Luca Pierelli

Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample

We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as little as 10,000 cells and to measure the expression levels of several target mRNAs from each sample. This procedure was optimized on the human erythroleukemia cell line TF-1 but was successfully used on primary cells and on different cell lines. We describe the detailed procedure for the analysis of Bcl-2 levels. Aldolase A was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. As for all quantitative techniques, great care must be taken in all optimization steps: the necessary controls to ensure a rough quantitative (semi-quantitative) analysis are described here, together with an example from a study on the effects of TGF-β1 in TF-1 cells.

Cells with Characteristics of Cancer Stem/Progenitor Cells Express the CD133 Antigen in Human Endometrial Tumors

Purpose: Cancer stem cells represent an attractive therapeutic target for tumor eradication. The present study aimed to determine whether CD133 expression may identify cells with characteristics of cancer stem/progenitor cells in human endometrial tumors. Experimental Design: We analyzed 113 tumor samples for CD133/1 expression by flow cytometry, immunohistochemistry, and semiquantitative reverse transcription–PCR. CD133+ cells were isolated and used to assess phenotypic characteristics, self-renewal capacity, ability to maintain CD133 expression and form sphere-like structures in long-term cultures, sensitivity to chemotherapeutic agents, gene expression profile, and ability to initiate tumors in NOD/SCID mice. Results: Primary tumor samples exhibited a variable degree of immunoreactivity for CD133/1, ranging from 1.3% to 62.6%, but stained negatively for other endothelial and stem cell–associated markers. Isolated CD133+ cells expanded up to 4.6-fold in serum-replenished cultures and coexpressed the GalNAcα1-O-Ser/Thr MUC-1 glycoform, a well-characterized tumor-associated antigen. Dissociated bulk tumors formed sphere-like structures; cells grown as tumor spheres maintained CD133 expression and could be propagated for up to 12 weeks. CD133+ cells purified from endometrioid adenocarcinomas were resistant to cisplatin-induced and paclitaxel-induced cytotoxicity and expressed a peculiar gene signature consisting of high levels of matrix metalloproteases, interleukin-8, CD44, and CXCR4. When serially transplanted into NOD/SCID mice, CD133+ cells were capable of initiating tumor formation and recapitulating the phenotype of the original tumor. Conclusions: CD133 is expressed by human endometrial cancers and might represent a valuable tool to identify cells with cancer stem cell characteristics.

Granulocyte colony‐stimulating factor promotes the generation of regulatory DC through induction of IL‐10 and IFN‐α

We have recently demonstrated that G‐CSF promotes the generation of human T regulatory (TREG) type 1 cells. In this study, we investigated whether the immunomodulatory effects of G‐CSF might be mediated by DC. CD14+ monocytes were cultured with serum collected after clinical administration of G‐CSF (post‐G), which contained high amounts of IL‐10 and IFN‐α. Similar to incompletely matured DC, monocytes nurtured with post‐G serum acquired a DC‐like morphology, expressed high levels of costimulatory molecules and HLA‐DR, and exhibited diminished IL‐12p70 release and poor allostimulatory capacity. Importantly, post‐G DC‐like cells were insensitive to maturation stimuli. As shown by neutralization studies, IFN‐α and, even more pronounced, IL‐10 contained in post‐G serum inhibited IL‐12p70 release by post‐G DC‐like cells. Furthermore, phenotypic and functional features of post‐G DC‐like cells were replicated by culturing post‐G monocytes with exogenous IL‐10 and IFN‐α. Post‐G DC‐like cells promoted Ag‐specific hyporesponsiveness in naive allogeneic CD4+ T cells and orchestrated a TREG response that was dependent on secreted TGFβ1 and IL‐10. Finally, neutralization of IL‐10 and IFN‐α contained in post‐G serum translated into abrogation of the regulatory features of post‐G DC‐like cells. This novel mechanism of immune regulation effected by G‐CSF might be therapeutically exploited for tolerance induction in autoimmune disorders.

Quercetin inhibits the growth of a multidrug-resistant estrogen-receptor-negative MCF-7 human breast-cancer cell line expressing type II estrogen-binding sites

SummaryIt has been demonstrated that the flavonoid quercetin (3,3′,4′,5,7-pentahydroxyflavone; Q) inhibits the growth of several cancer cell lines. There is evidence suggesting that the antiproliferative activity of this substance is mediated by the so-called type II estrogen-binding, site (type II EBS). We looked for the presence of type II EBS and the effect of Q on the proliferation of an Adriamycinresistant estrogen-receptor-negative human breast-cancer cell line (MCF-7 ADRr). By whole-cell assay using estradiol labelled with 6,7-tritium ([3H]-E2) as a tracer, we demonstrated that MCF-7 ADRr cells contain type II EBSs. Competition analysis revealed that diethylstilbestrol (DES) and Q competed with similar potency for [3H]-Es binding to type II EBSs. The antiestrogen tamoxifen (TAM) competed for type II EBSs, albeit to a lesser extent than either DES or Q. Growth experiments demonstrated that Q and DES exerted a dose-dependent inhibition of cell proliferation in the range of concentrations between 10 nM and 10 μm, whereas TAM was less effective. Q could also inhibit colony formation in a clonogenic assay. Our results indicate that multidrug-resistant estrogen-receptor-negative MCF-7 cells express, type II EBSs and are sensitive to the inhibitory effect of Q. This substance could be the parent compound of a novel class of anticancer agents.

The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs.

Immune reconstitution after transplantation of autologous peripheral CD34+ cells: analysis of predictive factors and comparison with unselected progenitor transplants

The recovery of lymphocyte count, CD4+ and CD8+ T‐cell subsets, natural killer (NK) cells and CD19+ B‐cells was evaluated in a cohort of 15 patients receiving autologous CD34+ peripheral blood progenitor cells (PBPCs; group A) for haematological malignancies and in 20 patients transplanted with autologous unselected PBPCs (group B). Lymphocyte count recovered in both patient cohorts, being significantly lower in group A than in group B 1 (P = 0·008) and 2 months (P = 0·0035) after progenitor cell infusion. The repopulation of CD3+ T‐cells occurred more rapidly in group B than in group A (P = 0·034 on week 4); CD19+ B‐lymphocytes did not return to reference ranges in either group of patients. The count of CD4+ T‐lymphocytes remained < 200/μl during the study period in patients transplanted with CD34+ PBPCs, significantly lower than group B levels (P = 0·034 and P = 0·021 on weeks 4 and 8 respectively). CD8+ T‐cells increased rapidly in both groups; thus, the CD4 to CD8 ratio was severely reduced. CD4+ and CD8+ T‐cells displayed an activated phenotype in both groups of patients, co‐expressing the HLA‐DR antigen throughout the study period. NK cells followed a similar repopulation kinetics in both study groups, although their expansion was greater in group B than in group A (P = 0·014 on week 4). In the CD34+ group, post‐transplant administration of granulocyte colony‐stimulating factor predicted a faster lymphocyte recovery in multivariate analysis (P = 0·025); interestingly, the amount of passively transferred lymphocytes correlated inversely with time to achieve a lymphocyte count > 0·5 × 109/l (r = –0·63, P = 0·01). Further investigations are necessary to characterize T‐cell competence after transplantation of CD34+ PBPCs.

CD105 (Endoglin) Expression on Hematopoietic Stem/Progenitor Cells

Endoglin (CD105) is a component of the transforming growth factor-β (TGF-β) receptor (TGF-βR) complex. Together with betaglycan, CD105 is considered as a TGF-βR accessory molecule (also called TGF-βRIII), but its functions in the receptor-ligand interactions are still poorly understood. A small subset of human CD34+ hematopoietic stem/progenitor cells that has phenotypic and functional features suggestive of very primitive hematopoietic cells expresses the CD105 antigen. CD34+/CD105+ cells recirculate in the peripheral blood of mobilized subjects and can be purified by immunomagnetic isolation strategies. The hematopoietic potential of these CD34+/CD105+ cells appears to be sustained by a combination of hematopoietic and non-hematopoietic cytokines, which comprises Flt3 ligand, erythropoietin, interleukin-15 and vascular endothelial growth factor. Endogenous TGF-β1 is a crucial factor for the maintenance of CD34+/CD105+ immaturity acting through positive modulation of both CD105 and CD34 molecules in the absence of relevant effects on the cell cycle profile. CD105 is absent on very primitive CD34-/lineage-/CD45+ (CD34-Lin-) human hematopoietic cells isolated from cord blood. However, in vitro exposure of CD34-Lin-cells to exogenous TGF-β1 causes the appearance of a discrete population of CD34+/CD105+ cells. Collectively, available data on CD105 expression and function in primitive hematopoiesis indicate that this molecule could cooperate with the dissociation of TGF-β1 cell cycle effects from its other effects on cell survival and differentiation.

CD34+/CD105+ cells are enriched in primitive circulating progenitors residing in the G0 phase of the cell cycle and contain all bone marrow and cord blood CD34+/CD38low/− precursors

A subset of circulating CD34+ cells was found to express CD105 antigen. Sorting experiments showed that most granulocyte–macrophage colony‐forming units (GM‐CFU) and burst‐forming units — erythroid (BFU‐E) were retained in the CD34+/CD105− fraction, whereas rare GM‐CFU/BFU‐E were generated from CD34+/CD105+ cells. Megakaryocytic aggregates were entirely retained in the CD34+/CD105+ fraction. Neutralizing doses of an anti‐TGF‐β1 antibody demonstrated CD34+/CD105+ cells capable of colony‐forming activity without any significant effect on CD34+/CD105− cells. Cloning of secondary colonies revealed that CD34+/CD105+ cells had a significantly higher secondary cloning efficiency than CD34+/CD105− cells. CD34+/CD105+ cells had a significantly higher long‐term culture‐initiating cell (LTC‐IC) frequency than CD34+/CD105− cells. Kinetic analysis showed that 75% of CD34+/CD105+ cells consisted of DNA 2n G0Ki‐67− cells whereas 82% of CD34+/CD105− were DNA 2n G1Ki‐67+ cells, and this latter subset showed a RNA content consistently higher than CD34+/CD105+ cells. CD34+/CD105+ progenitors were CD25+, whereas CD34+/CD105− contained a small CD25+ subset. Three‐colour analysis of bone marrow and cord blood CD34+ cells demonstrated that all the CD34+/CD38low/− primitive precursors were contained in CD34+/CD105+ cells. Extensive characterization of these CD105+ precursors indicated that they have biological properties associated with primitive haematopoietic precursors.

Human cord blood CD133+ cells immunoselected by a clinical-grade apparatus differentiate in vitro into endothelial- and cardiomyocyte-like cells

BACKGROUND: Recent findings on human hematopoietic stem cell (HSC) properties suggest a possible therapeutic role of human umbilical cord blood (UCB) HSC‐based cellular therapies in the treatment of myocardial infarction.

Targeting of macrophage galactose-type C-type lectin (MGL) induces DC signaling and activation

Dendritic cells (DCs) sense the microenvironment through several types of receptors recognizing pathogen‐associated molecular patterns. In particular, C‐type lectins, expressed by distinct subsets of DCs, recognize and internalize specific carbohydrate antigen in a Ca2+‐dependent manner. Targeting of these receptors is becoming an efficient strategy of delivering antigens in DC‐based anticancer immunotherapy. Here we investigated the role of the macrophage galactose type C‐lectin receptor (MGL), expressed by immature DCs (iDCs), as a molecular target for α‐N‐acetylgalactosamine (GalNAc or Tn)‐carrying tumor‐associated antigens to improve DC performance. MGL expressed by ex vivo‐generated iDCs from healthy donors was engaged by a 60‐mer MUC19Tn‐glycopeptide as a Tn‐carrying tumor‐associated antigen, and an anti‐MGL antibody, as a specific MGL binder. We demonstrated that MGL engagement induced homotrimers and homodimers, triggering the phosphorylation of extracellular signal‐regulated kinase 1,2 (ERK1,2) and nuclear factor‐κB activation. Analysis of DC phenotype and function demonstrated that MGL engagement improved DC performance as antigen‐presenting cells, promoting the upregulation of maturation markers, a decrease in phagocytosis, an enhancement of motility, and most importantly an increase in antigen‐specific CD8+ T‐cell activation. These results demonstrate that the targeting of MGL receptor on human DCs has an adjuvant effect and that this strategy can be used to design novel anticancer vaccines.