Antigenic profile of human thymus in concurrence with "Clusters of Thymic Epithelial Staining" classification.

We examined the expression of various CD coded or not yet defined antigens in human thymus samples using indirect immunoperoxidase and immunoflourescent techniques. Data obtained are presented in concurrence with Clusters of Thymic Epithelial Staining (CTES) classification for various monoclonal antibodies recognizing CD antigens (CD1, CD1a, CD6, CD9, CD14, CD16, CD29, CD30, CD32, CD44, CD45RB, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD53, CD54, CD56, CD57, CD63, CD85, CD95, CD98, CD102, CD103, CD106, CD109, CD146, CD147, CD148, CD151, CD152, CD158a, CD158b, CD164, CD165, CD166) and for monoclonal antibodies 1B10, 5G7, A4, BD46, BLTZ, HP1C5, IND.64, M72, WU947 whose specifities are not yet defined. Some of the mAbs such as CD49f, IND.64 and BD46 are detected as good markers for specific cell types or compartments. Significance of the presence of these antigens on thymic epithelial cells at certain locations is briefly discussed.     

Expression of homing receptors and related molecules on human mast cells and basophils: a comparative analysis using multi-color flow cytometry and toluidine blue/immunofluorescence staining techniques

Mast cells (MC) and blood basophils (Ba) are multifunctional effector cells of the immune system and accumulate in areas of ongoing disease. However, despite of similar morphology, MC and Ba differ from each other in terms of cell surface receptor expression, mediator content, and tissue distribution. In order to gain new insights into mechanisms and molecules responsible for the distribution and accumulation of MC and Ba, we have investigated expression of homing receptors on primary human MC (lung, n=28; uterus, n=17), Ba (healthy donors, n=64), the mast cell line HMC-1, and the basophil line KU-812. Expression of cell surface antigens on MC and Ba was analyzed by mAb and indirect immunofluorescence staining techniques. In addition to previous findings, Ba were found to react with mAb against the selectin-ligands sLe(x) (CD15s) and PSGL-1 (CD162), L-selectin (CD62L), beta7-integrin, the 'matrix-receptor' neurothelin (CD147), platelet-endothelial cell tetraspan antigen-3 (PETA-3=CD151), and BST-1 (CD157). Novel antigens detectable on MC (lung and uterus) were CD147, CD151, CD157 and CD49c (VLA-3alpha). By contrast, MC were not recognized by mAb to sLe(x), PSGL-1, L-selectin, or beta7 integrin. No reactivity of Ba or MC with mAb to syndecan-1 (CD138), VE-cadherin (CD144), MUC18/MCAM (CD146), MGC-24 (CD164), or ALCAM (CD166) was found. The cell lines HMC-1 and KU-812 expressed a similar profile of antigens when compared to primary cells. In summary, Ba and MC express a unique profile of homing molecules. Apparently, Ba differ from MC in expression of recognition receptors relevant for binding to endothelium and consecutive transmigration.     

Immunophenotypic analysis of human spleen compartments.

Microanatomical compartments of the human spleen are yet under evaluation as most of the present information comes from experiments on animals with different anatomical structures. Immune staining of stromal and blood-born cells by cell surface antigens facilitates the differentiation of functional microanatomical compartmentalization of immune organs, including the spleen. Twenty-two specimens from healthy adult subjects with the average age of 35.6 +/- 13.8 (Range 17 to 58) years were included in this study. Monoclonal antibodies used in this study were supplied from the 5th, 6th and 7th International Workshops and Conferences on Human Leukocyte Differentiation Antigens. Tetraspan antigens presented a rather unique staining pattern in the human spleen, suggesting special roles for each (CD9, CD53, CD63, CD151 and CD231) in certain locations. Sinus lining cells presented a distinctive antigenic profile, sharing both endothelial cell (CD31, CD36, CD54, CD62P, CD102, CD105, CD106 and CD146) and macrophage lineage characteristics. The sheathed capillaries were not restricted to the perifollicular zone alone. Extracellular matrix receptors (CD49 a, CD49 b, CD49 c, CD49 e, CD49f, CD29 and CD44) stained the penicillary arterioles and vascular smooth muscle. These molecules were also found on the vascular endothelium. Leukocyte antigens (CD11a, CD11b, CD22, CD43, CD45, CD45RB, CD45RO and CD50) were mainly expressed in the white and red pulp of the spleen at different intensities, excluding the penicillary arterioles. Activation antigens (CD26, CD71 and CD98) presented a diffuse and broad staining pattern. In conclusion, microanatomical compartmentalization, microcirculation and function of the human spleen were evaluated using a wide panel of monoclonal antibodies.     

Immunologic phenotype of cultured endothelial cells: quantitative analysis of cell surface molecules

Endothelial cells express a wide spectrum of surface molecules involved in multiple vascular functions. We quantitatively determined an extensive immunologic phenotype of endothelial cells through a large panel of antibodies directed against i) well-known endothelial molecules CD31, CD34, CD49b, e, f, CD51, CD54, CD55, CD62E and P, CD105, CD106, HLA class I and HLA class II; ii) molecules defined by monoclonal antibodies newly clustered during the 6th workshop of Human Leukocyte Differentiation Antigen (HLDA) CD109, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146 and CD147; iii) molecules defined by unclustered monoclonal antibodies. The expression of these molecules was quantified on human umbilical vein endothelial cells (HUVEC) cultured in resting conditions and after stimulation with IL-ip (10 U/ml), TNF-a (10 ng/ml) and phorbol myristate acetate (60 ng/ml). Some molecules were constitutively expressed, and others were negative, which served to determine the basal phenotype. After cell stimulation, the molecules showed weak or strong expression modulation, leading to the definition of an activated phenotype. Changes in the kinetics and the amplitude of expression served to characterize poorly defined molecules and may be useful to determine their physiologic role. Also, we compared the phenotypes of endothelial cell lines EA.hy 926 and ECV 304 to that of HUVEC to assess their reliability as an endothelial cell model. Each cell line displayed a specific repertoire of molecules expressed at different levels, which could have significant im-I plications for cell line behavior as endothelial cells.     

人羊膜间充质干细胞的生物学特性鉴定

目的分离提纯的人羊膜间充质干细胞的生物学特性鉴定。方法剥去38～40周孕妇剖腹产术后羊膜,用消化酶消化后制备人羊膜间充质细胞（hAMCs）悬浮液。利用流式细胞分选技术从hAMCs中分离提纯干细胞（hAMC-SP细胞）,并对于细胞抗原进行鉴定。结果从hAMCs中分离的hAMC-SP细胞,约占AMCs总数的0．3％。传代培养4～10代后的hAMC-SP细胞表达Nestin、Vimentin、整合素家属成员（CD49b、CD49c、CD49d、CD49e）、CD9、CD13、CD19、CD29、CD44、CD46、CD51、CD59、CD166及干细胞相关的Oct-3／4抗原。HLA-ABC、TRA-1-81及SSEA-4为弱表达;相反CD34、CD45、CD117、CD56、CD90、CD105、CD106、CD133、Fit-1、Musashi 1及HLA-DR无阳性结果,同样TRA-1-60及SSEA-3也表达阴性。体外转化实验表示hAMC-SP细胞在琼脂糖培养基上不形成细胞集落,但阳性对照组的HepG2形成多数细胞集落。结论hAMC-SP细胞表面标记符合骨髓及脂肪间充质等来源的干细胞特点;hAMC-SP细胞迅速而稳定扩增,无致瘤性。由于来源充足,hAMC-SP细胞在消除HLA-ABC阳性细胞亚群下,同种异体移植在内的广泛的再生医学领域中能成为理想的细胞来源。     

Cell surface membrane antigen phenotype of human gastrointestinal mast cells

BACKGROUND: Mast cells (MC) are important effector cells of allergic and inflammatory reactions in diverse organs. These cells interact with a number of other immune cells and structural cells in the tissues as well as with proinflammatory mediators and cytokines. The various interactions are considered to be mediated through distinct cell surface membrane receptors on MC. METHODS: In the present study, we have established the cell surface membrane phenotype of human gastrointestinal MC (HGMC) using a panel of monoclonal antibodies and indirect immunofluorescence staining techniques. RESULTS: HGMC were found to react with antibodies against CD29, CD33, CD44, CD45, CD47, CD54, CD55, CD58, CD63, CD117, CD147, CD151, CD172a, and CD203c. By contrast, HGMC did not express detectable amounts of CD1, CD2, CD4, CD5, CD14, CD15, CD16, CD22, CD24, CD25, CD26, CD27, CD28, CD31, CD32, CD34, CD35, CD88, or CD116. The alpha-chain of the IL-3 receptor (CD123) was detectable neither in resting HGMC nor in HGMC exposed to stem cell factor and interleukin-4. CONCLUSIONS: HGMC express a unique profile of surface antigens including the receptor for mast cell growth factor, adhesion-related molecules, and activation-linked membrane antigens.     

Detection of differentiation- and activation-linked cell surface antigens on cultured mast cell progenitors

BACKGROUND: Mast cells (MC) are multifunctional effector cells of the immune system. They derive from uncommitted CD34(+) hemopoietic progenitor cells (HPC). Depending on the stage of maturation and the environment, MC variably express differentiation- and activation-linked antigens. Little is known, however, about the regulation of expression of such antigens in immature human MC. METHODS: We analyzed expression of CD antigens on human MC grown from cord blood-derived CD34(+) HPC. The HPC were isolated by magnetic cell sorting (MACS) and FACS to >97% purity, and were cultured in stem cell factor (SCF) and interleukin (IL)-6 with or without additional cytokines (IL-4 or IL-10) in serum-free medium. The cell surface phenotype of MC was determined by monoclonal antibodies and flow cytometry. RESULTS: Cultured MC progenitors were found to react with antibodies against various CD antigens including CD58, CD63, CD117, CD147, CD151, CD203c, and CD172a, independent of the growth factors used and time-point investigated (days 14-42). CD116 [granulocyte-macrophage colony-stimulating factor receptor alpha (GM-CSFRalpha)] and CD123 (IL-3Ralpha) were expressed on MC precursors on day 14, but disappeared thereafter. Cultured MC did not express CD2, CD3, CD5, CD10, CD19, or CD25. Addition of IL-10 to MC cultures showed no effect on expression of CD antigens. However, IL-4 was found to promote expression of CD35 and CD88 on cultured MC without changing expression of other CD antigens. CONCLUSIONS: Most MC antigens may already be expressed at an early stage of mastopoiesis. Whereas IL-3R and GM-CSFRs are lost during differentiation of MC, these cells may acquire complement receptors (CD35, CD88) under the influence of distinct cytokines.     

Acquisition and alteration of adhesion molecules during cultured human mast cell differentiation

BACKGROUND: Mature human mast cells express several types of adhesion molecules on their surface. Interactions between extracellular matrix (ECM) and adhesion molecules may be important for the migration and localization of mast cells and their precursors in tissues. Little is known about the regulation of adhesion molecules on mast cells during their differentiation. OBJECTIVES: To clarify the evolution of adhesion phenotype and function, we examined the expression of adhesion molecules during cultured human mast cell (CHMC) differentiation and tested adhesion of mature CHMCs to various ECM proteins. METHODS: CHMCs were obtained by culturing human cord blood-derived CD34(+) cells in the presence of stem cell factor and IL-6. Indirect immunofluorescence and flow cytometry was used to study cell surface expression of adhesion molecules and other markers. Mature CHMCs were tested for adhesion molecule function with immobilized matrix proteins. RESULTS: At 1 week of culture, cells expressed CD11a, CD18, CD29, CD49d, and CD49e. At 14 weeks of culture, more mature CHMCs expressed CD11b, CD11c, CD29, CD49b, CD49c, CD49d, CD49e, CD51, CD61, and CD54 and weakly expressed CD18 and CD11a. CD11c, CD51, and CD61 appeared de novo by 4 weeks of culture, whereas CD49b and CD49c appeared by 8 weeks. CD29 decreased at 4 weeks but returned to the identical levels of 1-week-old cells by 8 weeks. Compared with levels at week 1, the levels of CD11a, CD18, CD49d, and CD49e at 4 weeks and beyond decreased during culture. Expression of CD49a, CD49f, and alphad integrin was never detectable during CHMC differentiation. Fourteen-week-old CHMCs significantly adhered to the leucine-aspartic acid-valine-containing connecting segment 1 fragment of fibronectin, the 120-kd argine-glycine-aspartic acid-containing fragment of fibronectin, vitronectin, and laminin through specific integrins. CONCLUSION: Expression of integrins and CD54 is differentially regulated during CHMC differentiation, and mature CHMCs can adhere to many ECM proteins. These changes may facilitate emigration from the bone marrow into the circulation and ultimately contribute to the tissue homing and localization pattern seen with mature mast cells.     

Differential expression of cell adhesion molecules in the functional compartments of lymph nodes and tonsils

Aims—To analyse the topographical distribution of adhesion molecules involved in lymphocyte recirculation in human lymph nodes and tonsils. The study focused on the expression of LECAM-1 (CD62L), VLA-α4 (CD49d), VLA-β1 (CD29), LFA-1 αL (CD11a), LFA-β2 (CD18), VCAM-1 (CD106), ICAM-1 (CD54), and H-CAM (CD44).     

Vital dye Dil and fluorescent magnetic microparticles do not affect the phenotype of mesenchymal stem cells from human amnion and their differentiation capacity

We present a method of labeling of mesenchymal stem cells from human amnion with a fluorescent dye Dil and microspheres (Bangs Laboratories). The possibility of administration of loaded cell culture was verified and comparative analysis of the phenotype of mesenchymal stem cells by the expression of fibronectin, nestin, CD13, CD29, CD34, CD44, CD54, CD90, CD105, CD106, HLA-ABC, HLA-DR, and PCNA was carried out. The labeled cells retained osteogenic differentiation capacity. The results suggest that fluorescent dye Dil and microspheres from Bangs Laboratories can be used for monitoring of mesenchymal stem cells from human amnion in in vivo experiments. __________ Translated from Kletochnye Tehnologii v Biologii i Meditsine, No. 2, pp. 73–79, April, 2008     

Adhesion receptors of intimal and subintimal cells of the normal synovial membrane.

The distribution of cell adhesion molecules (CAMs) and matrix proteins in the normal synovium of four subjects was studied by immunohistology in order to determine the factors governing the cellular and tissue organization of the intimal and subintimal compartments. Basement membrane proteins, laminin, and collagen type IV, as well as vitronectin and fibronectin, were identified in the intima and there was corresponding expression of integrin and non-integrin receptors (e.g., CD29, CD49b, CD49d, CD49e, CD49f, CD51, CD61, CD44) for these matrix proteins. There were notable differences in CAM expression between intimal, subintimal, and vascular compartments of the synovial membrane. Phenotypic heterogeneity for CAMs involved in cell-cell interactions, particularly CD11a, CD11b, ICAM-1, and HLA-DR, was also present. The range of CAMs expressed by synovial and endothelial cells not only indicates a structural role for these antigens, but also suggests that they may control leucocyte traffic into the membrane, including recruitment of cells into the synovial lining.     

Mesenchymal stem cell characteristics of human anterior cruciate ligament outgrowth cells

When ruptured, the anterior cruciate ligament (ACL) of the human knee has limited regenerative potential. However, the goal of this report was to show that the cells that migrate out of the human ACL constitute a rich population of progenitor cells and we hypothesize that they display mesenchymal stem cell (MSC) characteristics when compared with adherent cells derived from bone marrow or collagenase digests from ACL. We show that ACL outgrowth cells are adherent, fibroblastic cells with a surface immunophenotype strongly positive for cluster of differentiation (CD)29, CD44, CD49c, CD73, CD90, CD97, CD105, CD146, and CD166, weakly positive for CD106 and CD14, but negative for CD11c, CD31, CD34, CD40, CD45, CD53, CD74, CD133, CD144, and CD163. Staining for STRO-1 was seen by immunohistochemistry but not flow cytometry. Under suitable culture conditions, the ACL outgrowth-derived MSCs differentiated into chondrocytes, osteoblasts, and adipocytes and showed capacity to self-renew in an in vitro assay of ligamentogenesis. MSCs derived from collagenase digests of ACL tissue and human bone marrow were analyzed in parallel and displayed similar, but not identical, properties. In situ staining of the ACL suggests that the MSCs reside both aligned with the collagenous matrix of the ligament and adjacent to small blood vessels. We conclude that the cells that emigrate from damaged ACLs are MSCs and that they have the potential to provide the basis for a superior, biological repair of this ligament.     

Serotypes,virulence genes,and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from healthy sheep in Spain

Fecal swabs obtained from 1,300 healthy lambs in 93 flocks in Spain in 1997 were examined for Shiga toxin-producing Escherichia coli (STEC). STEC O157:H7 strains were isolated from 5 (0.4%) animals in 4 flocks, and non-O157 STEC strains were isolated from 462 (36%) lambs in 63 flocks. A total of 384 ovine STEC strains were characterized in this study. PCR showed that 213 (55%) strains carried the stx(1) gene, 10 (3%) possessed the stx(2) gene, and 161 (42%) carried both the stx(1) and the stx(2) genes. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 106 (28%) and 23 (6%) of the STEC strains, respectively. The STEC strains belonged to 35 O serogroups and 64 O:H serotypes (including 18 new serotypes). However, 72% were of 1 of the following 12 serotypes: O5:H-, O6:H10, O91:H-, O117:H-, O128:H-, O128:H2, O136:H20, O146:H8, O146:H21, O156:H-, O166:H28, and ONT:H21 (where NT is nontypeable). Although the 384 STEC strains belonged to 95 different seropathotypes (associations between serotypes and virulence genes), 49% of strains belonged to only 11. O91:H- stx(1) stx(2) (54 strains) was the most common seropathotype, followed by O128:H- stx(1) stx(2) (33 strains) and O6:H10 stx(1) (25 strains). Three strains of serotypes O26:H11, O156:H11, and OX177:H11 had intimin type beta1; 5 strains of serotype O157:H7 possessed intimin type gamma1; and 15 strains of serotypes O49:H-, O52:H12, O156:H- (12 strains), and O156:H25 had the new intimin, intimin type zeta. The majority (82%) of ovine STEC strains belonged to serotypes previously found to be associated with human STEC strains, and 51% belonged to serotypes associated with STEC strains isolated from patients with hemolytic-uremic syndrome. Thus, this study confirms that healthy sheep are a major reservoir of STEC strains pathogenic for humans.     

Expansion of mesenchymal stem cells from human pancreatic ductal epithelium

Fibroblast-like cells emerging from cultured human pancreatic endocrine and exocrine tissue have been reported. Although a thorough phenotypic characterization of these cells has not yet been carried out, these cells have been hypothesized to be contaminating fibroblasts, mesenchyme and/or possibly beta-cell progenitors. In this study, we expanded fibroblast-like cells from adult human exocrine pancreas following islet isolation and characterized these cells as mesenchymal stem cells (MSCs) based on their cell surface antigen expression and ability to differentiate into mesoderm. Analysis by flow cytometry demonstrated that pancreatic MSCs express cell surface antigens used to define MSCs isolated from bone marrow such as CD13, CD29, CD44, CD49b, CD54, CD90 and CD105. In addition, utilizing protocols used to differentiate MSCs isolated from other somatic tissues, we successfully differentiated pancreatic MSCs into: (1) osteocytes that stained positive for alkaline phosphatase, collagen, mineralization (calcification) and expressed osteocalcin, (2) adipocytes that contained lipid inclusions and expressed fatty acid binding protein 4 and (3) chondrocytes that expressed aggrecan. We also demonstrated that pancreatic MSCs are multipotent and capable of deriving cells of endodermal origin. Pancreatic MSCs were differentiated into hepatocytes that stained positive for human serum albumin and expressed endoderm and liver-specific genes such as GATA 4 and tyrosine aminotransferase. In addition, preliminary protocols used to differentiate these cells into insulin-producing cells resulted in the expression of genes necessary for islet and beta-cell development such as Pax4 and neurogenin 3. Therefore, multipotent MSCs residing within the adult exocrine pancreas could represent a progenitor cell, which when further manipulated could result in the production of functional islet beta-cells.     

Human mesenchymal stem cells at the single-cell level: simultaneous seven-colour immunofluorescence

Extracellular, intracellular or surface proteins can be used as putative markers to characterize human mesenchymal stem cells (hMSC). However, these markers are also expressed by other cell types and primary cell pools reveal considerable heterogeneity. Therefore, the simultaneous detection of several markers on a single cell appears to be an attractive approach to identify hMSC. Here we demonstrate the specific distinction of human MSC from human osteoblasts via seven-colour fluorescence on the single cell level with simultaneous marker detection of CD44, CD105/endoglin, CD106/VCAM-1, collagen-IV, fibronectin, actin and DAPI nuclear staining. We performed spectral image acquisition using a Sagnac-type interferometer. Subsequent linear unmixing allowed for decomposition of each pixel in its spectral components. Our approach reveals a typical expression profile of the adherent singular cells, allowing the specific distinction between hMSC and osteoblasts on the single cell level.     

八种粘附分子在急性淋巴细胞性白血病异常表达的研究

本文分析了３８名正常人和２５例急性淋巴细胞性白血病（ＡＬＬ）细胞表面ＣＤ１１ａ、ＣＤ１１ｂ、ＣＤ５４、ＣＤ４４等的表达。与正常人相比，肿瘤细胞上表达的ＣＤ１１ｂ、ＣＤ１８和ＣＤ５４降低，ＣＤ４４升高；ＣＤ１１ａ在Ｂ－ＡＬＬ和混合型ＡＬＬ表达减弱，在Ｔ－ＡＬＬ增强。３例急性淋巴细胞性白血病骨髓基质细胞表面ＣＤ５４、ＣＤｗ４９ｂ表达亦降低。认为粘附分子可能参与了急性淋巴细胞性白血病的发病机制。     

人骨髓间充质干细胞的分离培养及血管内皮生长因子对其体外诱导分化作用

目的：分离和培养人骨髓间充质干细胞(mcsenchymal stem cells，MSCs)并检测其免疫学表型，探讨血管内皮生长因子(vascular endothelial growth factor，VECF)对其体外诱导分化作用。方法：利用Pcreoll梯度分离、贴壁筛选法及单克隆培养法分离、培养、扩增MSCs；采用免疫荧光和流式细胞术检测MSCs免疫学表型；通过VFGF-165基因转染分析MSCs的表型变化。结果：体外分离、培养出高度同源性的MSCs；MSCs具有独特的细胞免疫学表型，即CD44，CD29，c-kit阳性，CD34，CD31，CD54阴性；VEGF-165诱导后CD44表达明显降低，CD31显著升高，MSCs向内皮分化。结论：成功建立人MSCs分离培养方法，探讨了MSCs细胞免疫学表型及VEGF对它的内皮诱导分化作用，为MSCs的应用提供理论基础。     

Human term placenta-derived mesenchymal stromal cells are less prone to osteogenic differentiation than bone marrow-derived mesenchymal stromal cells

Mesenchymal stromal cells (MSC) can be isolated from different tissues. They are capable of differentiating in vitro, for example, to osteoblasts, chondrocytes, or adipocytes. In contrast to CD34 for hematopoietic stem cells, a distinct MSC-defining antibody is not available. Further, for hematopoietic cells lineage-defining antigens such as CD3 or CD20 are known. In contrast, for MSC-derived cells lineage-associated cell surface markers are far from being established. We therefore investigated expression of cell surface antigens on human term placenta-derived MSC (pMSC) in more detail and correlated expression pattern to the osteogenic differentiation capacity of the MSC. We report that pMSC expressed the typical cell surface antigens at levels comparable to bone marrow-derived MSC (bmMSC), including CD73, CD90, and CD105, but did not express CD11b, CD34, and CD45. Further, CD164, TNAP, and the W5C5 antigens were detected on pMSC, whereas CD349 was not observed. Some pMSC expressed CD146 at low or moderate levels, and their osteogenic differentiation potential was weak. In contrast, bmMSC expressed CD146 at high levels, expression of alkaline phosphatase was significantly higher, and they presented a pronounced osteogenic differentiation potential. We conclude that MSC from different sources differ in their expression of distinct markers, and that this may correlate in part with their lineage determination. Thus, a higher percentage of bmMSC expressed CD146 at prominent levels and such cells may be better suited for bone repair. In contrast, many pMSC expressed CD146 at low or moderate levels. They, therefore, may be suitable for applications in which osteogenic differentiation is undesirable.