Apoptosis Of Mesenchymal Stem Cells Research Articles

Protective effects of hepatocyte growth factor gene overexpression against hydrogen peroxide-induced apoptosis in mesenchymal stem cells.

Hepatocyte growth factor (HGF) has recently been reported to exhibit antioxidant and antiapoptotic effects. Therefore, we investigated the effect of overexpression of HGF gene in H2 O2 -treated mesenchymal stem cells (MSCs). HGF-overexpression increased the cell viability from 50% to 84%, decreased the population of apoptotic cells from 20% to 16%, and decreased the intracellular reactive oxygen species (ROS) levels from 127% to 100% in cells treated with H2 O2 . HGF suppression decreased the cell viability from 58% to 36%, increased the population of apoptotic cells from 23 to 81%, and increased the intracellular ROS levels from 181% to 240% in cells exposed to H2 O2 . HGF-overexpression also reduced the expression levels of proapoptotic proteins in MSCs treated with H2 O2 . Phosphorylation of extracellular signal-regulated kinases, c-Jun amino-terminal kinases, and p38, which was induced by H2 O2 , decreased in MSCs overexpressing the HGF gene. Taken together, our results suggest that HGF has a protective effect on H2 O2 -induced apoptosis in MSCs.

LncRNA MALAT1 inhibits osteogenic differentiation of mesenchymal stem cells in osteoporosis rats through MAPK signaling pathway.

The aim of this study was to explore whether long non-coding ribonucleic acid metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1) could lead to osteoporosis (OP) by stimulating the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. The OP model was first successfully established in rats. The expression of lncRNA MALAT1 in OP rats and normal rats was detected via quantitative Polymerase Chain Reaction (qPCR). Bone marrow mesenchymal stem cells (BMSCs) were cultured and transfected to establish the MALAT1 knockdown model. Subsequently, the apoptosis of mesenchymal stem cells in MALAT1 siRNA group and NC siRNA group was detected via flow cytometry. Meanwhile, the expressions of the MAPK signaling pathway proteins related to OP were detected via Western blotting. After alkaline phosphatase (ALP) staining in cells of both groups, early osteogenic differentiation of BMSCs was observed. The results of qPCR showed that the expression of lncRNA MALAT1 in OP rats was significantly lower than that of normal rats. It was observed under a fluorescence microscope that there were a large number of siRNA particles in BMSCs. The expression of lncRNA MALAT1 in cells was detected via Real Time-fluorescence qPCR as well. The results indicated that siRNA transfection could effectively inhibit the expression of lncRNA MALAT1, indicating successful transfection. Flow cytometry revealed that no significant difference was observed in the apoptosis of BMSCs between the MALAT1 siRNA group and NC siRNA group. Besides, the results of Western blotting showed that the expression levels of the MAPK signaling pathway-related proteins extracellular signal-regulated kinase 1/2 (ERK1/2) and P38 in MALAT1 siRNA group were significantly higher than those of the NC siRNA group. This indicated that inhibiting the expression of lncRNA MALAT1 might promote the activation of the OP-related MAPK pathway. According to the results of ALP staining, the depth of staining in MALAT1 siRNA group was markedly declined when compared with the NC siRNA group. Quantification of ALP activity demonstrated that ALP activity in the MALAT1 siRNA group was markedly declined compared with the NC siRNA group. The above results suggested that suppressing the expression of lncRNA MALAT1 could reduce the ALP activity of BMSCs. Furthermore, lncRNA MALAT1 inhibited osteogenic differentiation of BMSCs. LncRNA MALAT1 was lowly expressed in OP rats. Moreover, it inhibited osteogenic differentiation of BMSCs by enhancing the activation of the MAPK signaling pathway, thereby promoting OP progression.

Dysregulated megakaryocyte distribution associated with nestin+ mesenchymal stem cells in immune thrombocytopenia

AbstractImpaired megakaryocyte (MK) maturation and reduced platelet production are important causes of immune thrombocytopenia (ITP). However, MK distribution and bone marrow (BM) niche alteration in ITP are unclear. To investigate the maturation and distribution of MKs in the BM niche and examine the components of BM niche regulation of MK migration, BM and peripheral blood were obtained from 30 ITP patients and 28 healthy donors. Nestin+ mesenchymal stem cells (MSCs) and CD41+ MKs were sorted by fluorescence-activated cell sorting. The components of the BM niche and related signaling were analyzed via immunofluorescence, flow cytometry, enzyme-linked immunosorbent assay, reverse transcription polymerase chain reaction, and western blot analysis. The number of MKs in the BM vascular niche was reduced in ITP. Moreover, the concentrations of CXCL12 and CXCR4+ MKs in the BM were decreased in ITP. Further investigation demonstrated that nestin+ MSCs and CXCL12 messenger RNA (mRNA) in nestin+ MSCs were both reduced whereas the apoptosis of nestin+ MSCs was significantly increased in ITP. Sympathetic nerves, Schwann cells, the proportion of β3-adrenoreceptor (β3-AR)+ nestin+ MSCs, and β3-AR mRNA in nestin+ MSCs were all markedly reduced in ITP. Moreover, matrix metalloproteinase 9, vascular endothelial growth factor (VEGF), and VEGF receptor 1 were significantly reduced in ITP. Our data show that impaired MK distribution mediated by an abnormal CXCL12/CXCR4 axis is partially involved in reduced platelet production in ITP. Moreover, sympathetic neuropathy and nestin+ MSC apoptosis may have an effect on the alterations of BM CXCL12 in ITP.

Norepinephrine protects against apoptosis of mesenchymal stem cells induced by high glucose.

In diabetes, the number of bone mesenchymal stem cells (MSCs) decreases and their differentiation is impaired. However, the exact mechanism is unclear. Patients with diabetes often experience sympathetic nerve injury. Norepinephrine (NE), a major mediator of the sympathetic nervous system, influences rat MSC migration in culture and in vivo. The present study aimed to investigate the effect of NE on MSCs under high glucose conditions; therefore MSCs were treated with high glucose and NE. High glucose-induced MSCs apoptosis, which was reversed by NE. To verify the effect of NE, mice underwent sympathectomy and were used to establish a diabetic model. Diabetic mice with sympathectomy had a higher apoptosis rate and higher levels of reactive oxygen species in their bone marrow-derived cells than diabetic mice without sympathectomy. High glucose inhibited p-AKT production and B-Cell CLL/Lymphoma 2 expression, and promoted BAX and caspase-3 expression. NE reversed these effects of high glucose. An AKT inhibitor enhanced the effects of high glucose. Thus, NE had a protective effect on MSC apoptosis induced by high glucose, possibly via the AKT/BCL-2 pathway.

Self-assembling peptide modified with QHREDGS as a novel delivery system for mesenchymal stem cell transplantation after myocardial infarction.

The lower cell survival and retention in the hostile microenvironment after transplantation has been implicated as a major bottleneck in the advancement of stem cell therapy for myocardial infarction (MI). In this study, we designed a novel self-assembling peptide (SAP) by attaching prosurvival peptide QHREDGS derived from angiopoeitin-1 to the known SAP, RADA16-I. The mesenchymal stem cells (MSCs) were harvested from male rats and cytoprotective effect of this designer SAP (DSAP) on cultured MSCs was detected by Hoechst 33342 staining after being exposed to oxygen and glucose deprivation (OGD). The cytoprotective effect of MSCs seeded in DSAP (DSAP-MSCs) on OGD treated cardiomyocytes was examined by TUNEL staining, phosphorylated (p-) protein kinase B (Akt) level, and ELISA. The therapeutic potential of MSC transplantation carried in DSAP was evaluated in a female rat MI model. PBS, MSCs alone, MSCs seeded in SAP (SAP-MSCs), or DSAP-MSCs were transplanted into the border of the infarcted area, respectively. DSAP not only increased the proliferation of MSCs and decreased apoptosis of MSCs after OGD treatment but also promoted the secretion of IGF-1 and HGF in MSCs. Treatment with culture supernatant of DSAP-MSCs markedly reduced the percentage of apoptotic cardiomyocytes and increased the level of p-Akt. Compared with the MSC group and SAP-MSC group, DSAP-MSC injection improved cardiac function and reduced infarct size, collagen content, and cell apoptosis. The number of Y chromosome-positive cells and microvessels in the DSAP-MSC group was higher than those in the MSC group and SAP-MSC group. Moreover, DSAP-MSC transplantation down-regulated the expression of IL-6 and IL-1β and up-regulated the level of VEGF and HGF. Interestingly, miR-21 was enriched in DSAP-MSC-derived exosomes (DSAP-MSC-Exos) and the protection against cardiomyocyte apoptosis by DSAP-MSC-Exos was inhibited when miR-21 was knocked down. Furthermore, miR-21 contributed to the improvement of cardiac function after DSAP-MSC-Exo injection in a rat model of MI. Additionally, the combination of DSAP and cardiotrophin-1 (Ctf1) pretreatment further improved the survival of MSCs and the efficiency of MSC transplantation. We proposed QHREDGS-modified SAP as an effective cell delivery system and demonstrated that MSC transplantation in this DSAP promoted angiogenesis and paracrine, thereby reducing scar size and cell apoptosis as well as improving cardiac function probably via exosome-mediated miR-21 after MI. Furthermore, for the first time, we proposed that DSAP, especially working together with Ctf1 pretreatment, could be a valuable way to improve the survival of MSCs and the efficiency of MSC transplantation after MI.-Cai, H., Wu, F.-Y., Wang, Q.-L., Xu, P., Mou, F.-F., Shao, S.-J., Luo, Z.-R., Zhu, J., Xuan, S.-S., Lu, R., Guo, H.-D. Self-assembling peptide modified with QHREDGS as a novel delivery system for mesenchymal stem cell transplantation after myocardial infarction.

Mesenchymal stem cell therapy modulates synovial macrophages in a murine model of osteoarthritis

Purpose: Inflammation is one of the major hallmarks of osteoarthritis (OA), with participation of synovial macrophages and infiltrated lymphocytes. Mesenchymal stem cells (MSCs) are known to modulate the response of all cells of the immune system, with a central role for apoptosis becoming evident in many disease models. Intra-articular (IA) injections of MSCs are known to protect from cartilage degeneration and modulate local inflammation, although the lack of engraftment suggests that they exert their action via paracrine mechanisms. It is also hypothesised that locally-delivered MSCs undergo apoptosis or necrosis.The aim of this study was to examine the mechanism of action of IA-delivered MSCs in an animal model of OA. Macrophages were investigated as recipients of the MSC action through ex vivo cultures. Also, the role of apoptosis was assessed with an in vitro model of cell death. Methods: Collagenase-induced OA (CIOA) was performed on C57BL/6 mice, with a saline-treated group (SHAM) used as a control. 2×10ˆ5 GFP+ MSCs were IA-injected in the animals’ knees after one week. Three days later, knee joints were digested into a single-cell suspension and MSCs retrieved by cell sorting, based on cell fluorescence intensity, and expanded for two passages in standard culture conditions. Capacity to form colonies in vitro was assessed by CFU-F assay and expression of surface markers characteristic of MSCs was measured by flow cytometry. Conditioned medium (CM) of retrieved cells was tested on syngeneic bone marrow-derived macrophages (BMDMs) activated with lipopolysaccharides (LPS) for 4 hours. Expression of major histocompatibility complex class II (MHC-II) and co-stimulatory molecule CD86 were measured by flow cytometry, while secretion of cytokines interleukin (IL)-10 and tumor necrosis factor (TNF)-α was quantified by enzyme-linked immunosorbent assay (ELISA). Apoptosis of MSCs was induced in vitro with staurosporine (STS) and confirmed by pre-treatment with the pan-caspase inhibitor Q-VD-OPh, while necrosis was induced by heat treatment at 90°C. Cell death was evaluated by Annexin V/Sytox Blue staining, analysed by flow cytometry. CM were collected and tested on BMDMs as previously described. Results: 1.63% of injected cells were retrieved and proliferated in culture. Compared to naïve (CTRL) MSCs, retrieved cells maintained morphology, proliferation rates, clonogeneicity and surface marker expression profile. CM of CIOA-retrieved MSCs modulated activation of macrophages, significantly downregulating expression of MHC-II and CD86 and upregulating secretion of IL-10, compared to both SHAM-retrieved MSCs and CTRL MSCs. TNF-α was downregulated in a similar fashion, but not significantly. STS induced apoptosis, which was completely prevented by pre-treatment with Q-VD-OPh, thus confirming initiation of caspase pathway. Necrosis was also confirmed by Annexin V/Sytox staining. Apoptotic but not necrotic MSC-CM prevented activation of BMDMs, with suppressed secretion of TNF-α and significantly increased production of IL-10. Conclusions: MSCs can be administered and retrieved from murine knees by cell sorting. Retrieval yield was low, consistently with previous in vivo cell tracking studies, confirming that most therapeutic action is performed via a hit-and-run mechanism. MSCs were licensed from the OA joint to produce an immunosuppressive milieu that modulated macrophages ex vivo, inducing an M2-like (anti-inflammatory) switch. In vitro, apoptotic MSCs showed an increased immunomodulatory potential in a way that reflected the mechanism observed in OA-challenged MSCs. This set of data suggests that the OA synovial environment licensed the MSCs to secrete immunomodulatory factors, but also that apoptosis is a contributing factor to the effects of cell therapy in OA. As expected, macrophages have a crucial role in propagating the MSC-mediated immunomodulation, by acquiring an M2-like phenotype, with inhibition of stimulatory and co-stimulatory molecules and further release of anti-inflammatory cytokines. These findings are key to the understanding of OA and the development of future cell therapies, which could be focused on apoptosis rather than integration in the degenerated tissues.

Alteration of Apoptosis during Differentiation in Human Dental Pulp-Derived Mesenchymal Stem Cell

Because mesenchymal stem cells (MSCs) maintain distinct capacities with respect to self-renewal, differentiation ability and immunomodulatory function, they have been highly considered as the therapeutic agents for cell-based clinical application. Of particular, differentiation condition alters characteristics of MSCs, including cellular morphology, expression of gene/protein and cell surface molecule, immunological property and apoptosis. However, the previous results for differentiation-related apoptosis in MSCs have still remained controversial due to varied outcomes. Therefore, the present study aimed to disclose periodical alterations of pro- and anti-apoptosis in MSCs under differentiation inductions. The human dental pulp-derived MSCs (DP-MSCs) were differentiated into adipocytes and osteoblasts during early (1 week), middle (2 weeks) and late (3 weeks) stages, and were investigated on their apoptosis-related changes by Annexin V assay, qRT-PCR and western blotting. The ratio of apoptotic cell population was significantly (p < 0.05) elevated during the early to middle stages of differentiations but recovered up to the similar level of undifferentiated state at the late stage of differentiation. In the expression of mRNA and protein, whereas expressions of pro-apoptosis-related makers (BAX and BAK) were not altered in any kind and duration of differentiation inductions, anti-apoptosis marker (BCL2) was significantly (p < 0.05) elevated even at the early stage of differentiations. The recovery of apoptotic cell population at the late stage of differentiation is expected to be associated with the response by elevation of anti-apoptotic molecules. The present study may contribute on understanding for cellular mechanism in differentiation of MSCs and provide background data in clinical application of MSCs in the animal biotechnology to develop effective and safe therapeutic strategy.

Apoptosis of mesenchymal stem cells is regulated by Rspo1 via the Wnt/β-catenin signaling pathway

ObjectiveThe aim of this study was to investigate the effect and possible mechanism of action of roof plate-specific spondin1 (Rspo1) in the apoptosis of rat bone marrow mesenchymal stem cells (BMSCs). MethodsOsteogenic and adipogenic differentiation of BMSCs was identified by Alizarin Red and Oil Red O staining, respectively. BMSC surface markers (cluster of differentiation 29 [CD29], CD90, and CD45) were detected using flow cytometry. BMSCs were transfected with an adenoviral vector encoding Rspo1 (BMSCs-Rspo1 group). The expression levels of Rspo1 gene and Rspo1 protein in the BMSCs-Rspo1 group and the two control groups (untransfected BMSCs group and BMSCs-green fluorescent protein [GFP] group) were analyzed and compared by quantitative polymerase chain reaction and Western blot. The occurrence of apoptosis in the three groups was detected by flow cytometry and acridine orange-ethidium bromide (AO-EB) double dyeing. The activity of the Wnt/β-catenin signaling pathway was evaluated by measuring the expression levels of the key proteins of the pathway (β-catenin, c-Jun N-terminal kinase [JNK], and phospho-JNK). ResultsOsteogenic and adipogenic differentiation was confirmed in cultured BMSCs by the positive expression of CD29 and CD90 and the negative expression of CD45. Significantly increased expression levels of Rspo1 protein in the BMSCs-Rspo1 group compared to those in the BMSCs (0.60 ± 0.05 vs. 0.13 ± 0.02; t=95.007, P=0.001) and BMSCs-GFP groups (0.60 ± 0.05 vs. 0.10 ± 0.02; t=104.842, P=0.001) were observed. The apoptotic rate was significantly lower in the BMSCs-Rspo1 group compared with those in the BMSCs group ([24.06 ± 2.37]% vs. [40.87 ± 2.82]%; t = 49.872, P = 0.002) and the BMSCs-GFP group ([24.06 ± 2.37]% vs. [42.34 ± 0.26]%; t = 62.358, P = 0.001). In addition, compared to the BMSCs group, the protein expression levels of β-catenin (2.67 ± 0.19 vs. 1.14 ± 0.14; t = −9.217, P = 0.000) and JNK (1.87 ± 0.17 vs. 0.61 ± 0.07; t = −22.289, P = 0.000) were increased in the BMSCs-Rspo1 group. Compared to the BMSCs-GFP group, the protein expression levels of β-catenin (2.67 ± 0.19 vs. 1.44 ± 0.14; t = −5.692, P = 0.000) and JNK (1.87 ± 0.17 vs. 0.53 ± 0.06; t = −10.589, P = 0.000) were also upregulated in the BMSCs-Rspo1 group. Moreover, the protein expression levels of phospho-JNK were increased in the BMSCs-Rspo1 group compared to those in the BMSCs group (1.89 ± 0.10 vs. 0.63 ± 0.09; t = −8.975, P = 0.001) and the BMSCs-GFP group (1.89 ± 0.10 vs. 0.69 ± 0.08; t = −9.483, P = 0.001). ConclusionThe Wnt/β-catenin pathway could play a vital role in the Rspo1-mediated inhibition of apoptosis in BMSCs.

Ulinastatin inhibits apoptosis induced by serum deprivation in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have exhibited great potential in the therapy of cardiovascular disease. However, the application of MSCs is hampered by apoptosis, which reduces the number of cells in the host cardiac microenvironment. Ulinastatin (UTI), a broad‑spectrum protease inhibitor that can be purified from human urine, has attracted attention for its protective effects through its immunomodulatory and anti‑inflammatory properties. The present study aimed to evaluate the effects of UTI on serum deprivation‑induced apoptosis of MSCs and investigate its molecular mechanisms. Cell viability was determined by the MTT assay. Apoptosis was assessed by flow cytometric analysis with Annexin V/propidium iodide staining. The protein levels of cleaved caspase‑3, B‑cell lymphoma‑2 (Bcl‑2) family proteins, total‑Akt and phospho‑Akt were evaluated by western blot. The results of the present study demonstrated that UTI exhibited a protective effect in serum deprived MSCs, as indicated by increased cell viability, and a reduction in the rate of apoptosis and caspase‑3 activation. In addition, treatment with UTI significantly decreased the expression levels of Bcl‑2, Bcl‑extra large and Bcl‑associated X protein. Furthermore, activation of the Akt signaling pathway was involved in the UTI‑induced anti‑apoptotic effects. The present findings indicated that UTI is able to promote the survival of MSCs under serum deprivation conditions. The present study may be helpful in improving the therapeutic efficacy of MSC transplantation used to cure chronic ischemic heart disease.

Downregulation of long noncoding RNA HOTAIRM1 variant 1 contributes to osteoarthritis via regulating miR-125b/BMPR2 axis and activating JNK/MAPK/ERK pathway

Objective: Osteoarthritis (OA) is a leading cause of pain and disability worldwide, which is characterized by degenerative and inflammatory processes affecting joints and surrounding tissues. This study aimed to elucidate the role and possible mechanism of long non-coding RNA HOX antisense intergenic RNA myeloid 1 variant 1 (HOTAIRM1-1) in the development of osteoarthritis (OA).Material and methods: The expressions of HOTAIRM1-1 and miR-125b in OA cartilages and normal articular cartilages were determined. Moreover, human mesenchymal stem cells (MSCs) were cultured in MSC growth medium for 8 days to induce chondrogenic differentiation. The effects of aberrant expression of HOTAIRM1-1 on MSCs viability, apoptosis and differentiation were investigated. Furthermore, the regulatory relationships between HOTAIRM1-1 and miR-125b as well as the target of miR-125b were explored. Besides, the association between HOTAIRM1-1 and JNK/MAPK/ERK pathway was detected.Results: Inverse expression of HOTAIRM1-1 (downregulated) and miR-125b (upregulated) was revealed in OA cartilages. In addition, HOTAIRM1-1 expression increased at 4 days after induction of chondrogenic differentiation and further enhanced after 8 days, while miR-125b had opposite expression changes. Suppression of HOTAIRM1-1 inhibited MSCs viability, induced apoptosis and suppressed cell differentiation. Moreover, HOTAIRM1-1 negatively regulated the expression of miR-125b, and the effects of HOTAIRM1-1 suppression on cell viability, apoptosis and differentiation were achieved by negative regulation of miR-125b. Furthermore, miR-125b regulated MSCs viability, apoptosis and differentiation via targeting bone morphogenetic protein receptor 2 (BMPR2). Besides, overexpression of HOTAIRM1-1 inhibited the activation of JNK/MAPK/ERK pathway.Conclusions: Our results indicate that HOTAIRM1-1 is downregulated in OA cartilages, and its downregulation may inhibit MSCs viability, induce apoptosis and suppress differentiation via regulating miR-125b/BMPR2 axis. JNK/MAPK/ERK pathway may be a possible downstream mechanism to mediate the role of HOTAIRM1-1 in OA development.

Growth arrest-specific gene 6 transfer promotes mesenchymal stem cell survival and cardiac repair under hypoxia and ischemia via enhanced autocrine signaling and paracrine action

Poor cell viability after transplantation has restricted the therapeutic capacity of mesenchymal stem cells (MSCs) for cardiac dysfunction after myocardial infarction (MI). Growth arrest-specific gene 6 (Gas6) encodes a secreted γ-carboxyglutamic acid (Gla)-containing protein that functions in cell growth, adhesion, chemotaxis, mitogenesis and cell survival. In this study, we genetically modified MSCs with Gas6 and evaluated cell survival, cardiac function, and infarct size in a rat model of MI via intramyocardial delivery. Functional studies demonstrated that Gas6 transfer significantly reduced MSC apoptosis, increased survival of MSCs in vitro and in vivo, and that Gas6-engineered MSCs (MSCGas6)-treated animals had smaller infarct size and showed remarkably functional recovery as compared with control MSCs (MSCNull)-treated animals. Mechanistically, Gas6 could enhance phosphatidylinositol 3-kinase (PI3K)/Akt signaling and improve hypoxia-inducible factor-1 alpha (HIF-1α)-driven secretion of four major growth factors (VEGF, bFGF, SDF and IGF-1) in MSCs under hypoxia in an Axl-dependent autocrine manner. The paracrine action of MSCGas6 was further validated by coculture neonatal rat cardiomyocytes with conditioned medium from hypoxia-treated MSCGas6, as well as by pretreatment cardiomyocytes with the specific receptor inhibitors of VEGF, bFGF, SDF and IGF-1. Collectively, our data suggest that Gas6 may advance the efficacy of MSC therapy for post-infarcted heart failure via enhanced Gas6/Axl autocrine prosurvival signaling and paracrine cytoprotective action.

Pioglitazone Protects Mesenchymal Stem Cells against P-Cresol-Induced Mitochondrial Dysfunction via Up-Regulation of PINK-1.

Mesenchymal stem cells (MSC) could be a candidate for cell-based therapy in chronic kidney disease (CKD); however, the uremic toxin in patients with CKD restricts the therapeutic efficacy of MSCs. To address this problem, we explored the effect of pioglitazone as a measure against exposure to the uremic toxin P-cresol (PC) in MSCs. Under PC exposure conditions, apoptosis of MSCs was induced, as well as PC-induced dysfunction of mitochondria by augmentation of mitofusion, reduction of mitophagy, and inactivation of mitochondrial complexes I and IV. Treatment of MSCs with pioglitazone significantly inhibited PC-induced apoptosis. Pioglitazone also prevented PC-induced mitofusion and increased mitophagy against PC exposure through up-regulation of phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK-1). Furthermore, pioglitazone protected against PC-induced mitochondrial dysfunction by increasing the cytochrome c oxidase subunit 4 (COX4) level and activating complexes I and IV, resulting in enhancement of proliferation. In particular, activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) regulated the pioglitazone-mediated up-regulation of PINK-1. These results indicate that pioglitazone protects MSCs against PC-induced accumulated mitochondrial dysfunction via the NF-κB–PINK-1 axis under P-cresol exposure conditions. Our study suggests that pioglitazone-treated MSCs could be a candidate for MSC-based therapy in patients with CKD.

GRAPHENE OXIDE CONTAINING CHITOSAN BASED HYDROGEL IMPROVES THE SURVIVAL OF TRANSPLANTED ALLOGENEIC STEM CELLS IN INFARCTED HEART

Bone marrow derived allogeneic mesenchymal stem cells (MSCs) are in clinical trials for myocardial repair after a cardiac injury. However, recent reviews of allogeneic MSCs based clinical trials reported that transplanted cells were not able to survive for long term in the heart. Therefore, strategies to improve survival of transplanted cells would preserve the benefits of allogeneic MSCs therapy for heart repair. Recently, graphene oxide (GO) based biomaterials are being widely investigated for their ability to improve survival of transplanted MSCs in the heart. However, the presence of reactive oxygen functional groups (-OH, -COOH) in the structure of GO increases oxidative stress and activate cell death pathways, which impairs the benefits of GO based biomaterials. Therefore, to improve benefits of GO for stem cell delivery, we synthesized a thermosensitive hydrogel by conjugating GO with chitosan (CS). Our fourier transformed infrared spectrum (FTIR) analysis revealed an interaction between oxygen functional groups of GO and amino (-NH2) groups of chitosan. Our data demonstrate that CS masked the reactive functional groups of GO and prevented GO mediated ROS induction. This novel chitosan-graphene oxide (CS-GO) composite exhibited optimal porosity for cell conjugation and retention. Furthermore, CS-GO biomaterial was cyto-compatible as there was no cytotoxicity caused by CS-GO to MSCs. The CS-GO also prevented hypoxia induced apoptosis of mesenchymal stem cells. We employed this novel biomaterial to deliver allogeneic mesenchymal stem cells to the infarcted heart in a rat model of myocardial infarction. After five weeks of cell transplantation, there was a significant improvement in the survival of implanted MSCs and cardiac function in CS-GO group. Therefore, conjugation of GO with chitosan prevented GO mediated ROS generation and cell death. Also, CS-GO mediated delivery of allogeneic MSCs to infarcted heart improved the survival of transplanted cells and preserved cardiac function.

The regulatory roles of VEGF-Notch signaling pathway on aplastic anemia with kidney deficiency and blood stasis.

Vascular endothelial growth factor (VEGF)-Notch signaling pathway plays an important role in aplastic anemia (AA). This study aimed to evaluate the regulatory roles of VEGF-Notch signaling pathway on mesenchymal stem cells (MSCs) isolated from AA patients with kidney deficiency and blood stasis (KB) (AA MSCs). Expression of VEGF-Notch signaling related factors, including VEGF, VEGFR, Notch-1, Jagged1, Delta-like1, and hes1 was detected in bone marrow (BM) tissues and AA MSCs by Western blot analysis. VEGF (100 ng/mL) and γ-secretase inhibitor (DAPT) (10 μM) was used to active and inhibit VEGF-Notch signaling in AA MSCs, respectively. After treatment, the proliferation, apoptosis, and adipogenic differentiation of AA MSCs was detected by Cell Counting Kit-8, flow cytometry, and Oil red O staining, respectively. Lentivirus short hairpin RNA (shRNA) were constructed to downregulate Notch-1 and VEGF in normal bone marrow mesenchymal stem cells (BMSCs), and the effects of VEGF/Notch-1 shRNA transfected BMSCs on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated. Significantly lower expression of VEGF, VEGFR, Notch-1, Jagged1, Delta-like1, and hes1 was revealed in AA BM tissues and AA MSCs when compared with the normal control (P < 0.05). The intervention of DAPT significantly inhibited the proliferation, and promoted the apoptosis and adipogenic differentiation of AA MSCs, while VEGF intervention exhibited opposite results (P < 0.05). Meanwhile, the proliferation, migration, and angiogenesis of HUVECs were significantly promoted by normal BMSCs, while inhibited by VEGF/Notch-1 shRNA transfected BMSCs (P < 0.05). The activation of VEGF-Notch signaling pathway may be a potential therapeutic target for AA with KB.

Co-expression of Akt1 and Wnt11 promotes the proliferation and cardiac differentiation of mesenchymal stem cells and attenuates hypoxia/reoxygenation-induced cardiomyocyte apoptosis.

Mesenchymal stem cells (MSCs) transplantation has emerged as a promising therapeutic strategy for acute myocardial infarction. However, there are still limitations for this therapy, such as low survival rate and poor cardiac differentiation potential of MSCs. In this study, we genetically engineered MSCs using ex vivo adeno-associated virus (AAV) transduction to overexpress Akt1 and Wnt11, which are well-characterized genes involved in MSC proliferation and cardiac differentiation. Our results showed that infection with AAV-Akt1-Wnt11 significantly upregulated the growth and proliferation of MSCs, as compared with those infected with AAV-Akt1 or AAV-Wnt11. In addition, co-expression of Akt1 and Wnt11 markedly promoted the expression of cardiac markers including NK2 transcription factor related 5, GATA-binding protein 4, α-myosin MHC and brain natriuretic protein. Notably, co-expression of Akt1 and Wnt11 increased cell survival and reduced cell apoptosis of MSCs under hypoxia/reoxygenation (H/R) treatment; however, these effects were blocked by Wnt11 neutralizing antibodies or Akt1 inhibitor. Moreover, co-culture of cardiomyocytes with MSCs infected with AAV-Akt1-Wnt11, in a dual chamber system, significantly reduced H/R-induced cell apoptosis compared with those co-cultured with MSCs infected with AAV-Akt1 or AAV-Wnt11. Overall, our results showed that MSCs, co-expressing Akt1 and Wnt11, showed greater survival and cardiac differentiation under H/R conditions and effectively ameliorated H/R-induced cardiomyocyte apoptosis in vitro. Our study suggests that transplantation of MSCs genetically engineered with AAV-Akt1-Wnt11 is a promising therapeutic strategy for treatment of acute myocardial infarction.

Antimicrobial effects of mesenchymal stem cells primed by modified LPS on bacterial clearance in sepsis.

Mesenchymal stem cells (MSCs)-based regenerative therapy is now considered as an alternative approach to revive infectious diseases, including sepsis. Nevertheless, the efficiency of MSC application is limited by the poor survival rate of engrafted MSCs. Hence, preconditioning was established as a strategy to increase the cells' efficiency. MSCs were preconditioned with 1 μg/ml of three different lipopolysaccharides (LPSs) of Pseudomonas (Pse-LPS), Acinetobacter (Ac-LPS), and Acinetobacter inactivated lipid A by PagL (Ac-LPS-PagL). Then, preconditioned MSCs were exposed to oxidative stress and serum deprivation followed by evaluation of the antibacterial activity, survival, and apoptosis of MSCs. Then, the murine sepsis model treated with 100 μl phosphate-buffered saline (control group, sepsis group), 100 μl of 1 × 10 6 wild MSCs (MSC group), and three remained groups received 100 μl of 1 × 10 6 LPS-preconditioned MSCs (Pse-LPS-MSCs group: LPS purified from Pseudomonas, or Ac-LPS-MSCs group: LPS purified from Acinetobacter, and Ac-PagL-LPS-MSCs group: detoxified LPS Pagl). After 4 days, LPS-preconditioned MSC transplantation modulated the immune response and reduced inflammation in septic mice. Apoptosis of Pse-LPS/Ac-LPS-preconditioned-MSCs was obviously reduced in vitro, and the survival rate of engrafted mice was evidently elevated in Pse-LPS-MSCs and Ac-LPS-MSCs groups compared with other three groups. LPS preconditioning provides an innovative strategy for evolving functional and biological properties of MSCs and ameliorates the survival rate of the mouse model of sepsis after MSC transplantation, protects cells from apoptosis and organ damages, and evaluates therapeutic properties, including immunemodulatory.

Abstract 331: Conjugation of Graphene Oxide With Chitosan Improves Survival of Transplanted Allogeneic Stem Cells and Preserves Ventricular Function in the Infarcted Heart

Allogeneic mesenchymal stem cells (MSCs) derived from bone marrow are considered to ideal candidate cell type for myocardial repair after a cardiac injury. However, recent reviews of allogeneic MSCs based clinical trials reported that transplanted cells were not able to survive for long term in the heart. Therefore, strategies to improve survival of transplanted cells would preserve the benefits of allogeneic MSCs therapy for heart repair. Recently, graphene oxide (GO) based biomaterials are being widely investigated for their ability to improve survival of transplanted MSCs in the heart. However, the presence of reactive oxygen functional groups (-OH, -COOH) in the structure of GO increases oxidative stress and activate cell death pathways, which impairs the benefits of GO based biomaterials. Therefore, to improve benefits of GO for stem cell delivery, we synthesized a thermosensitive hydrogel by conjugating GO with chitosan (CS). Our fourier transformed infrared spectrum (FTIR) analysis revealed an interaction between oxygen functional groups of GO and amino (-NH2) groups of chitosan. Our data demonstrate that CS masked the reactive functional groups of GO and prevented GO mediated ROS induction. This novel chitosan-graphene oxide (CS-GO) composite exhibited optimal porosity for cell conjugation and retention. Furthermore, CS-GO biomaterial was cyto-compatible as there was no cytotoxicity caused by CS-GO to MSCs. Furthermore, CS-GO also prevented hypoxia induced apoptosis of MSCs. We employed this novel biomaterial to deliver allogeneic MSCs to the infarcted heart in a rat model of myocardial infarction. After 5 weeks of cell transplantation, there was a significant improvement in the survival of implanted MSCs and cardiac function in CS-GO group. Therefore, conjugation of GO with chitosan prevented GO mediated ROS generation and cell death. Also, CS-GO mediated delivery of allogeneic MSCs to infarcted heart improved the survival of transplanted cells and preserved cardiac function.

Biochemical Effects of Some Endoplasmic Reticulum Stress Inducers on Mesenchymal Stem Cells in vitro

Our studies aimed the effects of some endoplasmic reticulum stress inducers (thapsigargin, a Ca2+-ATP-ase inhibitor; tunicamycin, a protein N-glycosylation inhibitor; brefeldin A, a protein transport inhibitor; paraquat, an enhancer of reactive oxygen species production; A23187, a Ca2+ ionophore), as well as some antioxidants (N-acetylcysteine; dithiothreitol, a disulfide bond formation inhibitor) on apoptosis of cultured rat mesenchymal stem cells. The analyze of obtained results evidenced that paraquat, a common and effective herbicide, induced the apoptosis of the isolated rat mesenchymal stem cells in a larger proportion as compared to other chemicals as follows: paraquat ] thapsigargin ] tunicamycin @A23187 ] brefeldin A. Dithiothreitol was effective as a reducer of mesenchymal stem cells apoptosis when was administered as co-treatment for paraquat for 24 h. In contrast, N-acetylcysteine, another potent antioxidant, had no protective effects against paraquat apoptotic effects.

Role of lincRNA‑p21 in the protective effect of macrophage inhibition factor against hypoxia/serum deprivation‑induced apoptosis in mesenchymal stem cells.

Stem cell transplantation is a promising clinical strategy for curing ischemic cardiomyopathy. However, its efficacy is impaired by low cell survival following transplantation, partly caused by insufficient resistance of the transplanted stem cells to severe oxidative stress at the injury site. In the current study, it was demonstrated that the small‑molecule macrophage migration inhibitory factor (MIF) enhanced the defense of bone marrow‑derived mesenchymal stem cells (MSCs) against hypoxia/serum deprivation (SD)‑induced apoptosis invitro. MIF significantly suppressed apoptosis and caspase family activities through inhibition of long intergenic noncoding (linc) RNA‑p21 to maintain activation of the Wnt/β‑catenin signaling pathway. The regulatory loop between MIF and the lincRNA‑p21‑Wnt/β‑catenin signaling pathway was identified to be associated with the inhibition of oxidative stress. The involvement of the lincRNA‑p21‑Wnt/β‑catenin signaling pathway in the effects of MIF in MSCs by overexpression of lincRNA‑p21and silencing β‑catenin using small interfering RNA was also demonstrated, both of which abolished the anti‑apoptotic and anti‑oxidative effects of MIF in MSCs under hypoxia/SD conditions. In conclusion, MIF protected MSCs from hypoxia/SD‑induced apoptosis by interacting with lincRNA‑p21, leading to activation of the downstream Wnt/β‑catenin signaling pathway and decreased oxidative stress. Thus, treatment with MIF may have important therapeutic implications in improving MSC survival and therapeutic efficiency.

Anti-apoptotic effects of adipose-derived adherent stromal cells in mesenchymal stem cells exposed to oxidative stress.

We investigated the expression levels of proteins associated with oxidation, inflammation, and apoptosis in MSCs co-cultured with isolated ADASs from 6 donors' SVFs under oxidative stress conditions. Our results imply that isolated ADASs from SVFs may inhibit oxidative stress-induced cell cycle arrest and/or apoptosis in MSCs via a p53-dependent pathway. Furthermore, we identified an anti-apoptotic mechanism involving oxidative stress-induced apoptosis by adipose-derived ADASs in MSCs for the first time. Our findings suggest that the positive effects of SVF implantation into damaged heart tissue may be attributed to the various actions of MSCs.