A feasibility study of an in vitro differentiation potential toward insulin-producing cells by dental tissue-derived mesenchymal stem cells

Dental tissue-derived mesenchymal stem cells have been proposed as an alternative source for mesenchymal stem cells. Here, we investigated the differentiation ability toward insulin producing cells (IPCs) of human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs). These cells expressed mesenchymal stem cell surface markers and were able to differentiate toward osteogenic and adipogenic lineages. Upon 3 step-IPCs induction, hDPSCs exhibited more colony number than hPDLSCs. The mRNA upregulation of pancreatic endoderm/islet markers was noted. However, the significant increase was noted only for PDX-1, NGN-3, and INSULIN mRNA expression of hDPSCs. The hDPSCs-derived IPCs expressed PRO-INSULIN and released C-PEPTIDE upon glucose stimulation in dose-dependent manner. After IPCs induction, the Notch target, HES-1 and HEY-1, mRNA expression was markedly noted. Notch inhibition during the last induction step or throughout the protocol disturbed the ability of C-PEPTIDE release upon glucose stimulation. The results suggested that hDPSCs had better differentiation potential toward IPCs than hPDLSCs. In addition, the Notch signalling might involve in the differentiation regulation of hDPSCs into IPCs.     

The differentiation of mesenchymal stem cells by mechanical stress or/and co-culture system

Differentiation of mesenchymal stem cells (MSCs) into anterior cruciate ligament (ACL) cells is regulated by many factors. Mechanical stress affects the healing and remodeling process of ACL after surgery in important ways. Besides, co-culture system had also showed the promise to induce MSCs toward different kinds of cells on current research. The purpose of this study was to investigate the gene expression of ACL cells' major extracellular matrix (ECM) component molecules of MSCs under three induction groups. In addition, to follow our previous study, cell electrophoresis technique and mRNA level gene expression of MSC protein were also used to analyze the differentiation of MSCs. The results reveal that specific regulatory signals which released from ACL cells appear to be responsible for supporting the selective differentiation toward ligament cells in co-culture system and mechanical stress promotes the secretion of key ligament ECM components. Therefore, the combined regulation could assist the development of healing and remolding of ACL tissue engineering. Furthermore, this study also verifies that cell electrophoresis could be used in investigation of cell differentiation. Importantly, analysis of the data suggests the feasibility of utilizing MSCs in clinical applications for repairing or regenerating ACL tissue.     

Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid- treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the differential recognition of MHC I molecules of xeno-endothelial cells by human NK cells could be the major reason for higher NK cytotoxicity to PAEC. KIR might be the primary molecule that transduced inhibitory signals when endothelial cells were injured by NK cells.     

Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid-treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the different     

Lysis of endothelial cells by autologous lymphokine-activated killer cells

The mechanisms of lysis of endothelial cells derived from human umbilical vein (HUVEC) by autologous lymphokine-activated killer (LAK) cells, generated from cord blood lymphocytes of the same donor, were investigated. Freshly isolated HUVEC as well as HUVEC cultured for several passages were efficiently lysed by autologous LAK cells, and their susceptibility to the LAK cells was almost the some as that of allogenic HUVEC. Complement-depletion experiments revealed that the lysis was mainly dependent on CD16-natural killer (NK) LAK cells. Pretreatment of HUVEC with recombinant interferon (rIFN) for 24 h made them resistant to lysis by autologous LAK cells, while pretreatment with either rIL-1. rTNF, or acidic or basic fibroblast growth factor did not alter the lytic sensitivity of HUVEC. The resistance of rIFN-treated HUVEC was specific to lysis by CD16⁺ NK LAK cells, and their lysis by CD3⁺ T-LAK cells was not significantly altered. Moreover, in comparison with control HUVEC or rIL-1-treated HUVEC, rIFN-treated HUVEC had a significantly less potent inhibitory effect on the lysis of untreated HUVEC, when used as an unlabeled target. This suggests that rIFN treatment may down-regulate the recognition of some molecules on HUVEC by rIL-2-activated NK cells. These data suggest that damage of the endothelium during LAK therapy is mainly dependent on LAK cells with a NK phenotype that can specifically recognize a certain molecule on autologous endothelial cells.     

膝关节内干细胞/祖细胞在软骨再生中作用研究进展

关节软骨(AC)由于缺乏血管、神经和淋巴,一旦损伤无法自我修复。虽然以外源性细胞为基础的治疗策略在一定程度上能够再生关节软骨,但仍然存在手术间隔长、供体有限、细胞体外培养易去分化和病原体传播等风险。成人膝关节存在许多类型干细胞/祖细胞(SCPCs),当软骨损伤时,就会被动员,迁移到损伤部位,参与再生修复。因此,基于趋化内源性SCPCs到损伤部位的AC原位再生修复策略,充分利用机体自我修复潜力,同时避免了外源性细胞策略的缺点,已经成为研究的热点。本综述主要介绍膝关节内SCPCs类型、迁移路径以及其在软骨损伤修复中的作用。重点介绍内源性AC再生修复策略的研究现状,以期吸引更多的研究人员参与这一有前景的研究领域。     

Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells

Abstract In vitro derivation of oocytes from embryonic stem (ES) cells has the potential to be an important tool for studying oogenesis as well as advancing the field of therapeutic cloning by providing an alternative source of oocytes. Here, we demonstrate a novel, two-step method for inducing mouse ES cells to differentiate into oocyte-like cells using mouse ovarian granulosa cells. First, primordial germ cells (PGCs) were differentiated within the embryonic body (EB) cells around day 4 as defined by the expression of PGC-specific markers and were distinguished from undifferentiated ES cells. Second, day 4 EB cells were co-cultured with ovarian granulosa cells. After 10 days, these cells formed germ cell colonies as indicated by the expression of the two germ cell markers Mvh and SCP3. These cells also expressed the oocyte-specific genes Fig α, GDF-9 , and ZP1-3 but not any testis-specific genes by RT-PCR analysis. EB cultured alone or cultured in granulosa cell-conditioned medium did not express any of these oocyte-specific markers. In addition, EB co-cultured with Chinese hamster ovary (CHO) cells or cultured in CHO cell-conditioned medium did not express all of these oocyte-specific markers. Immunocytochemistry analysis using Mvh and GDF-9 antibodies confirmed that some Mvh and GDF-9 double-positive oocyte-like cells were generated within the germ cell colonies. Our results demonstrate that granulosa cells were effective in inducing the differentiation of ES cell-derived PGCs into oocyte-like cells through direct cell-to-cell contacts. Our method offers a novel in vitro system for studying oogenesis; in particular, for studying the interactions between PGCs and granulosa cells.     

Malolactic fermentation in chardonnay wine by immobilised Lactobacillus casei cells

The kinetics of malolactic fermentation in Chardonnay wine by immobilised Lactobacillus casei cells has been studied. Calcium pectate gel and chemically modified chitosan beads were used as supports for immobilisation. Repeated batch fermentations were carried out with different wine samples, some of which were treated with sulfur dioxide (free 19–25 mg/litre and total 80–88 mg/litre), in shake flask at 36, 25 and 20°C without any loss of activity. The degradation of malic acid obtained using immobilised cells was twice as high as that obtained with free cells. At an initial pH 3·2, decrease of malic acid of about 30% was observed at 25°C in one hour using L. casei cells immobilised either in pectate gel or on chitosan. Among the physico-chemical parameters studied, temperature was the main factor affecting metabolism of the organic acids as well as the rate of the malolactic fermentation. Operational stability of calcium pectate gel beads and chemically modified chitosan beads was 6 months after eight fermentations and 2 months after five fermentations, respectively, which proved the possibility of industrial application of the chosen supports in wine making.     

Entrapment of Lavandula vera cells and production of pigments by entrapped cells

Finely suspended cells of Lavandula vera were obtained by cultivating the cells in the presence of 50 mM CaCl₂ and entrapped homogeneously in gels formed from natural polysaccharides, agar, alginate and κ-carrageenan. The gel-entrapped cells grew well inside the gel matrices, the growth being confirmed by the increases in oxygen uptake, cell number and chlorophyll content. Blue pigments were synthesized de novo in the presence of l-cysteine by the gel-entrapped cells as well as by the free counterparts. Calcium alginate gel-entrapped cells were employed for the repeated production of the pigments for over 7 months by alternating growth and production phases. Entrapment with adequate gels stabilized markedly the viability and the pigment productivity of the plant cells.     

Slight up-regulation of Kir2.1 channel promotes endothelial progenitor cells to transdifferentiate into a pericyte phenotype by Akt/mTOR/Snail pathway

It was shown that endothelial progenitor cells (EPCs) have bidirectional differentiation potential and thus perform different biological functions. The purpose of this study was to investigate the effects of slight up-regulation of the Kir2.1 channel on EPC transdifferentiation and the potential mechanism on cell function and transformed cell type. First, we found that the slight up-regulation of Kir2.1 expression promoted the expression of the stem cell stemness factors ZFX and NS and inhibited the expression of senescence-associated β-galactosidase. Further studies showed the slightly increased expression of Kir2.1 could also improve the expression of pericyte molecular markers NG2, PDGFRβ and Desmin. Moreover, adenovirus-mediated Kir2.1 overexpression had an enhanced contractile response to norepinephrine of EPCs. These results suggest that the up-regulated expression of the Kir2.1 channel promotes EPC transdifferentiation into a pericyte phenotype. Furthermore, the mechanism of EPC transdifferentiation to mesenchymal cells (pericytes) was found to be closely related to the channel functional activity of Kir2.1 and revealed that this channel could promote EPC EndoMT by activating the Akt/mTOR/Snail signalling pathway. Overall, this study suggested that in the early stage of inflammatory response, regulating the Kir2.1 channel expression affects the biological function of EPCs, thereby determining the maturation and stability of neovascularization.     

Production of trehalose by permeabilized Micrococcus QS412 cells

Permeabilized Micrococcus QS412 cells were used to produce trehalose from starch through catalysis of maltooligosyl trehalose synthase and maltooligosyl trehalose trehalohydrolase in the cells. The permeabilized cells could omit the enzyme purification and simplify the immobilization of intracellular enzymes. The reagent, reagent dosage and time of cell permeabilization treatment were determined. The maximum trehalose biosynthesis activity was obtained after the cells were treated with 5% (w/v) of toluene at 30 °C for 40 min. Reaction conditions of trehalose synthesis of permeabilized cells were optimized. The yield of trehalose was up to 188 mg/g wet permeabilized cells in pH 8.0, 100 mmol/l phosphate buffer at 30 °C after 12 h reaction. Batch reactions showed that the permeabilized cells could be reused for 16 cycles in the biosynthesis reaction. The total trehalose yield was up to 2.5 g/g wet permeabilized cells. Development of permeabilized cells provide a new cheaply alternative technology for trehalose production.     

Immunomodulation by mesenchymal stem cells:Interplay between mesenchymal stem cells and regulatory lymphocytes

Mesenchymal stem cells(MSCs) possess immunomodulatory properties, which confer enormous potential for clinical application. Considerable evidence revealed their efficacy on various animal models of autoimmune diseases, such as multiple sclerosis, systemic lupus erythematosus and uveitis. MSCs elicit their immunomodulatory effects by inhibiting lymphocyte activation and proliferation, forbidding the secretion of proinflammatory cytokines, limiting the function of antigen presenting cells, and inducing regulatory T(Treg) and B(Breg) cells. The induction of Treg and Breg cells is of particular interest since Treg and Breg cells have significant roles in maintaining immune tolerance. Several mechanisms have been proposed regarding to the MSCs-mediated induction of Treg and Breg cells. Accordingly, MSCs induce regulatory lymphocytes through secretion of multiple pleiotropic cytokines, cell-to-cell contact with target cells and modulation of antigen-presenting cells. Here, we summarized how MSCs induce Treg and Breg cells to provoke immunosuppression.     

Alterations of the cytoplasmic organization of WIRL cells induced by trifluoperazine

The antipsychotic drug trifluoperazine (TFP) causes a reversible rounding of cells of the rat liver epithelial cell line, WIRL. We have investigated the cytoplasmic organization of these cells after TFP treatment using SEM, TEM and immunofluorescence and have observed significant differences between the control and treated cells. Mitochondria are converted to the condensed configuration with distended cristae and the endoplasmic rcticulum becomes tubular with distended cisternae. Intermediate filaments, visualized with a monoclonal antibody, are aggregated to a cap on the nucleus in an arrangement different from that induced by colcemid.     

Helicobacter-stimulated IL-10-producing B cells suppress differentiation of lipopolysaccharide/Helicobacter felis-activated stimulatory dendritic cells

Regulatory B cells (Bregs) produce antiinflammatory cytokines and inhibits proinflammatory response. Recently, immunosuppressive roles of Bregs in the effector functions of dendritic cells (DCs) were demonstrated. However, cross talk between Bregs and DCs in Helicobacter infection remains unknown. Here, we showed that direct stimulation of bone marrow-derived DCs (BM-DCs) with Helicobacter felis (H. felis) antigen upregulates their CD86 surface expression and causes the production of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and interleukin-10 (IL-10). Furthermore, prestimulation of DCs with supernatants derived from both Helicobacter-stimulated IL-10^– B (Hf_stim-IL-10^– B) or IL-10⁺ B (Hf_stim-IL-10⁺) cells suppresses the secretion of TNF-α and IL-6, but does not affect the expression of CD86 and secretion of IL-12 by lipopolysaccharide (LPS) or H. felis-activated BM-DCs. Remarkably, soluble factors secreted by Hf_stim-IL-10^– B cells, but not by Hf_stim-IL-10⁺ B cells, suppress the secretion of IL-10 by BM-DCs upon subsequent LPS stimulation. In contrast, prestimulation with BM-DCs with supernatants of Hf_stim-IL-10⁺ B cells before H. felis antigen stimulation induces significantly their IL-10 production. Collectively, our data indicated that prestimulation with soluble factors secreted by Hf_stim-IL-10⁺ B cells, DCs exhibit a tolerogenic phenotype in response to LPS or Helicobacter antigen by secreting high levels of IL-10, but decreased levels of IL-6 and TNF-α.     

Effective expansion of umbilical cord blood hematopoietic stem/progenitor cells by regulation of microencapsulated osteoblasts under hypoxic condition

The expansion of hematopoietic stem/progenitor cells (HSPCs) from umbilical cord blood (UCB) with the support of microencapsulated osteoblasts under hypoxia environment was investigated. The expansion of HSPCs was evaluated through the total number of UCB mononuclear cells (MNCs) produced, their repopulating potential with the colony-forming unit assay (CFU-Cs) and CD34⁺ phenotypic analysis with flow cytometry. At the end of 7 days of culture, the UCB-MNCs, CFU-Cs and CD34⁺ cells had achieved 18.7 ± 1.6, 11.6 ± 0.9 and 23.4 ± 2-fold expansions, respectively, in the test groups. These were significantly different from those in control groups. Microencapsulated osteoblasts under hypoxia conditions had therefore a significant effect on the expansion potential of HSPCs in vitro.     

胚胎干细胞向造血干/祖细胞定向诱导分化的研究进展

胚胎干细胞(embryonic stem cell,ES细胞)是指由胚胎内细胞团(inner cell mass,ICM)细胞经体外抑制培养而筛选得到的细胞,具有无限增殖潜能,在体外可以向造血细胞分化,有可能为造血干细胞移植和血细胞输注开辟新的来源．此外,ES细胞向造血干/祖细胞的定向诱导分化也为阐明哺乳动物造血发育的细胞和分子机制提供了良好的体外模型．对ES细胞向造血干/祖细胞定向分化的研究进展进行了综述．