Differentiation of endothelial cells from human umbilical cord blood AC133−CD14+ cells

Endothelial progenitor cells (EPCs) participate in neovascularization and are consistent with postnatal vasculogenesis. In vitro, they differentiate into endothelial cells (ECs). Prior reports have suggested that circulating human AC133(+) cells have the capacity to differentiate into ECs as progenitor cells. However, recent studies have demonstrated that circulating CD34(-)CD14(+) cells also have EPC-like properties in vitro and in vivo. We tested whether AC133(-)CD14(+) cells from human umbilical cord blood (HUCB) have the potential to differentiate into ECs. The AC133(-)CD14(+) cells were isolated from HUCB by magnetic bead selection and cultured on fibronectin-coated six-well trays in M199 medium supplemented with fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin growth factor (IGF-1). The AC133(-)CD14(+) cells adhered slightly within 1 day of culture and subsequently underwent a distinct process of morphological transformation to spindle-shaped cells that sprouted from the edge of the cell clusters. After 14 days, the cells formed cord- and tubular-like structures. The AC133(-)CD14(+) cells showed a strong increase in the endothelial marker P1H12 over time, whereas CD14 decreased, and CD45 did not change, respectively. In addition, the cells expressed endothelial markers von Willebrand's factor (vWF), platelet/endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial growth factor receptor-1 (VEGFR-1)/Flt-1, VEGFR-2/Flk-1, eNOS, and VE-cadherin, but did not express Tie-2 after 7 days of culture. The present data indicate that AC133(-)CD14(+) cells from HUCB are able to develop endothelial phenotype with expression of endothelial-specific surface markers and even form cord- and tubular-like structures in vitro as progenitor cells.     

Regulatory dendritic cells program B cells to differentiate into CD19hiFcγIIbhi regulatory B cells through IFN-β and CD40L

Regulatory dendritic cells (DCs) play important roles in the induction of peripheral tolerance and control of adaptive immune response. Our previous studies demonstrate that splenic stroma can drive mature DCs to proliferate and further differentiate into a unique subset of CD11b(hi)Ia(low) regulatory DCs, which could inhibit T-cell response, program generation of immunosuppressive memory CD4 T cells. However, the effect of regulatory DCs on B-cell function remains unclear. Here, we report that regulatory DCs can induce splenic B cells to differentiate into a distinct subtype of IL-10-producing regulatory B cells with unique phenotype CD19(hi)FcγIIb(hi). CD19(hi)FcγIIb(hi) B cells inhibit CD4 T-cell response via IL-10. CD19(hi)FcγIIb(hi) B cells have enhanced phagocytic capacity compared with conventional CD19(+) B cells, and FcγRIIb mediates the uptake of immune complex by CD19(hi)FcγIIb(hi) B cells. We found that regulatory DC-derived IFN-β and CD40 ligand are responsible for the differentiation of CD19(hi)FcγIIb(hi) B cells. Furthermore, an in vivo counterpart of CD19(hi)FcγIIb(hi) B cells in the spleen and lymph nodes with similar phenotype and regulatory function has been identified. Our results demonstrate a new manner for regulatory DCs to down-regulate immune response by, at least partially, programming B cells into regulatory B cells.     

C-kit+ CD45− cells found in the adult human heart represent a population of endothelial progenitor cells

Although numerous reports support the existence of stem cells in the adult heart, few studies have been conducted using human cardiac tissue. Therefore, cells from human cardiac atrial biopsies were analyzed regarding progenitor properties. Expression of stem cell markers was analyzed using fluorescence-activated cell sorting. This identified a small population of C-kit+ cells, which could be further subdivided based on expression of CD45. The C-kit+ CD45+ population was determined to be of mast cell identity, while the C-kit+ CD45− population expressed mRNA of the endothelial lineage. Since the number of cells obtainable from biopsies was limited, a comparison between directly isolated and monolayer and explant cultured cells, respectively, was carried out. While both cultures retained a small population of mast cells, only monolayer culture produced a stable and relatively high percentage of C-kit+ CD45− cells. This population was found to co-express endothelial progenitor cell markers such as CD31, CD34, CXCR4, and FLK-1. The mRNA expression profile was similar to the one from directly isolated cells. When sorted cells were cultured in endothelial differentiation medium, the C-kit+ CD45− population retained its expression of endothelial markers to a large extent, but downregulated progenitor markers, indicating further differentiation into endothelial cells. We have confirmed that the human cardiac atrium contains a small C-kit+ CD45− population expressing markers commonly found on endothelial progenitor cells. The existence of an endothelial progenitor population within the heart might have future implications for developing methods of inducing neovascularization after myocardial infarction.     

Circulating human B and plasma cells. Age-associated changes in counts and detailed characterization of circulating normal CD138? and CD138+ plasma cells

Generation of B and plasma cells involves several organs with a necessary cell trafficking between them. A detailed phenotypic characterization of four circulating B-cell subsets (immature-, naïve-, memory- B-lymphocytes and plasma cells) of 106 healthy adults was realized by multiparametric flow cytometry. We show that CD10, CD27 and CD38 is the minimal combination of subsetting markers allowing unequivocal identification of immature (CD10⁺CD27⁻CD38⁺, 6±6 cells/μL), naïve (CD10⁻CD27⁻CD38⁻, 125±90 cells/μL), memory B lymphocytes (CD10⁻CD27⁺CD38⁻, 58±42 cells/μL), and plasma cells (CD10⁻CD27⁺⁺CD38⁺⁺, 2.1±2.1 cells/μL) within circulating CD19⁺ cells. From these four subsets, only memory B lymphocytes and plasma cells decreased with age, both in relative and absolute counts. Circulating plasma cells split into CD138⁻ (57±12%) and CD138⁺ (43±12%) cells, the latter displaying a more mature phenotypic profile: absence of surface immunoglobulin, lower CD45 positivity and higher amounts of cytoplasmic immunoglobulin, CD38 and CD27. Unlike B lymphocytes, both populations of plasma cells are KI-67⁺ and show weak CXCR4 expression.     

Diurnal variation and effect of insulin on circulating high molecular weight (HMW) adiponectin and NF-κB activity in human endothelial cells

ObjectiveTo study the diurnal variation and the effect of insulin on adiponectin multimers and nuclear factor-kappaB (NF-κB) activity in human endothelial cells.Methods and resultsWe utilized a prolonged insulin–glucose infusion in six healthy human subjects. HMW and total adiponectin levels were higher in the morning and lower at night; NF-κB activities in serum treated human microvascular endothelial cells (HMEC-1) cells were lower in the morning and higher at night. Hyperinsulinemic induction significantly decreased HMW and total adiponectin levels but increased NF-κB activity in serum treated HMEC-1 cells (P < 0.05, P < 0.01). There were no significant changes to MMW and LMW adiponectin levels (P > 0.05).ConclusionCircadian rhythm of HMW adiponectin and NF-κB activity are altered by hyperinsulinemia providing novel insights adiponectin and NF-κB biology, which may be pertinent to insulin resistant states, e.g. obesity and type 2 diabetes mellitus.     

Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β1

AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-7721 cells and its contribution to this adhesive course. METHODS: Adhesive force of SMMC-7721 cells to endothelial cells was measured using micropipette aspiration technique. Synchronous G1 and S phase SMMC-7721 cells were achieved by thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronous rates of SMMC-7721 cells and expression of integrinβ1 in SMMC-7721 cells were detected by flow cytometer. RESULTS: The percentage of cell cycle phases of general SMMC-7721 cells was 11.01% in G2/M phases, 53.51% in G0/G1 phase, and 35.48% in S phase. The synchronous rates of G1 and S phase SMMC-7721 cells amounted to 74.09% and 98.29%, respectively. The adhesive force of SMMC-7721 cells to endothelial cells changed with the variations of adhesive time and presented behavior characteristics of adhesion and de-adhesion. S phase SMMC-7721 cells had higher adhesive forces than G1 phase cells [(307.65&#177;92.10)&#215;10^-10N vs (195.42&#177;60.72)&#215;10^-10N, P&lt;0.01]. The expressive fluorescent intensity of integrinβ1 in G1 phase SMMC-7721 cells was depressed more significantly than the values of S phase and general SMMC-7721cells. The contribution of adhesive integrinβ1 was about 53% in this adhesive course. CONCLUSION: SMMC-7721 cells can be synchronized preferably in G1 and S phases with thymine-2-deoxyriboside and colchicines. The adhesive molecule integrinβ1 expresses a high level in SMMC-7721 cells and shows differences in various cell cycles, suggesting integrin β1 plays an important role in adhesion to endothelial cells. The change of adhesive forces in different cell cycle SMMC-7721 cells indicates that S phase cells play predominant roles possibly while they interact with endothelial cells.     

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 pancreaticductal stem cells in vitro,and to observe the potency of thesemultipotential cells differentiation into insulin-producingcells.METHODS: The human fetal pancreas was digested by 1 g/Lcollagease type Ⅳ and then 2.5 g/L trypsin was used toisolate the pancreatic ductal stem cells,followed by culturein serum-free,glucose-free DMEM media with some additionalchemical substrates in vitro (according to the differentstage).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 usingimmunocytochemical staining.RESULTS: The shape of human fetal pancreatic ductal stemcells cultured in vitro was firstly fusiform in the first 2 wk,and became monolayer and cobblestone pattern afteranother 3 to 4 wk.After induced and differentiated by theglucose of different concentrations for another 1 to 2 wk,the cells formed the pancreatic islet-like structures.Theidentification and potency of these cells were then identifiedby using the pancreatic ductal stem cell marker,cytokeratin-19 (CK-19),pancreatic β cell marker,insulin and pancreaticα cell marker,glucagons with immunocytochemical staining.At the end of the second week,95.2% of the cells werepositive for CK-19 immunoreactivity.Up to 22.7% of thecells induced by glucose were positive for insulinirnrnunoreactivity,and less than 3.8% of the cells werepositive for glucagon immunoreactivity in pancreatic islet-like structures.The positive ratio of immunoreactive stainingwas dependent on the concentration of glucose,and itwas observed that the 17.8 mmol/L glucose stimulatedeffectively to produce insulin- and glucagons-producing cells.CONCLUSION: The human fetal pancreatic ductal stemcells are capable of proliferation in vitro.These cells havemultidifferentiation potential and can be induced by glucoseand differentiated into insulin-producing cells in vitro.     

TFPIβ is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes

Tissue factor pathway inhibitor (TFPI) produces factor Xa-dependent feedback inhibition of factor VIIa/tissue factor-induced coagulation. Messages for 2 isoforms of TFPI have been identified. TFPIα mRNA encodes a protein with an acidic N-terminus, 3 Kunitz-type protease inhibitor domains and a basic C-terminus that has been purified from plasma and culture media. TFPIβ mRNA encodes a form in which the Kunitz-3 and C-terminal domains of TFPIα are replaced with an alternative C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPIβ protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment is TFPIα, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPIβ based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and (3) it contains a GPI anchor. Immunoassays demonstrate that, although endothelial cells secrete TFPIα, greater than 95% of the TFPI released by PIPLC treatment from the surface of endothelial cells and from placental microsomes is TFPIβ.     

Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β_1

AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-7721 cells and its contribution to this adhesive course. METHODS: Adhesive force of SMMC-7721 cells to endothelial cells was measured using micropipette aspiration technique. Synchronous G1 and S phase SMMC-7721 cells were achieved by thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronous rates of SMMC-7721 cells and expression of integrinβ1 in SMMC-7721 cells were detected by flow cytometer. RESULTS: The percentage of cell cycle phases of general SMMC-7721 cells was 11.01% in G2/M phases, 53.51% in G0/G1 phase, and 35.48% in S phase. The synchronous rates of G1 and S phase SMMC-7721 cells amounted to 74.09% and 98.29%, respectively. The adhesive force of SMMC-7721 cells to endothelial cells changed with the variations of adhesive time and presented behavior characteristics of adhesion and de-adhesion. S phase SMMC-7721 cells had higher adhesive forces than d phase cells [(307.65±92.10)×10-10N vs(195.42±60.72)×10-10N, P<0.01]. The expressive fluorescent intensity of integrinβ1 in G1 phase SMMC-7721 cells was depressed more significantly than the values of S phase and general SMMC-7721cells. The contribution of adhesive integrinβ1 was about 53% in this adhesive course. CONCLUSION: SMMC-7721 cells can be synchronized preferably in d and S phases with thymine-2-deoxyriboside and colchicines. The adhesive molecule integrinβ1 expresses a high level in SMMC-7721 cells and shows differences in various cell cycles, suggesting integrin β1 plays an important role in adhesion to endothelial cells. The change of adhesive forces in different cell cycle SMMC-7721 cells indicates that S phase cells play predominant roles possibly while they interact with endothelial cells.     

Characterization of CD4 and CD8 T cells producing IFN-γ in human latent and active tuberculosis

Patients with pulmonary tuberculosis (PTB) frequently have reduced IFN-γ production in response to mycobacterial antigens, compared to individuals with latent Mycobacterium tuberculosis infection (LTBi). However, it is not clear whether this reduced responsiveness is restricted to a particular T cell subset. Herein, PBMCs from 26 PTB patients, 30 household contacts (HHCs) of PTB, and 30 tuberculin positive (TST+) healthy subjects not recently exposed to PTB, were stained with CFSE and stimulated non-specific (PPD) for 120?h, and specific (CFP-10/ESAT-6) and latency (HSpX) mycobacterial antigens for 144?h and the percentage of CD4(+) and CD8(+)IFN-γ(+) T cells responding determined by flow cytometry, in addition to their memory phenotype by the CD45RO and CD27 expression. PTB had decreased frequency of both CD4(+) and CD8(+) precursor cells, as well as decreased number of CD4(+)IFN-γ(+) cells in response to all antigens, whereas CD8(+)IFN-γ(+) cells were decreased in response to PPD and ESAT-6, but not to CFP-10 and HSpX. HHCs exhibited the highest precursor frequencies and IFN-γ responses, irrespective of the antigen employed. The CD4(+)/CD8(+) cell ratios showed that in response to PPD CD4(+) precursor and IFN-γ-producer cells are more frequent than their CD8(+) counterparts, and that PTB have a decreased CD4(+)IFN-γ(+)/CD8(+)IFN-γ(+) ratio in response to PPD, CFP-10, and ESAT-6. CD4(+)IFN-γ(+) and CD8(+)IFN-γ(+) cells exhibited a central memory phenotype (CD45RO(+)CD27(+)), irrespective of the group of subjects and the antigen used for stimulation. In conclusion, PTB patients had a decreased percentage of CD4(+) and CD8(+) precursor cells and CD4(+)IFN-γ(+). HHCs exhibited the highest frequency of CD4(+) and CD8(+) precursors and CD4(+)IFN-γ(+)-producing cells.     

ISHAGE-based single-platform flowcytometric analysis for measurement of absolute viable T cells in fresh or cryopreserved products: CD34/CD133 selected or CD3/CD19 depleted stem cells, DLI and purified CD56+CD3− NK cells

Considering the growing use of immunotherapeutic strategies in paediatric stem cell transplantation associated with risk of graft-versus-host disease, an accurate method for the enumeration of residual T cells/kg recipient’s body weight is of paramount importance. Therefore, we propose a multi colour-flow cytometric strategy for correct absolute vital T cell enumeration in manipulated cell preparations for clinical use. The gating strategy is based on the ISHAGE single-platform stem cell enumeration method in combination with experiences from lymphocyte subtyping, using low scatter, high expression of CD3 and CD45 antigens and 7-AAD staining in a no-wash-preparation with counting beads. In spiking experiments, the detection limit was determined to be at 0.7 ± 0.5 CD3⁺ cells/μl with a minimum of 50 T cell events acquired. The cell preparations analysed contained a median absolute CD3⁺ T cell number of 221 × 10³ (0.09%, CD34 selected grafts, n = 187), 900 × 10³ (0.004%, CD3/CD19 depleted grafts, n = 15) and 283 × 10³ (0.012%, CD3 depleted/CD56 enriched NK-cells, n = 14), respectively. The results differed of those from conventional T cell measurement in cell products after extensive manipulation. Our method provides reliable residual T cell enumeration even at extremely low concentrations. U. Koehl and K. Bochennek contributed equally to the work.     

Lymphotropic HCV strain can infect human primary na?ve CD4+ cells and affect their proliferation and IFN-�� secretion activity

Background

Lymphotropic hepatitis C virus (HCV) infection of B and T cells might play an important role in the pathogenesis of hepatitis C. Recently, we showed that a lymphotropic HCV (SB strain) could infect established T-cell lines and B-cell lines. However, whether HCV replication interferes with cell proliferation and function in primary T lymphocytes is still unclear.

Aim

The aim of this study was to analyze whether HCV replication in primary T lymphocytes affected their development, proliferation, and Th1 commitment.

Methods

SB strain cell culture supernatant (2?×?10⁴?copies/ml HCV) was used to infect several kinds of primary lymphocyte subsets. Mock, UV-irradiated SB-HCV, JFH-1 strain, and JFH-1 NS5B mutant, which could not replicate in T cells, were included as negative controls. Carboxyfluorescein succinimidyl ester (CFSE) and CD45RA double staining was used to evaluate the proliferative activity of CD4⁺CD45RA⁺CD45RO^? na?ve CD4⁺ cells. Interferon (IFN)-?? and interleukin (IL)-10 secretion assays magnetic cell sorting (MACS) were carried out.

Results

Negative strand HCV RNA was detected in CD4⁺, CD14⁺, and CD19⁺ cells. Among CD4⁺ cells, CD4⁺CD45RA⁺RO^? cells (na?ve CD4⁺ cells) were most susceptible to replication of the SB strain. The levels of CFSE and CD45RA expression gradually declined during cell division in uninfected cells, while HCV-infected na?ve CD4⁺ cells expressed higher levels of CFSE and CD45RA than Mock or UV-SB infected na?ve CD4⁺ cells. Moreover, the production of IFN-?? was significantly suppressed in SB-infected na?ve CD4⁺ cells.

Conclusions

Lymphotropic HCV replication suppressed proliferation and development, including that towards Th1 commitment, in human primary na?ve CD4⁺ cells.