CD36 and macrophage scavenger receptor a modulate foam cell formation via inhibition of lipid-laden platelet phagocytosis

CD34 (+) progenitor cells are a promising source of regeneration in atherosclerosis or ischemic heart disease. However, as recently published, CD34(+) progenitor cells have the potential to differentiate not only into endothelial cells but also into foam cells upon interaction with platelets. The mechanism of platelet-induced differentiation of progenitor cells into foam cells is as yet unclear. In the present study we investigated the role of scavenger receptor (SR)-A and CD36 in platelet-induced foam cell formation. Human CD34(+) progenitor cells were freshly derived from human umbilical veins and were co-incubated with platelets (2 x 10(8)/mL) up to 14 days resulting in large lipid-laden foam cells. Developing macrophages expressed SR-A, CD36, and Lox-1 as measured by fluorescent-activated cell sorting analysis. The presence of a blocking anti-CD36 or anti-SR-A antibody nearly abrogated foam cell formation, whereas anti-Lox-1 did not affect foam cell formation. Consistently blocking either anti-CD36 or anti-SR-A antibody significantly reduced the phagocytosis of lipid-laden platelets by macrophages. We conclude that CD36 and SR-A play an important role in platelet-induced foam cell formation from CD34(+) progenitor cells and thus represent a promising target to inhibit platelet-induced foam cell formation.

Controlled angiogenesis in the heart by cell-based expression of specific vascular endothelial growth factor levels

Vascular endothelial growth factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell because it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput fluorescence-activated cell sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose tissue-derived stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8) or CD8 alone as control (CTR). VEGF-expressing cells were FACS-purified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intramyocardial injection of 10(7) cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF, and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.

Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense

Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl(4)-induced ascitic cirrhosis and 2-day portal vein-ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth cell α-cryptdins and β-defensins was determined by real-time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence-activated cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth cell α-cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver-gut axis including the underlying mechanisms could help us to find new treatment avenues.

Cardiac c-kit+AT2+ cell population is increased in response to ischemic injury and supports cardiomyocyte performance

The expression pattern of angiotensin AT2 receptors with predominance during fetal life and upregulation under pathological conditions during tissue injury/repair process suggests that AT2 receptors may exert an important action in injury/repair adaptive mechanisms. Less is known about AT2 receptors in acute ischemia-induced cardiac injury. We aimed here to elucidate the role of AT2 receptors after acute myocardial infarction. Double immunofluorescence staining showed that cardiac AT2 receptors were mainly detected in clusters of small c-kit+ cells accumulating in peri-infarct zone and c-kit+AT2+ cells increased in response to acute cardiac injury. Further, we isolated cardiac c-kit+AT2+ cell population by modified magnetic activated cell sorting and fluorescence activated cell sorting. These cardiac c-kit+AT2+ cells, represented approximately 0.19% of total cardiac cells in infarcted heart, were characterized by upregulated transcription factors implicated in cardiogenic differentiation (Gata-4, Notch-2, Nkx-2.5) and genes required for self-renewal (Tbx-3, c-Myc, Akt). When adult cardiomyocytes and cardiac c-kit+AT2+ cells isolated from infarcted rat hearts were cocultured, AT2 receptor stimulation in vitro inhibited apoptosis of these cocultured cardiomyocytes. Moreover, in vivo AT2 receptor stimulation led to an increased c-kit+AT2+ cell population in the infarcted myocardium and reduced apoptosis of cardiomyocytes in rats with acute myocardial infarction. These data suggest that cardiac c-kit+AT2+ cell population exists and increases after acute ischemic injury. AT2 receptor activation supports performance of cardiomyocytes, thus contributing to cardioprotection via cardiac c-kit+AT2+ cell population.

High aldehyde dehydrogenase activity identifies tumor-initiating and metastasis-initiating cells in human prostate cancer

Metastatic progression of advanced prostate cancer is a major clinical problem. Identifying the cell(s) of origin in prostate cancer and its distant metastases may permit the development of more effective treatment and preventive therapies. In this study, aldehyde dehydrogenase (ALDH) activity was used as a basis to isolate and compare subpopulations of primary human prostate cancer cells and cell lines. ALDH-high prostate cancer cells displayed strongly elevated clonogenicity and migratory behavior in vitro. More strikingly, ALDH-high cells readily formed distant metastases with strongly enhanced tumor progression at both orthotopic and metastatic sites in preclinical models. Several ALDH isoforms were expressed in human prostate cancer cells and clinical specimens of primary prostate tumors with matched bone metastases. Our findings suggest that ALDH-based viable cell sorting can be used to identify and characterize tumor-initiating and, more importantly perhaps, metastasis-initiating cells in human prostate cancer.

Placental mesenchymal stem cells as potential autologous graft for pre- and perinatal neuroregeneration

OBJECTIVE: Mesenchymal stem cells (MSCs) have a broad differentiation potential. We aimed to determine if MSCs are present in fetal membranes and placental tissue and to assess their potential to differentiate into neurogenic and mesodermal lineages. STUDY DESIGN: MSCs isolated from first and third trimester chorion and amnion and first trimester chorionic villi and characterized morphologically and by flourescence-activated cell sorting analysis. Their ability to mature under different culture conditions into various cells of mesodermal and neuroectodermal cell lines was assessed by immuno- and cytochemical staining. RESULTS: Independent of gestational age, cells isolated from fetal membranes and placenta showed typical MSC phenotype (positive for CD166, CD105, CD90, CD73, CD49e, CD44, CD29, CD13, MHC I; negative for CD14, CD34, CD45, MHC II) and were able to differentiate into mesodermal cells expressing cell markers/cytologic staining consistent with mature chondroblasts, osteoblasts, adipocytes, or myocytes and into neuronal cells presenting markers of various stages of maturation. The differentiation pattern was mainly dependent on cell type. CONCLUSION: Mesenchymal cells from chorion, amnion, and villous stroma can be differentiated into neurogenic, chondrogenic, osteogenic, adipogenic, and myogenic lineage. Placental tissue obtained during prenatal chorionic villous sampling or at delivery might be an ideal source for autologous stem cell graft for peripartum neuroregeneration and other clinical issues.

Pregnancy induces numerical and functional changes of CD4+CD25 high regulatory T cells in patients with rheumatoid arthritis

OBJECTIVE: In a prospective study we investigated whether numerical and functional changes of CD4+CD25(high) regulatory T cells (Treg) were associated with changes of disease activity observed during pregnancy and post partum in patients with rheumatoid arthritis (RA). METHODS: The frequency of CD4+CD25(high) T cells was determined by flow cytometry in 12 patients with RA and 14 healthy women during and after pregnancy. Fluorescence-activated cell sorting (FACS) was used to sort CD4+CD25(high) T cells and CD4+CD25- T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies alone or in co-culture to investigate proliferation and cytokine secretion. RESULTS: Frequencies of CD4+CD25(high) Treg were significantly higher in the third trimester compared to 8 weeks post partum in patients and controls. Numbers of CD4+CD25(high) Treg inversely correlated with disease activity in the third trimester and post partum. In co-culture experiments significantly higher amounts of IL10 and lowered levels of tumour necrosis factor (TNF)alpha and interferon (IFN)gamma were found in supernatants of the third trimester compared to postpartum samples. These findings were independent from health or disease in pregnancy, however postpartum TNFalpha and IFN gamma levels were higher in patients with disease flares. CONCLUSION: The amelioration of disease activity in the third trimester corresponded to the increased number of Treg that induced a pronounced anti-inflammatory cytokine milieu. The pregnancy related quantitative and qualitative changes of Treg suggest a beneficial effect of Treg on disease activity.

Pregnancy in patients with ankylosing spondylitis: do regulatory T cells play a role?

OBJECTIVE: To investigate the numerical and functional changes of CD4+CD25(high) regulatory T (Treg) cells during pregnancy and postpartum in patients with ankylosing spondylitis (AS). METHODS: The frequency of CD4+CD25(high) T cells was determined by flow cytometry in 10 pregnant and 5 nonpregnant patients with AS as well as in 14 pregnant and 4 nonpregnant healthy controls. Pregnant individuals were investigated at the third trimester and 8 weeks postpartum. Treg cells and CD4+CD25- effector T (Teff) cells separated by fluorescence-activated cell sorting were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies, alone or in coculture, to investigate proliferation and cytokine secretion. RESULTS: The frequency of CD4+CD25(high) Treg cells was significantly higher during pregnancy than postpartum in both healthy control subjects and patients with AS. In contrast to Treg cells in healthy pregnant women, Treg cells in pregnant women with AS secreted only small amounts of interleukin-10 and showed lower suppression of tumor necrosis factor alpha and interferon-gamma secretion by CD4+CD25- Teff cells. At the postpartum time point, proinflammatory cytokine levels in the Treg/Teff cell cocultures and Teff cell monocultures were significantly higher in patients with AS than in healthy controls. CONCLUSION: Pregnancy influenced the expansion and cytokine secretion of Treg cells in both patients with AS and control subjects. However, the Treg cells of pregnant patients with AS failed to support an antiinflammatory cytokine milieu, thereby possibly contributing to the persistent disease activity of AS during pregnancy.

Isotype-specific detection of ABO blood group antibodies using a novel flow cytometric method

Several methods to detect anti-A/B antibodies based on haemagglutination and haemolysis have been described. These methods measure predominantly anti-A/B immunoglobulin (Ig)M, whereas anti-A/B IgG and IgG subclasses are less well examined. We established a flow cytometry method (ABO-fluorescence-activated cell sorting; ABO-FACS) to quantify binding of anti-A/B IgM, IgG and IgG subclasses to human A or B red blood cells. Anti-A/B IgM were present in the majority of 120 blood donors, as expected from blood group typing. The sensitivity and specificity of anti-A/B IgM to predict the blood group was 93% and 96% respectively. Anti-A/B IgG was found in 34/38 blood group O samples (89%). Anti-B IgG in blood group A or anti-A IgG in blood group B was present in 4/28 (14%) and 1/28 (4%) samples, respectively, and absent in 26 AB sera. IgG2 was the predominant IgG subclass. The correlation of anti-A/B IgM and IgG in the ABO-FACS with haemagglutination titres was 0.870 and 0.783, respectively (n = 240; P < 0.001) whereas the comparison of ABO-FACS with ABO-enzyme-linked immunosorbent assay was less significant. In conclusion, ABO-FACS is a valid method to quantify anti-A/B IgM, IgG and IgG subclasses. It opens the possibility of isotype-specific monitoring of anti-A/B antibodies levels after ABO-incompatible solid organ and stem cell transplantation.

Identification of small Sca-1(+), Lin(-), CD45(-) multipotential cells in the neonatal murine retina

OBJECTIVE: Bone marrow contains a subset of stem cells that give rise to nonhematopoietic lineages. These nonhematopoietic stem cells appear heterogeneous and contain cells committed to mesenchymal and endothelial lineages, as well as more primitive multipotential cells resembling progenitors of germ cells and very small embryonic/epiblast-like stem cells (VSELs). Nonhematopoietic stem cells can be mobilized from the bone marrow in response to tissue injury, and cells with similar properties have been found in cord blood and normal adult organs. However, the relationship between bone marrow cells and these adult organ stem cells is still unclear. The differentiation potential of some adult stem cells is organ-restricted, but other populations appear to retain multipotential capacity. MATERIALS AND METHODS: A population of small Sca-1(+), lineage-negative (Lin(-)), CD45(-) cells resembling VSELs were isolated from neonatal mouse retina by cell sorting. Differentiation of the cells in culture was achieved by exposure to embryonic stem cell differentiation protocols. RESULTS: VSEL-like cells comprise 1.5% of the neonatal mouse retina. They remain quiescent during retinal differentiation, and thus they do not contribute to normal retinal development. However, they display eye cell differentiation potential in culture and they are also multipotential and can give rise to cells representative of all three embryonic layers. CONCLUSIONS: The neonatal retina is an abundant postnatal source of multipotential VSEL-like cells that can differentiate in culture into a variety of lineages.

Isolation of myeloid dendritic cells and epithelial cells from human thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c(+)), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45(hi)) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

Characterization and Clinical Relevance of ALDHbright Populations in Prostate Cancer

PURPOSE High aldehyde dehydrogenase (ALDH) has been suggested to selectively mark cells with high tumorigenic potential in established prostate cancer cell lines. However, the existence of cells with high ALDH activity (ALDH(bright)) in primary prostate cancer specimens has not been shown so far. We investigated the presence, phenotype, and clinical significance of ALDH(bright) populations in clinical prostate cancer specimens. EXPERIMENTAL DESIGN We used ALDEFLUOR technology and fluorescence-activated cell-sorting (FACS) staining to identify and characterize ALDH(bright) populations in cells freshly isolated from clinical prostate cancer specimens. Expression of genes encoding ALDH-specific isoforms was evaluated by quantitative real-time PCR in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer tissues. ALDH1A1-specific expression and prognostic significance were assessed by staining two tissue microarrays that included more than 500 samples of BPH, prostatic intraepithelial neoplasia (PIN), and multistage prostate cancer. RESULTS ALDH(bright) cells were detectable in freshly excised prostate cancer specimens (n = 39) and were mainly included within the EpCAM((+)) and Trop2((+)) cell populations. Although several ALDH isoforms were expressed to high extents in prostate cancer, only ALDH1A1 gene expression significantly correlated with ALDH activity (P < 0.01) and was increased in cancers with high Gleason scores (P = 0.03). Most importantly, ALDH1A1 protein was expressed significantly more frequently and at higher levels in advanced-stage than in low-stage prostate cancer and BPH. Notably, ALDH1A1 positivity was associated with poor survival (P = 0.02) in hormone-naïve patients. CONCLUSIONS Our data indicate that ALDH contributes to the identification of subsets of prostate cancer cells of potentially high clinical relevance.

Overexpression of EphB4 in the mammary epithelium shifts the differentiation pathway of progenitor cells and promotes branching activity and vascularization

Postnatally, the mammary gland undergoes continuous morphogenesis and thereby is especially prone to malignant transformation. Thus, the maintenance of the epithelium depends on a tight control of stem cell recruitment. We have previously shown that epithelial overexpression of the EphB4 receptor results in defective mammary epithelial development and conferred a metastasizing tumor phenotype on experimental mouse mammary tumors accompanied by a preponderance of progenitor cells. To analyze the effect of EphB4 overexpression on mammary epithelial cell fate, we have used Fluorescence Activated Cell Sorting (FACS) analyses to quantify epithelial sub-populations and repopulation assays of cleared fat pads to investigate their regenerative potential. These experiments revealed that deregulated EphB4 expression leads to an augmentation of bi-potent progenitor cells and to a shift of the differentiation pathway towards the luminal lineage. The analyses of the ductal outgrowths indicated that EphB4 overexpression leads to enforced branching activity, impedes ductal differentiation and stimulates angiogenesis. To elucidate the mechanisms forwarding EphB4 signals, we have compared the expression profile of defined cell populations between EphB4 transgene and wild type mammary glands concentrating on the wnt signaling pathway and on genes implicated in cell migration. With respect to wnt signaling, the progenitor cell population was the most affected, whereas the stem cell-enriched population showed the most pronounced deregulation of migration-associated genes. Thus, the luminal epithelial EphB4 signaling contributes, most likely via wnt signaling, to the regulation of migration and cell fate of early progenitors and is involved in the determination of branching points along the ductal tree.

Fluorescence-encoded gold nanoparticles: library design and modulation of cellular uptake into dendritic cells.

In order to harness the unique properties of nanoparticles for novel clinical applications and to modulate their uptake into specific immune cells we designed a new library of homo- and hetero-functional fluorescence-encoded gold nanoparticles (Au-NPs) using different poly(vinyl alcohol) and poly(ethylene glycol)-based polymers for particle coating and stabilization. The encoded particles were fully characterized by UV-Vis and fluorescence spectroscopy, zeta potential and dynamic light scattering. The uptake by human monocyte derived dendritic cells in vitro was studied by confocal laser scanning microscopy and quantified by fluorescence-activated cell sorting and inductively coupled plasma atomic emission spectroscopy. We show how the chemical modification of particle surfaces, for instance by attaching fluorescent dyes, can conceal fundamental particle properties and modulate cellular uptake. In order to mask the influence of fluorescent dyes on cellular uptake while still exploiting its fluorescence for detection, we have created hetero-functionalized Au-NPs, which again show typical particle dependent cellular interactions. Our study clearly prove that the thorough characterization of nanoparticles at each modification step in the engineering process is absolutely essential and that it can be necessary to make substantial adjustments of the particles in order to obtain reliable cellular uptake data, which truly reflects particle properties.

Retinal differentiation of human bone marrow-derived stem cells by co-culture with retinal pigment epithelium in vitro.

The goal of this study was to assess the in vitro differentiation capacity of human bone marrow-derived stem cells (hBMSCs) along retinal lineages. Mononuclear cells (MNC) were isolated from bone marrow (BM) and mobilized peripheral blood (mPB) using Ficoll-Paque density gradient centrifugation, and were sorted by magnetic-activated cell sorting (MACS) for specific stem cell subsets (CD34(+)CD38(+)/CD34(+)CD38(-)). These cells were then co-cultured on human retinal pigment epithelial cells (hRPE) for 7 days. The expression of stem cell, neural and retina-specific markers was examined by immunostaining, and the gene expression profiles were assessed after FACS separation of the co-cultured hBMSCs by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, in vitro functionality of the differentiated cells was analyzed by quantifying phagocytosis of CY5-labeled photoreceptor outer segments (POS). After 7 days of co-culture, hBMSCs adopted an elongated epithelial-like morphology and expressed RPE-specific markers, such as RPE65 and bestrophin. In addition, these differentiated cells were able to phagocytose OS, one of the main characteristics of native RPE cells. Our data demonstrated that human CD34(+)CD38(-) hBMSC may differentiate towards an RPE-like cell type in vitro and could become a new type of autologous donor cell for regenerative therapy in retinal degenerative diseases.