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Objective:To examine the possibility of the differentiation into islet-like cell clusters from the co-culture system of bone marrow mesenchymal stem cells (BMSCs) and islet cells.Methods:Rat BMSCs from the femur and tibia of Wistar rats were isolated and purified taken under aseptic conditions; the surface markers CD 44 and CD 90 expressions of BMSCs were detected by flow cytometry; and alizarin red staining and oil red O staining were used to identify the cells induced in the osteogenic direction and adipogenic direction, respectively. Rat islet cells from the pancreas of Wistar rats were isolated and purified; and dithiazone staining was performed for validation. The basal insulin level of the culture was detected by ELISA method. 5.6mmol/L (low glucose) and 25.0 mmol/L (high glucosa) glucose were added to the culture, respectively, and insulin release was detected by ELISA. 5-generation BMSCs and islet cells were collected and divided randomly into stem cell culture alone group (stem cell group), stem cell-islet co-culture group (co-culture group), and islet culture alone group (islet group). The morphological changes of BMSCs during co-culture were observed using an inverted phase contrast microscope; basal insulin secretion and insulin secretion stimulated by low and high glucose were tested by ELISA. Insulin protein expression in induced islet-like cell masses in co-culture group were detected by immunocytochemical staining. The ultrastructure of islet-like cells was observed by using transmission electron microscopy. Results:The positive rates of CD 44 and CD 90 were 99.48% and 99.50%, respectively; BMSCs were induced the formation of multiple calcium nodules outside the differentiation cells in the osteogenic direction, and many lipid droplets in the cytoplasm of differentiated cells in the adipogenic direction. Dithiazone staining showed that β cells in pancreatic islet were brown red and about 450 islets could be obtained per pancreas with a mean purity up to 80%. The insulin release in the low sugar group and the high sugar group were (7.105±1.551) mIU/ml and (20.231±1.592) mIU/ml, respectively, with a statistically significant difference ( P<0.05). It can be seen that local stem cells began to gather and grow upward into small clumps in the budding manner until finally forming a spherical islet-like cell cluster structure after 7 days of culture in the co-culture group. The basal insulin secretion in the stem cell group was <0.5 mIU/L. In the islet group, insulin secretion peaked on the 5th day and then gradually decreased to about 20% of the highest value on the 13th day. The insulin level of the co-culture group peaked on the 5th day, and the 13th day remained at about 40% of the peak level. There were statistically significant differences on basal insulin secretion on the 8th, 10th and 13th day between islet group and co-culture group (all P value >0.05). There was no statistically significant difference between the insulin release by islet in islet group under the stimulation of low and high sugar and that by islet-like cell cluster in co-culture group. There were a large number of brownish-yellow granules in the islet-like cell clusters after the co-culture for 14 days; and there were more secretory granules and coarse endoplasmic reticulum in the ultrastructure, showing more active protein secretion functions. Conclusions:The co-culture system of BMSCs and islet cells could induce BMSCs into differentiating into islet-like cell clusters, which can express insulin protein and had relatively mature function of insulin secretion.
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Objective:To investigate the effect of KRT5 knockdown in keratinocytes on melanin content in co-cultured melanocytes, and to explain mechanisms underlying formation of hyperpigmented lesions in reticulate pigmented anomaly of the flexures (Dowling-Degos disease, DDD) .Methods:HaCaT cells with heterozygous mutations in the KRT5 gene were obtained by using clustered regularly interspaced short palindromic repeats (CRISPR) -CRISPR-associated protein 9 (Cas9) technology (experimental group) , and HaCaT cells transfected with non-targeting single guide RNA:Cas9 protein complex served as control group, both of which were in vitro co-cultured with primary human melanocyte cells (HEMn) separately. Immunofluorescence study was conducted to determine the expression of cytokeratin and melanosomes in co-cultured cells; melanin content was detected in melanocytes in different co-culture groups, which were obtained by differential trypsinization. Immunohistochemical study was performed to determine the expression of melanocyte-specific premelanosome protein 17 (Pmel17) in skin lesions in a patient with DDD carrying a KRT5 mutation and normal skin tissues in a healthy control. Results:Sanger sequencing showed a heterozygous mutation (c.1delA) at the initiation codon of exon 1 of the KRT5 gene in HaCaT cells in the experimental group, but no mutation in the KRT5 gene in the control group. Western blot analysis showed that the KRT5 protein expression was significantly lower in the experimental group (0.60 ± 0.05) than in the control group (1.00 ± 0.00, t = 32.38, P = 0.001) . Compared with the co-culture system in the control group, the number of Pmel17-labeled melanosomes markedly increased with the melanin content elevated by 52.5% ( t = -3.48, P = 0.025) in the HEMn cells co-cultured with HaCaT cells in the experimental group. Immunohistochemical study showed that the Pmel17 expression increased in the skin lesions in the DDD patient with KRT5 mutation compared with the normal skin tissues in the healthy control. Conclusion:The effect of HaCaT cells with CRISPR-Cas9-induced KRT5 mutation on the co-cultured HEMn melanocytes was verified by the successfully established in vitro co-culture system, which provides a primary cell model for further studies on interaction mechanisms between keratinocytes and melanocytes, and on pathogenesis of skin pigmentation abnormalities.
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Objective:To develop an in vitro neuroinflammation model by establishing a microglia-neuron co-culture system. Methods:Mouse microglia (BV-2), motor neurons (NSC34) and hippocampal neurons (HT-22) were selected.This experiment was performed in two parts.Experiment Ⅰ BV-2 microglia were stimulated with different concentrations of lipopolysaccharide (LPS, 10, 100, 500 and 1 000 ng/ml). Microglia culture supernatant(Conditioned Medium) was extracted and two types of neurons were cultured separately.The concentration of LPS that resulted in a significant 50% decrease in neuronal viability was selected using the CCK-8 method for establishment of the Transwell co-culture system.Experiment Ⅱ Microglia were cultured in the upper chamber of Transwell, and neurons were seeded in the lower chamber.Microglia were divided into 2 groups ( n=12 each) using the random number table method: control group and LPS group.In control group and LPS group, microglia were cultured for 6 h with cell culture medium and LPS, respectively, then the medium was replaced with fresh medium, microglia were continuously incubated for 12 h, and then the cells in the upper and lower chambers were combined.The cells were incubated using the BV-2-NSC34 Transwell co-culture system for 12 h and using the BV-2-HT-22 Transwell co-culture system for 24 h. The concentrations of interleukin-1beta (IL-1β) and IL-18 in neuronal culture supernatant were measured by enzyme-linked immunosorbent assay, the apoptotic rate of neurons was determined by flow cytometry, the expression of Bcl-2 and Bax mRNA in neurons was detected by quantitative real-time polymerase chain reaction, and the expression of cleaved caspase-3, Bcl-2 and Bax in neurons was detected by Western blot. Results:Experiment Ⅰ LPS concentration for stimulation was 10 ng/ml in BV-2-NSC34 Transwell co-culture system and 1, 000 ng/ml in BV-2-HT-22 Transwell co-culture system.Experiment Ⅱ Compared with control group, the concentrations of IL-1β and IL-18 and apoptotic rate of neurons were significantly increased, Bax protein and mRNA expression was up-regulated, Bcl-2 protein and mRNA expression was down-regulated, and cleaved caspase-3 expression was up-regulated in LPS group ( P<0.05 or 0.01). Conclusions:The microglia-neuron co-culture system is successfully established by the conditioned medium technique and Transwell co-culture system, which provides an experimental protocol for establishment of neuroinflammation models associated with postoperative cognitive dysfunction.
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Objective To study the changes of acetylation level of smooth muscle marker gene after the differentiation of bone marrow mesenchymal stem cells (BMSCs) in smooth muscle microenvironment, and explore the role and mechanism of histone acetylation modification in the differentiation of stem cells. Methods BMSCs and bladder smooth muscle cells (BSMCs) were cultured in vitro. The third generation BMSCs of the same batch were selected, and BMSCs co-cultured with BSMCs for 3 days were set as the experimental group and the BMSCs without co-culture as the control group. RT-PCR was performed to compare the expression abundance between the two groups of α-smooth muscle actin (α-SMA), calponin and smooth muscle myosin heavy chain (SM-MHC) in BMSCs. Ultrasound of power 80%, 20 times, 0.5 s and 8 cycles were used to break the BMSCs DNA of both experimental and control group. Antibody H3K9 was used to bind to the specific acetylation sites. The acetylation site genes of histone in BMSCs were precipitated by chromatin immunoprecipitation (ChIP). The genes obtained was amplified by adaptor PCR. The expression levels of the three kinds of target genes (α-SMA, calponin, SM-MHC) were detected by Real-time PCR. Results RT-PCR showed that the expression levels of mRNA of smooth muscle marker genes (α-SMA, calponin, SM-MHC) in BMSCs were significantly higher in experimental group than in control group (0.176±0.003 vs. 0.070±0.002; 0.079±0.002 vs. 0.051±0.003 and 0.091±0.004 vs. 0.034±0.001, respectively) with significant differences. Test results of spectrophotometer showed that the amount of DNA obtained by precipitation of H3K9 acetylated antibody was higher than that of IgG antibody, and was higher in experimental group than in control group (P<0.05). Real-time PCR used to analyze the acetylation level of H3K9 before and after the differentiation of BMSCs showed that the mRNA transcription levels of smooth muscle marker genes (α-SMA, calponin, SM-MHC) were significantly higher in experimental group than in the control group (9.26±5.03 vs. 1.01±0.05, 2.33±0.65 vs. 0.99±0.05, 2.63±0.37 vs. 1.00±0.03, respectively) after BMSCs differentiation with statistically significant differences. Conclusion The increase of acetylation level of H3K9 specific site of BSMCs in smooth muscle microenvironment promotes the differentiation of BMSCs into BSMCs.
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Objective: To investigate the effects of cytokine Fractalkine (FKN) combined with M2-type macrophages on the proliferation, invasion, and migration of human pancreatic cancer PANC-1 cells. Methods: The recombinant lentivirus HBLV-h-FKN-GFP-PURO carrying FKN gene was constructed and infected into PANC-1 cells. THP-1 cells, a kind of monocytes of human leukemia, were induced into M2-type macrophages by phorbol 12-myristate 13-acetate (PMA) and interleukin-4 (IL-4) in suquence. Then the non-contacting co-culture model of pancreatic cancer cells and M2-type macrophages with different expression level of FKN was established by Transwell chamber system. The proliferation, invasion and migration of human pancreatic cancer PANC-1 cells were detected by CCK-8 method, Transwell chamber test and wound-healing assay, respectievely. Results: As compared with the uninfected control and empty lentivirus infected groups, the expression level of FKN protein was significantly increased in human pancreatic cancer PANC-1 cells infected with recombinant lentivirus HBLV-h-FKN-GFP-PURO (both P < 0.001). THP-1 cells were successively induced to become M2-type macrophages with high expression of arginase-1 (Arg-1) and low expression of inducible nitric oxide synthase (iNOS) protein (PArg-1 < 0.001, PiNOS < 0.01). The recruiting ability of PANC-1 cells to M2 type macrophages was enhanced after FKN over-expression (P < 0.001). The proliferation, invasion and migration abilities of PANC-1 cells were increased after FKN over-expression (all P < 0.001), and were more significantly increased after co-culture with M2-type macrophages (all P < 0.01). There were interactions between M2-type macrophages and FKN for proliferation, invasion and migration abilities of PANC-1 cells (P < 0.01, P < 0.01, P < 0.05). Conclusion: The chemokine FKN promotes the recruitment of PANC-1 cells to M2-type macrophages. Both FKN and M2-type macrophages can enhance the proliferation, invasion and migration abilities of PANC-1 cells, and there is synergistical interaction between them.
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Objective To assess the osteogenic ability after co-culture BMSC and ADSC in vivo and in vitro.Methods ADSC and BMSC were obtained by adherent screening method and enzymatic digestion method.Flow cytometry was used to confirm the phenotypes of ADSC and BMSC.Oil red O was used to induce MSC to fat.Alkaline phosphatase (ALP) and alizarin red staining were used in osteogenic group.This sample was divided into four groups,no-induced stem cells group;BMSC osteogenic induction group;ADSC osteogenic induction group;co-culture of BMSC and ADSC osteogenic induction group.ALP activities and Calcium absorbance were determined during different periods of osteogenic introduction.OCN and Runx2 expression level were tested via RT-PCR and western blot methods after osteogenic induction for 2 weeks.Furthermore,cells in each group were seeded on HA/CS/PLLA composite scaffolds,and the scaffolds with cells were planted into bone defects in rat models.The rats were sacrificed by overdose anesthesia at 8 weeks after surgery and the scaffolds were removed for further analysis.Results Oil red O staining demonstrated red after adipogenic induction.Alkaline phosphatase and Alizarin red staining showed flaky red under condition of osteogenic induction.There had no statistical change among each group after osteogenic induction for 3 days,and ALP activity significantly increased after osteogenic induction for 5 days.Meanwhile,the ALP activity in co-culture of BMSC and ADSC group was markedly higher than the other three groups.However,there had no significant change in A value of calcium absorbance among each group after osteogenic induction for 7 days,while it increased at 14th day and ALP activity in co-culture of BMSC and ADSC group was significantly higher than the other three groups.After osteogenic induction for 2 weeks,the mRNA expression of OCN and Runx2 in co-culture of BMSC and ADSC group was 78.24±8.11 and 1 180.13±121.16 respectively,and the protein expression of OCN and Runx2 was 6.54±0.59 and 4.43±0.51.These mRNA and protein expression level in co-culture of BMSC and ADSC group enhanced significant compared with the other 3 groups.Histological assay demonstrated that the new bone tissues formed in co-culture of BMSC and ADSC group were 497.75±7.44 μm2,which was larger than that in the other 3 groups at 8 weeks after implantation.Conclusion Co-culture BMSC and ADSC may up-regulated the osteogenic ability in vivo and in vitro.
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OBJECTIVE: This study was conducted to compare the effects of static culture, dynamic culture, and the combination of dynamic culture with specialized surfaces involving co-culture on human embryonic development. Embryos cultured using conventional static culture (SC) techniques served as a control group. We compared dynamic culture using micro-vibration culture (MVC) and micro-vibration with co-culture (MCoC), in which autologous cumulus cells were used as a specialized surface. METHODS: We conducted a chart review of patients who were treated between January 2011 and November 2014 in order to compare embryonic development rates and pregnancy rates among the groups. Zygotes were cultured in micro-droplets, and embryos were subsequently selected for transfer. Some surplus embryos were cryopreserved, and the others were cultured for blastocyst development. A micro-vibrator was set at the frequency of 42 Hz for duration of 5 seconds per 60 minutes to facilitate embryo development. RESULTS: No significant differences among the groups were present in patient's characteristics. However, the clinical pregnancy rates were significantly higher in the MVC group and the MCoC group than in the SC group. No significant differences were found in the blastocyst development rate between the SC group and the MVC group, but the blastocyst development rate in the MCoC group was significantly higher than in the SC and MVC groups. CONCLUSION: The clinical pregnancy rate was significantly increased by the application of micro-vibration to the embryonic cultures of poor responders. The blastocyst development rate was significantly increased by the application of MCoC to surplus embryos.
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Female , Humans , Pregnancy , Blastocyst , Coculture Techniques , Cumulus Cells , Embryo Culture Techniques , Embryonic Development , Embryonic Structures , Pregnancy Rate , ZygoteABSTRACT
Objective To investigate the influence of chondrocytes originating from different source on early chondrogenic differentiation of bone marrow mesenchymal stem cells (MSCs) in isolated co-culture system.Methods We applied hanging cell culture system to culture chondrocytes of different origin (osteoarthritis chondrocyte cells,nomal chondrocyte cells,infant chondrocyte cells) and controls.These chondrocytes and MSCs of the same origin were cultured in the common medium in a separated condition,and observed by microscope at 3,6,9,12 day after co culture.Expression levels of aggrecan,collagen type Ⅱ (Col 2),cartilage-specific transcription factor (Sox-9) in MSCs of different origin were determined by Real-time PCR.Results MSCs showed obviously morphological differentiation induced by chondrocytes of different origin at 12 day after coculture as compared with controls.Real-time PCR analysis showed that SOX9 mRNA level was stimulated by 1.7-fold,1.6-fold and 1.2-fold (all P＜0.05) and aggrecan mRNA level was increased by 2.8-fold,2.2-fold and 1.3-fold (all P＜0.05) in infant chondrocytes group,nomal chondrocytes group,osteoarthritis chondrocytes group respectively as compared with controls while COL2 mRNA level had no significant differences among the four groups.Corresponding protein signal level had obvious differences among the four groups,especially in infant chondrocytes as compared with osteoarthritis chondrocytes and nomal chondrocytes.Conclusions Isolated co-culture system may indirectly promote MSCs differentiation to chondrocytes by local micro-environment regulation.Chondrocytes of different origin have different effects on MSCs differentiation,but they could promote MSCs differentiation to chondrocytes.
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BACKGROUND:Mesenchymal stem cels can secrete a variety of cytokines and growth factors that promote the survival of surrounding cels and play a paracrine role. OBJECTIVE:To investigate the effect of human umbilical cord mesenchymal stem cels on the proliferation and apoptosis of ectopic endometrial cels. METHODS:After isolation and culture, human umbilical cord mesenchymal stem cels and ectopic endometrial cels were co-cultured as observation group, and ectopic endometrial cels cultured alone served as control group. At 24, 48, 72 hours of culture, the proliferation and apoptosis of ectopic endometrial cels were detected by MTT and flow cytometry, respectively; RT-PCR was used to measure the expression ofPTEN gene in ectopic endometrial cels. RESULTS AND CONCLUSION:At 24, 48 and 72 hours, the proliferation of ectopic endometrial cels in the observation was inhibited significantly as compared with the control group, and the hypodiploid peak ratio also increased significantly (P < 0.05). Over time, the cel inhibition rate was gradualy declined, and there were significant differences at different time points (alP < 0.05). Compared with the control group, the expression of PTEN gene was up-regulated significantly in the observation group (P < 0.05). In the observation group, the expression ofPTEN gene at 48 and 72 hours was significantly higher than that at 24 hours (P < 0.05). These findings indicate that in the human umbilical cord mesenchymal stem cels can inhibit the proliferation of ectopic endometrial celsin vitro and promote their apoptosis by up-regulation ofPTEN mRNA expression.
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BACKGROUND:Human umbilical cord blood-derived mesenchymal stem cels can be induced through the co-culture to differentiate into other cels, but how to get more seed cels for tissue engineering is one of the most difficult problems. OBJECTIVE: To investigate the role of the different concentrations of thyroxine in chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cels by co-culture with rabbit chondrocytes. METHODS:Human umbilical cord blood-derived mesenchymal stem cels were co-cultured with rabbit chondrocytes at 2:1, and stimulated by medium containing different concentrations of thyroxine (0, 0.01, 0.1 and 1, 10 μmol/L). Co-cultured cels with no thyroxine served as control group. After 14 days of co-culture, the cel RNA and protein were extracted, mRNA expressions of aggrecan and colagen type II were detected by real-time PCR, and protein expression of aggrecan and colagen type II were detected by western blot assay. RESULTS AND CONCLUSION:After intervention with 0.01, 0.1, 1, 10 μmol/L thyroxine, the mRNA and protein expressions of aggrecan and colagen type II were enhanced with the increase of thyroxine concentration, which were significantly different from those in the control group (P < 0.05). Experimental findings indicate that high levels of thyroxine can enhance the chondrogenic ability of human umbilical cord blood-derived mesenchymal stem cels co-cultured with rabbit chondrocytes.
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BACKGROUND:Imatinib resistance is a key issue in treatment of chronic myeloid leukemia. It is confirmed that the leukemia cels can obtain drug resistance phenotype mediated by adhesion to the bone marrow stromal cels (BMSCs). But, the role of BMSCs in imatinib resistance is unclear because chronic myeloid leukemia is deficient in adhesion function. OBJECTIVE: To in vitro simulate the bone marrow microenvironment of patients with chronic myeloid leukemia and to explore its influences on imatinib sensitivity as wel as possible mechanisms. METHODS:BMSCs isolated from patients with chronic myeloid leukemia but not undergoing treatment were co-cultured with K562 cels to construct the BMSCs-K562 cel co-culture model in chronic myeloid leukemia patients, then exposed to 0.2-3.2 μmol/L imatinib for 48 hours, and the proliferation inhibition rate of K562 cels was studied by MTT assay. The apoptosis rate of K562 cels and the expression of the CXCR4 in K562 cels exposed to 0.5 μmol/L imatinib for 72 hours were detected by flow cytometry. The K562 cels were exposed to 0.5 μmol/L imatinib for 4 hours and labeled by Calcein-AM fluorescent labeling sytem, and then, the adhesion rate of the K562 cels was calculated based on fluorescence intensity. RESULTS AND CONCLUSION: The suppressing effect of imatinib (0.2-3.2μmol/L) on the proliferation of K562 cels was weakened significantly by co-culture with the bone marrow stromal cels from patients with chronic myeloid leukemia. The apoptosis rate of K562 cels exposed to 0.5 μmol/L imatinib for 72 hours in co-culture group was significantly lower than that in the suspension culture group (P=0.020). The positive rates of CXCR4 in the co-culture group and suspension culture groups were both increased after exposure to 0.5 μmol/L imatinib for 72 hours (P=0.001). The adhesion rate of the K562 cels to the BMSCs was elevated from (32.18±6.17)% to (68.97±11.08)% when the K562 cels were exposed to 0.5 μmol/L imatinib for 4 hours, and the difference had statistical significance (P=0.022). These findings indicate that the co-culture with the BMSCs from patients with chronic myeloid leukemia can mediate K562 cels resistance to imatinib, which may be related to that the co-culture with BMSCs and exposure to imatinib can induce the K562 cels to express CXCR4. But the mechanism needs in-depth studies.
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Background Researches showed that stem cells can rescue damaged cells through mitochondrial transfer.This mode has been used to regenerative cell-based therapy.Retinal pigment degeneration is an eye disease of retinal pigment epithelial (RPE) cell apoptosis as pathogenetic mechanism.Whether stem cells can repair the target cells by above mechanism has not been clarified.Objective This study was to investigate the influence of mitochondrial transfer on the function of RPE cells.Methods The RPE cells of Long-Evans rats were isolated and cultured and the third generation of cells were used in sequential experiment.The cells were identified by detecting the expressions of RPE65 and Bestrophin proteins with immunofluorescence stain.Mouse neural stem cells (NSCs) (C17.2 strain) with green fluorescence protein (GFP) and without GFP were cultured.Mitotracker-green and Mitotracker-red staining were separately used to labeled the mitochondria of RPE cells and NSCs.RPE cells were cocultured with NSCs,and wheat germ agglutinin (WGA) was used to mark the tunneling nanotubes (TNT) between the two kinds of cells,and then the mitochondrial migration in TNT was exhibited by the laser scanning confocal microscope.The proportion of RPE cells in different cycles was assayed after marked with propidium iodide (PI) by flow cytometry.The contents of ATP,ADP and AMP in RPE cells were detected by high performance liquid chromatography (HPLC).Results The third-generation of RPE cells grew well with the RPE65-and Bestrophinpositive rate ＞85％.The Mitotracker-red-labeled rates of NSCs and RPE cells were no less than 95％.TNT structure was seen to appear the blue fluorescence between RPE cells and NSCs 24 hours after co-cultured and the red dye mitochondria from NSCs migrated toward red dye mitochondria from RPEs with the lapse of time.The RPE cell proportion reduced in G1 phase and increased by 5％ and 2％ in the S phase and G2/M phase respectively after mitochondrial transfer than before (P=0.016,0.114,0.189).The contents of ATP,ADP and AMP in the RPE cells were (8.77 ±3.68),(2.76±0.92) and (1.07 ±0.65) μg/mg after cell co-culture,and those before co-culture were (11.29±2.29),(3.12±0.95) and (1.59± 1.22) μg/mg,without significant differences between them (P =0.370,0.668,0.553).Conclusions NSCs can transfer normal mitochondria to co-cultured RPEs via TNT structure.Mitochondrial exchange might be one of therapeutic mechanisms of NSCs recuing damaged RPE cells.
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Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells are a promising cell source for drug screening and toxicity tests. Thus, various hepatic differentiating protocols have been developed, leading to a hepatic differentiation efficiency of approximately 90%. However, HLC drug metabolizing ability remains very low compared to human primary hepatocytes. In order to overcome this problem, several alternative methods, such as, co-culture, three-dimensional (3D) culture, bioreactor, nanochip-based, etc., have been developed, but optimization to produce fully functional HLCs is ongoing. Recently, our group reported that repeated exposure of HLCs to xenobiotics can improve the expression of hepatic metabolizing enzymes such as cytochrome P450s (CYPs) and glutathione S-transferases (GSTs). These data suggest that we should develop strategies for differentiating cells into mature HLCs by more closely mimicking in vivo fetal and postnatal liver development. Here, we review the current development of alternative methods for enhancing the drug metabolizing functions of HLCs derived from human embryonic stem cells, human-induced pluripotent stem cells, and mesenchymal stem cells as used for drug screening and toxicity tests.
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Humans , Bioreactors , Coculture Techniques , Cytochrome P-450 Enzyme System , Drug Evaluation, Preclinical , Embryonic Stem Cells , Glutathione , Hepatocytes , Liver , Mesenchymal Stem Cells , Pluripotent Stem Cells , Stem Cells , Toxicity Tests , XenobioticsABSTRACT
Objective To investigate influences of co-culture with the bone marrow stromal cells (BMSCs) on imatinib sensitivity,and the role of stromal cell-derived factor-1 (SDF-1)/chemokine receptor 4 (CXCR4) axis in imatinib resistance of K562 cells in the co-culture model.Methods The model was constructed by co-culturing K562 cells with BMSCs isolated and cultured from the patients with chronic myeloid leukemia.The apoptosis rate and the CXCR4 expressing rate of the K562 cells exposed to 0.5 μmol/L imatinib for 72 hours were detected by fluorescent-activated cell scanning (FACS) machine.The K562 cells were exposed to 0.5 μmol/L imatinib for 4 hours,and labelled by calckin-AM fluorescent labeling sytem.The adhesion rate of the K562 cells co-cultured with BMSCs for 24 hours was calculated with fluorescence intensity.The IC50 value of K562 cells exposed to imatinib was detected by methyl thiazolyl tetrazolium (MTT) assay while the SDF-1/CXCR4 axis was blocked by 10 μg/ml monoclonal antibody of CXCR4.Results The apoptosis rate of K562 cells exposed to 0.5 μmol/L imatinib for 72 hours in co-culture group and suspension culture group was (15.48 ±4.17) ％ and (32.01 ±6.83) ％,respectively.The apoptosis rates of K562 cells in the two groups were significantly different (t =5.587,P =0.001).For the co-culture group,the CXCR4 expressing rates of K562 cells unexposed and exposed to 0.5 μmol/L imatinib for 72 hours were (20.31 ± 3.76) ％ (suspension cultured:11.28％ ± 3.44％) and (53.64 ± 5.35) ％ (suspension cultured:25.34％ ± 3.21％),respectively.Those results showed that co-culture with BMSCs and exposure to imatinib induced the K562 cells to express CXCR4.The adhesion rates of the K562 cells to the BMSCs were elevated from (42.18 ± 6.17) ％ to (68.97 ± 11.08) ％ when the K562 cells were exposed to 0.5 μmol/L imatinib for 4 hours.The IC50 values of block group (the SDF-1/CXCR4 axis was blocked by 10 μg/ml monoclonal antibody of CXCR4) and unblock group were (0.68 ± 0.04) μmol/L and (1.27 ± 0.05) μmol/L,respectively.The IC50 values of two groups were significantly different(t =4.869,P =0.001).Conclusions The K562 cells co-cultured with the BMSCs from the patients with chronic myeloid leukemia can obtain resistance to imatinib,which was related with that co-culture with the BMSCs and exposure to imatinib can induce the K562 cells to express CXCR4.To a certain extent,the imatinib resistance mediated by co-culture with BMSCs can be reversed by monoclonal antibody of CXCR4.
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BACKGROUND:Recent studies have found that adipose mesenchymal stem cells can inhibit immune responses of T cells, B cells and dendritic cells. But it is unclear whether adipose mesenchymal stem cells are able to regulate the immune responses of macrophages. OBJECTIVE:To observe the expression of interleukin-6 and tumor necrosis factorαin J774.1 cells co-cultured with adipose mesenchymal stem cells co-cultured. METHODS:Adipose mesenchymal stem cells at a density of 2×107 per wel were inoculated into the upper chamber of the transwel and 24-wel plates. Then, J774.1 cells were suspended with cellculture medium containing 1 mg/L lipopolysaccharide and planted into the lower chamber of the transwel and 24-wel plates containing adipose mesenchymal stem cells as co-culture group. Meanwhile, inactive J774.1 cells to lipopolysaccharide-activat J774.1 cells served as negative and positive control groups, respectively. After 48 hours, the J774.1 cells were col ected to extract the RNA samples. Then, mRNA expressions of interleukin-6 and tumor necrosis factorαwere measured by real-time quantitative PCR. RESULTS AND CONCLUSION:Compared with the positive control group, direct-contact co-culture of adipose mesenchymal stem cells and J774.1 cells significantly reduced expressions of interleukin-6 and tumor necrosis factorαin J774.1 cells, but non-contact co-culture only reduced interleukin-6 expression. These findings indicate that adipose mesenchymal stem cells can inhibit the immune response of J774.1 cells activated by lipopolysaccharide.
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Objective To detect supernatant of SD rat bone marrow mesenchymal stem cells(BMSC)and liver cells,and explore the role of cytokines,and provide a theoretical basis for the biological artificial liver and clinical treatment of liver failure.Methods BMSCs and primary hepatocytes of SD rat were isolated.BMSCs with its third generation and liver cells by the ratio of 1∶10 were co-cultured.Hepatocytes and BMSC were treated as control group.Observation the cells′survival and morphology and analysis of cytokine after 48 h were made by RayBiotech.Results BMSC-hepatocyte in the co-culture group grow and proliferated rapidly,and hepatocyte could survive up to 14 days;while hepatocyte cultured alone,it grow slowly and survived only 9 days.Expression of BM-SC-liver cells supernatant changed obviously:interleukin 1,interleukin 6,interleukin 10 were higher than those in the control group,above 2 times,which showed significant difference (P<0.01).Conclusion Co-culture of BMSC-liver cell could induce BM-SC secrete paracrine and autocrine:IL-1 and IL-6 and IL-10,which could promote the growth of hepatocyte and extend the liver cell survival time.In-vitro BMSC-liver cells could provide biological artificial liver cell sources,as well as provide theory basis for using cytokine treatment for liver failure.
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BACKGROUND:Coculture of bone marrow mesenchymal stem cel s and human umbilical vein endothelial cel s can improve both osteogenic and angiogenic outcomes and provide a promising strategy for bone tissue engineering and osteanagenesis. OBJECTIVE:To summarize recent researches and related progresses in coculture of human umbilical vein endothelial cel s and bone marrow mesenchymal stem cel s. METHODS:A computer-based online search of CNKI database from January 2000 to March 2012, PubMed database and Web of Knowledge database from January 1980 to March 2012, was performed with the keywords of“human umbilical vein endothelial cel s, bone mesenchymal stem cel s, coculture, tissue engineering”both in Chinese and English. A total of 135 articles were screened out, 103 of them were excluded due to unrelated study objective and repeated contents, and final y 32 articles were involved in further analysis. RESULTS AND CONCLUSION:At present, studies on coculture of bone marrow mesenchymal stem cel s and human umbilical vein endothelial cel s mainly focus on mimicking in vivo environments, the interactions between cel s, and the influence of different cel ratios and culture media. Most of these researches play important roles in bone tissue engineering and bone regeneration therapy, but the mechanism of action and concrete regulation in vivo between bone marrow mesenchymal stem cel s and human umbilical vein endothelial cel s stil need further research and analysis.
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BACKGROUND:Articular chondrocytes with the ability of autocrine and paracrine can provide the growth factors and microenvironment for synovial mesenchymal stem cels differentiating into the chondrocyte. The three-dimensional scaffold could provide space for cels adhesion, proliferation and differentiation. OBJECTIVE: To study the ability of chondrogenesis by co-culturing synovial mesenchymal stem cels and chondrocytes under the three-dimensional condition. METHODS:The synovial membrane and articular cartilage were harvested from rat knee joint. The synovial mesenchymal stem cels and chondrocytes were obtained through the method of enzyme digestion. The passage 3 synovial mesenchymal stem cels and passage 2 chondrocytes were co-cultured in the chitosan/I colagen composite scaffolds at the ratio of 1:2. Then, the cels/scaffold composite was harvested to be examined morphologicaly, histologicaly and immunohistochemicaly after being cultured 21 days. The confocal laser was also employed to detect the cels distribution in the scaffold. RESULTS AND CONCLUSION: After being cultured 72 hours, it could be observed from the cels/scaffold composite examined through the scanning electron microscope that the cels adhered on the surface of the scaffold and extracelular matrix surrounding the cels was seen on the scaffold. After being cultured 21 days, it could be found through the confocal laser scanning that the cels were wel-distributed on the scaffold, and cels decreased gradualy. Type II colagen was positive in the extracelular matrix immunohistochamicaly. It suggested from this study that the synovial mesenchymal stem cels could be co-cultured with chondrocytes in the chitosan/I colagen composite scaffolds and have the ability of chondrogenesis differentiation.
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Objective To establish the condition cultrue cell system and co-culture cell system with SKOV3/PM4,HUVEC and to study the changes of their biological characteristics. Methods The cells of SKOV3/PM4 and HUVEC were labeled with green and red fluorescent respectively. The cell supernatant of SKOV3/PM4 and HUVEC were collected respectively as the condition medium(e.g:the cell supernatant of HUVEC cells was used as SKOV3/PM4 condition medium)and to establish the condition cultrue cell system and the co-culture cell system of the two cell lines. In the condition cultrue cell system, The morphological changes of cells were observed by HE staining to calculate the mitotic index. The ultrastructural changes of the two cells were observed by transmission electron microscopy(TEM). The growth curve of the cells was determined by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry was used to analyzed the cell cycles.In the co-culture cell system, the interaction of the two cells were detected by laser scanning confocal microscope(LSCM). The expression of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9 (MMP-9) were detected by gelatin zymography. Results Compared with the single culture SKOV3/PM4, the cells which cultured in HUVEC condition medium showed the increase of pseudopodia and nuclear division,the mitotic index respectively were [(4.8 ± 0.8)%,(11.2 ± 0.3)%;P<0.05]. The growth rate was significantly increased. In cell cycles, it showed the declined cell ratio of G0/G1 phase, respectively[(69.4±3.6)%, (48.4±4.6)%;P<0.05] and the raised cell ratio of G2/M phase, respectively [(5.2±1.6)%, (24.9±2.2)%;P<0.05]. Compared with the single culture HUVEC,the cells which cultured in SKOV3/PM4 condition medium showed the significant morphological change and vacuolization in the cytoplasm, Nuclear division was increased and the mitotic index respectively were [(2.7±0.5)%, (5.7±0.6)%;P<0.05]. The growth rate was slightly declined. In cell cycles, it showed the raised cell ratio in G0/G1 phase, respectively [(51.4 ± 2.2)%,(79.0 ± 4.1)%;P<0.05] and the declined cell ratio in G2/M phase, respectively [(19.1±1.2)%, (3.3±0.5)%;P<0.05]. After co-culture for 48 hours, spontaneous fusion between SKOV3/PM4 and HUVEC cell was observed by the laser confocal microscope. Gelatin zymography assay showed that MMP-2 was not expressed in HUVEC cells, low-expressed in SKOV3/PM4 cells and high-expressed in the co-culture SKOV3/PM4+HUVEC cells. The expression of MMP-2 in co-culture SKOV3/PM4+HUVEC cells and SKOV3/PM4 cells respectively were 1 885 ± 84 and 1 209 ± 114 (P<0.05). But there were no MMP-9 expression in HUVEC cells, SKOV3/PM4 cells, and the co-culture SKOV3/PM4+HUVEC. Conclusion The characteristics of SKOV3/PM4 and HUVEC show significant changes after condition culture and co-culture, it may involve in the microenvironment of the cells and the intercellular crosstalk pathway.
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Objective To observe the cellular phenotype conversion of human mesenchymal stem cells (MSCs) cocultured indirectly with heat-shocked human sweat gland cells (SGCs) in vitro and explore the relative mechanism. Methods MSCs and SGCs were isolated and amplified in vitro. First,primary confluent cultures of SGCs were heat-shocked at 47℃. Then, the supernatants were collected immediately and 24 hours before applied to the third generation of MSCs. After seven days, the MSCs expressing CK7, CK18 and CEA were examined by two-step immunocytochemistry and flow cytometry and compared with the control group. Results MSCs treated with the supernatants of SGCs proliferated slowly, with no obvious morphological changes during seven days. Two-step immunocytochemistry demonstrated positive staining of CK7 and CEA in some cells. Additionally, the positive rate of CK7 and CEA was 5.76% and 2.01% by flow cytometry, much higher than that of the control sample, which was only 1.12% and 0.51% respectively (P ＜ 0.01 ). Conclusions There are some signal moleculars in the supernatants of heat-shocked SGCs, which benefits the transdifferentiation of MSCs.