SDF-1/CXCR4 axis modulates bone marrow mesenchymal stem cell apoptosis,migration and cytokine secretion

Bone marrow mesenchymal stem cells (MSCs) are considered as a promising cell source to treat the acute myocardial infarction. However, over 90% of the stem cells usually die in the first three days of transplantation. Survival potential, migration ability and paracrine capacity have been considered as the most important three factors for cell transplantation in the ischemic cardiac treatment. We hypothesized that stromal-derived factor-1 (SDF-1)/CXCR4 axis plays a critical role in the regulation of these processes. In this study, apoptosis was induced by exposure of MSCs to H₂O₂ for 2 h. After re-oxygenation, the SDF-1 pretreated MSCs demonstrated a significant increase in survival and proliferation. SDF-1 pretreatment also enhanced the migration and increased the secretion of pro-survival and angiogenic cytokines including basic fibroblast growth factor and vascular endothelial growth factor. Western blot and RT-PCR demonstrated that SDF-1 pretreatment significantly activated the pro-survival Akt and Erk signaling pathways and up-regulated Bcl-2/Bax ratio. These protective effects were partially inhibited by AMD3100, an antagonist of CXCR4. We conclude that the SDF-1/CXCR4 axis is critical for MSC survival, migration and cytokine secretion.     

Alpinate Oxyphyllae extracts enhance the longevity and homing of mesenchymal stem cells and augment their protection against senescence in H9c2 cells

Adipose-derived mesenchymal stem cells (ADMSCs) are easily accessible and are attractive mesenchymal stem cells for use in regenerative medicine; however their application is frequently restricted due to various challenges present in the environment they are administered. Therefore ADMSCs are preferably preconditioned with various stimulating factors to overcome the barriers developed in any pathological conditions. Here we used ADMSCs from rat adipose based on the abundance of positive markers and preconditioned the cells with extracts from Alpinate Oxyphyllae Fructus (AOF), a traditional Chinese herb used for antiaging, associated various health benefits. The preconditioned stem cells were tested for their potential to drive H9c2 from doxorubicin (Dox)-induced aging. The AOF-treated stem cells enriched stemness in ADMSCs with respect to their stem cells' positive marker, and enhanced their longevity mechanism and elevated the stem cell homing-associated C-X-C chemokine receptor type 7 (CXCR7). The AOF preconditioned stem cells, when cocultured with H9c2 cells, showed effective protection to Dox-induced senescence and stem cell homing to damaged H9c2 cells. The presence of AOF provided greater protective effects in the Dox environment. In addition, AOF-pretreated ADMSCs showed enhanced migration than those treated with AOF in Dox environment. Therefore, our results show that administration of AOF preconditioned stem cells is potentially an effective strategy in the management of aging-associated cardiac disorders.     

HIV glycoprotein gp120 enhances mesenchymal stem cell migration by upregulating CXCR4 expression

Background

HIV infection and/or the direct pathogenic effects of circulating HIV proteins impairs the physiological function of mesenchymal stem cells (MSCs), and contribute to the pathogenesis of age-related clinical comorbidities in people living with HIV. The SDF-1/CXCR4 pathway is vital for modulating MSC proliferation, migration and differentiation. HIV glycoprotein gp120 inhibits SDF-1 induced chemotaxis by downregulating the expression and function of CXCR4 in monocytes, B and T cells. The influence of gp120 on CXCR4 expression and migration in MSCs is unknown.

Snail基因修饰对骨髓间充质干细胞CXCR4表达水平及向SDF-1趋化能力的影响

为研究Snail基因修饰对骨髓间充质干细胞(MSCs)CXCR4表达水平及向SDF-1趋化能力影响, 将重组真核表达载体(pCAGGSneo-snail-HA)及对照空质粒(pCAGGSneo)转染MSCs, 采用免疫荧光细胞化学染色、荧光标记流式细胞仪技术及RT-PCR检测细胞CXCR4表达水平; 体外跨膜趋化实验评价MSCs向SDF-1趋化能力, 观察抗CXCR4中和抗体的干预作用。MSCs-Sna的CXCR4表达水平明显高于MSCs-neo。MSCs-Sna在SDF-1诱导下细胞迁移量较MSCs-neo显著增加(P<0.05)。抗CXCR4中和抗体可显著减少SDF-1a诱导的MSCs-Sna趋化运动。研究提示通过上调Snail表达而提高MSCs向正调节表达SDF-1的受损组织迁移效率的可行性, 为优化MSCs迁移力的研究提供了实验基础。     

Surface expression of CXCR4 in unrestricted somatic stem cells and its regulation by growth factors

Umbilical cord blood‐derived USSCs (unrestricted somatic stem cells) have recently been considered as a potential source for stem cell therapy and transplantation due to their characteristics such as easy accessibility, low immunogenicity, self‐renewing and multilineage differentiation potential. Stem cell homing is a key factor in successful transplantation, which is regulated by CXCR4 in stem cells. In this study, we evaluated the expression of CXCR4 in USSCs different passages. Moreover, the effect of VEGF (vascular endothelial growth factor) and IGF‐1 (insulin‐like growth factor 1) on its expression was assessed. It was shown that the expression of CXCR4 in USSCs decreased with the increase in passage number. It was also revealed that VEGF increased surface expression and mRNA level of CXCR4 in USSCs, while IGF‐1 decreased its expression. When VEGF and IGF‐1 were administered simultaneously, CXCR4 expression was increased, but the expression level was less than VEGF alone. Finally, it was shown that over‐expression of CXCR4 enhanced the migratory capacity of USSCs. The increase of CXCR4 expression, here caused by VEGF in USSCs, can improve the efficacy of stem cell therapy and transplantation after long‐term culture of stem cells before clinical use.     

Characterization of bone-marrow-derived rat mesenchymal stem cells depending on donor age

It is generally accepted that autologous transfers, as non-immunogenic, constitute the safest approach in cellular transplantations. However, this attitude is often associated with the need for isolation and extracorporeal propagation of cells derived from aged patients. Thus the knowledge about relationship between aging and the properties of MSCs (mesenchymal stem cells) is crucial in developing new clinical strategies. The aim of this study was to perform complex comparison of MSC derived from young and aged individuals, which included phenotype, proliferating rate, osteogenic and adipogenic potential and secretory activity. Evaluated populations were isolated from bone marrow of 3-month-old and 24-month-old rats. There was no significant difference in membrane antigen expression and PDT (population doubling time). Additionally, the adipogenic and osteogenic potential did not vary between studied populations. The reaction of MSCs to either mitogen [bFGF (basic fibroblas t growth factor)] or oxidative stress (H2O2) in vitro displayed a very similar pattern in both analysed populations. There was no difference in TGFβ1 (transforming growth factor β1) and VEGF (vascular endothelial growth factor) secretion measured by ELISA test and gene expression evaluated by real-time PCR. However, the expression of the gene for IL-1α (interleukin-1α) was 8-fold lower in oMSC (MSC isolated from old rats). These results indicate that aging individuals can be considered as candidates for autologous transplantation of bone-marrow-derived MSCs.     

Hypermethylation of microRNA-149 activates SDF-1/CXCR4 to promote osteogenic differentiation of mesenchymal stem cells

MicroRNAs (miRs) involve in osteogenic differentiation and osteogenic potential of mesenchymal stem cells (MSCs). Accordingly, the present study aimed to further uncover role miR-149 plays in osteogenic differentiation of MSCs with the involvement of the stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor-4 (CXCR4) pathway. Initially, the osteogenic differentiation model was induced. Next, the positive expression of STRO-1 in periosteum, alkaline phosphatase (ALP) activity, osteocalcin (OCN) protein content, and the calcium deposition in MSCs were determined. MSCs were treated with DNA methyltransferase inhibitor 5-aza-CdR, SDF-1 neutralizing antibody, or CXCR4 antagonist AMD3100 to investigate their roles in osteogenic differentiation; with the expression of CD44, CD90, CD14, and CD45 detected. Furthermore, the levels of SDF-1 and CXCR4, and the genes related to stemness (Nanog, Oct-4, and Sox-2) were measured to explore the effects of miR-149. The obtained data revealed the upregulation of STRO-1 in the periosteum. miR-149 could specifically bind to SDF-1. Besides, increased miR-149 methylation, higher ALP activity and OCN content, decreased positive rates of CD44 and CD90, and increased positive rates of CD14 and CD45 were found in osteogenic differentiation of MSCs. Subsequently, 5-Aza-CdR treatment reversed the above-mentioned effects. MSCs were finally treated with SDF-1 neutralizing antibody or AMD3100 to decrease Nanog, Oct-4, and Sox-2 expression. Taken together these results, miR-149 hypermethylation has the potential to activate the SDF-1/CXCR4 pathway and further promote osteogenic differentiation of MSCs.     

Systematic parameter optimization of a Me(2)SO- and serum-free cryopreservation protocol for human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have great potential for clinical therapy and regenerative medicine. One major challenge concerning their application is the development of an efficient cryopreservation protocol since current methods result in a poor viability and high differentiation rates. A high survival rate of cryopreserved cells requires an optimal cooling rate and the presence of cryoprotective agents (CPA) in sufficient concentrations. The most widely used CPA, dimethylsulfoxide (Me₂SO), is toxic at high concentrations at temperatures >4 °C and has harmful effects on the biological functionality of stem cell as well as on treated patients.Thus, this study investigates different combinations of non-cytotoxic biocompatible substances, such as ectoin and proline, as potential CPAs in a systematic parametric optimization study in comparison to Me₂SO as control and a commercial freezing medium (Biofreeze®, Biochrom). Using a freezing medium containing a low proline (1%, w/v) and higher ectoin (10%, w/v) amount revealed promising results although the highest survival rate was achieved with the Biofreeze® medium. Cryomicroscopic experiments of hMSCs revealed nucleation temperatures ranging from −16 to −25 °C. The CPAs, beside Me₂SO, did not affect the nucleation temperature. In most cases, cryomicroscopy revealed intracellular ice formation (IIF) during the cryopreservation cycle for all cryoprotocols. The occurence of IIF during thawing increased with the cooling rate. In case of hMSC there was no correlation between the rate of IIF and the post-thaw cell survival. After thawing adipogenic differentiation of the stem cells demonstrated cell functionality.     

Modification of the alkaline comet assay with human mesenchymal stem cells

MSCs (mesenchymal stem cells) are planned foruse in regenerative medicine to offset age-dependent alterations. However, MSCs are affected by replicative senescence associated with decreasing proliferation potential, telomere shortening and DNA damage during in vitro propagation. To monitor in vitro senescence, we have assessed the integrity of DNA by the alkaline comet assay. For optimization of the comet assay we have enhanced the stability of comet slides in liquid and minimized the background noise of the method by improving adhesion of agarose gels on the comet slides and concentrating cells on a defined small area on the slides. The modifications of the slide preparation increase the overall efficiency and reproducibility of the comet assay and minimize the image capture and storage. DNA damage of human MSCs during in vitro cultivation increased with time, as assessed by the comet assay, which therefore offers a fast and easy screening tool in future efforts to minimize replicative senescence of MSCs in vitro.     

CXCR4‐SF1 bifunctional adipose‐derived stem cells benefit for the treatment of Leydig cell dysfunction‐related diseases

Stem cell transplantation is a candidate method for the treatment of Leydig cell dysfunction‐related diseases. However, there are still many problems that limit its clinical application. Here, we report the establishment of CXCR4‐SF1 bifunctional adipose‐derived stem cells (CXCR4‐SF1‐ADSCs) and their reparative effect on Leydig cell dysfunction. CD29⁺ CD44⁺ CD34^? CD45^? ADSCs were isolated from adipose tissue and purified by fluorescence‐activated cell sorting (FACS). Infection with lentiviruses carrying the CXCR4 and SF1 genes was applied to construct CXCR4‐SF1‐ADSCs. The CXCR4‐SF1‐ADSCs exhibited enhanced migration and had the ability to differentiate into Leydig‐like cells in vitro. Furthermore, the bifunctional ADSCs were injected into BPA‐mediated Leydig cell damage model mice via the tail vein. We found that the CXCR4‐SF1‐ADSCs were capable of homing to the injured testes, differentiating into Leydig‐like cells and repairing the deficiency in reproductive function caused by Leydig cell dysfunction. Moreover, we investigated the mechanism underlying SF1‐mediated differentiation and testosterone synthesis in Leydig cells, and the B‐box and SPRY Domain Containing Protein (BSPRY) gene was proposed to be involved in this process. This study provides insight into the treatment of Leydig cell dysfunction‐related diseases.     

Co-culture with human synovium-derived mesenchymal stem cells inhibits inflammatory activity and increases cell proliferation of sodium nitroprusside-stimulated chondrocytes

Rheumatoid arthritis (RA) and osteoarthritis (OA) are primarily chronic inflammatory diseases. Mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the mesodermal lineage, and to regulate immunomodulatory activity. Specifically, MSCs have been shown to secrete insulin-like growth factor 1 (IGF-1). The purpose of the present study was to examine the inhibitory effects on inflammatory activity from a co-culture of human synovium-derived mesenchymal stem cells (hSDMSCs) and sodium nitroprusside (SNP)-stimulated chondrocytes. First, chondrocytes were treated with SNP to generate an in vitro model of RA or OA. Next, the co-culture of hSDMSCs with SNP-stimulated chondrocytes reduced inflammatory cytokine secretion, inhibited expression of inflammation activity-related genes, generated IGF-1 secretion, and increased the chondrocyte proliferation rate. To evaluate the effect of IGF-1 on inhibition of inflammation, chondrocytes pre-treated with IGF-1 were treated with SNP, and then the production of inflammatory cytokines was analyzed. Treatment with IGF-1 was shown to significantly reduce inflammatory cytokine secretion in SNP-stimulated chondrocytes. Our results suggest that hSDMSCs offer a new strategy to promote cell-based cartilage regeneration in RA or OA.     

GATA-4 induces changes in electrophysiological properties of rat mesenchymal stem cells

Background

Transplanted mesenchymal stem cells (MSC) can differentiate into cardiac cells that have the potential to contribute to heart repair following ischemic injury. Overexpression of GATA-4 can significantly increase differentiation of MSC into cardiomyocytes (CM). However, the specific impact of GATA-4 overexpression on the electrophysiological properties of MSC-derived CM has not been well documented.

Methods

Adult rat bone marrow MSC were retrovirally transduced with GATA-4 (MSC^GATA-4) and GFP (MSC^Null) and subsequently co-cultured with neonatal rat ventricular cardiomyocytes (CM). Electrophysiological properties and mRNA levels of ion channels were assessed in MSC using patch-clamp technology and real-time PCR.

Results

MSC^GATA-4 exhibited higher levels of the TTX-sensitive Na⁺ current (I_Na.TTX), L-type calcium current (I_Ca.L), transient outward K⁺ current (I_to), delayed rectifier K⁺ current (IK_DR) and inwardly rectifying K⁺ current (I_K1) channel activities reflective of electrophysiological characteristics of CM. Real-time PCR analyses showed that MSC^GATA-4 exhibited upregulated mRNA levels of Kv1.2, Kv2.1, SCN2a1, CCHL2a, KV1.4 and Kir1.1 channels versus MSC^Null. Interestingly, MSC^GATA-4 treated with IGF-1 neutralizing antibodies resulted in a significant decrease in Kir1.1, Kv2.1, KV1.4, CCHL2a and SCN2a1 channel mRNA expression. Similarly, MSC^GATA-4 treated with VEGF neutralizing antibodies also resulted in an attenuated expression of Kv2.1, Kv1.2, Kv1.4, Kir1.1, CCHL2a and SCN2a1 channel mRNAs.

Conclusions

GATA-4 overexpression increases I_to, IK_DR, I_K1, I_Na.TTX and I_Ca.L currents in MSC. Cytokine (VGEF and IGF-1) release from GATA-4 overexpressing MSC can partially account for the upregulated ion channel mRNA expression.

General significance

Our results highlight the ability of GATA4 to boost the cardiac electrophysiological potential of MSC.     

The effects of cyclin-dependent kinase inhibitors on adipogenic differentiation of human mesenchymal stem cells

The molecular mechanisms that couple growth arrest and cell differentiation were examined during adipogenesis. Here, to understand the cyclin-dependent kinase inhibitor (CKI) genes involved in the progression of adipogenic differentiation, we examined changes in the protein and mRNA expression levels of CKI genes in vitro. During the onset of growth arrest associated with adipogenic differentiation, two independent families of CKI genes, p27Kip1 and p18INK4c, were significantly increased. The expressions of p27Kip1 and p18INK4c, regulated at the level of protein and mRNA accumulation, were directly coupled to adipogenic differentiation. This finding was supported by the inhibition of adipogenic differentiation caused by short interfering RNA (siRNA). In this study, we investigated the regulatory effects of transforming growth factor beta-1 (TGFβ-1) on CKI genes involved in adipogenic differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). Only the up-regulation of p18INK4c during adipogenic differentiation, and not that of the p27Kip1 gene was prevented by treatment with TGFβ-1, one of the factors that inhibit adipogenesis in vitro. This finding indicates a close correlation between adipogenic differentiation and p18INK4c induction in hMSCs. Thus, these data demonstrate a role for the differentiation-dependent cascade expression of cyclin-dependent kinase inhibitors in regulating adipogenic differentiation, thereby providing a molecular mechanism that couples growth arrest and differentiation.