Cell-cell Contact Induces Mesenchymal Stem Cells Differentiation To Smooth Muscle Cells

There are over 200 million people worldwide with incontinence, a condition that is associated with social impact and a reduced quality of life. Stress urinary incontinence (SUI) is one of the most common forms of incontinence and it can be grouped into two major categories: urethral hyper mobility and intrinsic sphincter deficiency (ISD). The periurethral injection of bμlking agents including polytetrafluoroethylene, bovine collagen, silicone particles, carbon beads, and autologous ear chondrocytes has yielded short-term success in the treatment of SUI. However, use of bulking agents has leading to chronic inflammatory reactions, foreign body giant cell responses, periurethral abscess, and particle migration, erosion of the urinary bladder or the urethra, obstruction of the lower urinary tract with urinary retention, severe voiding dysfunction, and pulmonary embolism. Smooth muscle cells (SMCs) is an essential component of sphincter muscle in urinary system. It plays a key role in SUI, so the ideal substance for such a procedure is considered to be one that is improve sphincter muscle function, readily available, safe, economic, efficacious, and durable, the effecting improvements sphincter contractility is critical for treatment SUI. A major obstacle for such an approach has been finding a reliable source of healthy SMC that can be safely harvested and that requires minimal wound. Cellular injection therapy displays an ability to undergo self-renewal and multipotent differentiation, leading to sphincter regeneration. Mesenchymal stem cells (MSCs) have been reported to differentiation toward SMCs; In addition, stem cells may release neurotrophins with subsequent paracrine recruitment of endogenous host cells to concomitantly promote a regenerative response of nerve-integrated muscle. Bladder tissue engineering pointed out that using MSCs show better results than by using differentiated cells, MSCs may migrate to the bladder grafts and differentiate into smooth muscle cells.Although the Bone marrow cells containing MSCs were most widely studied in fundamental and clinical trials; however, the underlying mechanisms have not been fully clearly, the differentiation of MSCs into smooth muscle cells (SMCs) might not completely explain. Stem cells microenvironment or'niche'is one current line of research have been put forward to explain stem cell lineage determination. Adult stem cells that are implanted into a totally different niche (different germ layer) can potentially differentiate into cell types similar to those found in the new environment. A niche consists of signaling molecules, intercellular communication and the interaction between stem cells and their neighboring extracellular matrix. Gap junctions provide a pathway for direct intercellular communication. As numerous physiological processes are mediated by regulatory molecules that are exchanged via gap junctions, gap junction is considered a key mechanism in the control of virtually all aspects of the cellular life cycle.In this study, we conducted co-culture experiments to investigate whether BMSCs differentiation to SMCs is the effect of soluble chemical factors signal on BMSCs or direct contact between BMSCs and SMCs, meanwhile elucidating the potential role of gap junction in differentiation of BMSCs.Results1. The characteristics of Cultured BMSCPassage 3rd rat BMSC cell-surface antigen profile was ascertained by staining with rat-specific monoclonal antibodies by flow cytometry. CD90, CD44 expression was positive , but CD31 and CD45 were negative. This result suggests that these BMSCs are undifferentiated stem cells and they different from hematopoietic stem cells and endothelial cell. In the experiment of osteogenic, and adipogenic differentiation. they were assessed after induced 3 weeks, osteogenic differentiation show calcium deposition, and BMSCs formation adipose in adipogenic differentiation. From the results of experiment can be seen that these BMSCs had potential of Multilineage Differentiation.2. Co-culture induce MSC differentiate into SMC2.1 Immunofluorescence analysisIn CON. group, NO-CON. group, and CON.+H group, after co-culture 8days,the rat BMSCs expression of a-SMA, Calponin and SM-MHC of SMCs was examined by immunofluorescence. Untreated rat BMSCs and adult SMCs were used as negative and positive controls, respectively. we observed a-SMA expression on three groups. Only in direct contacted culture group revealed an increased expression of Calponin but all of three groups have no expression of SM-MHC. 2.2 Western analysisTotal protein was isolated from rat BMSCs of CON. group, NO-CON. group, and CON.+H group, on co-culture 4 days and 8days respectively. At the same time , a total protein was isolated from untreated rat BMSCs as control. This experiment confirmed the immunofluorescence Data, NO-CON. group and CON.+H group have no discrepancy by statistical evaluation after co-culture. BMSCs in CON. group with increases in the expression of ASMA and Calponin compared to untreated rat BMSCs , and expression of SM-MHC only in cells grown in CON. group at 8 days. In CON.+H group which present the increase of a-SMA and decrease of Calponin.2.3 Quantitative Real-Time PCRRT-PCR analyses of CON. group were performed as previously describe.[1] The expression of ASMA, Calponin, and SM-MHC was quantified for differentiation conditions described above. mRNA of a-SMA, Calponin and SM-MHC increases in the expression were confirmed with real-time PCR.DiscussionCell transplantation therapy offers hope for the treatment of the regenerative repair of the intrinsic sphincter deficiency (ISD) of urinary incontinence. The use of autologous multipotent stem cells becomes the first choice of Cell-based therapies and tissue engineering. Stem cell therapy might replace, repair, or enhance the biological function of damaged tissue or organs. One commonly described source of SMCs is the muscle derived stem cells (MDSC). Although muscle stem cells and satellite cells can be isolated from biopsy of adult, the number of cells that can be harvested may be limited. Bone marrow stem cells are an idea source of autologous adult stem cells. BMSCs are easy to isolate and expand rapidly from patients without leading to major ethical and technical problems; they have great potential as therapeutic agents. But the application to ISD depends on the ability to control their differentiation into SMCs with high efficiency and purity.In our study we conduct a direct co-culture system via used a semi- permeable membrane to investigate the effect of local environment on BMSCs differentiation. We sowed the SMCs under the membrane, and sowed BMSCs in the upper of membrane when SMCs confused. The semi- permeable membrane has more 1um diameter pores which would not permit cell through the pores of semi- permeable membrane but can form cell-cell contact and allow chemical factors to diffuse. Through this method to establish whether rat BMSCs is differentiation to SMC after direct co-culture with adult rat SMCs, we assessed the expression of certain unique markers of SMCs, include a-SMA, Calponin and SM-MHC by real-time PCR, confocal laser microarray scanner and western blot. We not only detected the expression of a-SMA and Calponin which are early and middle marker of developing smooth muscle respectively, but also tested the present of SM-MHC, a highly restricted to differentiated smooth muscle at western blot experiment. In this study we didn't add any exogenous cytokine or signaling molecules, only rely on SMCs secreted. signaling molecules and interaction between stem cells with their neighboring cell are two important factor of local environment, Chiu et al. have been suggested that milieu-induced differentiation play an important role in induced myoblasts to acquire a cardiac-like phenotype. The''milieu-induced differentiation''hypothesis focuses on the local microenvironment. Our experiment demonstrated local environment and resident cellular populations are likely to be major factors in determining MSCs fates.In NO-CON. group, We sowed the SMCs up the six well culture plate, and sowed BMSCs in the upper of membrane when SMCs confused. through this method, BMSCs wouldn't contact with SMCs, but the SMCs might secreted cytokine or signaling molecules diffuse into DMEM and permeate the semi- permeable membrane affect the BMSCs. after co-culture ,We found that there was no expression of a-SMA, Calponin and SM-MHC of BMSCs. This means cytokine or signaling molecules released by SMCs can not induce BMSCs to differentiate into SMCs. This result suggested that the cell-cell contact is dominant in the differentiation of BMSCs at the condition without artificially add exogenous cytokine or signaling molecules.Our data suggest that contact with SMCs would render BMSCs differentiate into SMCs lineage. There has been report that differentiation of bone marrow stromal cells into cells with cardiac phenotype requires intercellular communication with myocytes. so, whether intercellular communication is essential for SMCs differentiation is unknown. To confirm that whether gap junction contribute to stem cell differentiation, on the basis of the direct contact culture experiment we added the gap junction uncoupler heptanol which is a selectively block gap junction communication. The data showed, the inductive effect in direct contact culture group was inhibition by heptanol. The expression of Calponin decrease, and not expression SM-MHC the late marker of SMCs .However, in this group the expression of a-SMA was increased indicates that gap junction is not inherently and invariably linked to the differentiation of BMSCs. This suggests that the relationship between gap junction and BMSCs differentiation depends upon the specific cascades activated by the particular growth factor involved. It has also been reported that the signals in local environment are likely to be major factors determining stem cells fates, which exert influence through cell surface receptors and ligands, the formation of structures. The possibility that other singnal path way reported in differerntiation process might also be involved at the transcript or protein level in BMSCs. Gap junctions allow exchange of small molecules, including secondary messengers between adjacent cells might be one of potential route for BMSCs to differentiation.In this study, we found that the direct contact co-culture with SMCs facilitate BMSCs to SMCs lineage. This inductive effect mainly mediated through gap junction. The direct contact co-culture system serves as an important tool for the study of the communications between cells, because the method is simple and easy to master and the material is easily obtainable. Treatment of SUI with MSCs might be a long-term application toward cell replacement therapies in the future. it can increase the function of deficient rhabdosphincter , which has a significant clinical impact.