-3 MSCs were condensed into small pellets and cultivated in the following groups based on the supplementation of chondrogenic medium: transforming growth factor (TGF)-β1, TGF-β1 + LiCl, TGF-β1 + SB216763, TGF-β3, TGF-β3 + LiCl, and TGF-β3 + SB216763. The cultures were maintained for 21 days and then analyzed for expression of Sox9, aggrecan, collagen II, β-catenin, and axin genes. Deposition of glycosaminoglycan (GAG) in the cartilage matrix was also measured for certain cultures. The presence of both LiCl and SB216763 along with TGF-β in the MSC chondrogenic culture led to the up-regulation of cartilage-specific genes. TGF-β3 appeared much better than TGF-β1. Based on our findings, SB216763 was more effective in up-regulation of cartilage-specific genes. These chondrogenic effects appeared to be mediated through the Wnt signaling pathway since β-catenin and axin tended to be up-regulated and down-regulated, respectively. In the culture with SB216763 + TGF-β3, significantly more GAG was deposited (P < 0.05). In conclusion, addition of either SB216763 or LiCl to hMSC chondrogenic culture up-regulates cartilage-specific gene expression and enhances GAG deposition in the culture.     

Effects of Mesenchymal Stem Cell Treatment on the Expression of Matrix Metalloproteinases and Angiogenesis during Ischemic Stroke Recovery

Background

The efficacy of mesenchymal stem cell (MSC) transplantation in ischemic stroke might depend on the timing of administration. We investigated the optimal time point of MSC transplantation. After MSC treatment, we also investigated the expression of matrix metalloproteinases (MMPs), which play a role in vascular and tissue remodeling.

Methods

Human bone marrow-derived MSCs (2 × 10⁶, passage 5) were administrated intravenously after permanent middle cerebral artery occlusion (MCAO) was induced in male Sprague-Dawley rats. First, we determined the time point of MSC transplantation that led to maximal neurological recovery at 1 h, 1 day, and 3 days after MCAO. Next, we measured activity of MMP-2 and MMP-9, neurological recovery, infarction volume, and vascular density after transplanting MSCs at the time that led to maximal neurological recovery.

Results

Among the MSC-transplanted rats, those of the MSC 1-hour group showed maximal recovery in the rotarod test (P = 0.023) and the Longa score (P = 0.018). MMP-2 activity at 1 day after MCAO in the MSC 1-hour group was significantly higher than that in the control group (P = 0.002), but MMP-9 activity was not distinct. The MSC 1-hour group also showed smaller infarction volume and higher vascular density than did the control group.

Conclusions

In a permanent model of rodent MCAO, very early transplantation of human MSCs (1 h after MCAO) produced greater neurological recovery and decreased infraction volume. The elevation of MMP-2 activity and the increase in vascular density after MSC treatment suggest that MSCs might help promote angiogenesis and lead to neurological improvement during the recovery phase after ischemic stroke.     

Change in hepatocyte growth factor concentration promote mesenchymal stem cell-mediated osteogenic regeneration

Mesenchymal stem cells (MSCs) play a crucial role in tissue repair by secretion of tissue nutrient factors such as hepatocyte growth factor (HGF). However, studies examining the effects of HGF on the proliferation and differentiation of MSCs used different concentrations of HGF and reported conflicting conclusions. This study aimed to determine the mechanisms by which different concentrations of HGF regulate MSC proliferation and osteogenic differentiation, and validate the mechanism in an animal model of early stage avascular necrosis of femoral head (ANFH). Our results demonstrate that a low concentration of HGF (20 ng/ml) preferentially promotes MSC osteogenic differentiation through increased c-Met expression and phosphorylation, Akt pathway activation, and increased expression of p27, Runx2 and Osterix. In contrast, a high concentration of HGF (100 ng/ml) strongly induced proliferation by inducing strong activation of the ERK1/2 signalling pathway. As validated by animal experiments, high localized expression of HGF achieved by transplantation of HGF transgenic MSCs into ANFH rabbits increased the number of MSCs. Subsequently, 2 weeks after transplantation, HGF levels decreased and MSCs differentiated into osteoblasts and resulted in efficient tissue repair. Our results demonstrate that sequential concentration changes in HGF control the proliferation and osteogenic differentiation of MSCs in vivo. This phenomenon can be exploited therapeutically to induce bone regeneration and, in turn, improve the efficacy of pharmacological intervention for ANFH treatment.     

Effect of bone marrow–derived mesenchymal stromal cells on hepatoma

Background aimsThe aim of the study was to evaluate the effect of mesenchymal stromal cells (MSCs) on tumor cell growth in vitro and in vivo and to elucidate the apoptotic and anti-proliferative mechanisms of MSCs on a hepatocellular carcinoma (HCC) murine model.MethodsThe growth-inhibitory effect of MSCs on the Hepa 1–6 cell line was tested by means of methyl thiazolyl diphenyl-tetrazolium assay. Eighty female mice were randomized into four groups: group 1 consisted of 20 mice that received MSCs only by intrahepatic injection; group 2 consisted of 20 HCC mice induced by inoculation of Hepa 1–6 cells into livers without MSC treatment; group 3 consisted of 20 mice that received MSCs after induction of liver cancer; group 4 consisted of 20 mice that received MSCs after induction of liver cancer on top of induced biliary cirrhosis.ResultsMSCs exhibited a growth-inhibitory effect on Hepa 1–6 murine cell line in vitro. Concerning in vivo study, decreases of serum alanine transaminase, aspartate transaminase and albumin levels after MSC transplantation in groups 2 and 3 were found. Gene expression of α-fetoprotein was significantly downregulated after MSC injection in the HCC groups. We found that gene expression of caspase 3, P21 and P53 was significantly upregulated, whereas gene expression of Bcl-2 and survivin was downregulated in the HCC groups after MSC injection. Liver specimens of the HCC groups confirmed the presence of dysplasia. The histopathological picture was improved after administration of MSCs to groups 2 and 3.ConclusionsMSCs upregulated genes that help apoptosis and downregulated genes that reduce apoptosis. Therefore, MSCs could inhibit cell division of HCC and potentiate their death.