BACKGROUND:In vitro isolation and purity technique of stem cells mostly depends on the identification of cell surface marker,such as monoclonal antibody adherent spreading method,flow cell sorting method and immunomagnetic beads sorting method,but the operation was complicated and the price was high.OBJECTIVE:To observe the biological characteristics of human amniotic fluid-derived embryonic mesenchymal stem cells,which were isolated and cultured using the two-step method.DESIGN,TIME AND SETTING:The opening study was conducted at the Stem Cell Research Room of Xinjiang Medical University from March 2008 to March 2009.MATERIALS:Totally 10 amniotic fluid specimens were obtained from pregnant women who underwent prenatal diagnosis following 16-22 weeks of gestation or voluntarily induced abortion.With ultrasonic guidance,amniocentesis was performed to collect 20-40 mL amniotic fluid.METHODS:Human amniotic fluid-derived embryonic mesenchymal stem cells were isolated and cultured using the two-step method.Amniotic fluid was first centrifuged and incubated till spindle-shape cells were seen,with the presence of flbroblast-tike cell colonies.Supematant was moved to a new 25 cm~2 culture flask for further culture till spindle-shape fibroblast-like mesenchymal stem cell colonies.When 70% confluence,cells were digested,and incubated in α-MEM,supplemented with basic fibroblast growth factor,served as the first passage.MAIN OUTCOME MEASURES:Morphological changes in human amniotic fluid-derived embryonic mesenchymal stem cells of primary culture and subculture were measured.Karyotype,cycle,growth curve and colony formation ability of human amniotic fluid-derived embryonic mesenchymal stem cells were measured.Surface antigen and cytokine were examined using flow cytometry,immunofluorescence and RT-PCR.RESULTS:Human amniotic fluid-derived embryonic mesenchymal stem cells were successfully isolated and subcultured.During metaphase,primarily cultured amniotic fluid cells presented scattered spindle cells and flbroblast-like mesenchymal stem cell colonies every 7 days.Passaged cells completely adhered in 12 hours.Following 1 or 2 days of latent period,cells proliferated rapidly.About 90% confluence was observed following 6 or 7 days of culture.Cell arranged regularly,showing whirlpool-shape,radiated shape.Cells were spindle-shape,with unclear boundary.Chromosome karyotype of human amniotic fluid-derived embryonic mesenchymal stem cells was normal diploid.Growth curve showed "S" shape,but the two-step method reached a peak at (6.1±0.5) days,which was significantly rapid compared with the one-step method (7.2±0.6) days (P=0.035).Flow cytometry analyses showed that P3 cells at S phase took up (14±2.3)% using the two-step method,which was more than the one-step method (9.0±1.4)% (P=0.031).Low-density human amniotic fluid-derived embryonic mesenchymal stem cells were incubated for 7 days prior to cells formed scattered cell colonies.However,colony forming efficiency using the two-step method (15.0±2.3)% were significantly more than the one-step method (10.0±1.8)% (P=0.021).Flow cytometry results showed that human amniotic fluid-derived embryonic mesenchymal stem cells expressed CD44,CD29 and CD105,but were negatively for CD45,CD34,HLA-DR.Immunofluorescence suggested that Oct-4-positive cells were observed in amniotic fluid.However,the proportion of Oct-4-positive cells using two-step method (1.2±0.3)% was significantly greater than the one-step method (0.9±0.2)% (P=0.041).RT-PCR suggested that human amniotic fluid-derived embryonic mesenchymal stem cells obtained using the two methods expressed Oct-4.CONCLUSION:Human multipotent mesenchymal stem cells are present in human amniotic fluid.The two-step culture protocol could be a kind of high performance and simple protocol which may not interfere with the normal prenatal diagnosis procedure.

BACKGROUND:Can adipose-derived mesenchymal stem cells be used to treat sensorineural deafness caused by hair celldegeneration and loss? OBJECTIVE:To investigate the effect of adipose-derived mesenchymal stem cells from guinea pigs transplanted via the scala tympani on the recovery from sensorineural hearing in an animal model. METHODS:Intraperitoneal injection of gentamicin was performed to prepare sensorineural deafness models in guinea pigs. Then, adipose-derived mesenchymal stem cells from guinea pig were transplanted via the scala tympani. After 1 and 3 weeks, auditory brainstem responses were detected to observe the hearing variation after adipose-derived mesenchymal stem celltransplantation. Meanwhile, the migration and distribution of EDU-labeled adipose-derived mesenchymal stem cells in the cochlea were traced. RESULTS AND CONCLUSION:After 1 and 3 weeks of implantation, the results from auditory brainstem response showed that the hearing was gradual y improved. At 1 week of implantation, most of the cells were distributed in the perilymph and some cells migrated to the Corti organ and attached to the basement membrane. At 3 weeks of implantation, cells migrated and attached to the basement membrane of Corti organ, furthermore, some cells migrated to the cochlear nerve. The longer the time of implantation, the less cellsurvived. The results indicate that adipose-derived mesenchymal stem cells from guinea pig that are transplanted via the scala tympani can directly migrate and survive ultimately to improve the hearing.

BACKGROUND:Sensorineural hearing loss is mainly caused by missing or damaged hair cells in the inner ear. Application of adipose-derived mesenchymal stem cells to regenerate inner ear hair cells is an effective treatment for hearing loss. OBJECTIVE:To explore the feasibility of in vitro inducing adipose-derived mesenchymal stem cells to differentiate into inner ear hair cel-like cells in guinea pigs. METHODS:Adipose-derived mesenchymal stem cells from guinea pigs were isolated and cultured to the 3rd generation. cellphenotype was detected using flow cytometry. Cytokines were added for induction and differentiation by stages, including epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor-1, al-trans retinoic acid, brain-derived neurotrophic factor, neurotrophin 3. RESULTS AND CONCLUSION:Adipose-derived mesenchymal stem cells of guinea pigs cultured in vitro were fusiform and showed a swirled adherent growth. Passage 3 cells were positive for CDCD29 and CD44, but negative for CD34 and CD45. After induction, the cells were positive for nestin and GFAP positive at early stage;after 10-day continuous induction, the cells expressed Myosin VIIa and Math1, specific markers of hair cells, indicating that cytokines can directly induce adipose-derived mesenchymal stem cells to differentiating into inner ear hair cells in guinea pigs.

BACKGROUND:Under mitomycin C treatment, feeder cells appear to have restricted proliferation, but they are stil able to secret different cytokines. Non-mesenchymal stem cells from the bone marrow and secreted factors in plasma maintain the micro-environment suitable for the growth of mesenchymal stem cells that can improve the yield of mesenchymal stem cells.
OBJECTIVE:To study the biological characteristics of C57 mouse bone marrow mesenchymal stem cells isolated using a whole bone marrow adherent culture technique.
METHODS:Using the whole bone marrow adherent culture technique, purified and amplified C57 mouse bone marrow mesenchymal stem cells were harvested. cellproliferation kinetics, immune cellsurface markers, multiple differentiation potential and cellcycle were detected.
RESULTS AND CONCLUSION:Using the whole bone marrow culture, mouse bone marrow mesenchymal stem cells were harvested and capable of adhering to the plastic culture vessel. The obtained cells expressed CD45, CD105 and Sca-1, but were negative for CD34, CD33 and C-kit. The doubling time was (57.11±1.5) hours. The cells could be induced to differentiate into osteoblasts, adipocytes and chondrocytes. The cellcycle analysis showed that 64%of cells were in G 0-G 1 phase. These indicates that C57 mouse bone marrow mesenchymal stem cells isolated using a whole bone marrow adherent culture technique have biological characteristics of mesenchymal stem cells.

Objective To observe the effect of Yingliu mixture combined with methimazole on autoantibodies and traditional Chinese medical syndromes of patients with Graves’ disease ( GD) . Methods A randomized, paralleled and controlled trial was carried out in 92 GD patients. The patients were evenly randomized into treatment group and control group, and were separately treated with Yingliu mixture combined with methimazole, and methimazole. The treatment for both groups lasted 12 weeks, and forty patients in each group completed the whole treatment. The changes of thyroid function, thyroid autoantibodies, and traditional Chinese medical syndrome scores were observed before and after treatment, and the clinical efficacy was also evaluated. Results (1) After treatment for 12 weeks, serum levels of thyroid peroxidase antibody (TPOAb), thyroglobulin antibody ( TGAb) and thyrotrophin receptor antibody ( TRAb) were decreased, and thyroid stimulating hormone ( TSH) was increased obviously in both groups after treatment ( P<0.01 compared with those before treatment) . The treatment group has better effect on improving TGAb and TRAb than the control group ( P<0.05). ( 2) Compared with those before treatment, the total scores of clinical symptoms and signs were decreased in the two groups at different time points of treatment course ( P<0.001) , and the decrease value in the treatment group was larger than that in the control group ( P<0.05) . ( 3) The total effective rate of the treatment group was 92.50%, higher than 82.50% of the control group ( P<0.05) . Conclusion Yingliu mixture combined with methimazole is effective on improving thyroid function, decreasing autoantibodies levels and relieving clinical symptoms and signs, and has better effect than methimazole alone for the treatment of GD.

BACKGROUND:The cultivation of mammary gland stem cel s is of great significance for the development of mammary gland and breast cancer.
OBJECTIVE:To seek an easy method to isolate and culture mammary gland stem cel in vitro, and verify the safety of cel s.
METHODS:Mammary epithelial cel s were isolated from normal tissues surrounding breast cancer, and CD49f-and EPCAM-positive cel s were sorted using flow cytometry fol owed by surface marker analysis and cel colony formation ability analysis. Afterwards, real-time fluorescent quantitative PCR was used to detect C-erbB-2 and Maspin mRNA expression in mammary gland stem cel s, breast cancer tissues and normal tissues surrounding breast cancer.
RESULTS AND CONCLUSION:Human mammary gland stem cel s were successful y cultured and highly expressed CD49f and EPCAM, with the presence of mixed colony, pleural epithelial cel colony, and myoepithelial cel colony. c-erbB-2 was lowly expressed while Maspin highly expressed in mammary gland stem cel s. Our experimental findings indicate that the mammary gland stem cel s derived from normal tissue surrounding breast cancer have biological safety.

BACKGROUND:Endothelial progenitor cells from the peripheral blood and umbilical cord blood are an essential source to repair vascular endothelial cells damaged by various diseases.
OBJECTIVE:To compare the biological characteristics of endothelial progenitor cells from peripheral blood and umbilical cord blood in vitro.
METHODS:Mononuclear cells were isolated from the umbilical cord blood and peripheral blood with density gradient centrifugation method and 6%hydroxyethyl starch precipitation combined with density gradient centrifugation method, respectively. Mononuclear cells were counted. Then the cells were implanted on the culture plate pre-paved with rat tail col agen at the concentration of 1×106/cm2, and cultured in an endothelial cellculture medium for 7 days.
RESULTS AND CONCLUSION:Isolated and cultured endothelial progenitor cells from the peripheral blood and umbilical cord blood had similar morphological characteristics in vitro. Under the optical microscope, with the increase of culturing days, most adherent cells were changed from round to spindle-shaped. Peripheral blood endothelial progenitor cells had cellcolony formation, and spindle cells from the umbilical cord blood arranged typical y in a line structure. After trypan blue staining and drawing of cellgrowth curves, the number of mononuclear cells and endothelial progenitor cells, survival rate and proliferative ability of endothelial progenitor cells from the peripheral blood were al lower than those from the umbilical cord blood (P<0.05). At day 3 after incubation, the proliferation of endothelial progenitor cells from the peripheral blood and umbilical cord blood reached the peak, and then showed a decreased tendency. Flow cytometry and Immunofluorescence staining showed that endothelial progenitor cells from the peripheral blood and umbilical cord blood could express CD34, CD133, and vascular endothelial cellfactor receptor 2. The endothelial progenitor cells from the peripheral blood and umbilical cord blood could swal ow acetylated low density lipoprotein and be combined with ulex europaeus agglutinin Ⅰ. The results confirmed that the endothelial progenitor cells from the peripheral blood and cord blood have similar biologicla characteristics, but the proliferation ability of endothelial progenitor cells from the cord blood is higher.

BACKGROUND:Cryopreservation of human umbilical cord mesenchymal stem cel s has been a hot research issue currently, but the studies concerning their effects on expansion of hematopoietic stem/progenitor cel s after cryopreservation are seldom. OBJECTIVE:To investigate the effects of human umbilical cord mesenchymal stem cel s before and after cryopreservation as feeder layer on expansion of human bone marrow mononuclear cel s in vitro. METHODS:2.5g/L mitomycin C processed human umbilical cord mesenchymal stem cel s and bone marrow mesenchymal stem cel s at passage 3 were used as the feeder layer to expand adult al ogeneic bone marrow mononuclear cel s in culture. Up to day 35, methylcel ulose assay was used to detect hematopoietic stem/progenitor cel colony proliferation. RESULTS AND CONCLUSION:There were no differences in the morphology and size of colonies in the cryopreserved human umbilical cord mesenchymal stem cel group, bone marrow mesenchymal stem cel group and non-cryopreserved human umbilical cord mesenchymal stem cel group. However, these parameter described above were significantly higher in these three groups than the blank control group (P<0.05). There were fewer colonies in the cryopreserved human umbilical cord mesenchymal stem cel group than the non-cryopreserved human umbilical cord mesenchymal stem cel group (P<0.05). These findings indicate that human umbilical cord mesenchymal stem cel s before and after cryopreservation have the ability as feeder layer on expansion of bone marrow mononuclear cel s in vitro similar to bone marrow mesenchymal stem cel s. But this ability of human umbilical cord mesenchymal stem cel s may decrease after cryopreservation.

Abstract: Objective　To prepare a microparticle delivery system that regulates the release rate of extracellular vesicles (EVs), and to exert long-term enhancement of liver cell proliferation after only one intervention. Methods EVs was extracted by differential centrifugation. The structure of the EVs was observed by transmission electron microscopy and the membrane marker protein of EVs was detected by Western blotting. EVs-PLA microspheres with "core-shell" structure were prepared by emulsion-solvent evaporation method. Scanning and transmission electron microscopy were used to detect the morphology of EVs-PLA microspheres and EVs. The release test detected the release behavior of EVs in EVs-PLA microspheres. Scanning electron microscopy was used to detect the morphological changes of EVs-PLA microspheres at 8 weeks of release. EVs-PLA microspheres were co-cultured with hepatocytes, and Phalloidin/DAPI staining was used to observe the cell morphology and evaluate the cytotoxicity of the microspheres. CCK8-test was used to evaluate the cell proliferation activity. Western blot analysis was used to detect extracellular vesicles membrane marker protein expression. Results Comparing the ability of hepatocyte proliferation in the group treated with EVs-PLA microspheres and the control group, it was found that EVs-PLA microspheres did not cause cell apoptosis and mutation in cell structure, had biocompatibility and no cytotoxicity. The EVs-PLA microspheres with "core-shell" structure regulated the release behavior of EVs, which can continuously release EVs, exerting a continuous biological role in promoting hepatocyte proliferation after a single intervention. Conclusions The EVs-PLA microspheres can control-release EVs and promote hepatocyte proliferation continuously after a single intervention, providing a reference for further exploration of EVs-loaded delivery systems in promoting liver regeneration.

BACKGROUND:Bone marrow mesenchymal stem cels are crucial for bone and cartilage development and regeneration at a celular level. Insufficient quantity and functional impairment of bone marrow mesenchymal stem cels is widely considered to be one of osteoarthritis causes. OBJECTIVE: To explore the relationship between the functional status of bone marrow mesenchymal stem cels and disease progression in osteoarthritis patients.METHODS: Thirty patients with osteoarthritis were enroled from July 2013 to October 2014, and divided into control, mild osteoarthritis, and severe osteoarthritis groups, with 10 cases in each group. 5 mL bone marrow from the femur or tibia was extracted from each patient to isolate and culture bone marrow mesenchymal stem cels. Proliferation ability of cels at passage 3 was detected using cel counting kit-8; toluidine blue staining was performed at 14 days after chondrogenic induction; real-time PCR was used to detect the mRNA expression of Aggrecan and Col2A1 in the control group after chondrogenic induction. RESULTS AND CONCLUSION:Afterin vitro culture, bone marrow mesenchymal stem cels grew adherently in polygonal and fusiform shape with multiple processes at uniform size. The cytoplasm contained larger particles and the nuclei were ovoid. Most of cels were in cel division phase. The proliferation ability was strongest in the control group and weakest in the severe osteoarthritis group. Cels from the three groups were al at plateau phase after 1 week culture. At 14 days after chondrogenic induction, the cels were polygonal and quasi-circular, and purple metachromatic granules distributed outside of the cytoplasm. The expression of Aggrecan and Col2A1 in the control group displayed an overexpression trend. These findings indicate that the functional status of bone marrow mesenchymal stem cels from osteoarthritis patients is negatively correlated with the severity of disease, which can influence the disease progression in osteoarthritis patients.