Identification Of The Multi-directional Differentiation Potential Of Muscle Derived Stem Cells In The Rat Penile Corpus Cavernosum In Vitro

Objective: To isolate and culture muscle derived stem cells （MDSCs） of the ratpenile corpus cavernosum and detect the expression of stem cell markers. The primarycells are gained and carried out subculture. And the aim of this study is to observe theirdifferentiation potential to adipose cells, osteogenic cells and endothelial cells in definiteculture.Method: The penile corpus cavernosum were taken under sterile condition from2-month-old Sprague-Dawley rats, MDSCs were purified through density gradientcentrifugation and preplate technique. PP6cells were obtained and detected expression ofstem cell marker Sca-1and myoblast marker Desmin by immunofluorescence. The primaryPP6cells were gained and carried out subculture in vitro. After that, the third passages PP6cells were induced to differentiate to adipose cells in21days in Adipose DifferentiationCulture Medium stimulation and others third passages PP6cells were induced todifferentiate to osteogenic cells in21days in Osteogenesis Differentiation Culture Mediumstimulation, while the control group used stem cells culture medium. Induced cells weredetected by oil red O staining at0d,7d,14d and21d respectively in order to observedwhether there were lipid droplets. Others induced cells were detected by Alizarin Red Sstaining at0d,7d,14d and21d respectively in order to demonstrated differentiation intoosteogenic cells. The third passages PP6cells were induced to differentiate into endothelialcells within21days after Vascular endothelial growth factor（VEGF） and Basic fibroblastgrowth factor （bFGF） stimulation. The differentiating MDSCs were assessed by usingimmunofluorescence of ECs markers [CD31, fetal liver kinase-1（Flk-1）], reversetranscriptase-polymerase chain reaction （RT-PCR） of ECs markers [CD31, endothelialnitric oxide synthase（eNOS）, Flk-1] and stem cell markers（Oct-4）at0,5,10,15,21days. Dil-Ac-LDL uptake tests were used to test the ability of cells taking up acetylated LDL,and scratch tests were used to observe the scratch space distance after24h.Results: PP6cells expressed stem cell markers Sca-1and myoblast marker Desmin.In the course of adipose cells differentiation, on the first three days, we observed that theshape of cells had changed from long spindle-shape to circular or triangular, closelyarranging as imbricate, meanwhile nucleus clarity and proliferation ability of cells hadsignificantly slowed down. Both of groups had not changed by oil red O staining. On theseventh day, we observed that a small amount of high refractive small lipid droplets was incytoplasm under phase contrast microscope, uniform size, which mainly concentratedaround the cells nucleus, and was dyed red by oil red O staining. On the fourteenth day, thenumber of lipid droplets in cells was increased, lipid droplets blended into large, and thecells nucleus were crowded to one side or disappeared. On the twenty-one day, celldifferentiation, lipid droplets volume and number in cytoplasm reached to peak, and evencontinued to induce, but lipid droplets did not increased. In control group, however, Cellmorphology did not change and oil red O staining was negative. In the course ofosteogenic cells differentiation, on the seventh day, we observed that the shape of cells hadchanged from long spindle-shape to circular or triangular, closely arranging, meanwhilenucleus clarity and proliferation ability of cells had significantly slowed down. Both ofgroups showed none calcium nodules formation by Alizarin red staining. On the fourteenthday, cells gradually appear stacked aggregation growth trend, closely arranging asimbricate like change, and cell size enlarge, shape polygonal, large nucleus clarity,increased intracellular particles and a small amount of matrix secretion. Alizarin redstaining showed small calcium nodules was scattered distribution. On the twenty-one day,we observed through the microscope that the particles increase in intracellular,extracellular matrix secretion exuberant, alizarin red staining detected that intercellularmatrix precipitated and the calcified nodules informed. In control group, however, Cellmorphology did not change and Alizarin red staining was negative. In the course ofendothelial cells differentiation, we observed that the differentiating MDSCs expressedECs markers （CD31,Flk-1） within21days after VEGF and bFGF stimulation. Ascompared with0d, the expression of Oct-4was declined by0.445±0.025times, and thatof ECs-specific marker mRNA was significantly increased in the differentiated MDSCs at21days by2.541±0.146,2.364±0.175and2.538±0.100times for CD31, eNOS and Flk-1, respectively （P<0.05）. An increased uptake of Dil-Ac-LDL in MDSCs-derived ECs wasobserved at21days compared to undifferentiated MDSCs, and the migration ability ofMDSCs-derived ECs was significant increased. The scratch space distance ofdifferentiated MDSCs was282.01±3.84and that of undifferentiated MDSCs was450.35±1.86with the difference between statistically significant （P <0.05）.Conclusion: The cells from rat penile corpus cavernosum had the cell phenotype ofMDSCs, and have potential to differentiate into adipose cells, osteogenic cells andendothelial cells in vitro. That is given the further evidence that there are endogenousMDSCs in the penis.