Effect of all-trans-retinoic acid on the differentiation, maturation and functions of dendritic cells derived from cord blood monocytes

We investigated the effects of all-trans-retinoic acid on dendritic cells derived from human cord blood monocytes to clarify how vitamin A affects immune function in children. Monocytes were separated from 18 cord blood samples, and dendritic cells were differentiated by culture. The percentage of dendritic cells was markedly lower in all-trans-retinoic acid treated cells than in untreated cells. After exposure to tumour necrosis factor-alpha for 3 days, all-trans-retinoic acid treated dendritic cells showed a reduced capacity to activate alloreactive T cells compared to untreated cells. In addition, all-trans-retinoic acid-treated dendritic cells could drive T cells towards T-helper cell type 2 responses with decreased secretion of interleukin-12, interferon-gamma, and increased production of interleukin-10 and interleukin-4. However, when Ro 41-5253, a selective retinoic acid receptor alpha antagonist, was add to culture, all the above actions were reversed. Thus, all-trans-retinoic acid may act at the first step of the immune response by inhibiting the differentiation of dendritic cells, maturation and induction of the T-helper cell type-2 response. The actions of all-trans-retinoic acid on dendritic cells were mediated through retinoic acid receptor alpha.     

Gangliosides from human melanoma tumors impair dendritic cell differentiation from monocytes and induce their apoptosis

Gangliosides are ubiquitous membrane-associated glycosphingolipids, which are involved in cell growth and differentiation. Most tumor cells synthesize and shed large amounts of gangliosides into their microenvironment, and many studies have unraveled their immunosuppressive properties. In the present study we analyzed the effects of GM3 and GD3 gangliosides, purified from human melanoma tumors, on the differentiation of monocyte-derived dendritic cells (MoDC). At concentrations close to those detected in the sera from melanoma patients, both gangliosides dose-dependently inhibit the phenotypic and functional differentiation of MoDC, as assessed by a strong down-regulation of CD1a, CD54, CD80, and CD40 Ags and impaired allostimulatory function on day 6 of culture. Furthermore, GM3 and GD3 gangliosides decreased the viable cell yield and induced significant DC apoptosis. Finally, addition of GD3 to differentiating DC impaired their subsequent maturation induced by CD154. The resulting DC produced low amounts of IL-12 and large amounts of IL-10, a cytokine pattern that might hamper an efficient antitumor immune response. In conclusion, the results demonstrate that gangliosides impair the phenotypic and functional differentiation of MoDC and induce their apoptosis, which may be an additional mechanism of human melanoma escape.     

Renal ischemia/reperfusion injury inhibits differentiation of dendritic cells derived from bone marrow monocytes in rats

Dendritic cells (DCs) are impacted by surgical injury, exercise, and other physiological stressors. This study aims to determine whether renal I/R injury affects 1) the differentiation of myeloid DCs from bone marrow monocytes (BMMos) and the maturation and activation state of these DCs and 2) DC infiltration of kidney. Sprague-Dawley rats were subjected to I/R injury or sham-operated. Creatinine clearance was monitored daily during the 14 d of reperfusion that followed the ischemic insult. At 2 and 14 d of reperfusion, the following were assessed 1) properties of BMMo-derived DCs (i.e., the amount of generated DCs, differentiation state markers [CD11c, CD80, CD86, and Ia], and functional state [MLR and amount of IL-12 produced]), and 2) the presence of DCs in the kidney. Numbers of BMMo-derived DCs were significantly decreased in the I/R injured group (compared with the sham-operated group) at 2 d but not 14 d. A comparison of the their functionality found mixed lymphocyte response [MLR] and IL-12 production were similar in the two groups at both time points. Also, immunohistochemistry showed infiltrating DCs in the outer medulla of the I/R injured kidney at 2 d but not 14 d of reperfusion. Thus, I/R stress reduces the number of DCs differentiated from BMMos but not the functional activity of these DCs. This decrease may reflect a stress-induced downshift in the capacity of BMMos to differentiate into DCs and a parallel upshift in the capacity of DCs to infiltrate the kidney.     

Neural precursors derived from human embryonic stem cells

Human embryonic stem (hES) cells provide a promising supply of specific cell types for transplantation therapy. We presented here the method to induce differentiation of purified neural precursors from hES cells. hES cells (Line PKU-1 and Line PKU-2) were cultured in suspension in bacteriological Petri dishes, which differentiated into cystic embryoid bodies (EBs). The EBs were then cultured in N₂ medium containing bFGF in poly-L-lysine-coated tissue culture dishes for two weeks. The central, small cells with 2–3 short processes of the spreading outgrowth were isolated mechanically and replated. The resulting neurospheres were cultured in suspension for 10 days, then dissociated into single cell suspension with a Pasteur pipette and plated. Cells grew vigorously in an attached way and were passed every 4–5 days. Almost all the cells were proved nestin positive by immunostaining. Following withdrawal of bFGF, they differentiated into neurons expressing β-tubulin isotype_III, GABA, serotonin and synaptophysin. Through induction of PDGF-AA, they differentiated into astrocytes expressing GFAP and oligodendrocytes expressing O₄. The results showed that hES cells can differentiate into typical neural precursors expressing the specific marker nestin and capable of generating all three cell types of the central nervous system (CNS)in vitro.     

Neural precursors derived from human embryonic stem cells

Before the successful isolation of human embryonic stem (hES) cells, many investigations had shown that mouse embryonic stem (mES) cells can be induced to differentiate into neural precursors which could be purified and differentiated to mature dopamine, motor, serotonin, GABA neurons, and oligodendrocytes and astrocytes in vitro[1―3]. mES cell-derived dopamine neurons have been shown capable of integrating into host brains after transplanting to the rodents of Park-inson’s disease model …     

Endothelial-like cells derived from human CD14 positive monocytes

In the present study, we show that endothelial-like cells (ELCs) can develop from human CD14-positive mononuclear cells (CD14 cells) in the presence of angiogenic growth factors. The CD14 cells became loosely adherent within 24 h of culture and subsequently underwent a distinct process of morphological transformation to caudated or oval cells with eccentric nuclei. After 1 week in culture the cells showed a clear expression of endothelial cell markers, including von Willebrand factor (vWF), CD144 (VE-cadherin), CD105 (endoglin), acetylated low-density lipoprotein (AC-LDL)-receptor, CD36 (thrombospondin receptor), FLT-1, which is vascular endothelial cell growth factor (VEGF) receptor-1, and, to a weaker extent, KDR (VEGF receptor-2). Furthermore, in these cells structures resembling Weibel-Palade bodies at different storage stages were identified by electron microscopy, and upon culturing on three-dimensional fibrin gels the cells build network-like structures. In addition, cell proliferation and vWF expression was stimulated by VEGF, and the endothelial cell adhesion molecules CD54 (ICAM-1), and CD106 (VCAM-1) became transiently inducible by tumor necrosis factor-alpha (TNF-alpha). In contrast, the dendritic markers CD1a, and CD83 were not expressed to any significant extent. The expression of CD68, CD80 (B7-1), CD86 (B7-2), HLA-DR and CD36 may also suggest that ELCs might be related to macrophages, sinus lining or microvascular endothelial cells. Taken together, our observations indicate that ELCs can differentiate from cells of the monocytic lineage, suggesting a closer relationship between the monocyte/macrophage- and the endothelial cell systems than previously supposed.     

Accessory cells, dendritic cells, or monocytes, are required for the lymphokine-activated killer cell induction from resting T cell but not from natural killer cell precursors

In this study we have investigated the role of accessory cells in the development of lymphokine-activated killer cells (LAK) from highly purified human NK and small resting T cell progenitors. As accessory cells we used autologous, as well as allogeneic, monocytes, and dendritic cell enriched cells. Both NK and T cells were able to generate LAK activity, but their activation requirements were different. NK cells were activated merely by IL-2, and accessory cells did not enhance their lytic activity in the presence or absence of IL-2. Conversely, T cells were practically unresponsive to even high concentrations of IL-2 having a strict requirement for accessory cells for the development of lytic activity and proliferation. Accessory cells differed in their ability to activate T cells presumably depending on their ability to induce IL-2 synthesis, allogeneic dendritic cells being the most effective accessory cells and IL-2 synthesis stimulators. Allogeneic accessory cells could induce lytic activity in T cells even in the absence of exogenous IL-2. Thus, accessory cells play a central role in expanding the LAK effector cell population.     

Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain

Human embryonic stem (hES) cells provide a potentially unlimited cell source for regenerative medicine. Recently, differentiation strategies were developed to direct hES cells towards neural fates in vitro. However, the interaction of hES cell progeny with the adult brain environment remains unexplored. Here we report that hES cell-derived neural precursors differentiate into neurons, astrocytes and oligodendrocytes in the normal and lesioned brain of young adult rats and migrate extensively along white matter tracts. The differentiation and migration behavior of hES cell progeny was region specific. The hES cell-derived neural precursors integrated into the endogenous precursor pool in the subventricular zone, a site of persistent neurogenesis. Like adult neural stem cells, hES cell-derived precursors traveled along the rostral migratory stream to the olfactory bulb, where they contributed to neurogenesis. We found no evidence of cell fusion, suggesting that hES cell progeny are capable of responding appropriately to host cues in the subventricular zone.     

A subset of myeloid dendritic cells derived from peripheral blood monocytes represented a predominant subset characterized by their potential tumor-inhibiting activity

Besides their role as potent antigen-presenting cells, myeloid dendritic cells (MDCs), but not plasmacytoid dendritic cells (PDCs), have been reported to have cytotoxic or cytostatic activity on some tumor cells. In this article, we analyzed the tumoristatic potential of a distinct peripheral blood monocyte-derived MDC subset which co-expressed PDC-specific marker CD123. CD123⁺ MDCs represented a subset of small-sized DCs and accounted for 45–60% of peripheral blood monocytes cultured with granulocyte-macrophage colony-stimulating factor and interleukine-4 (IL-4) for 7 d. They exhibited more significant antiproliferative activity toward hematological tumor cell lines of Jurkat, HL60, and myelodysplastic syndromes over-leukemia than CD123⁻ MDCs even at a low effecter/target ratio. Pretreatment of MDC and their supernatant with TRAIL-R2:Fc significantly reduced the tumoristatic effect of CD123⁺ MDCs but not of CD123⁻ MDCs and their supernatant. CD123⁺ MDCs expressed higher level of cytoplasmic TNF-α-related apoptosis-inducing ligand (TRAIL) than CD123⁻ MDCs, whereas both expressed very little surface and soluble TRAIL. These results reveal that CD123⁺ cells represented a predominant subset of MDCs generated from peripheral blood monocytes in vitro, characterized by their potential tumoristic activity partially via cytoplasmic TRAIL.     

Biophysical studies on the differentiation of human CD14+ monocytes into dendritic cells

Dendritic cells (DCs), which are the most efficient antigen-presenting cells (APCs) currently known, can be derived from CD14⁺ monocytes (DC predecessor cells) in vitro. Immature DCs actively take up antigens and pathogens, generate major histocompatability complex-peptide complexes, and migrate from the sites of antigen acquisition to secondary lymphoid organs to become mature dendritic cells that interact with and stimulate T-lymphocytes. During this process, the cells must undergo deformation to translocate through several barriers, including the basement membrane and interstitial connective tissue in the blood vessel wall. To further understand the mechanisms of the activation of immunological responses and the migration from peripheral tissue to secondary lymphoid organs, we have applied biophysical and microrheological methods to study the development processes of DCs in vitro. The results showed that membrane fluidity, osmotic fragility, membrane viscoelastic properties, infrared spectroscopy, and cytoskeleton organization of DCs exhibit significant differences in different developmental stages. These authors contributed equally to this work.     

Identification of novel dendritic cell subset markers in human blood

Human dendritic cells (DC) are key regulators of innate and adaptive immunity that can be divided in at least three major subpopulations: plasmacytoid DC (pDC), myeloid type 1 DC (mDC1) and myeloid type 2 DC (mDC2) exhibiting different functions. However, research, diagnostic and cell therapeutic studies on human DC subsets are limited because only few DC subset markers have been identified so far. Especially mDC2 representing the rarest blood DC subset are difficult to be separated from mDC1 and pDC due to a paucity of mDC2 markers. We have combined multiparameter flow cytometry analysis of human blood DC subsets with systematic expression analysis of 332 surface antigens in magnetic bead-enriched blood DC samples. The initial analysis revealed eight novel putative DC subset markers CD26, CD85a, CD109, CD172a, CD200, CD200R, CD275 and CD301 that were subsequently tested in bulk peripheral blood mononuclear cell (PBMC) samples from healthy blood donors. Secondary analysis of PBMC samples confirmed three novel DC subset markers CD26 (dipeptidyl peptidase IV), CD85a (Leukocyte immunoglobulin-like receptor B3) and CD275 (inducible costimulator ligand). CD85a is specifically expressed in mDC1 and CD26 and CD275 represent novel mDC2 markers. These markers will facilitate human DC subset discrimination and additionally provide insight into potentially novel DC subset-specific functions.     

EPN: a novel epithelial cell line derived from human prostate tissue

This work reports the isolation and characterization of a line of human, nontransformed and differentiated prostate epithelial cells (EPN) in continuous culture. Primary cultures of epithelial prostate cells were set up using normal tissue isolated from a prostate sample collected after radical prostatectomy for cancer. After 70 passages, EPN cells did not undergo "Hayflike crisis" and were free of fibroblast contamination and were thus subcloned and characterized. EPN cells in culture, as prostate epithelial cells in vivo, express high-molecular weight cytokeratin and Pyk2, whereas they do not express desmin. EPN cells are nontransformed because they do not form colonies in semisolid medium and do not form tumors once injected into nude mice. EPN cells express the functional androgen receptor, which can mediate the mitogenic activity of testosterone. Finally, clonal production of the prostate-specific antigen could be detected in EPN cells. The availability of a line of epithelial nontransformed prostate cell in culture will be useful in investigating the complex process regulating normal prostate physiology as well as the development and progression of prostate tumors.     

Human CD141+ dendritic cells generated from adult peripheral blood monocytes

Human CD141⁺ dendritic cells (DCs), specialized for cross-presentation, have been extensively studied in the development of DC-based therapy against cancer. A series of attempts was made to generate CD141⁺ DCs from cord blood CD34⁺ hematopoietic progenitors to overcome the practical limitation of in vivo rareness. In the present study, we identified a culture system that generates high CD141⁺ DCs. After culture of CD14⁺ monocytes in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 8 days, CD141 was detected on cells that adhered to the bottom of the culture plate. The attached cells exhibited typical features of immature monocyte-derived DCs (moDCs), except for higher CD86 expression, more dendrites and higher granularity compared with those that did not attach. With 3 additional days of culture, increased CD141 expression on the cells was retained along with adhesion ability and partial expression of CLEC9A, a c-type lectin receptor. Furthermore, the cells exhibited effective uptake of dead cells. Interestingly, the attached moDCs differently responded to polyinosinic:polycytidylic acid (poly I:C) stimulation as well as a mixed lymphocyte reaction. Collectively, our findings show that human CD141⁺ DCs can be sufficiently generated from peripheral blood CD14⁺ monocytes, potentiating further investigation into generation of higher yields of cross-priming human DCs in vitro.     

HLA-DR-presented peptide repertoires derived from human monocyte-derived dendritic cells pulsed with blood coagulation factor VIII

Activation of T-helper cells is dependent upon the appropriate presentation of antigen-derived peptides on MHC class II molecules expressed on antigen presenting cells. In the current study we explored the repertoire of peptides presented on MHC class II molecules on human monocyte derived dendritic cells (moDCs) from four HLA-typed healthy donors. MHC class II-bound peptides could be routinely recovered from small cultures containing 5 × 10(6) cells. A fraction of the identified peptides were derived from proteins localized in the plasma membrane, endosomes, and lysosomes, but the majority of peptides that were presented on MHC class II originate from other organelles. Subsequently, we studied the antigen-specific peptide repertoire after endocytosis of a soluble antigen. Blood coagulation factor VIII (FVIII) was chosen as the antigen since our current knowledge on MHC class II presented peptides derived from this immunogenic therapeutic protein is limited. Analysis of the total repertoire of MHC class II-associated peptides revealed that per individual sample 20-50 FVIII-derived peptides were presented on FVIII-pulsed moDCs. Repertoires of FVIII-derived peptides eluted from moDCs derived from a panel of four HLA typed donors revealed that some MHC class II-presented FVIII peptides were presented by multiple donors, whereas the presentation of other FVIII peptides was donor-specific. In total 32 different core peptides were presented on FVIII-pulsed moDCs from four HLA-typed donors. Together our findings provide an unbiased approach to identify peptides that are presented by MHC class II on antigen-loaded moDCs from individual donors.     

Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood

Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells, with practical and ethical advantages. To date, the presence of other stem cells in UCB remains to be established. We investigated whether other stem cells are present in cryopreserved UCB. Seeded mononuclear cells formed adherent colonized cells in optimized culture conditions. Over a 4- to 6-week culture period, colonized cells gradually developed into adherent mono-layer cells, which exhibited homogeneous fibroblast-like morphology and immunophenotypes, and were highly proliferative. Isolated cells were designated 'multipotent progenitor cells (MPCs)'. Under appropriate conditions for 2 weeks, MPCs differentiated into neural tissue-specific cell types, including neuron, astrocyte, and oligodendrocyte. Differentiated cells presented their respective markers, specifically, NF-L and NSE for neurons, GFAP for astrocytes, and myelin/oligodendrocyte for oligodendrocytes. In this study, we successfully isolated MPCs from cryopreserved UCB, which differentiated into the neural tissue-specific cell types. These findings suggest that cryopreserved human UCB is a useful alternative source of neural progenitor cells, such as MPCs, for experimental and therapeutic applications.