Stem cells are known to maintain stemness at least in part through secreted factors that promote stem-like phenotypes in resident cells. Accumulating evidence has clarified that stem cells release nano-vesicles, known as exosomes, which may serve as mediators of cell-to-cell communication and may potentially transmit stem cell phenotypes to recipient cells, facilitating stem cell maintenance, differentiation, self-renewal, and repair. It has become apparent that stem cell-derived exosomes mediate interactions among stromal elements, promote genetic instability in recipient cells, and induce malignant transformation. This review will therefore discuss the potential of stem cell-derived exosomes in the context of stromal remodeling and their ability to generate cancer-initiating cells in a tumor niche by inducing morphologic and functional differentiation of fibroblasts into tumor-initiating fibroblasts. In addition, the immunosuppressive potential of stem cell-derived exosomes in cancer immunotherapy and their prospective applications in cell-free therapies in future translational medicine is discussed.
Apoptosis ; Cell Communication ; Cell Transformation, Neoplastic ; Disease Progression ; Exosomes ; physiology ; Humans ; Immunotherapy ; methods ; Mesenchymal Stromal Cells ; physiology ; Neoplasms ; blood supply ; pathology ; therapy ; Neoplastic Stem Cells ; ultrastructure ; Neovascularization, Pathologic ; pathology ; Organelle Biogenesis ; Tumor Microenvironment

BACKGROUNDThis study characterized the cardiac telocyte (TC) population both in vivo and in vitro, and investigated its telomerase activity related to mitosis.

METHODSUsing transmission electron microscopy and a phase contrast microscope, the typical morphological features of cardiac TCs were observed; by targeting the cell surface proteins CD117 and CD34, CD117 + CD34 + cardiac TCs were sorted via flow cytometry and validated by immunofluorescence based on the primary cell culture. Then the optimized basal nutrient medium for selected population was examined with the cell counting kit 8. Under this conditioned medium, the process of cell division was captured, and the telomerase activity of CD117 + CD34 + cardiac TCs was detected in comparison with bone mesenchymal stem cells (BMSCs), cardiac fibroblasts (CFBs), cardiomyocytes (CMs).

RESULTSCardiac TCs projected characteristic telopodes with thin segments (podomers) in alternation with dilation (podoms). In addition, 64% of the primary cultured cardiac TCs were composed of CD117 + CD34 + cardiac TCs; which was verified by immunofluorescence. In a live cell imaging system, CD117 + CD34 + cardiac TCs were observed to enter into cell division in a short time, followed by an significant invagination forming across the middle of the cell body. Using a real-time quantitative telomeric-repeat amplification assay, the telomerase concentration in CD117 + CD34 + cardiac TCs was obviously lower than in BMSCs and CFBs, and significantly higher than in CMs.

CONCLUSIONSCardiac TCs represent a unique cell population and CD117 + CD34 + cardiac TCs have relative low telomerase activity that differs from BMSCs, CFBs and CMs and thus they might play an important role in maintaining cardiac homeostasis.

Animals ; Antigens, CD34 ; metabolism ; Fibroblasts ; enzymology ; ultrastructure ; Flow Cytometry ; Mesenchymal Stromal Cells ; enzymology ; ultrastructure ; Mice ; Mice, Inbred C57BL ; Microscopy, Confocal ; Microscopy, Electron, Transmission ; Microscopy, Phase-Contrast ; Myocytes, Cardiac ; enzymology ; ultrastructure ; Proto-Oncogene Proteins c-kit ; metabolism ; Telomerase ; metabolism ; Vimentin ; metabolism

PURPOSE: Clinical studies have reported a correlation between pelvic ischemia and voiding dysfunction in elderly men. The aim of this study was to identify and compare prostate structural modifications in cultured cells and in a rabbit model after exposure to hypoxia, oxidative stress, and chronic ischemia. MATERIALS AND METHODS: Cultured human prostate smooth muscle cells (SMCs), epithelial cells (ECs), and stromal cells (SCs) were incubated under normoxia, hypoxia, and oxidative stress conditions by use of a computerized oxycycler system. We developed a rabbit model of chronic prostate ischemia by creating aorto-iliac arterial atherosclerosis. Markers of oxidative stress were examined by using fluorometric analysis and enzyme immunoassay. Prostate structure was examined by using Masson's trichrome staining and transmission electron microscopy (TEM). RESULTS: Lipid peroxidation was found in SMCs exposed to hypoxia and in all cell types exposed to oxidative stress. We identified protein oxidation in ECs exposed to hypoxia and in all cell types exposed to oxidative stress. Markers indicating oxidative damage were present in chronically ischemic rabbit prostate tissue. These reactions were associated with DNA damage. Prostate ischemia resulted in epithelial atrophy, loss of smooth muscle, and diffuse fibrosis. TEM showed swollen mitochondria with degraded cristae, loss of membrane, loss of Golgi bodies, degenerated nerves, and disrupted cell-to-cell junctions. CONCLUSIONS: Human prostate cells exhibited differential reactions to hypoxia and oxidative stress with widespread DNA damage. Structural modifications in ischemic prostate tissue were similar to those in cells exposed to oxidative stress. Structural changes due to ischemia and oxidative stress may contribute to prostatic noncompliance in aging men.
Animals ; Anoxia/*complications ; Atherosclerosis/complications ; Biomarkers ; Cells, Cultured ; DNA Damage ; Disease Models, Animal ; Epithelial Cells/ultrastructure ; Fibrosis ; Humans ; Ischemia/*complications ; Lipid Peroxidation ; Male ; Myocytes, Smooth Muscle/ultrastructure ; Nerve Degeneration ; *Oxidative Stress ; Prostate/*anatomy & histology/*cytology ; Rabbits ; Stromal Cells/ultrastructure ; Urinary Bladder Neck Obstruction/complications

BACKGROUNDThe survival ratio of implanted mesenchymal stem cells (MSCs) in the infarcted myocardium is low. Autophagy is a complex "self-eating" process and can be utilized for cell survival. We have found that atorvastatin (ATV) can effectively activate autophagy to enhance MSCs survival during hypoxia and serum deprivation (H/SD). The mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway is a non-canonical autophagy pathway. We hypothesized that the MEK/ERK pathway mediated ATV-induced autophagy of MSCs under H/SD.

METHODSMSCs were pretreated with ATV (0.01-10 µmol/L) under H/SD for three hours. For inhibitor studies, the cells were pre-incubated with the MEK1/2 inhibitor U0126. Cell autophagy was assessed by acidic vesicular organelles (AVO)-positive cells using flow cytometry, autophagy related protein using Western blotting and autophagosome using transmission electron microscopy.

RESULTSAutophagy was elevated in the H/SD group compared with the normal group. ATV further enhanced the autophagic activity as well as the phosphorylation of ERK1/2 evidenced by more AVO-positive cells ((8.63 ± 0.63)% vs. (5.77 ± 0.44)%, P < 0.05), higher LC3-II/LC3-I ratio (4.36 ± 0.31 vs. 2.52 ± 0.18, P < 0.05) and more autophagosomes. And treatment with U0126 downregulated the phosphorylation of ERK1/2 and attenuated ATV-induced autophagy.

CONCLUSIONThe MEK/ERK pathway participates in ATV-induced autophagy in MSCs under H/SD, and modulation of the pathway could be a novel strategy to improve MSCs survival.

Animals ; Atorvastatin Calcium ; Autophagy ; drug effects ; Cell Hypoxia ; physiology ; Cells, Cultured ; Flow Cytometry ; Heptanoic Acids ; pharmacology ; MAP Kinase Signaling System ; drug effects ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; ultrastructure ; Microscopy, Electron, Transmission ; Pyrroles ; pharmacology ; Rats

The purpose of this study was to observe the ultrastructure of human umbilical cord mesenchymal stem cells (hUCMSC). hUCMSC from full-term newborn umbilical cord were isolated and cultured by collagenase digestion, and then subcultured, amplification, and cell morphology was observed by microscopy. The immunophenotype and trilineage differentiation potential of hUCMSCs at passage 3 were analyzed. Transmission electron microscopy and scanning electron microscopy were used to observe the ultrastructure of hUCMSC. The results indicated that appearance of hUCMSC was spindle-shaped and polygonal, and nuclei were observed. hUCMSC expressed immunophenotype CD44, CD73, CD105, did not express CD34, CD45, CD31 and human leukocyte antigen HLA-DR. hUCMSC were capable of adipogenic, osteogenic, and cartilage differentiation; the short and thick microvilli processes were seen at the surface of hUCMSC by scanning electron microscope. Two different cell morphologies of hUCMSC were seen under transmission electron microscope, the one was a quiescent period in which a large and round or oval nucleus only one nucleolus were seen, cytoplasmic organelles were less; the other was in a relatively active period in which one or two nuclei in the same one cell were observed, the organelles were rich, structure was clear, expansion of the mitochondria was visible. It is concluded that the cells successfully isolated and cultured from umbilical cord, which possess biological characteristics of MSC and display two different states of ultrastructure.
Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; ultrastructure ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Umbilical Cord ; cytology

BACKGROUNDOsteochondral lesion repair is a challenging area of orthopedic surgery. Here we aimed to develop an extracellular matrix-derived, integrated, biphasic scaffold and to investigate the regeneration potential of the scaffold loaded with chondrogenically-induced bone marrow-derived mesenchymal stem cells (BMSCs) in the repair of a large, high-load-bearing, osteochondral defect in a canine model.

METHODSThe biphasic scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and characterized by scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). Osteochondral constructs were fabricated in vitro using chondrogenically-induced BMSCs and a biphasic scaffold, then assessed by SEM for cell attachment. Osteochondral defects (4.2 mm (diameter) × 6 mm (depth)) were created in canine femoral condyles and treated with a construct of the biphasic scaffold/chondrogenically-induced BMSCs or with a cell-free scaffold (control group). The repaired defects were evaluated for gross morphology and by histological, biochemical, biomechanical and micro-CT analyses at 3 and 6 months post-implantation.

RESULTSThe osteochondral defects of the experimental group showed better repair than those of the control group. Statistical analysis demonstrated that the macroscopic and histologic grading scores of the experimental group were always higher than those of the control group, and that the scores for the experimental group at 6 months were significantly higher than those at 3 months. The cartilage stiffness in the experimental group (6 months) was (6.95 ± 0.79) N/mm, 70.77% of normal cartilage; osteochondral bone stiffness in the experimental group was (158.16± 24.30) N/mm, 74.95% of normal tissue; glycosaminoglycan content of tissue-engineered neocartilage was (218 ± 21.6) µg/mg (dry weight), 84.82% of native cartilage. Micro-CT analysis of the subchondral bone showed mature trabecular bone regularly formed at 3 and 6 months, with no significant difference between the experimental and control groups.

CONCLUSIONThe extracellular matrix-derived, integrated, biphasic scaffold shows potential for the repair of large, high-load-bearing osteochondral defects.

Animals ; Bone Marrow Cells ; cytology ; Bone Regeneration ; physiology ; Cartilage, Articular ; surgery ; Dogs ; Extracellular Matrix ; chemistry ; Mesenchymal Stromal Cells ; cytology ; ultrastructure ; Microscopy, Electron, Scanning ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry ; X-Ray Microtomography

OBJECTIVETo isolate and culture human umbilical cord mesenchymal stem cells (MSCs) and explore their biological features and ultrastructure.

METHODSAfter isolating MSCs from the human umbilical cord, the proliferation, cycle, and apoptosis were observed. The cell ultrastructure was observed under transmission electron microscope. The cytokines including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1) were detected using enzyme-linked immunosorbent assay.

RESULTSHuman umbilical cord MSCs had fibroblast-like morphology and increased proliferation capability. Ultrastructural analysis showed that the MSCs had active cellular metabolism and strong migration and differentiation capabilities. Meanwhile, they could secrete anti-apoptotic cytokines such as VEGF, IGF-1, and HGF.

CONCLUSIONHuman umbilical cord MSCs can secrete many anti-apoptotic cytokine and have good biological features.

Apoptosis ; Cell Cycle ; Cell Proliferation ; Cells, Cultured ; Hepatocyte Growth Factor ; metabolism ; Humans ; Insulin-Like Growth Factor I ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; ultrastructure ; Umbilical Cord ; cytology ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo study the compatibility between human adipose-derived mesenchymal stem cells and porcine bone scaffolds.

METHODSPorcine bone tissues were co-cultured with adipose-derived mesenchymal stem cells, and the complex was observed under scanning electron microscope. The viability and alkaline phosphatase (ALP) activity of the cells were examined with the cells co-cultured with human bone scaffold as the control.

RESULTSAt 4 and 10 days after the co-culture, the adipose-derived mesenchymal stem cells were observed to extend pseudopodia to adhere to the two scaffold materials. MTT assay showed that the cell proliferation on both of the materials increased with time, and the two cell complexes exhibited similar pattern of changes in ALP activity.

CONCLUSIONAs the seed cells, human adipose-derived mesenchymal stem cells exhibit good comparability with porcine bone scaffold, suggesting their potential of constructing tissue-engineered bone graft.

Adipose Tissue ; cytology ; Adult ; Alkaline Phosphatase ; metabolism ; Animals ; Bone and Bones ; cytology ; metabolism ; Cell Proliferation ; Coculture Techniques ; Humans ; Materials Testing ; Mesenchymal Stromal Cells ; cytology ; enzymology ; metabolism ; ultrastructure ; Microscopy, Electron, Scanning ; Swine ; Time Factors ; Tissue Scaffolds

OBJECTIVETo investigate the expression and distribution of extracellular matrix (ECM) in the co-culture of porcine primary hepatocytes and bone marrow mesenchymal stem cells (MSCs) in vitro.

METHODSMononuclear cells were isolated from bone marrow of swine by density gradient centrifugation. MSCs of passage 3 and primary hepatocytes harvested by a two-step in situ collagenase perfusion technique were co-cultured, and the morphological and functional changes of heterotypic interactions were characterized. Immunocytochemical analysis was performed to monitor the expression and distribution of ECM.

RESULTSThe purity of the third passage MSCs and primary hepatocytes was more than 90% and 99%, respectively. More than 95% of the hepatocytes were viable. Compared to hepatocytes culture, co-culture with MSCs significantly enhanced hepatic function: including albumin secretion and urea synthesis (P < 0.01). The best hepatic function level was achieved on day 2 and gradually decreased in the following co-culture days. Immunocytochemical staining suggested that higher amounts of naturally occurring ECM proteins including fibronectin, laminin, and several kinds of collagens were produced in co-culture group compared to hepatocyte homo-culture (P < 0.01). RNAi experiments verified that there was a correlation between ECM and hepatic functions.

CONCLUSIONECM may indeed play a key role in the up-regulation of hepatocyte functions in MSC/hepatocytes co-culture.

Albumins ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; Cell Separation ; methods ; Cell Survival ; Cells, Cultured ; Coculture Techniques ; Extracellular Matrix ; metabolism ; Female ; Hepatocytes ; cytology ; metabolism ; ultrastructure ; Immunohistochemistry ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Microscopy, Confocal ; Microscopy, Electron, Scanning ; Swine ; Urea ; metabolism

Bone Marrow Cells ; cytology ; Cell Cycle ; Cell Differentiation ; Cell Nucleus ; genetics ; Cells, Cultured ; Diploidy ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Immunophenotyping ; Mesenchymal Stromal Cells ; cytology ; metabolism ; ultrastructure ; Transfection