Umbilical Cord-Mesenchymal Stem Cell-Conditioned Medium Improves Insulin Resistance in C2C12 Cell.

Background: Umbilical cord-mesenchymal stem cell-conditioned medium (UC-MSC-CM) has emerged as a promising cell-free therapy. The aim of this study was to explore the therapeutic effects of UC-MSC-CM on insulin resistance in C2C12 cell. Methods: Insulin resistance was induced by palmitate. Effects of UC-MSC-CM on insulin resistance were evaluated using glucose uptake, glucose transporter type 4 (GLUT4) translocation, the insulin-signaling pathway, and mitochondrial contents and functions in C2C12 cell. Results: Glucose uptake was improved by UC-MSC-CM. UC-MSC-CM treatment increased only in membranous GLUT4 expression, not in cytosolic GLUT4 expression. It restored the insulin-signaling pathway in insulin receptor substrate 1 and protein kinase B. Mitochondrial contents evaluated by mitochondrial transcription factor A, mitochondrial DNA copy number, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha were increased by UC-MSC-CM. In addition, UC-MSC-CM significantly decreased mitochondrial reactive oxygen species and increased fatty acid oxidation and mitochondrial membrane potential. There was no improvement in adenosine triphosphate (ATP) contents, but ATP synthesis was improved by UC-MSC-CM. Cytokine and active factor analysis of UC-MSC-CM showed that it contained many regulators inhibiting insulin resistance. Conclusion: UC-MSC-CM improves insulin resistance with multiple mechanisms in C2C12 cell.

Assessing the effects of cyclosporine A on the osteoblastogenesis, osteoclastogenesis, and angiogenesis mediated by human periodontal ligament stem cells.

BACKGROUND: This study was performed to investigate the effects of cyclosporine A (CsA) on the osteogenic differentiation, osteoclastogenic-supporting ability, and angiogenic potential of human periodontal ligament stem cells (hPDLSCs). METHODS: hPDLSCs were isolated from the extracted teeth of orthodontic patients. Cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and alizarin red (ARS) staining. Real-time polymerase chain reaction (PCR) was used to quantify transcripts. Tartrate-resistant acid phosphatase staining of bone marrow-derived macrophages (BMMs) and tube formation assays on human umbilical vein endothelial cells (HUVECs) were performed after treating cells with the conditioned media from CsA-exposed or non-exposed hPDLSCs. Signaling pathways mediating the angiogenic activity were investigated using western blotting. RESULTS: CsA suppressed the proliferation of hPDLSCs but enhanced osteogenic differentiation as determined by ALP and ARS staining and PCR of osteogenic transcripts. The expressions of osteoclastogenic transcripts in hPDLSCs and the differentiation of BMMs treated with conditioned medium from CsA-exposed hPDLSCs were unaffected by CsA. However, the expressions of angiogenic transcripts and the transcripts known to support angiogenesis-phosphorylation of extracellular signal p-regulated kinase (ERK) and p38, and c-fos-were inhibited. Conditioned medium from CsA-exposed hPDLSCs suppressed the tube forming abilities of HUVECs. CONCLUSIONS: CsA enhanced the osteogenic differentiation and reduced angiogenesis by blocking the ERK and p38/c-fos pathway in hPDLSCs. It is necessary to confirm whether this phenomenon is also observed in vivo in subsequent animal experiments.

Inflammation activated bone marrow mesenchymal stem cell conditioned medium repairs radiation-induced acute injury to intestinal epithelial stem cells / 中国组织工程研究

BACKGROUND: Our previous findings indicate that inflammation-activated bone marrow mesenchymal stem cell conditioned medium (MSC-CM) contribute to repairing the structure and function of the small intestine after radiation-induced acute intestinal injury. However, it is unclear whether the repair effect can be achieved by regulating small intestinal stem cells. OBJECTIVE: To investigate the effects of inflammation-activated bone marrow MSC-CM on the small intestinal epithelial stem cells after acute radiation-induced intestinal injury and to further discuss the repairing mechanism. METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats were separated, cultured and identified. Then, the bone marrow mesenchymal stem cells were co-cultured with normal or radiation-induced IEC-6 cell lines in the Transwell system for 24 hours. Inflammation-activated bone marrow mesenchymal stem cells were cultured alone for 48 hours. Non-activated MSC-CM (MSC-CMNOR) and MSC-CM under radiation-induced inflammatory condition (MSC-CMIR) were collected. Adult Sprague-Dawley rats (provided by the Experimental Center of Sun Yat-Sen University North Campus) were randomly divided into four groups with 20 rats in each group: control group, radiation group, radiation+MSC-CMNOR group and radiation+MSC-CMIR group. The rats in the latter three groups were exposed to one-off 14 Gy whole abdominal radiation to make a rat model of acute radiation-induced small intestinal injury. Three-day continuous administration beginning within 4 hours after successful modeling was given via the tail vein and intraperitoneal implantation of Alzet micro-osmotic pumps: EMEM-F12 (200 μL/d) for the radiation group, MSC-CMNOR for radiation+MSC-CMNOR group and MSC-CMIR for radiation+MSC-CMIR group. There was 2 mL of concentrated conditioned medium in the pump which was released at a constant rate of 10 μL/h into the abdominal cavity after implantation. Intestinal samples were collected at 1, 3, 5, 7 days after radiation for immunochemistry staining, western blot and qRT-PCR detection. RESULTS AND CONCLUSION: (1) On the 3rd day after radiation, Lgr5 positive cells, which were actively proliferating on the base of crypts, became significantly reduced compared with the normal control group, and there was nearly no existing Lgr5 positive cells. However, after infusion of MSC-CMIR, Lgr5 positive intestinal stem cells were significantly increased compared with the radiation group, while in the radiation+MSCNOR group, there was no significant increase in Lgr5 positive intestinal stem cells. (2) On the 3rd day after radiation injury, Bmi1 positive intestinal stem cells were almost invisible. After infusion of MSC-CMIR, Bmi1 positive intestinal stem cells increased significantly, and it was observed not only in the +4 cell position but also in the common position used to be Lgr5 stem cells, indicating that Bmi1 stem cells could differentiate into Lgr5 positive cells to act its repairing effect. (3) Western blot and qRT-PCR further confirmed that the radiation+MSC-CMIR group was significantly higher on the Lgr5 expression level than the radiation group and the radiation+MSC-CMNOR group, and it returned to the normal level on the 7th day after the continuous high expression level. The repair effect of radiation+MSC-CMNOR group was weaker, and only on the 7th day, the expression level of Lgr5 was statistically different from the radiation group. To conclude, inflammation-activated bone marrow MSC-CM exert a protective effect on the small intestinal epithelial stem cells after acute radiation-induced intestinal injury

Cell Responses to Conditioned Media Produced by Patient-Matched Stem Cells Derived From Healthy and Inflamed Periodontal Ligament Tissues.

BACKGROUND: Periodontal ligament stem cells (PDLSCs) derived from clinically compromised teeth with periodontitis are considered a readily accessible cell source, but their impaired stem cell functionalities, as observed in various in vitro and in vivo models, necessitate further investigation of these inflamed cells before their translation into therapeutic applications. In this study, the effects of conditioned media (CM) produced by stem cells derived from human healthy periodontal ligament tissues (H-PDLSCs) or inflamed periodontal ligament tissues (I-PDLSCs), referred to as H-CM and I-CM, respectively, on the biologic properties of H-PDLSCs and I-PDLSCs from the same donor are compared to explore the extent to which inflamed cells can be rescued by their extrinsic environment (i.e., by H-CM). METHODS: H-CM and I-CM were prepared from in vitro cell cultures, and the cellular responses of H-PDLSCs and I-PDLSCs to patient-matched H-CM and I-CM were investigated in terms of colony-forming ability, cell proliferation, and adipogenic/osteogenic differentiation. RESULTS: In H-CM and I-CM, H-PDLSCs and I-PDLSCs exhibited similar adipogenic potential. However, when incubated in I-CM, both cell types demonstrated an increased capacity to proliferate but a decreased capacity to differentiate into osteoblasts. Significantly, the impaired osteogenic differentiation of I-PDLSCs was partially rescued by incubation in H-CM under osteo-inducing conditions. CONCLUSION: The CM of patient-matched H-PDLSCs and I-PDLSCs differed, and the impaired osteogenic differentiation of inflamed stem cells had the potential to be rescued, at least partially, for therapeutic use via changing the cell culture microenvironment in vitro.

Establishment and Optimization of Culture Technique for Enteroids / 胃肠病学

Background:Enteroids are considered to be the best tool for studies on intestinal epithelium in vitro and have a widely application prospects,however,there are no associated reports in China. Aims:To establish and optimize the culture technique for enteroids and provide a fantastic platform for the basic research of small intestinal epithelial cells in China. Methods:L-WRN cells were cultured routinely and the conditioned medium with different concentrations of fetal bovine serum(FBS)was collected. Six to eight weeks old C57BL/ 6 mice were sacrificed and 15 cm small intestine from the terminal ileum was removed and cut longitudinally. Crypts were digested with EDTA and then collected and embedded in Matrigel? Matrix;after polymerization of Matrigel? Matrix,L-WRN conditioned medium at different concentration gradient was added. The budding ratio and length of buds were measured dynamically under microscope. The enteroids were re-embedded for subculture when certain length of buds was reached. Results:Compared with L-WRN conditioned medium containing 20% FBS,the conditioned medium containing 10% FBS was more favorable for enteroids culture in vitro. When conditioned medium accounted for 10% ,15% ,20% ,25% or 30% of the mixed medium,they all promoted the growth of enteroids and the 15% one seemed to yield better result. Conclusions:An enteroids culture technique was successfully established for the first time in China. When the L-WRN conditioned medium containing 10% FBS accounts for 15% of the mixed medium,it might promote budding better than the others.

Formation of cell sheet on acellular porcine corneal stroma with human corneal endothelial cells cocultured by mouse embryonic stem cell conditioned medium / 中华实验眼科杂志

Background Corneal transplantation faces a great challenge because of the shortage of corneal donors and difficulty of human corneal endothelial cells (HCECs) regeneration in vitro.So the study on tissue engineering cornea is still a main topic.Previous research showed that mouse embryonic stem cell conditioned medium (ESC-CM) improved the proliferative capacity of HCECs in vitro,and acellular porcine corneal stroma (APCS) was a good saffold material.However,whether HECEs cultured by mouse ESC-CM can form cell sheet in vitro were rarely studied.Objective This study was to investigate the potential that HCECs cultured by mouse ESC-CM form a monolayer cell sheet.Methods The supernatant of ESC-CM was collected after mouse ES-E14 cells were cultured,and the cultured medium was centrifuged and mixed with 75％ human corneal endothelium medium (CEM)at a proportion of 1 ∶ 3 to prepare the 25％ ESC-CM system.Primary cultures of HCECs were established from explants of corneal limbal with Descemet's membrane,and the cells were identified by using reverse-transcription PCR to determine the expressions of collagen Ⅷ (Col Ⅷ) mRNA and neuron-specific enolase (NSE) mRNA in the cells.APCS was prepared by decellularization with phospholipase A2 and bicarbonate solution,and the second generation of HCECs were inoculated on the sterilized APCS at a 800/mm2 density.The morphology of the cells was observed by hematoxylin-eosin staining under the phase-contrast microscope.The expressions of zona occludens protein-1 (ZO-1)and Na+-K+-ATPase in the cell sheet were detected by immunofluorescence staining.Results The second generation of HCECs cultured with 25％ ESC-CM in vitro showed the hexagon in shape with positive expressions for Col Ⅷ mRNA and NSE mRNA.Decellularization APCS was transparent,and no corneal cells were seen,the structures of corneal collagenous fibres were regular.HCECs attached closely to APCS and formed monolayer sheet 7 days after culture on the APCS with the cell density of (2 694±143)/mm2.ZO-1 and Na+-K+-ATPase were positively expressed on the HCECs sheet.Conclusions Twenty-five percent ESC-CM promotes the proliferation and maintains the normal morphology of HCECs.APCS provide a good scoffold and microenvironment for the formation of HCEC sheet.The HCEC sheet Possesses the pump function of HCECs and is a good corneal donor for transplantation.

Therapeutic Effects of Umbilical Cord Blood Derived Mesenchymal Stem Cell-Conditioned Medium on Pulmonary Arterial Hypertension in Rats.

BACKGROUND: Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) may have multiple therapeutic applications for cell based therapy including the treatment of pulmonary artery hypertension (PAH). As low survival rates and potential tumorigenicity of implanted cells could undermine the mesenchymal stem cell (MSC) cell-based therapy, we chose to investigate the use of conditioned medium (CM) from a culture of MSC cells as a feasible alternative. METHODS: CM was prepared by culturing hUCB-MSCs in three-dimensional spheroids. In a rat model of PAH induced by monocrotaline, we infused CM or the control unconditioned culture media via the tail-vein of 6-week-old Sprague-Dawley rats. RESULTS: Compared with the control unconditioned media, CM infusion reduced the ventricular pressure, the right ventricle/(left ventricle+interventricular septum) ratio, and maintained respiratory function in the treated animals. Also, the number of interleukin 1α (IL-1α), chemokine (C-C motif) ligand 5 (CCL5), and tissue inhibitor of metalloproteinase 1 (TIMP-1)-positive cells increased in lung samples and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL)-positive cells decreased significantly in the CM treated animals. CONCLUSIONS: From our in vivo data in the rat model, the observed decreases in the TUNEL staining suggest a potential therapeutic benefit of the CM in ameliorating PAH-mediated lung tissue damage. Increased IL-1α, CCL5, and TIMP-1 levels may play important roles in this regard.

Conditioned medium of demineralized freeze-dried bone activates gene expression in periodontal fibroblasts in vitro.

BACKGROUND: Demineralized bone matrix (DBM) is used for the treatment of osseous defects. Conditioned medium from native bone chips can activate transforming growth factor (TGF)-ß signaling in mesenchymal cells. The aim of this study is to determine whether processing of native bone into DBM affects the activity of the conditioned medium. METHODS: Porcine cortical bone blocks were subjected to defatting, different concentrations of hydrochloric acid, and various temperatures. DBM was lyophilized, ground, and placed into culture medium. Human gingiva and periodontal fibroblasts were exposed to the respective conditioned medium obtained from DBM (DBCM). Changes in the expression of TGF-ß target genes were determined. RESULTS: DBCM altered the expression of TGF-ß target genes (e.g., adrenomedullin, pentraxin 3, KN motif and ankyrin repeat domains 4, interleukin 11, NADPH oxidase 4, and BTB [POZ] domain containing 11) by at least five-fold. The response was observed in fibroblasts from both sources. Defatting lowered the activity of DBCM. The TGF-ß receptor type I kinase inhibitor SB431542 [4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide] but not the inhibitor of bone morphogenetic protein receptor dorsomorphin, blocked the effects of DBCM on gene expression. Moreover, conditioned medium obtained from commercial human DBM modulated the expression of TGF-ß target genes. CONCLUSION: The findings suggest that the DBCM can activate TGF-ß signaling in oral fibroblasts.

Therapeutic Effects of Umbilical Cord Blood Derived Mesenchymal Stem Cell-Conditioned Medium on Pulmonary Arterial Hypertension in Rats

BACKGROUND: Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) may have multiple therapeutic applications for cell based therapy including the treatment of pulmonary artery hypertension (PAH). As low survival rates and potential tumorigenicity of implanted cells could undermine the mesenchymal stem cell (MSC) cell-based therapy, we chose to investigate the use of conditioned medium (CM) from a culture of MSC cells as a feasible alternative. METHODS: CM was prepared by culturing hUCB-MSCs in three-dimensional spheroids. In a rat model of PAH induced by monocrotaline, we infused CM or the control unconditioned culture media via the tail-vein of 6-week-old Sprague-Dawley rats. RESULTS: Compared with the control unconditioned media, CM infusion reduced the ventricular pressure, the right ventricle/(left ventricle+interventricular septum) ratio, and maintained respiratory function in the treated animals. Also, the number of interleukin 1alpha (IL-1alpha), chemokine (C-C motif) ligand 5 (CCL5), and tissue inhibitor of metalloproteinase 1 (TIMP-1)-positive cells increased in lung samples and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL)-positive cells decreased significantly in the CM treated animals. CONCLUSIONS: From our in vivo data in the rat model, the observed decreases in the TUNEL staining suggest a potential therapeutic benefit of the CM in ameliorating PAH-mediated lung tissue damage. Increased IL-1alpha, CCL5, and TIMP-1 levels may play important roles in this regard.

Effects of bone marrow mesenchymal stem cell conditioned medium with and without inflammatory activation on radiation-induced intestinal injury / 中国组织工程研究

BACKGROUND:Conditioned medium from mesenchymal stem cels (MSC-CM) may represent a promising alternative to MSCs transplantation. Previous studies have shown that inflammatory activation can strengthen the multiple biological potencies of MSCs; however, normal MSCs with insufficiency of immunocompetence and migration ability are not effective for tissue damage repair. OBJECTIVE:To investigate differential effects of MSC-CM with and without inflammatory activation on radiation-induced intestinal injury.METHODS:MSCs from the bone marrow of SD rats were separated, cultured and identified, and then co-cultured with non-irradiated IEC-6 or irradiated IEC-6 in a transwel system for 24 hours. Then, MSCs with inflammatory activation were cultured alone for another 48 hours. After that, the supernatant was colected as non-activated MSC-CM (MSC-CMNOR) and MSC-CM under radiation-induced inflammatory condition (MSC-CMIR). Rats were exposed to 14 Gy whole abdominal irradiation and randomly divided into four groups: control group, radiation injury group (DMEM/F12), MSC-CMNOR group and MSC-CMIR group. Continuous administration was givenvia tail vein and intraperitoneal implantation of Alzet microosmotic pumps. Intestinal samples were colected at 1, 3, 7 days after radiation for analysis of short circuit variation, at 3 days after radiation for analysis of intestinal epithelium ultrastructure, and at 1, 3, 5, 7, 14 days after radiation for histological observation of the intestinal epithelium using hematoxylin-eosin staining. Blood samples were colected at 1, 3, 7 days after radiation for analysis of serum xylose levels. In addition, the survival state and survival time of rats were observed and recorded. RESULTS AND CONCLUSION: The short circuit variation responding to electrical field stimulation was significantly reduced at al frequencies, but it was significantly improved in the MSC-CMIR group. Similarly, the intestinal absorption (serum xylose levels) was also significantly impaired by irradiation, but improved by delivery of MSC-CMIR (P < 0.05). At 3 days after MSC-CMIR infusion, the intestinal epithelium exhibited an increase in crypt size and vilous length (P < 0.05). Under the electron microscope, a reduction in intestinal microvili and open tight junctions in irradiated intestinal epithelium was found, and the intestine from rats treated with MSC-CMIR had more obvious tight junctions. In addition, treatment with MSC-CMIR dramaticaly improved the survival rate and mean survival time of irradiated rats as compared to those treated with DMEM/F12 or MSC-CMNOR (P < 0.05). Taken together, the present study demonstrated that MSC-CMIR , but not non-activated MSC-CM, improves the structural and functional restoration of the smal intestine after radiation-induced intestinal injury.

Bone marrow mesenchymal stem cell conditioned medium with hypoxic activation enhances its effects on radiation-induced intestinal epithelial cell injuryin vitro / 中国组织工程研究

BACKGROUND:Conditioned medium from mesenchymal stem cels (MSC-CM) that contains abundant MSCs paracrine substances may represent a promising alternative to MSCs transplantation. However, normal MSC-CM with insufficient paracrine ability is not effective for tissue damage repair. OBJECTIVE:To investigate the effects of MSC-CM with (MSC-CMHyp) and without hypoxic activation (MSC-CMNor) on the proliferation and apoptosis of radiation-induced injured intestinal epithelial cels (IEC-6) and to further discuss the paracrine mechanisms. METHODS: IEC-6 cels were exposed to 10 Gy irradiation and cultured in MSC-CMHyp, MSC-CMNor, and DMEM-F12 medium, respectively. RESULTS AND CONCLUSION: Findings from trypan blue staining, flow cytometry and western blot assay showed that, compared with the DMEM-F12 medium group, treatment with MSC-CMHyp significantly enhanced IEC-6 viability proliferation after radiation-induced injury, as wel as significantly decreased cel apoptosis and expression of Caspases-3/8 (P 0.05). On the other hand, the increased levels of vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factor-1, and interleukin-10 were detected in the MSC-CMHyp group compared to the MSC-CMNor group (P < 0.05). These results suggest that the MSC-CMHyp improves the viability and proliferative capacity of IEC-6 cels after radiation-induced injuryvia up-regulating secretion of cytokines and down-regulating apoptotic signaling.

Advances in serum-free culture systems of human embryonic stem cells / 中国组织工程研究

BACKGROUND:Human embryonic stem cels are able to self-renew indefinitely and have the capacity to differentiate into al three germ layers (ectoderm, endoderm and mesoderm). These properties imply great potential in the basic research and clinical application, including regenerative medicine, drug screening and toxins, early human embryo, cel transplantation, gene therapy,etc. However, it is a substantial chalenge to develop efficient techniques for their large-scale culture under defined conditions, and for controling and directing their differentiation. For therapeutic purposes, many scholars are trying to establish methods for maintaining pluripotency in defined xeno-free conditions and scalable culture systems. OBJECTIVE:To discuss the progress of serum-free culture systems in human embryonic stem cel research reported in recent years and to highlight the chalenges and advances being made towards the development of serum-free and xeno-free culture systems suitable for therapeutic applications. METHODS:A computer-based search of CNKI and PubMed academic database was performed for articles addressing serum-free culture systems of human embryonic stem cels published from 2008 to 2015. Repetitive and old articles were excluded. Finaly, 58 articles were summarized. RESULTS AND CONCLUSION:Several groups have attempted to exclude individual animal components by using feeder-free matrices, feeder cels of human origin, or defined xeno-free media, aiming to select a suitable matrix and medium that can minimize or not use heterologous components, in order to obtain cel lines at clinical level. However, the current cel products are far from clinical application. There are stil many problems to be solved, such as standardization, normalization and individualization of cel products. With the normative development of stem cel research and industry, human embryonic stem cel products are expected to be widely used in clinic.

Paracrine effects of bone marrow mesenchymal stem cells on biological function of osteoblasts / 中国组织工程研究

OBJECTIVE:To investigate the paracrine effects of bone marrow mesenchymal stem cells on osteoblast biological function. METHODS:Bone marrow mesenchymal stem cells were isolated using standard Ficol-Paquedensity gradient centrifugation. Mesenchymal stem cellconditioned medium was prepared to cultivate osteoblasts, MG63. Proliferation of MG63 cells was analyzed by cellcounting kit-8. Migration of MG63 cells was analyzed by cellscratch method. Alkaline phosphatase activity of MG63 cells was analyzed by microplate test kit. Real-time PCR was performed to evaluate osteoblast differentiation markers, alkaline phosphatase, col agen type I and osteocalcin. Alizarin red staining was performed to evaluate osteoblast mineralization. RESULTS AND CONCLUSION:The cells were strongly positive for CD44, CD73 and CD90, but negative for CD34. MG63 cells cultured in the conditioned medium showed better proliferation and migration than those cultured in the Dulbecco’s modified Eagle’s medium. The activity and mRNA expression of alkaline phosphatase were much higher after induction of 4, 7 days (P<0.01). There was no significant difference in expression of col agen type I and osteocalcin after induction of 4 days, but they were significantly higher than those in the control group after induction of 7 days (P<0.05). Alizarin red staining showed that the number of calcium nodules was increased and the mineral apposition was enhanced after induction of 21 days with the conditioned medium. These findings suggest that the paracrine substance of bone marrow mesenchymal stem cells can significantly promote osteoblast proliferation, migration, differentiation and mineralization.

Effects of bone marrow mesenchymal stem cell conditioned medium on bioactivity of scar fibroblasts / 中国组织工程研究

BACKGROUND:Mesenchymal stem celltransplantation promoted skin repair in trauma via various regulatory mechanisms and inhibited scar formation. At present, many scholars believed that bioactive factors secreted by mesenchymal stem cells played an important role. OBJECTIVE:To investigate the effects of bone marrow mesenchymal stem cellconditioned medium on the proliferation and col agen synthesis of hypertrophic scar fibroblasts. METHODS:Human bone marrow mesenchymal stem cells and hypertrophic scar fibroblasts were isolated and cultured, and bone marrow mesenchymal stem cellconditioned medium was prepared. Hypertrophic scar fibroblasts were cultured in vitro with 12, 24, and 48 hour-col ected conditioned medium for 24 hours, which was compared with blank control group. The proliferation of cells was determined by CCK-8. Type I and type III col agen expression in hypertrophic scar fibroblasts was detected using real-time PCR. RESULTS AND CONCLUSION:Compared with the blank control group, 24 and 48 hour-col ected conditioned medium significantly inhibited the proliferation of hypertrophic scar fibroblasts (P<0.01), and also suppressed col agen synthesis of hypertrophic scar fibroblasts (P<0.01). Results suggested that bone marrow mesenchymal stem cellconditioned medium inhibited the proliferation and col agen synthesis of hypertrophic scar fibroblasts by secreting anti-fibrotic bioactive factors, which may provide new theoretical supports for celltherapy to reduce cutaneous scarring.

Osteogenetic ability of mouse spermatogonial stem cells cultured in vitro / 中国组织工程研究

BACKGROUND:Spermatogonial stem cells are a kind of adult stem cells, which have self-renewal and differentiation potential, and can be differentiated into specific cells in vitro, suggesting that the spermatogonial stem cells may be possibly differentiated into osteoblasts. But the related research has not been reported. OBJECTIVE:To observe the biological characterization and osteogenic process of mouse spermatogonial stem cells cultured in vitro. METHODS:Spermatogonial stem cells were obtained from the testicle of mice aged 15-20 days, and were cultured on the feeder layer from bone marrow stroma cells in vitro. When cultured for 3 days, the cells were cultured in the conditioned medium (experimental group) and basic medium (control group). The cells proliferation capability and osteogenic property were examined by phase-contrast microscope, alkaline phosphatase activity and type I col agen immunofluorescence staining. RESULTS AND CONCLUSION:Spermatogonial stem cells proliferated faster in the experiment group than in the control group. cells grew rapidly in colony-like shape in the conditioned medium at 3-6 days, the three-dimensional feeling enhanced, cellmass and clusters continued to increase in size, the extracellular matrix was increased in number and the cytoplasmic bridge was not obvious. After culture for 15 days, cells in the two groups were positive for alkaline phosphatase staining that the cytoplasmic membrane was dyed black. Under the fluorescent microscope, green fluorescence was visible in the experimental group, suggesting the cells in the experimental group was positive for type I col agen, but negative in the control group, which is similar with the biological characteristics of osteoblasts. These findings indicate that spermatogonial stem cells possess the osteogenic capability under induction conditions, which are expected to provide seed cells for bone tissue engineering.

Avaliação do efeito de células-tronco mesenquimais humanas de várias origens na atividade de linhagem de células tumorais HepG-2 / Evaluation of the effect of human mesenchymal cells from several sources in the activity of tumor cells line

INTRODUÇÃO O câncer é uma das principais causas de morte no mundo, sendo responsável por cerca de 8 milhões de mortes por ano, segundo dados da OMS. As mortes por câncer são provocadas por tumores que se originam em órgãos como pulmão, fígado, estômago, intestino, mama e esôfago. As células-tronco mesenquimais (CTM) foram identificadas em vários órgãos e estudos da sua interação com células tumorais têm apresentado resultados indicando ação inibitória sobre alguns tipos de tumores. Para explorar essa questão foram analisados os efeitos sobre células tumorais de carcinoma hepatocelular humano (HepG-2) do meio condicionado (MC) obtido do cultivo de CTM isoladas de tecido adiposo (TA), líquido amniótico (LA) e geleia de Wharton (GW). MÉTODOS Os MCs foram coletados após 24 horas de incubação das CTM sub-confluentes com ?-MEM contendo 20% de soro fetal bovino (SFB). Os MCs foram centrifugados e passados através de filtros de 0,22 ?M e armazenadas a -20 °C. O MC da própria célula HepG-2 foi utilizado como controle. Os efeitos dos MCs sobre a proliferação de células HepG-2 foram testados por ensaio MTT, em várias concentrações após 24 h de incubação. O ciclo celular de células HepG-2 tratadas com MC a 25%, 50% ou 75% foi analisado por citometria de fluxo (coloração IP) utilizando o software Modfit LT. A expressão dos genes bcl-2, bcl-6, CCND1 foi analisada por RT-PCR. A proliferação celular foi avaliada pela expressão das proteínas survivina, Bcl-2, PCNA e Ki-67 e pela quantificação de mitocôndrias com corante MitoTracker, assim como pelo potencial de membrana mitocondrial por corante JC-1 Mitoscreen utilizando equipamento de high content analysis. RESULTADOS Os meios condicionados de células-tronco de tecido adiposo (MC-TA) não alteraram a proliferação de células tumorais HepG-2 e os meios condicionados de células-tronco de líquido amniótico (MC-LA) e de geleia de Wharton (MC-GW) provocam aumento da proliferação, confirmada pela contagem de células com núcleos...

INTRODUCTION Cancer is a leading cause of death worldwide, accounting for about 8 million deaths per year, according to WHO data. Cancer deaths are caused by tumors that originate in organs such as lung, liver, stomach, bowel, breast and esophagus. The mesenchymal stem cells (MSCs) have been identified in many organs and studies of their interaction with tumor cells have shown results indicating an inhibitory effect on some types of tumors. To explore this question the effects of the conditioned medium (CM) obtained from mesenchymal stem cell isolated from adipose tissue (AT), amniotic fluid (AF) and Wharton jelly (WJ) on tumor cells of human hepatocellular carcinoma (HepG-2) were analyzed. METHODS The MSC CM was collected after 24 hours incubation of sub confluent MSC with ?-MEM containing 20% fetal bovine serum (FBS). The MSC CM were centrifuged and passed through 0.22 ?M filter and stored at -20° C. The CM of HepG-2 cell itself was used as control. The effects of contrast media on proliferation of HepG-2 cells were tested by MTT assay at various concentrations after 24 h of incubation. The cell cycle HepG-2 cells treated with CM at 25%, 50% or 75% was analyzed by flow cytometry (PI staining) using Modfit software LT. The expression of the genes Bcl-2, Bcl-6, CCND1 was analyzed by RT-PCR. Cell proliferation was assessed by the expression of survivin, Bcl-2, Ki-67 and PCNA proteins, and the quantization of mitochondrial by MitoTracker dye, as well as the mitochondrial membrane potential by JC-1 Mitoscreen dye using high content analysis equipment. RESULTS The conditioned media of mesenchymal stem cells (MSC) from adipose tissue (AT-CM) did not alter the proliferation of tumor HepG-2 cells and conditioned media of MSC cells from amniotic fluid (AF-CM) and Wharton jelly (WJ-CM) caused increased proliferation, confirmed by counting cells with nuclei stained with Hoechst 33342. The cell cycle analysis showed that exposure of HepG-2 cells to AF-CM...

Overexpression of bone morphogenetic protein 4 in STO fibroblast feeder cells represses the proliferation of mouse embryonic stem cells in vitro

Embryonic stem cells (ESCs) can be propagated in vitro on feeder layers of mouse STO fibroblast cells. The STO cells secrete several cytokines that are essential for ESCs to maintain their undifferentiated state. In this study, we found significant growth inhibition of mouse ESCs (mESCs) cultured on STO cells infected with adenovirus containing a dominant-negative mutant form of IkappaB (rAd-dnIkappaB). This blockage of the NF-kappaB signal pathway in STO cells led to a significant decrease in [3H]thymidine incorporation and colony formation of mESCs. Expression profile of cytokines secreted from the STO cells revealed an increase in the bone morphogenetic protein4 (BMP4) transcript level in the STO cells infected with adenoviral vector encoding dominant negative IkappaB (rAd-dnIkappaB). These results suggested that the NF-kappaB signaling pathway represses expression of BMP4 in STO feeder cells. Conditioned medium from the rAd-dnIkappaB-infected STO cells also significantly reduced the colony size of mESCs. Addition of BMP4 prevented colony formation of mESCs cultured in the conditioned medium. Our finding suggested that an excess of BMP4 in the conditioned medium also inhibits proliferation of mESCs.

Factors Affecting on the Survival Rate for Shoot-tip Micro-grafting of Citrus medica L.var.sarcodactylis Swingle / 广州中医药大学学报

Objective To lay a foundation for the cultivation of disease-free plantlet of Citrus medica L.var.sarcodactylis Swingle(CMS) by discussion of the factors affecting survival rate for micro-grafted shoot-tip of CMS.Methods With citrus limon seedlings cultured in the dark as the rootstock,shoot-tip of CMS was grafted to the inverse T-shaped cut in the cotyledon of rootstocks.Then an investigation was carried out on the factors which affected the survival rate for micrografted shoot-tip of CMS.The factors included age of rootstocks,scion size,culture medium with different concentrations of phytohormones,and rootstocks with or without cotyledon.Results The survival rate for micro-grafted shoot-tip of CMS was closely related to the factors mentioned above.A higher survival rate was obtained when CMS shoot-tip with 4 pieces of leaf primordia was grafted to the inverse "T" shaped cut in the cotyledon of parental stock(citrus limon seedlings) which was cultured in the dark for 14 days,and when the citrus limon seedlings were cultivated in MT-based medium with ?-naphthylacetic acid 0.1 mg?L-1 added.Conclusion The optimized micro-grafting conditions for CMS shoot-tips improve the propagation of micro-grafted plantlet and the survival rate of micro-grafted shoot-tip of CMS.

Selection of Induction Factors for Adventitious Buds of Sarcandra glabra in Vitro / 广州中医药大学学报

Objective To establish the culture technology system for the in-vitro induction of adventitious buds of Sarcandra glabra(Thumb.) Nakai by investigating the induction factors of in-vitro adventitious buds.Methods Stem segments of Sarcandra glabra were cultured as explants.The effects of culture medium with different concentrations of 6-benzylaminopurine(6-BA) and indole butyric acid(IBA) added,lighting time,light intensity,and temperature on the induction of adventitious buds from the explants were observed.Results The medium containing both 6-BA and IBA was more effective to induce shoot formation of Sarcandra glabra than the medium only containing 6-BA or IBA;high concentration of plant hormones would block the growth of the plantlets.The scattering light or faint direct light was suitable for the shoot formation,while dark or strong light would induce the plantlets withered or even dead.Temperature in the range of 25 ℃～30 ℃ was beneficial for the growth of adventitious buds,and the bud induction rate arrived 100% at the temperature of 25 ℃.Conclusion At the temperature of 25 ℃,the faint sunlight or direct light less than 2000 lux,and the medium with 2.5 mg?L-1 6-BA and 0.1 mg?L-1 IBA added can stimulate significantly the adventitious bud formation of Sarcandra glabra,the induction rate up to 82.98%.

The Biological Activities of Conditioned Medium Derived from Human Dermal Papilla Cells Cultured In Vitro / 中华皮肤科杂志

Objective To investigate the biological activities of a conditioned medium for human dermal papilla. Methods Culture medium of the lower passage human dermal papilla cells was collected as the conditioned medium. The growth pattern and the growth curve of the higher passage human dermal papilla cells cultured with conditioned medium were observed in vitro. And the morphology of the co-culture of the higher passage human dermal papilla cells and the lower passage human dermal papilla cells was observed. Results The higher passage human dermal papilla cells, which was cultured with conditioned medium from the lower passage human dermal papilla cells, showed aggregative growth pattern. And the growth curve of the higher passage human dermal papilla cells was much better than that in the control groups (P