Mesenchymal stem cells (MSCs) can be obtained from a variety of human tissues. Placenta has become an attractive stem cell source for potential applications in regenerative medicine and tissue engineering. The aim of this study was to localize and characterize MSCs within human chorionic membranes (hCMSCs). For this purpose, immunofluorescence labeling with CD105 and CD90 were used to determine the distribution of MSCs in chorionic membranes tissue. A medium supplemented with a synthetic serum and various concentrations of neurotrophic factors and cytokines was used to induce hCMSCs to neural cells. The results showed that the CD90 positive cells were scattered in the chorionic membranes tissue, and the CD105 positive cells were mostly located around the small blood vessels. hCMSCs expressed typical mesenchymal markers (CD73, CD90, CD105, CD44 and CD166) but not hematopoietic markers (CD45, CD34) and HLA-DR. hCMSCs differentiated into adipocytes, osteocytes, chondrocytes, and neuronal cells, as revealed by morphological changes, cell staining, immunofluorescence analyses, and RT-PCR showing the tissue-specific gene presence for differentiated cell lineages after the treatment with induce medium. Human chorionic membranes may be the source of MSCs for treatment of nervous system injury.
Adipocytes ; Blood Vessels ; Cell Lineage ; Chondrocytes ; Chorion* ; Cytokines ; Fluorescent Antibody Technique ; HLA-DR Antigens ; Humans* ; Membranes* ; Mesenchymal Stromal Cells* ; Nerve Growth Factors ; Neurons ; Osteocytes ; Placenta ; Regenerative Medicine ; Stem Cells ; Tissue Engineering ; Trauma, Nervous System

10.3969/j.issn.2095-4344.2013.23.006

BACKGROUND: There have been no effective means for clinical treatment of large regions of bone defects.Nano-hydroxyapatite/collagen(nHAC)composite would provide a new pathway for repair of bone defects owing to its similar structure to natural skeleton and better biocompatibility.OBJECTIVE: To investigate the role of nHAC composite co-cultured with bone marrow-derived mesenchymal stem cells(BMSCs)in repair of bone defects.METHODS: Following isolation and culture,human BMSCs were co-cultured with nHAC composite.Gross observation,histological analysis,and electron microscope observation were performed to analyze osteogenesis for repair of bone defects in the clinic.RESULTS AND CONCLUSION: Human nHAC could greatly proliferate in vitro.X-ray photography revealed that bone defects well healed after implantation of nHAC/BMSCs composite.These findings indicate that BMSCs exhibit osteogenic potential and nHAC is a satisfactory scaffold material for construction of tissue-engineered bone.

BACKGROUND: Umbilical cord blood mesenchymal stem cells (UCBMSCs) have been shown to differentiate into nerve-like cells under the condition of in vitro culture. Brain-derived neurotrophic factors (BDNF) combined with ciliary neurotrophic factors (CNTF) at certain concentration can in vitro induce the differentiation of UCBMSCs into high proportion of neuronal-like cells. OBJECTIVE: To investigate the feasibility of BDNF, CNTF and their combination for in vitrodifferentiation of UCBMSCs into nerve-like cells.METHODS: UCBMSCs of passage 5 were induced to differentiate into nerve-like cells using 5,10, 20 ug/L BDNF, 5,10, 20 ug/L CNTF or their combination. A blank control group was set, with no any interventions. Cell morphology was observed under inverted phase contrast microscope. At 1, 3, and 6 days of experiment, immunocytochemical staining for neuron specific enolase and glial fibrillary acidic protein was performed. The proportion of differentiated neuronal-like cells and glial cell-like cells was calculated.RESULTS ANDCONCLUSION:After induced differentiation into nerve-like cells, human UCBMSCs exhibited changeable morphology with contracted cell body, enhanced refraction of nucleus, and dendrite- and axon-like structure. Compared with blank control group, BDNF and CNTF couldsignificantly enhance the differentiation proportion of UCBMSCs into nerve-like cells. 20 ug/LBDNF combined with 20 ug/L CNTF yielded highest differentiation proportion of human UCBMSCs into nerve-like cells. These findings indicate that human UCBMSCs can differentiate into nerve-like cells after in vitro induction of BDNF, CNTF or their combination.

BACKGROUND:As a kind of undifferentiated precursor cells,the phenotypic differentiation of bone marrow mesenchymal stem cells (BMSCs) remains immaturity,thus it presents weak rejection following transplantation.However,the in vitro directional differentiation of BMSCs into neuronal cells is easy affected by various factors.OBJECTIVE:To observe the immunomodulatory effect and the potential of BMSCs differentiate into neuronal-like cells.DESIGN,TIME AND SETTING:A contrast observation was conducted at the Department of Cytology,Third People's Hospital of Wuxi from January 2008 to March 2009.MATERIALS:Bone marrow was harvested from chips of cancellous or ilium bone dudng hip joint surgery.METHODS:Firstly,the BMSCs were separated and cultured to establish mixed lymphocyte reaction (MLR) system.Secondly,2 samples of peripheral blood mononuclear cells (1×10~5/well) were added into 96-well plate,and then BMSCs treated by mitomycin were added according to different ratios (BMSCs/peripheral blood monouclear cells).At the end,the cells were cultured as follows:Method 1:DMEM+10% fetal calf serum+1 μmol/L RA +20 μg/L basic fibroblast growth factor+20 μg/L epidermal growth factor.Method 2:DMEM+2% dimethyl sulfoxide +100 μmol/L butylated hydroxyanisole.MAIN OUTCOME MEASURES:The growth rate of lymphocyte was measured by ~3H-Thymidine,and the effect of BMSCs on lymphocyte proliferation was observed.Additionally,the differentiation potential of BMSCs into neuronel cells was determined by immunofluorescenca and immunohistochemistrical staining.RESULTS:①The BMSCs inhibited lymphocyte proliferation in MLR system and the influence on proliferation of lymphocyte was direct related to ratio of BMSCs.②Under a light microscope,cytoplasm of BMScs were shrinkd,which presented typical perikaryon morphology at hour 2 after culture with method 1.The majority of BMSCs were formed neuronal-like cells without number changes at hours 3-5,which turned to be dipolar or multipolar neuronal shapes at day 3.There were 60%-70% neuronspecific enolase,45%-50% glial fibrillary acidic protein were positive expressed.However,the positive rate of nidogen was decreased 3.4%.Cells cultured with method 2 became smaller after 2 hours,formed dipolar or multipolar body cells,and most of cells were died after 48 hours.The 40%-50% neuronspecific enolase,35%-40% glial fibrillary acidic protein was found to be positive.The positive rate of nidogen was temporary increased to 63% at hour 2 after culture；however,it was decreased to 1.6% after 48 hours.CONCLUSION:BMSCs can differentiate into neuronal-like cells,as well as inhibit lymphocyte proliferation in MLR system,which possess down regulation effect on alloimmunity-reaction.

BACKGROUND:Several studies have demonstrated that stem cells can differentiate into vascular endothelial cells, and then further differentiate and form blood capillary. Based on this principle, autologous bone marrow mesenchyrnal stem cell (BMSC) transplantation promotes angiogenesis to treat ischemia in lower limb.OBJECTIVE: To evaluate the angiogenesis in rabbit ischemic limbs following autologous BMSC transplantation using high-frequency two-dimensional ultrasound detection in conjunction with Doppler color-flow imaging examination. DESIGN, TIME AND SEI-FING: A randomized, controlled, animal experiment was performed in the Third People's Hospital of Wuxi between March 2007 and April 2008.MATERIALS: Twenty-four New Zealand rabbits were randomized to a control group and a cell transplantation group, with 12 rabbits in each.METHODS: Ischemia in lower limbs was induced in all rabbits. One week following ischemia induction, the cell transplantation group was injected with 0.5 mL cell suspension, comprising 2×106 BrdU-labeled autologous BMSCs cultured in vitro, through multiple sites in the region of gastrocnemius muscle. Simultaneously, the control group received the same amount of physical saline in the same region.MAIN OUTCOME MEASURES: The initial segment of rabbit femoral artery and superficial femoral artery was subjected to high-frequency two-dimensional ultrasound and Doppler color-flow imaging examinations to measure femoral artery vascular internal diameter, blood flow peak velocity, and blood flow acceleration time. Ischemic muscular tissue was taken for immunohistochemical staining to detect transplanted cell distribution and for pathological examination of angiogenesis. RESULTS: Two weeks following autologous BMSC transplantation, high-frequency ultrasound results revealed that femoral artery internal diameter and blood flow peak velocity were greater, but blood flow acceleration time was shorter, in the cell transplantation group than in the control group (P<0.01). Immunohistochemical staining results demonstrated the presence of BrdU-positive cells. Pathological sections displayed that vascular density was significantly higher in the cell transplantation group than ih the control group. CONCLUSION: Autologous BMSC transplantation is a promising, simple, and effective method of treating ischemia in lower limbs owing to its promotion of angiogenesis. Meanwhile, high-frequency ultrasound detection of femoral artery is an effective, practical method to evaluate the clinical outcomes of autologous BMSC transplantation.

Objective To locally inject human umbilical cord blood (HUCB) mesenchymal stem cells (MSCs) to rat traumatic brain injury (TBI) model to investigate expression of neural markers and neurological functional improvement. Methods HUCB-MSCs were labeled by bis-benzimide for over 24 hours and stereotactically transplanted into the brain of the rats. All rats were divided into four groups, ie, sham injury group, TBI group, control (TBI + PBS) group and treatment (TBI + MSCs) group, Im-munohistochemical methods and immanofluorescence staining were used to observe the survival, migration and differentiation of the transplanted cells. The neurological functional improvement was evaluated by u-sing the neurological severity score (NSS). Results There existed a large number of MSCs survived in local region of the brain that received transplants, when some MSCs differentiated into neurons or astro-cytes and expressed the neurocyte markers including NSE and GFAP around the grafted site. Treatment group had significantly improved scores compared with sham injury group, TBI group and control group. Conclusions HUCB-MSCs transplantation can potentially improve neurological functional after TBI and may be a good alternative to bone marrow cells for stem cell transplantation or cell therapy.

Objective:To optimize the culture system for the mesenchymal stem cells from the Placenta Chorion (hCMSC) in vitro by adding the selected cytokines to amplify the hCMSC effectively,with furthermore some preliminary analysis of the hCMSC performed.Methods:Immunohistochemistry was applied to show the localization of the mesenchymal stem cells within the placenta chorion,then the corresponding part of chorion was digested by collagenase Ⅳ to get the cell suspension for primary culture and subculture;Flow Cytometry was used to detect the surface marker of hCMSC,whose proliferation was detected by direct cell counting and the MTT method;then Butylated Hydroxyanisole (BHA),and DMSO was used combinedly as the inducer in the differentiation of the hCMSC to the neuron like cells,which was then detected by Immunofluorescence staining for the expression of NSE;finally RT-PCR was used to detect the expression of flt3.Results:The result of immunohistochemistry showed CD105~-,CD90~- positive hCMSC was much easier to be found around the blood vessels within the villous of chorion,which then could be obtained by mechanical dissociation and collagenase digestion.bFGF was good at amplifying the hCMSC among all selected cytokines,but FL was showed even obviously effective on the proliferation of the hCMSC.The propagating cells were found to highly express CD73,CD90,CD105;and negative for CD14,CD34,CD45 and HLA-DR.We also found that the hCMSC happened to express PDL-1 the negative co-stimulatory molecules,while their potential to different into the neuron like cells was still well maintained.Conclusion::There is low immunogenicity mesenchymal stem cells within the placenta chorion,both bFGF,FL are shown good effects on the proliferation of the hCMSC,with maintaining their differentiation potential as well.

BACKGROUND: Longterm therapeutic effects of routine drug treatment, intervention or vascular bypass transplantation on lower extremity arterial occlusion are not ideal. During recent years, angiogenesis of stem cells possibly becones a new method to repair or rebuild an effective collateral circulation at infarct regions.OBJECTIVE: To verify the effect of autologous bone marrow mesenchymal stem cell transplantation on promoting angiogenesis in rabbit ischemic hind limbs.DESIGN, TIME AND SETTING: A randomized controlled animal study was performed in Jiangsu Institute of Schistosomiasis Control between August 2005 and November 2006. MATERIALS: Eight rabbits were used to prepare ischemic models of hind limbs, and then they were randomly divided into experimental group (n=4) and control group (n=4).METHODS: Bone marrow mesenchymal stem cells were isolated and cultured from New Zealand rabbits in the experimental group, and they were then marked 5-bromo-2-deoxyuridine (Brdu). Suspension of bone marrow mesenchymal stem cells was injected into the ischemic hind limbs in the experimental group, while the same volume of saline was injected into the controls. MAIN OUTCOME MEASURES: After two weeks, two-dimensional and color Doppler ultrasound detection was used on rabbit femoral artery to measure inner diameter of blood vessel, peak velocity and acceleration time of blood flow before and after transplantation. Muscle tissues were obtained from ischemic regions to observe distribution of transplant cells and state of angiogenesis using immunofluorescence staining and hematoxylin-eosin (HE) staining. RESULTS: Two weeks after transplantation, the inner diameter of femoral artery and the peak velocity of blood flow in the experimental group were higher than those in the control group (P < 0.01), but the acceleration time of blood flow was shorter than that in the control group (P < 0.01). Lmmunofluorescence staining showed that anti-Brdu-staining positive cells were found out in transplant part in the experimental group; while HE staining indicated that vessel density of ischemic region in the experimental group was higher than that in the control group. CONCLUSION: Bone marrow mesenchymal stem cells can promote angiogenesis, while autologous bone marrow mesenchymai stem cell transplantation will become a simple and effective method to treat lower limb ischemia.

BACKGROUND: The repair of periodontal tissue is dependent on the number and proliferation and differentiation abilities of periodontal ligament (PDL) cells. PDL cells have the potentiality of multi-directional differentiation such as cementoblast,osteoblast and fibroblast to fonn cement, alveolar bone and periodontal ligament and finally achieve periodontal tissue regeneration.OBJECTIVE:To observe the effects of Shuanghuangbu extract on the proliferation and differentiation of PDL cells.DESIGN:Observation trail.SETTING:Central Laboratory of Wuxi Third People's Hospital. MATERIALS: Periodontal tissue was provided voluntarily by the healthy young patients with deformity correction, and golden thread,skullcap,and rhizoma drynariae by the National Institute for the Control of Pharmaceutical and Biological Products.METHODS: The experiment was conducted in the Central Laboratory of Wuxi Third People's Hospital from July to October 2003.The crushed golden thread.skullcap,and rhizoma drynariae were mixed with distilled water at ratio of 1:10 (m:V),and refluxed in boiling water for 5 hours.The extract was collected,and after colation,the residue was refluxed in boiling water for another 3 hours. Both extract was blended, rotary evaporated and condensed, finally the liquid extract of 3 kg/L was obtained.There were 8 groups in the study including golden thread group, skullcap group,rhizoma drynariae group,golden thread plus skullcap group,golden thread plus rhizoma drynariae group, skullcap plus rhizoma drynariae group, Shuanghuangbu group and control group. PDL cells were cultured/n vitro assisted with Shuanghuangbu.The proliferation of cells was detected with MTT method and the ratio of collagen content in total protein was evaluated with hydroxyproline (HP).MAIN OUTCOME MEASURES:A value of proliferated PDL cells and the proportion of collagen protein in total protein.RESULTS:①Proliferation of PDL cells:Except golden thread group,all Chinese medicine promoted the proliferation of PDL cells significantly compared with control group (P＜0.05). The A value of Shuanghuangbu group was significantly increased.A value was increased with time and reached the peak on day 5.There were significant differences among each group at different time (P＜0.05).②Ratio of collagen content in total protein:Except golden thread group,the percentage was significantly increased by other Chinese medicines compared with control group (P＜0.05), especially Shuanghuangbu. The percentage was increased with time and reached the peak on day 5. There were significant differences among each group at different time (P＜0.05). CONCLUSION:The findings suggest that as a traditional Chinese herb,Shuanghuangbu can significantly stimulate the proliferation and differentiation of PDL cells.and increase the proportion of collagen content in total protein.It may act as an ideal Chinese medicine helper factor for the regeneration of PDL cells.