Indirect Contact Co-culture System Research Articles

Activation of Toll-like receptor 2 enhances peripheral and tumor-infiltrating CD8+ T cell cytotoxicity in patients with gastric cancer

BackgroundToll-like receptors (TLRs) play central roles in the initiation of innate immune response, and also control adaptive immunity activation. Thus, the aim of the study was to investigate the regulation of TLR activation to CD8+ T cells has not been fully elucidated in gastric cancer (GC).Materials and methodsThirty-two GC patients and twenty-three healthy controls were enrolled. Expression profile of TLRs in peripheral and tumor-infiltrating CD8+ T cells was investigated. Purified CD8+ T cells were stimulated with Pam3Csk4, an agonist of TLR2, and cytotoxic and co-inhibitory molecules in CD8+ T cells was measured. Direct and indirect contact coculture system between CD8+ T cells and AGS cells was set up. Modulation of TLR2 activation to CD8+ T cells was assessed by measuring lactate dehydrogenase release and cytokine secretion.ResultsTLR2 mRNA and TLR2+ cell percentage was down-regulated in GC derived peripheral and tumor-infiltrating CD8+ T cells. CD8+ T cells from GC patients showed exhausted phenotype, which presented as decreased perforin/granzyme B, increased programmed death-1, and reduced cytotoxicity to AGS cells. TLR2 activation by Pam3Csk4 enhanced perforin and granzyme B expression in CD8+ T cells, however, did not affect either proinflammatory cytokine production or co-inhibitory molecules expression. Pam3Csk4 stimulation enhanced cytolytic activation of peripheral and tumor-infiltrating CD8+ T cells from GC, but not those from healthy individuals.ConclusionThe present data revealed an important immunomodulatory activity of TLR2 to CD8+ T cells in GC patients.

Effect of tenofovir disoproxil fumarate antiviral therapy on virus-specific CD8+T Cells function in patients with chronic hepatitis B

Objective: To observe the effect of tenofovir disoproxil fumarate (TDF) antiviral therapy on HBV-specific CD8(+)T cell function in peripheral blood of patients with HBeAg-positive chronic hepatitis B, and to assess its correlation with HBeAg sero-negativeness. Methods: Sixty-three cases with HLA-A02 restricted HBeAg-positive chronic hepatitis B who received TDF (300 mg/d) antiviral therapy were enrolled from October 2016 to July 2018. The peripheral blood CD8(+)T cells were separated at baseline and 48 weeks after treatment. The peripheral blood T cells count were detected by flow cytometry. The frequency of HBV-specific CD8(+)T cells secreting perforin, granzyme B, and interferon-γ (IFN-γ) were detected by enzyme-linked immunoblotting test. Direct and indirect contact co-culture system was established between HBV-specific CD8(+)T cells and HepG2.2.15 cells. HBV DNA was detected in the culture supernatant. Target cell mortality was calculated by lactate dehydrogenase level. Cytokines expression was detected by enzyme-linked immunosorbent assay. Virus-specific CD8(+)T cells cytokilling and non-cytokilling functions were evaluated. Measurement data of the two groups were compared by t-test or paired t-test. Results: Viral response, biochemical response, and HBeAg seroconversion rate at 48 weeks of TDF treatment were 100%, 90.48% (57/63), and 25.40% (16/63), respectively. There was no statistically significant difference in peripheral blood T cell count when compared with baseline and control group at 48 weeks of TDF treatment (P > 0.05). At 48 weeks of TDF treatment, the frequency of HBV-specific CD8(+)T cells secreting perforin, granzyme B, and IFN-γ in CHB patients was significantly higher than baseline (P < 0.001). Furthermore, the frequency of HBV-specific CD8(+)T cells secreting perforin, granzyme B, and IFN-γ was also significantly higher in CHB patients with HBeAg negative than that of non-negative (P < 0.05). HBV-specific CD8(+)T cells had induced significant down-regulation of HBV DNA in the supernatant of HepG2.2.15 cell culture (P < 0.001) and remarkable IFN-γ and interleukin-2 secretion (P < 0.05) at 48 weeks of TDF therapy in direct and indirect contact co-culture system. However, HepG2.2.15 cells death rate induced by virus-specific CD8(+)T cells was increased only in the direct contact co-culture system (21.7% ± 6.18% vs. 16.1% ± 4.15%, P < 0.001). Compared with HBeAg non-negative patients, HBeAg negative CHB patients with HBV-specific CD8(+)T cells had induced a strong decrease in HBV DNA (P < 0.001) and an increase in IFN-γ secretion level (P < 0.05). However, the target cell death proportion difference between HBeAg negative and non-negative patients was not statistically significant (P > 0.05). Conclusion: During TDF treatment, with the viral load reduction, virus-specific CD8(+)T cells cytokilling and non-cytokilling functions are significantly enhanced, and are closely related to HBeAg negative.

Notch signaling suppresses CD14+ monocytes cells activity in patients with chronic hepatitis C

Hepatitis C virus (HCV) infection always leads to chronic hepatitis via dysregulation of host immunity. Notch signaling also modulates the response of monocytes/macrophages. Thus, we aimed to investigate the regulatory role of Notch signaling to CD14+ monocytes. Forty patients with chronic hepatitis C and twenty normal controls (NC) were enrolled. CD14+ monocytes and CD4+ T cells were purified from peripheral bloods. Notch receptors' mRNA expression in CD14+ monocytes was semi-quantified by real-time PCR. Cytokine production by CD14+ monocytes in response to γ-secretase inhibitor (GSI) was investigated by ELISA. GSI-induced CD14+ monocytes activity to HCV clearance in Huh7.5 cells and to CD4+ T cell differentiation was also assessed in direct and indirect contact co-culture system. Notch1 mRNA relative level was approximately 10-fold elevated in CD14+ monocytes from chronic hepatitis C patients when compared with NC. GSI stimulation resulted in enhanced cytokines production by CD14+ monocytes from chronic hepatitis C patients. GSI-stimulated CD14+ monocytes from chronic hepatitis C patients induced suppression of HCV RNA replication in both direct and indirect contact co-culture system of CD14+ monocytes and HCVcc-infected Huh7.5 cells, and this process was accompanied by elevation of interferon-γ production but not increased target cell death. Moreover, GSI stimulation also enhanced CD14+ monocytes-induced Th1 and Th17 cells activation, and this process required direct cell-to-cell contact. Effective antiviral therapy down-regulated Notch1 mRNA expression and promoted cytokine production by CD14+ monocytes from chronic hepatitis C. Current data revealed an important immunoregulatory property of Notch signaling to CD14+ monocytes in chronic HCV infection.

Transcription Factor MafB Suppresses Type I Interferon Production by CD14+ Monocytes in Patients With Chronic Hepatitis C.

Transcription factor MafB regulates differentiation and activity of monocytes/macrophage and is associated with the development of atherosclerosis and cancers. However, the role of MafB in modulation of CD14+ monocytes in chronic viral hepatitis was not fully elucidated. Thus, the aim of current study was to investigate the immunoregulatory function of MafB to type I interferon (IFN) secretion by CD14+ monocytes and its contribution to pathogenesis of chronic hepatitis C virus (HCV) infection. A total of 29 chronic hepatitis C patients and 21 healthy individuals were enrolled. Serum IFN-α1 and IFN-β was measured by ELISA, while MafB mRNA and protein expression were assessed by real-time PCR and Western blot. MafB siRNA or MafB expression plasmid was transfected into purified CD14+ monocytes to suppress or increase MafB expression. The function of MafB siRNA transfected CD14+ monocytes to HCV in cell culture (HCVcc)-infected Huh7.5 cells or CD4+ T cells was also investigated in direct and indirect contact co-culture system. Serum IFN-α1 and IFN-β was robustly reduced in chronic hepatitis C patients. By contrast, MafB was notably elevated in chronic hepatitis C patients and negatively correlated with serum IFN-α1. Overexpression of MafB reduced the IFN-α1 production by CD14+ monocytes from healthy individuals. However, MafB inhibition elevated IFN-α1 secretion by CD14+ monocytes and interferon regulatory factor 3 phosphorylation in chronic hepatitis C. MafB inhibition also promoted CD14+ monocytes-induced viral clearance in HCVcc-infected Huh7.5 cells by up-regulation of IFN-α1 and IFN-β without increasingly destroying hepatocytes, however, did not affect CD14+ monocytes-induced CD4+ T cells differentiation in chronic hepatitis C patients. The current data revealed that overexpression of MafB in chronic hepatitis C patients might suppress type I IFN production by CD14+ monocytes, leading to the viral persistence. MafB might be a potential therapeutic target for treatment of chronic hepatitis C.

Influence of Toll-like receptor 7 on CD8(+) T lymphocytes in patients with breast cancer

Objective: To investigate the effect of Toll-like receptor 7 (TLR7) in CD8(+) T cells activity from patients with breast cancer. Methods: Thirty-three patients with breast cancer, twenty-three patients with benign breast tumor, and twenty healthy individuals were collected from The First Affiliated Hospital of Xinxiang Medical University between December 2017 and March 2018. Peripheral blood mononuclear cells (PBMCs) were isolated, and CD8(+) T cells were purified. TLR7 protein and mRNA relative expression in CD8(+) T cells was measured using flow cytometry and real-time PCR, respectively. mRNA relative expressions corresponding to perforin, granzyme B, and FasL in CD8(+) T cells were measured in response to TLR7 agonist stimulation. Direct/indirect contact co-culture system of CD8(+) T cells and breast cancer cell line MCF-7 was also used to assess cytolytic and noncytolytic function in response to TLR7 agonist CL097 stimulation. Results: The mean fluorescence intensity corresponding to TLR7 protein in CD8(+) T cells from breast cancer patients was 124.0±15.32, which was significantly down-regulated in comparison with benign breast tumor patients (255.5±54.91) and healthy individuals (261.9±68.65) (P<0.000 1). TLR7 mRNA relative level was also remarkably reduced in CD8(+) T cells from breast cancer patients (1.97±1.18) in comparison with benign breast tumor patients (4.84±1.01) and healthy individuals (4.75±1.40) (P<0.000 1). TLR7 agonist CL097 stimulation notably increased mRNA relative levels of perforin and granzyme B mRNA in CD8(+) T cells (P<0.01), but not elevated FasL mRNA (P>0.05).Furthermore, TLR7 agonist CL097 stimulation enhanced the cytolytic and noncytolytic function of CD8(+) T cells to MCF-7 cells, which presented as the elevation of target cell death and increase of interferon-γ production in direct and indirect contact co-culture system. Conclusion: TLR7 agonist promoted CD8(+) T cells function from breast cancer patients.

Clinical significance of ascitic interleukin-7 expression levels in cirrhotic patients complicated with spontaneous bacterial peritonitis

Objective: To observe ascitic interleukin-7 expression level in cirrhotic patients complicated with spontaneous bacterial peritonitis, and to detect the effect of recombinant human IL-7 on CD4(+) and CD8(+)T lymphocyte function. Methods: A total of 84 patients with liver cirrhosis who were hospitalized from August 2017 to April 2018 were selected. Among them, 51 cases were complicated with cirrhosis and untainted ascites, and 33 cases were cirrhosis complicated with spontaneous bacterial peritonitis. Peripheral blood and ascites were collected routinely. The levels of IL-7 in peripheral blood and ascites were measured by enzyme-linked immunosorbent assay. CD4(+)T cells and CD8(+)T cells were purified from ascites, and were stimulated with recombinant IL-7. Cellular proliferation, key transcription factors for mRNA, and cytokines production by CD4(+)T cells in response to IL-7 stimulation was measured. mRNA expression corresponding to perforin, granzyme B, and granulysin as well as cytokines production by CD8(+)T cells was also measured in response to IL-7 stimulation. Cytolytic and non-cytolytic activity of CD8(+)T cells in response to IL-7 stimulation was also investigated in both direct and indirect contact co-culture system. Measurement data of the normal distribution were compared between the two groups by Student's t-test and the data before and after stimulation were compared by paired t-test. Measurements that did not conform to normal distribution were compared between the two groups using Mann-Whitney U test, and data before and after stimulation were compared using Wilcoxon paired test. Results: There was no significant statistical difference in serum IL-7 levels between the two groups [(5 001 ± 1 458) pg/ml vs. (4 768 ± 1 128) pg/ml, P = 0.41]. The level of ascitic IL-7 in cirrhotic patients complicated with SBP was significantly lower than cirrhosis patients with untainted ascites [(966.4 + 155.8) pg/ml vs. (792.1 + 126.4) pg/ml, P < 0.01]. Recombinant IL-7 stimulation promoted the proliferation of CD4(+) and CD8(+)T cells from ascites in patients with liver cirrhosis complicated by SBP. T-bet mRNA relative expression and IFN-γ secretion in CD4(+)T cells was also elevated in response to IL-7 stimulation in vitro. Moreover, IL-7 stimulation also increased the mRNA expressions of perforin, granzyme B, and granulysin as well as productions of IFN-γ and TNF-α by CD8(+)T cells. Recombinant IL-7 stimulation elevated cytolytic and non-cytolytic activity of CD8(+)T cells from ascites in patients with liver cirrohosis complicated by SBP, which manifested as increased target cell death and IFN-γ production in both direct and indirect contact co-culture system. Conclusion: Ascitic IL-7 promotes T lymphocyte function in patients with liver cirrhosis complicated with SBP.

Interleukin-7 promotes lung-resident CD14+ monocytes activity in patients with lung squamous carcinoma

Interleukin (IL)-7 enhances cytokines secretion by CD14+ monocytes, and induces recruitment of monocytes to endothelium. As an important regulator to different types of immune cells, the role of IL-7 in modulation of CD14+ monocytes is still not fully elucidated. Thus, the aim of current study was to investigate the immunoregulatory activity of IL-7 to peripheral and lung-resident CD14+ monocytes in lung squamous carcinoma patients. Thirty-seven lung squamous carcinoma patients and eighteen healthy individuals were enrolled. CD14+ monocytes and CD4+ T cells were purified from both peripheral bloods and bronchoalveolar lavage fluids (BALF). IL-7 expression in plasma and BALF was measured by ELISA, and CD127 expression in peripheral and lung-resident CD14+ monocytes was investigated by real-time PCR and flow cytometry, respectively. Cellular proliferation, cytokine production, and molecules in IL-7 signaling pathway was assessed in CD14+ monocytes in response to IL-7 stimulation. IL-7-induced CD14+ monocytes activity to CD4+ T cells was also assessed in direct and indirect contact co-culture system. There were no remarkable differences of plasma IL-7 concentration or CD127 level between healthy individuals and lung squamous carcinoma patients. However, IL-7 expression was significantly reduced in BALF from tumor site in squamous carcinoma patients, especially in stage III and IV. IL-7 stimulation not only promoted proliferation, cytokines secretion, and STAT-5 phosphorylation in lung-resident CD14+ monocytes, but also enhanced CD14+ monocytes-induced Th1 and T follicular helper cells activation, which presented as elevated interferon-γ and IL-21 secretion by CD4+ T cells. This process required direct cell-to-cell contact, and was dependent on IL-6 secretion. The current data revealed a potential immunopromotive property of IL-7 to lung-resident CD14+ monocytes in lung squamous carcinoma.

Influences of daclatasvir plus asunaprevir therapy on CD4+and CD8+T cell functions in patients with chronic hepatitis C

Objective To investigate the changes in CD4+ and CD8+ T cell functions in patients with chronic hepatitis C in response to daclatasvir plus asunaprevir therapy. Methods A total of 21 HLA-A2-restricted patients with chronic hepatitis C virus (HCV) genotype 1b infection were enrolled in this study. All patients were treated with daclatasvir plus asunaprevir for 24 weeks. Peripheral blood samples were collected at baseline, 4 and 24 weeks post-therapy. CD4+ and CD8+ T cells were sorted and purified. Cytokines secreted by CD4+ T cells were measured by flow cytometry. CD8+ T cells were co-cultured with HCV cell culture (HCVcc)-infected Huh7.5 cells in both direct and indirect contact co-culture systems. The cytolytic and non-cytolytic functions of CD8+ T cells were analyzed by measuring the levels lactate dehydrogenase and cytokines in the supernatants of cell culture. Results The virological and biochemical response rates were 71.43% (15/21) and 77.78% (14/18) at 4 weeks post-therapy, respectively. Both rates reached 100% at 24 weeks post-therapy. Secretion of IFN-γ, TNF-α and IL-17 by CD4+ T cells was significantly enhanced at 4 and 24 weeks in response to daclatasvir plus asunaprevir therapy. In contrast, IL-10 secretion by CD4+ T cells did not change notably post-therapy. However, no significant differences in cytokine secretion were found between patients with and without virological response at 4 weeks post-therapy. Daclatasvir plus asunaprevir therapy increased the percentage of dead cells in direct contact co-culture system of CD8+ T cells and HCVcc-infected Huh7.5 cells at 4 and 24 weeks post-therapy. However, it did not affect the cytotoxity of CD8+ T cells in indirect contact co-culture system. Moreover, IFN-γ expression in both direct and indirect contact co-culture systems was significantly increased at 4 and 24 weeks post-therapy. There was also a notable increase in the expression of TNF-α in direct contact co-culture system, while no remarkable change in TNF-α expression was detected in indirect contact co-culture system. No significant differences in cytolytic and non-cytolytic activities of CD8+ T cells were found between patients with virological and without virological response at 4 weeks post-therapy. Conclusion Daclatasvir plus asunaprevir therapy achieves high clinical cure rate in patients with chronic hepatitis C. Inhibition of HCV replication contributes to the improvement of CD4+ and CD8+ T cell functions. Key words: Chronic hepatitis C; Direct antiviral agent; T cell; Immunoregulation

Notch signaling pathway dampens tumor-infiltrating CD8+ T cells activity in patients with colorectal carcinoma

CD8+ T cells play critical role in controlling the metastasis and prognosis of cancer. Controversy remains as to the contribution of Notch signaling pathway in modulation of CD8+ T cells activity and development of tumorigenesis. Thus, the aim of the current study was to investigate the immunoregulatory role of Notch signaling pathway to peripheral and tumor-infiltrating CD8+ T cells in patients with colorectal carcinoma. A total of 46 patients with colorectal carcinoma and 20 health individuals were enrolled, and CD8+ T cells were purified from both peripheral bloods and carcinoma specimens. Cytolytic and noncytolytic functions of CD8+ T cells in response to Notch signaling inhibition were evaluated by measurements of lactate dehydrogenase release and proinflammatory cytokines production in both direct and indirect contact co-culture system to target HT29 cells. Cellular proliferation and inhibitory receptors expression in CD8+ T cells were also assessed by CCK-8 method and flow cytometry. There was no remarkable difference in percentage of CD8+ T cells between healthy individuals and patients with colorectal carcinoma. Notch1/2 and Hes1/5 mRNAs were elevated expressed in tumor-infiltrating CD8+ T cells in patients with colorectal carcinoma, however, did not correlated with tumor differentiation or stages. CD8+ T cells from healthy individuals presented stronger cytotoxicity, which was not affected by Notch signaling inhibitor. Inhibition of Notch signaling pathway not only promoted cytotoxicity of tumor-infiltrating CD8+ T cells, but also enhanced proinflammatory cytokines (including IFN-γ, TNF-α, IL-1β, IL-6, and IL-8) production by CD8+ T cells from patients with colorectal carcinoma. This process was accompanied by decreased expression of PD-1 in CD8+ T cells without influencing cellular proliferation. Our results indicated a potential immunosuppressive property of Notch signaling pathway, which dampened both cytolytic and noncytolytic functions of CD8+ T cells probably via induction of PD-1 in patients with colorectal carcinoma.

Establishment of invitro model of erectile dysfunction for the study of high-glucose-induced angiopathy and neuropathy.

Penile erection requires complex interaction between vascular endothelial cells, smooth muscle cells, pericytes, and autonomic nerves. Diabetes mellitus is one of the most common causes of erectile dysfunction (ED) and multiple pathogenic factors, such as cavernous angiopathy and autonomic neuropathy, are associated with diabetic ED. Although a variety of animal models of diabetic ED play an important role in understanding pathophysiologic mechanisms of diabetes-induced ED, these animal models have limitations for addressing the exact cellular or molecular mechanisms involved in ED. Therefore, we established an invitro model of ED for the study of high-glucose-induced angiopathy and neuropathy. We successfully isolated and cultivated mouse cavernous endothelial cells (MCECs) and mouse cavernous pericytes (MCPs). The cells were exposed to the normal-glucose (5mmoL) or high-glucose (30mmoL) condition for 48h. In vitro matrigel assay revealed impairments in tube formation in primary cultured MCECs or MCPs exposed to high-glucose condition. To study cellular interaction between MCECs and MCPs, co-culture systems including indirect contact, indirect non-contact, and direct mixed co-culture system, were established. We observed impaired tube formation and increased permeability in MCECs-MCPs co-culture exposed to high-glucose condition. To evaluate the effect of high-glucose on neurite sprouting, the mouse major pelvic ganglion (MPG) tissue was harvested and cultivated in matrigel. Neurite outgrowth and nNOS-positive nerve fibers were significantly lower in MPG tissues exposed to the high-glucose condition than in the tissues exposed to the normal-glucose condition. We believe that invitro model of ED will aid us to understand the role of each cellular component in the pathogenesis of diabetic ED, and also be a useful tool for determining the efficacy of candidate therapeutics targeting vascular or neuronal function. This model would present a new avenue for drug discovery and development of novel therapeutic modalities for erectile dysfunction.

Effects and potential mechanism of bone marrow-derived stem cells on hepatic stellate cells in two contact coculture system

Objective To investigate the effects of bone marrow-derived stem cells (BMMSC) on the expressions of inflammatory cytokines and Toll-like receptor (TLR) 4 pathway in primary hepatic stellate cells (HSC) with direct and indirect contact coculture system. Methods Purified HSC were separately treated with LPS in the concentrations of 0, 50, 100 and 150 g/L for 48 h. Proliferation ratio was tested with cell counting kit-8 to determine the optimal concentration. HSC in LPS were divided into three groups, including HSC alone group, cocultured with BMMSC at 1∶1 group and cocultured with transwell group at the optimal concentration. The supernatants were collected to detect the concentrations of interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α. Cells were further divided into seven groups, including BMMSC without LPS group, HSC without LPS group, BMMSC with LPS group, HSC with LPS group, BMMSC in transwell system group, HSC in transwell system group, all cells in transwell system group and direct contact system group. The mRNA expressions of α-smooth muscle actin (α-SMA) and collagen I were detected by quantitative real-time PCR, and protein expressions of TLR4, myeloid differentiation factor 88 (MyD88) and nuclear factor-κB (NF-κB) were analyzed by Western blot. Data were analyzed with one-way ANOVA analysis. Results The proliferation rate of HSC in 50, 100 and 150 g/L LPS were (129.77±11.26)%, (162.90±13.15)% and (55.12±11.6)%, respectively compared with HSC without LPS group. The differences were statistically significant (t=5.91, 10.70 and 8.65, respectively, all P<0.05). The concentrations of IL-6 and TNF-α in HSC alone group, directly/indirectly cocultured group were (252.02±30.94) ng/L and (148.00±10.27) ng/L, (88.52±6.61) g/L and (72.63±5.54) ng/L, (103.74±7.14) ng/L and (81.79±6.92) ng/L, respectively. The differences between HSC alone group and directly/indirectly cocultured group were significant (t=12.66 and 15.82, 11.81 and 12.34, respectively, all P<0.05). The directly and indirectly cocultured groups were significantly different (t=3.83 and 2.53, respectively, both P<0.05). The mRNA expressions of α-SMA and collagen I in HSC with LPS were remarkably increased compared with HSC without LPS (t=14.16 and 11.84, respectively, both P<0.05) and reversed by cocultured with BMMSC (t=11.98 and 4.47, respectively, P<0.05). All cells in transwell group expressed more α-SMA and collagen I than the direct contact group (t=3.70 and 3.19, respectively, P<0.05). The TLR pathway associated protein expressions, TLR4, MyD88, and NF-κB in HSC in transwell group were significantly down-regulated compared with HSC with LPS group. And all cells in transwell system had higher level of protein expressions compared with direct contact system (P<0.05). Conclusions BMMSC are effective in inhibiting HSC activation and inflammatory cytokines excretion, which may be modulated through TLR4 pathway and cell to cell contact. Key words: Mesenchymal stem cells; Hepatic stellate cells; Liver cirrhosis; Toll-like receptors; Co-culture system

Microfabrication of a tunable collagen/alginate-chitosan hydrogel membrane for controlling cell–cell interactions

Indirect cell contact co-culture system is increasingly becoming more attractable owing to their advantages of easy cell separation and desirable outcomes for cell–cell interactions. However, how to precisely control the spatial position of cells within multicellular co-cultures is still experimentally challenging due to the incapability of the conventional methods in vitro. In the present study, a tunable collagen/alginate-chitosan (Col/Alg-Chi) membrane was established, which was capable of controlling intercellular distance between the neighboring cells at a level of micrometer resolution. It was showed that intercellular distance between the hepatocytes and the fibroblasts exerted significant influence on hepatic function in vitro. In particular, maintenance of the functionality of primary hepatocytes requires direct contact between the hepatocytes and their supportive stromal cells, and their effective contact distance is within 30μm. This technical platform would potentially enable investigations of dynamic cell–cell interaction in a multitude of applications including organogenesis, development or even neoplastic transformation.

Apelin Enhances Brown Adipogenesis and Browning of White Adipocytes

Brown adipose tissue expends energy in the form of heat via the mitochondrial uncoupling protein UCP1. Recent studies showed that brown adipose tissue is present in adult humans and may be exploited for its anti-obesity and anti-diabetes actions. Apelin is an adipocyte-derived hormone that plays important roles in energy metabolism. Here, we report that apelin-APJ signaling promotes brown adipocyte differentiation by increasing the expressions of brown adipogenic and thermogenic transcriptional factors via the PI3K/Akt and AMPK signaling pathways. It is also found that apelin relieves the TNFα inhibition on brown adipogenesis. In addition, apelin increases the basal activity of brown adipocytes, as evidenced by the increased PGC1α and UCP1 expressions, mitochondrial biogenesis, and oxygen consumption. Finally, we provide both in vitro and in vivo evidence that apelin is able to increase the brown-like characteristics in white adipocytes. This study, for the first time, reveals the brown adipogenic and browning effects of apelin and suggests a potential therapeutic route to combat obesity and related metabolic disorders.

EXPERIMENTAL RESEARCH ABOUT EFFECT OF CELL–CELL CONTACT TO MESENCHYMAL STEM CELLS DIFFERENTIATE INTO VASCULAR ENDOTHELIAL CELLS

ObjectivesUse the method of HUVECs were cultured with human mesenchymal stem cells by cell–cell contact to simulate intravascular environment after angioplasty and stent implantation, and research the role of cell–cell...

Generation of induced pluripotent mouse stem cells in an indirect co-culture system

Typically, production of induced pluripotent stem cells requires direct contact with feeder cells. However, once the stem cells have reached the appropriate maturation point, it is difficult to separate them from feeder cells, which must be irradiated with γ-rays or treated with the antibiotic mitomycin-C. We used a microporous poly-membrane-based indirect contact co-culture system with mouse embryonic fibroblasts to induce mouse pluripotent stem cells without radiation or antibiotics. We found that induced pluripotent stem cells induced by this co-culture method had a reprogramming efficiency and time similar to those induced using traditional methods. Furthermore, strongly expressed pluripotent markers showed a normal karyotype and formation and contained all three germ layers in a teratoma.

Ex vivo differentiation of multipotent adult progenitor cells to skin epidermal cells

Objective By establishing the indirect contact co-culture system, we studied the in vitro condition for MAPCs differentiating into epidermal cells and the transformation of MAPCs into epidermal cell phenotype. Methods Cell culture insert membrane was used for substitute basal membrane and MAPCs, fibroblast cells (FCs) and mixture of MAPCs and epidermal cells and FCs were separately implanted into 2 sides of it. PKH26 was used to label cloned MAPCs; type IV collagen rapid adhering method was used to isolate and culture the skin epidermal cells from 1-day-old SD rat. Results Part of the MAPCs transformed into cells expressing keratin in the presence of peripheral epithelia and FCs. Type IV collagen rapid adhering method successfully selected rats' epidermal stem cells. The mixture of the 2 kinds of cells or indirect culture might promote the differentiation through mesenchymal factors secreted by dermis FC. Conclusion We were the first to have established the in vitro model of MAPCs differentiation into epidermal cells, in which MAPCs were transformed into epithelium-like cells.

Roles of Osteoclasts in Leukemic Cells

We have previously reported that zoledoronic acid (ZOL) augmented the in vivo effect of imatinib in a murine chronic myeloid leukemia (CML) model (Blood 2003). ZOL alone induces apoptosis in leukemic cells in vitro by inhibiting prenylation of the Ras-related proteins. In addition to this direct anti-leukemic effect, we hypothesized that ZOL also has some influence in leukemic cells in vivo indirectly by destroying osteoclasts (OCs), which is the primary therapeutic activity of ZOL in osteoporosis patients. Supporting this notion is that by mediating bone resorption, OCs release a variety of cytokines such as IGF- 1, TGF-β, etc. that have accumulated in the bone matrix. It has been reported that OCs play an important role in bone metastasis of solid tumor, especially in cancer stem cells. However, little is known about the role of OCs in leukemia. Therefore, we investigated it in vitro and in vivo. For this purpose, we established an in vitro osteoblasts (OBs) and OCs co-culture system. The stable co-culture system that we developed includes collagen gel and murine primary OBs and OCs. In addition, murine femoral bone sections were sometimes added to this culture system so that the OCs could release the cytokines from the bone matrix. Thus, the collagen gel and OBs were placed in 12-well plates with and without bone sections and/or OCs. The transwell chambers over the wells then received 1×104 Ba/F3 cells that had been transfected with wild type bcr-abl (Ba/F3/bcr-abl cells). OBs markedly enhanced the growth of Ba/F3/bcr-abl cells in this indirect contact coculture system whereas the presence of both OBs and OCs slightly suppressed cell growth. Intriguingly, when bone sections were added (OBs+OCs+bone), Ba/F3/bcr-abl cell proliferation was significantly suppressed compared to the effect of OBs alone or OBs+OCs (Figure). Cell cycle analysis revealed that the G0/G1 population was increased in Ba/F3/bcr-abl cells co-cultured with OBs+OCs+bones. We also observed that the p27 protein levels of Ba/F3/bcr-abl cells increased upon co-culture with OCs or OCs+bones, similar to their response to treatment with purified TGF-β. We performed ELISAs to determine the concentrations of cytokines in the supernatants of co-cultured OBs and OCs. There were higher levels of TGF-β1 in the OBs+OCs+bones supernatant than in the OBs+OCs supernatant. Furthermore, OBs produced high levels of IGF-1. These findings suggest that OBs and OCs affect the proliferation and the cell cycle arrest of leukemic cells by releasing soluble factors, respectively. To more comprehensively elucidate the roles OCs play in leukemia cells in vivo, we used reveromycin A (RM-A) which inhibits bone resorption by specifically inducing apoptosis in OCs (Woo et al, PNAS 2006). RM-A did not have any in vitro effects on the proliferation of Ba/F3/bcr-abl cells. Thus, we could know the unalloyed role of OCs in leukemia with RM-A compared with ZOL which inhibited directly both OCs and leukemic cells. Our preliminary data show that RM-A suppresses the engraftment of inoculated Ba/F3/bcr-abl cells to nude mice. We also present data from ongoing studies showing the effect of RM-A on leukemic cells in murine models. These findings suggested that OCs may be an important constituent of leukemia stem cell niche and destruction of OCs by either ZOL or RM-A is a novel strategy for leukemia treatment. [Display omitted]