Ligation of E-cadherin on in vitro-generated immature Langerhans-type dendritic cells inhibits their maturation

Epithelial tissues of various organs contain immature Langerhans cell (LC)-type dendritic cells, which play key roles in immunity. LCs reside for long time periods at an immature stage in epithelia before migrating to T-cell-rich areas of regional lymph nodes to become mature interdigitating dendritic cells (DCs). LCs express the epithelial adhesion molecule E-cadherin and undergo homophilic E-cadherin adhesion with surrounding epithelial cells. Using a defined serum-free differentiation model of human CD34(+) hematopoietic progenitor cells, it was demonstrated that LCs generated in vitro in the presence of transforming growth factor beta1 (TGF-beta1) express high levels of E-cadherin and form large homotypic cell clusters. Homotypic LC clustering can be inhibited by the addition of anti-E- cadherin monoclonal antibodies (mAbs). Loss of E-cadherin adhesion of LCs by mechanical cluster disaggregation correlates with the rapid up-regulation of CD86, neo-expression of CD83, and diminished CD1a cell surface expression by LCs-specific phenotypic features of mature DCs. Antibody ligation of E-cadherin on the surfaces of immature LCs after mechanical cluster disruption strongly reduces the percentages of mature DCs. The addition of mAbs to the adhesion molecules LFA-1 or CD31 to parallel cultures similarly inhibits homotypic LC cluster formation, but, in contrast to anti-E-cadherin, these mAbs fail to inhibit DC maturation. Thus, E-cadherin engagement on immature LCs specifically inhibits the acquisition of mature DC features. E-cadherin-mediated LC maturation suppression may represent a constitutive active epithelial mechanism that prevents the uncontrolled maturation of immature LCs. (Blood. 2000;96:4276-4284)     

5,5'-Diphenylhydantoin decreases the entry of 3,5,3'-triiodo-L-thyronine but not L-thyroxine in cultured GH-producing cells

We determined the effect of 5,5'-diphenylhydantoin (DPH) on the kinetics of T3 and T4 uptake in cultured GH-producing (GC) cells under serum-free conditions. GC cells accumulated [125I]T3 at a greater fractional rate than [125I]T4. The t 1/2 of exit of [125I]T4 and [125I]T3 previously equilibrated in GC cells was 28 min for T4 and 66 min for T3. T3 and T4 entry rates were not influenced by up to a 10,000-fold molar excess of nonradioactive T3, T4, d-T4, rT3, 3,5-T2 and diiodotyrosine. Thus, entry of T3 and T4 in GC cells appeared nonsaturable and was not influenced by various thyroid hormone analogues. DPH, 25-200 mumol/l, decreased the rate of T3 entry in a dose-dependent manner and did not influence the T3 exit rate. At 200 mumol/l DPH, T3 entry decreased by 40%. Rates of entry and exit of T4 were unaffected by DPH. DPH may affect T3 and T4 entry differentially at the level of the plasma membrane.     

Origin of monocytes and their differentiation to macrophages and dendritic cells

Activation of the immune system requires that the presence of an incipient infection first be detected. This essential step is carried out by tissue macrophages, which alert innate immunity, and by dendritic cells whih alert the adaptive immune system. Both of these sentinel cell types are derived from circulating precursors known as blood monocytes. Here we briefly review current work which has recently expanded our understanding of the origin of blood monocytes and of the factors governing their further differentiation into tissue sentinels.     

Studies of the proliferation and differentiation of immature myeloid cells in vitro. I. Chronic myelogenous leukemia

Immature cells prepared from chronic myelogenous leukemic patients in the chronic or blastic phase of their disease were placed into suspension culture in vitro and studied over a 2-week period. Regardless of the status of the disease, the immature cells proliferated and maintained good viability. In contrast, evolution of the disease toward blastic crisis was associated with a progressive loss in the ability of the immature cells to differentiate. Cell proliferation and differentiation were independent of added growth factors.     

Studies of the proliferation and differentiation of immature myeloid cells in vitro. II. Acute myelogenous leukemia

Immature myeloid cells prepared from patients with AML were placed into suspension culture and studied over a 2-week period. Cell numbers usually fell and viability was not well maintained. Some degree of differentiation was observed in most cultures. Considering these observations, together with those derived from parallel studies of immature CML cells, the data suggest that AML cells are more dependent than CML cells on environmental conditions for the maintenance of cell viability and proliferation. This is especially the case for AML cells obtained at the time of initial diagnosis. On the other hand, AML cells and myeloid blastic crisis CML cells are similar with respect to their apparent greater ability to differentiate in vitro than in vivo. The addition of recombinant hemopoietins to the suspension cultures of AML cells is associated with either increased proliferation or differentiation but not both. The cells of different patients respond differently to the different hemopoietins and the different hemopoietins produce different affects in cultures of cells obtained from the same patient.     

Generation of human natural killer cells from immature progenitors does not require marrow stromal cells

Human natural killer (NK) cells comprise 10% to 15% of peripheral blood mononuclear cells and have an important role in immune responses against tumors, viral infections, and graft rejection. NK cells originate in bone marrow (BM), but their progenitors and lineage development have not been completely characterized. We studied the generation of NK cells from purified CD34+HLADR- and CD34+HLADR+ BM progenitors and the influence of various cytokines on their production. We show that CD3-CD56+ cytotoxic NK cells can develop from both progenitors populations when interleukin-2 (IL-2) is present in an in vitro suspension culture system containing IL-1 alpha and stem cell factor. Up to 83.8% and 98.6% CD3-CD56+ cells were detected in CD34+HLADR- and CD34+DR+ cultures, respectively, after 5 weeks of culture; significant numbers of NK cells were first detected after 2 weeks. Cytotoxic activity paralleled NK cell numbers; up to 70% specific lysis at an effector:target ratio of 10:1 was observed at 5 weeks. IL-7 also triggered development of CD3-CD56+ cells from these immature progenitors (up to 24% and 55% appeared in CD34+HLADR- and CD34+HLADR+ cultures, respectively). Our data suggest that BM stromas are not necessary for NK cell development and that IL-2 remains essential for this lineage development and differentiation.     

Accumulation of MFG-E8/lactadherin on exosomes from immature dendritic cells

Exosomes are vesicles of endocytic origin secreted spontaneously by dendritic cells (DCs). We have shown previously that exosomes can transfer antigen or MHC-peptide complexes between DCs, thus potentially amplifying the immune response. We had also identified milk fat globule EGF/factor VIII (MFG-E8), also called lactadherin, as one of the major exosomal proteins. MFG-E8 has two domains: an Arg-Gly-Asp sequence that binds integrins alphavbeta3 and alphavbeta5 (expressed by human DCs and macrophages) and a phosphatidyl-serine (PS) binding sequence through which it associates to PS-containing membranes (among which exosomes). MFG-E8 is thus a good candidate molecule to address exosomes to DCs. Here, we show that MFG-E8 is expressed by immature bone-marrow-derived DCs (BMDCs) and secreted in association with exosomes in vitro. We have generated mice expressing an inactive form of MFG-E8, fused to beta-galactosidase. Analyzing these mice, we demonstrate that MFG-E8 is expressed in vivo in splenic DCs. In a mouse DC-dependent, antigen-specific, CD4 T cell-stimulation assay, exosomes produced by MFG-E8-deficient BMDCs were barely less efficient than exosomes bearing MFG-E8. We conclude that MFG-E8 is efficiently targeted to exosomes but is not essential to address exosomes to mouse BMDCs. Involvement of MFG-E8/lactadherin in exosome targeting to other DC subpopulations, or to human DCs, is still possible.     

Isolation, purification and culture of Sertoli cells from immature piglet testes

Several studies suggest a role of Sertoli cells in the control of Leydig cell steroidogenesis. In order to verify this hypothesis, we have developed a system for the purification of pig Sertoli cells. These cells were then characterized by their morphological appearance in light and electron microscopy, their ability to bind [125I]follicle stimulating hormone (FSH) and their functional capacity as evaluated by adenosine 3',5' monophosphate (cAMP) accumulation and lactate production when in primary culture under basal and FSH-stimulated conditions. Crude Sertoli cell suspensions from immature porcine testes were fractionated on discontinuous Percoll gradients (densities 1.025, 1.039, 1.055, 1.080 g/ml). Highly purified Sertoli cells were contained in the second band (d: 1.039) generated on the gradient. These cells demonstrated morphological and functional integrity as evidenced by binding specifically [125I] FSH and by responding to FSH stimulation (by an increased production of cAMP and lactate after 3 days in primary culture), but not to human chorionic gonadotrophin (hCG). This preparation represents a useful model for the study of Sertoli cell functions and their interation with Leydig cells in the regulation of testicular steroidogenesis.     

成人骨髓间充质干细胞体外诱导分化为神经元样细胞的研究

目的 研究成人骨髓间充质干细胞(hMSC)体外定向诱导可否分化为神经元样细胞。方法 Percoll分离液离心分离hMSC，体外扩增，采用两种方法：①含碱性成纤维细胞生长因子(bFGF)预诱导24小时，甲氧酚(BHA)和二甲亚枫(DMSO)联合诱导6小时；②2-巯基乙醇(2-ME)预诱导24小时，BHA和DMSO诱导6小时。免疫组化检测神经元样细胞表达神经元烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)。结果 hMSC在体外扩增传至5代后，流式细胞仪显示hMSC表面抗原CD29、CD44、CD90表达阳性率分别为99．5％，97．8％，98．8％。采用两种方法诱导hMSC后，胞体收缩，突起伸出，较密的部分神经元拉成网状；免疫组化显示诱导的神经元样细胞能特异性表达NSE、NF，不表达GFAP。结论 成人骨髓hMSC在体外可以分化为神经元样细胞。     

Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells

Cardiomyocyte regeneration is limited in adult life. Thus, the identification of a putative source of cardiomyocyte progenitors is of great interest to provide a usable model in vitro and new perspective in regenerative therapy. As adipose tissues were recently demonstrated to contain pluripotent stem cells, the emergence of cardiomyocyte phenotype from adipose-derived cells was investigated. We demonstrated that rare beating cells with cardiomyocyte features could be identified after culture of adipose stroma cells without addition of 5-azacytidine. The cardiomyocyte phenotype was first identified by morphological observation, confirmed with expression of specific cardiac markers, immunocytochemistry staining, and ultrastructural analysis, revealing the presence of ventricle- and atrial-like cells. Electrophysiological studies performed on early culture revealed a pacemaker activity of the cells. Finally, functional studies showed that adrenergic agonist stimulated the beating rate whereas cholinergic agonist decreased it. Taken together, this study demonstrated that functional cardiomyocyte-like cells could be directly obtained from adipose tissue. According to the large amount of this tissue in adult mammal, it could represent a useful source of cardiomyocyte progenitors.     

大鼠诱导多能干细胞向肝系细胞的诱导分化

目的本研究探讨利用诱导因子将大鼠诱导多能干细胞(induced pluripotent stem cells,iPSCs)快速高效诱导分化为肝细胞样细胞(hepatocyte-like cells,HLCs)。方法利用诱导因子将iPSCs向肝系细胞方向分化,采用免疫细胞化学染色和RT-PCR方法检测培养20 d时细胞各种肝系细胞标志表达。结果培养分化20 d的iPSCs形成肝细胞样集落,表达肝系细胞特征性标志,并且具有尿素合成和糖原贮备能力。结论利用诱导因子可有效地将大鼠iPSCs诱导分化为HLCs,有望为细胞移植治疗各种肝脏疾病提供种子细胞。     

T lineage differentiation from human embryonic stem cells

Harnessing the ability of genetically manipulated human embryonic stem cells (hESC) to differentiate into appropriate lineages could revolutionize medical practice. These cells have the theoretical potential to develop into all mature cell types; however, the actual ability to develop into all hematopoietic lineages has not been demonstrated. Using sequential in vitro coculture on murine bone marrow stromal cells, and engraftment into human thymic tissues in immunodeficient mice, we demonstrate that hESC can differentiate through the T lymphoid lineage. Stable transgene expression was maintained at high levels throughout differentiation, suggesting that genetically manipulated hESC hold potential to treat several T cell disorders.     

Neuroectodermal differentiation from mouse multipotent adult progenitor cells

We recently showed that a rare cell from murine bone marrow, which we termed multipotent adult progenitor cells (MAPCs), can be expanded for >120 population doublings. Mouse (m)MAPCs differentiate into mesenchymal lineage cells as well as endothelium and endoderm, and, when injected in the blastocyst, mMAPCs contribute to most if not all somatic cell lineages including the different cell types of the brain. Our results, reported herein, demonstrate that mMAPCs can also be induced to differentiate into cells having anatomical and electrophysiological characteristics similar to those of midbrain neurons. Differentiation to a neuronal phenotype was achieved by coculturing mMAPCs with astrocytes, suggesting that neuronal differentiation may require astrocyte-derived factors similar to what is required for the differentiation of embryonic stem cells and neural stem cells to neurons. Differentiation of mMAPCs to neuron-like cells follows similar developmental steps as described for embryonic stem cells and neural stem cells. MAPCs therefore may constitute a source of cells for treatment of central nervous system disorders.     

绿色荧光蛋白基因转染小鼠胚胎干细胞及向神经细胞的分化

目的 建立能够稳定表达绿色荧光蛋白(GFP)的小鼠胚胎干细胞(ESC),并诱导其向神经细胞分化,为临床移植治疗神经系统疾病提供方便追踪观察的神经细胞.方法 分别用脂质体、电穿孔转染法转染小鼠胚胎干细胞系R1,检测48 h转染效率;经G418筛选后,得到稳定高效表达GFP的ESC克隆,通过碱性磷酸酶(AKP)染色检测ESC的生物学特性;利用单层分化法诱导转染GFP的胚胎干细胞向神经细胞分化,通过免疫荧光检测神经细胞特异标记Toil.结果 转染48 h的GFP阳性细胞转染率脂质体组为65%,电穿孔组为79%,两组比较差异无统计学意义(X2=3.14,P0.05).转染GFP的胚胎干细胞AKP染色阳性,并可诱导分化为Tuj1阳性的神经细胞.结论 建立稳定表达GFP基因的胚胎干细胞克隆,获得的转染GFP的胚胎干细胞能够分化为神经细胞.     

In vitro cultivation of human fetal pancreatic ductal stem cells and their differentiation into insulin-producing cells

AIM: To isolate, culture and identify the human fetal pancreatic ductal stem cells in vitro, and to observe the potency of these multipotential cells differentiation into insulin-producing cells.METHODS: The human fetal pancreas was digested by i g/L collagease type IV and then 2.5 g/L trypsin was used to isolate the pancreatic ductal stem cells, followed by culture in serum-free, glucose-free DMEM media with some additional chemical substrates in vitro (according to the different stage). The cells were induced by glucose-free (control),5 mmol/L, 17.8 mmol/L and 25 mmol/L glucose, respectively.The cell types of differentiated cells were identified using immunocytochemical staining.RESULTS: The shape of human fetal pancreatic ductal stem cells cultured/n vitro was firstly fusiform in the first 2 wk,and became monolayer and cobblestone pattern after another 3 to 4 wk. After induced and differentiated by the glucose of different concentrations for another 1 to 2 wk,the cells formed the pancreatic islet-like structures. The identification and potency of these cells were then identified by using the pancreatic ductal stem cell marker, cytokeratin-19 (CK-19), pancreatic β cell marker, insulin and pancreatic cell marker, glucagons with immunocytochemical staining.At the end of the second week, 95.2% of the cells were positive for CK-19 immunoreactivity. Up to 22.7% of the cells induced by glucose were positive for insulin immunoreactivity, and less than 3.8% of the cells were positive for glucagon immunoreactivity in pancreatic isletlike structures. The positive ratio of immunoreactive staining was dependent on the concentration of glucose, and it was observed that the 17.8 mmol/L glucose stimulated effectively to produce insulin- and glucagons-producing cells.CONCLUSION: The human fetal pancreatic ductal stem cells are capable of proliferation in vitro. These cells have multidifferentiation potential and can be induced by glucose and differentiated into insulin-producing cells in vitro.     

Hepatocyte differentiation from embryonic stem cells and umbilical cord blood cells

With the development of regeneration medicine, many researchers have attempted hepatic differentiation from nonhepatic-origin cell sources. The differentiation of embryonic stem (ES) cells into hepatocyte-like cells has been reported in several papers. Mouse ES cells have shown a potential to develop into hepatocyte-like cells in vitro on the basis of hepatic gene expression after adding several growth factors. We transplanted cultured embryoid body (EB) cells (male) into female mice. A liver specimen of the recipient was examined by immunohistochemical staining for albumin and fluorescence in situ hybridization for the Y chromosome after transplantation. Both Y chromosome- and albumin-positive cells were recognized in the recipient female liver, and were considered to be hepatocyte-like cells derived from ES cells containing the Y chromosome. Many groups, including ourselves, have studied hepatocyte-like cell differentiation from umbilical cord blood cells (UBCs). We cultured nucleated cells isolated from UBCs. Using immunostaining, ALB-positive and CK-19-positive cells were recognized in the culture. Dual staining of ALB and CK-19 demonstrated that ALB was coexpresed with CK-19, suggesting the existence of hepatic progenitors. In this review, we consider recent studies of the differentiation of hepatocytes from nonhepatic origins, especially ES cells and umbilical cord blood.     

Identification of nuclear tri-iodothyronine receptors in Sertoli cells from immature piglet testes.

The existence of nuclear tri-iodothyronine (T3) receptors in both Sertoli and Leydig cells isolated from immature piglet testes was investigated. The results demonstrated the presence of high-affinity (Kd = 1.09 +/- 0.25 nM), low-capacity (185 +/- 24 pg T3/mg DNA) binding sites for T3 in nuclei from freshly isolated Sertoli cells. No specific binding for T3 was observed in nuclei isolated from Leydig cells. The localization of specific T3 receptors, which might mediate the onset of thyroid hormone action, at Sertoli cell level confirms that these cells are a target for thyroid hormone and strongly sustain the role of the thyroid in the regulation of testicular functions during postnatal development.