OBJECTIVETo further investigate the osteogenic potential of rabbit marrow stromal stem cells cultured in vitro.

METHODSRabbit marrow stromal stem cells were isolated by density gradient centrifugation method and amplified in the flasks, using the osteogenic inducing conditions (OGC) as the culture media. The osteogenic potential of marrow stromal stem cells were investigated by means of bone-seeking fluorescence (tetracycline) labelling, Alizarin red S (ARS) staining, Alcian blue-Sirius red (AS) staining, and scanning electron microscope.

RESULTSAfter being passaged, the marrow stromal stem cells increased in number, became confluent and formed multi-layer structure. The stromal stem cells excreted innumerable tiny granules, heaping up on the cell body and merging gradually into foggy substances. These foggy substances kept on enlarging and formed round, oval, or flake-like nodules. These nodules revealed bright golden yellow fluorescence under fluorescence microscope when labelled with tetracycline. Histochemical study with specific new bone staining with ARS revealed positive calcium reaction, both denoting that they were newly formed bone tissues. After they were stained with AS, collagen and acid mucopolysaccharide were shown. Under scanning electron microscope, three types of cells with different configurations were found. They were globular cells, spindle-shaped cells and polygonal or polygonal cells. Granules were excreted from the cells and heaped up on the cell body. Needle-shaped and irregularly rectangular crystals also appeared and agglomerated with the granules to form nodules and trabecula-like or flake-like structures.

CONCLUSIONSSequence of events of bone formation by rabbit marrow stromal stem cells cultured in vitro is fully depicted and confirmed, which provides the foundation for further investigating the mechanisms of osteoblast differentiation from marrow stromal stem cells and the possible application in orthopaedics.

Animals ; Bone Marrow Cells ; pathology ; ultrastructure ; Cells, Cultured ; Immunohistochemistry ; In Vitro Techniques ; Male ; Microscopy, Electron, Scanning ; Models, Animal ; Osteogenesis ; physiology ; Rabbits ; Sensitivity and Specificity ; Stromal Cells ; physiology ; ultrastructure

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

OBJECTIVETo explore the origin and differentiation of gastrointestinal stromal tumors (GISTs).

METHODSImmunohistochemistry staining and electron microscopy were adopted.

RESULTSIn 212 cases of primary GISTs, the positive rates of CD117, CD34, alpha-SMA, MSA, desmin, S-100, PGP9.5 were 96.7%, 77.3%, 19.3%, 15.6%, 1.9%, 16.3%, and 12.3% respectively. Among them, GISTs showed a diffuse and strong positivity for CD117. Electron microscopy of tumor cells demonstrated numerous mitochondria, prominent perinuclear Golgi complex, smooth and rough endoplasmical reticulum and intermediate filaments. Irregular caveolae, dense plaque, incontinuous basal lamina were observed occasionally. Cytoplasmic processes were often observed accompanying with local adhesion present between the processes or between the processes and the cell membrane.

CONCLUSIONSData from both immunophenotype and electron microscopy suggest that GIST might originate from the mesenchymal cells, differentiating to be ICC afterwards, and possessing myoid characteristics in various extent.

Cell Differentiation ; Gastrointestinal Stromal Tumors ; chemistry ; ultrastructure ; Golgi Apparatus ; ultrastructure ; Humans ; Immunohistochemistry ; Microscopy, Electron ; Proto-Oncogene Proteins c-kit ; analysis ; S100 Proteins ; analysis ; Stromal Cells ; chemistry ; ultrastructure ; Ubiquitin Thiolesterase ; analysis

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

OBJECTIVETo explore the differences of phenotype and biological characteristics between fetal and adult bone marrow derived mesenchymal stem cells.

METHODSMononuclear cells from 4-5 months old human aborted fetus and normal adult bone marrow were cultured in SF medium to obtain mesenchymal stem cells. The growth curve, cell cycle, immunophenotype, in vitro expansion potential, differentiation capacities were investigated.

RESULTSThe adherent fetal and adult bone marrow-derived cells cultured in the absence of differentiation stimuli gave rise to a population of cells with phenotypical features of mesenchymal stem cells (MSC). These MSCs were similar in cell morphology and antigenic phenotype. The proliferative and multilineage differentiation potential of the bone marrow derived MSC from the fetus is higher than that from the adult, but the adherent ability of the MSCs from the adult is higher than that from the fetus.

CONCLUSIONFetal bone marrow derived MSCs should be enough to sustain a steady supply of low differentiated cells for proliferation, hence an abundant and accessible cellular reservoir for stem cell bioengineering, whereas adult bone marrow derived MSCs are more useful in hematopoietic reconstitution in bone marrow transplantation.

Adult ; Adult Stem Cells ; cytology ; immunology ; ultrastructure ; Bone Marrow Cells ; cytology ; immunology ; ultrastructure ; Cell Cycle ; Cell Differentiation ; Cell Lineage ; Cell Proliferation ; Cells, Cultured ; Fetal Stem Cells ; cytology ; immunology ; ultrastructure ; Flow Cytometry ; Humans ; Immunophenotyping ; Mesenchymal Stromal Cells ; cytology ; immunology ; ultrastructure ; Microscopy, Electron

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the gastrointestinal tract (GIT). Although interstitial cells of Cajal has been suggested as origin of this tumor, the cytological and ultrastructural features of GISTs are heterogeneous and unclear. A total 10 cases of normal gastrointestinal tissue (control), 13 GISTs of the stomach (8), small intestine (3), mesocolon (1) and liver (1), and 2 gastrointestinal autonomic nervous tumor (GANT) of small intestine were ultrastructurally studied. Normal interstitial cells of Cajal (ICC) were abundantly present around the myenteric plexuses or individually scattered through the wall of GIT. ICC was characterized by slender cytoplasmic processes, well-developed endoplasmic reticulum (ER), mitochondria, Golgi apparatus, caveolae and intermediate filaments. The GISTs and GANTs had overlapping ultrastructures. The most common and important ultrastructural features of GISTs were rich villous cytoplasmic processes, dispersed intermediate filaments and abundant SER, and those of GANTs were neurosecretory granules and skenoid fibers. Compared with ICC, the GISTs and GANTs had remarkably reduced caveolae and gap junctions. Our study suggested that ultrastructural analysis gives much information to investigate lineage differentiation of neoplastic cells and make a differential diagnosis of these tumors from other mesenchymal tumors and between GISTs and GANTs.
Adult ; Aged ; Autonomic Nervous System/*pathology/ultrastructure ; Comparative Study ; Cytoplasm/pathology/ultrastructure ; Female ; Gastrointestinal Neoplasms/*pathology/ultrastructure ; Human ; Immunohistochemistry ; Male ; Microscopy, Electron ; Middle Aged ; Peripheral Nervous System Neoplasms/*pathology/ultrastructure ; Stromal Cells/*pathology/ultrastructure ; Support, Non-U.S. Gov't ; Tumor Markers, Biological ; Vacuoles/pathology/ultrastructure

OBJECTIVETo observe the ultrastructure of bone marrow stromal cells (BMSCs) cultured in coralline hydroxyapatite (CHA) and evaluate their biocompatibility.

METHODSBMSCs isolated from dogs were cultured with CHA as the scaffold, and the morphologies of the cells were observed with phase-contrast microscope and scanning electron microscope.

RESULTS AND CONCLUSIONBMSCs grew well with good attachment to the CHA scaffold and performed normal function, demonstrating CHA as one of useful biocarrier materials for bone tissue engineering.

Animals ; Bone Marrow Cells ; cytology ; ultrastructure ; Bone Substitutes ; chemistry ; Cell Culture Techniques ; Cells, Cultured ; Ceramics ; chemistry ; Dogs ; Hydroxyapatites ; chemistry ; Male ; Microscopy, Electron, Scanning ; Microscopy, Phase-Contrast ; Stromal Cells ; cytology ; ultrastructure ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

BACKGROUNDCardiomyocyte transplantation for the therapy of myocardial ischaemia is being paid close attention. However, how the microenvironment controls the differentiation of transplanted bone marrow stromal cells (BMSCs) is unknown. Endothelin-1 (ET-1), a cytokine, increases during myocardial infarction, but it is not known whether ET-1 is responsible for the fate of transplanted BMSCs. In the present study, we investigated the effects of ET-1 on differentiation and maturation of induced rabbit BMSCs, in vitro, to elucidate the cellular biological mechanisms.

METHODSThe proliferation of BMSCs isolated from femur of rabbits was induced by ET-1 only, by 5-azacytidine (5-aza) or ET-1 combined with 5-aza. After 4 weeks of induced culturing, the differentiation rate and the diameter of induced myocyte like cells were estimated and the expressions of GATA-4 protein and phosphorylation level were assayed by Western-blot, RT-PCR analysis of beta-myosin heavy chain (MHC). mRNA expression, levels of troponin-I by immunohistochemical staining and ultrastructure of induce-cultured BMSCs were also determined.

RESULTSBy induction with ET-1 and 5-aza, mean cell diameter of induced BMSCs was larger than induced with 5-aza [(6.26 +/- 0.22) microm cf (5.29 +/- 0.19) microm] (P < 0.001). There was no difference in rate of differentiation of myocyte like cells between the groups induced with 5-aza and ET-1 combined with 5-aza [(29.82 +/- 0.23)% cf (29.94 +/- 0.18)%] (P > 0.05). The expressions of GATA-4 protein and phosphorylation were enhanced significantly in groups induced with ET-1 combined with 5-aza (P < 0.05). In the group induced with ET-1 combined with 5-aza, expression of beta-MHC mRNA was higher than control [(0.122 +/- 0.008) cf (0.022 +/- 0.003)] (P < 0.01), and more troponin-I positive cells were also detected in this group. Differentiated BMSCs showed formations of myofilaments and primitive sarcomere, i.e., morphological characteristics of myocyte like cells.

CONCLUSIONSThis study suggests that induced culturing of BMSCs by ET-1 combined with 5-aza can express cardiomyocytic characteristics whereas ET-1 alone could not induce BMSCs to differentiate to myocyte like cells. ET-1 upregulates the expression of GATA-4 protein and phosphorylation level of induced BMSCs, and rapidly promotes the differentiation and maturation of myocyte like cells from BMSCs.

Animals ; Bone Marrow Cells ; cytology ; ultrastructure ; Cell Differentiation ; drug effects ; Cells, Cultured ; Endothelin-1 ; pharmacology ; GATA4 Transcription Factor ; analysis ; metabolism ; Myocytes, Cardiac ; cytology ; Myosin Heavy Chains ; genetics ; Phosphorylation ; RNA, Messenger ; analysis ; Rabbits ; Stromal Cells ; cytology ; ultrastructure

The present study aimed to determine the role of tissue injury in migration of mesenchymal stem cells (MSCs) intravenously transplanted into heart and to establish experimental basis for improving stem cell therapy in its targeting and effectiveness. MSCs were isolated from bone marrow of male Sprague-Dawley rats and purified by density centrifuge and adhered to the culture plate in vitro. Female rats were divided randomly into four groups. Myocardial ischemia (MI) transplanted group received MSCs infusion through tail vein 3 h after MI and compared with sham-operated group or normal group with MSCs infusion, or control group received culture medium infusion. MI was created in female rats by ligating the left anterior descending coronary artery. The heart was harvested 1 week and 8 weeks after transplantation. The characteristics of migration of MSCs to heart were detected with expression of sry gene of Y chromosome by using fluorescence in situ hybridization (FISH). Ultrastructural changes of the ischemic myocardium of the recipient rats were observed by transmission electron microscope (TEM). One week or 8 weeks after transplantation, sry positive cells were observed in the cardiac tissue in both of MI transplanted group and sham-operated group, the number of sry positive cells being significantly higher in MI transplanted group (P<0.01). No significant difference was found in the number of sry positive cells between 1 week and 8 weeks after transplantation. No sry positive cells were observed in the hearts of control and normal group. In addition, the ultrastructure of some cells located in the peri-infarct area of MI rats with MSCs transplantation was similar to that of MSCs cultured in vitro. These results indicate that MSCs are capable of migrating towards ischemic myocardium in vivo and the fastigium of migration might appear around 1 week after MI. The tissue injury and its degree play an important role in the migration of MSCs.
Animals ; Cell Movement ; Cell Tracking ; Female ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Myocardial Ischemia ; therapy ; Myocardium ; ultrastructure ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the histogenesis and neural differentiation of gastrointestinal stromal tumor (GIST).

METHODSThe ultrastructural morphology and neural differentiated antigen expression were studied in 20 cases of GIST using electron microscopy and immunohistochemistry.

RESULTSAll of the 20 cases mentioned were positive for c-kit expression. The ultrastructural features of neural differentiation were observed in 7 cases, while no neural or myogenic differentiation seen in 12 cases. Myogenic differentiation to smooth muscle was observed in one case. The ultrastructural features of neural differentiation included scattered or cluster distribution of dense core granules in cytoplasm and cytoplasmic processes; formation of synaptic construction of cell processes; and neurogenic-like processes. In some cases pinocytotic vesicles under the cell membrane and skenoid fibers were seen. Neural differentiation with dense core granules was seen in one case in benign, one case in borderline and five cases in malignant group. The positive reactivity of neural differentiated antigen NSE, CD99, S-100p and CD56 in cases of the neural differentiation group appeared in seven, seven, five and four cases respectively, which were significantly higher than that of the undifferentiated group.

CONCLUSIONSIt's rather difficult to differentiate GIST accompanying with neural differentiation from gastrointestinal autonomic nerve tumor if depending only on its histology and immunophenotype appearance, since many features were overlapping in both tumors. Examination of the neural ultrastructures and neural differentiated antigen in GIST might be helpful to clarify the neural differentiation and potential behavior of GIST.

12E7 Antigen ; Adolescent ; Adult ; Aged ; Antigens, CD ; metabolism ; Autonomic Nervous System Diseases ; metabolism ; pathology ; Biomarkers, Tumor ; metabolism ; CD56 Antigen ; metabolism ; Calcium-Binding Proteins ; metabolism ; Cell Adhesion Molecules ; metabolism ; Cell Differentiation ; Child ; Diagnosis, Differential ; Female ; Gastrointestinal Stromal Tumors ; metabolism ; ultrastructure ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Proto-Oncogene Proteins c-kit ; metabolism ; Stromal Cells ; ultrastructure ; Synapses ; ultrastructure