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At present, injuries or rupture of tendons are treated by surgical repair or conservative approaches with unpredictable clinical outcome. Alternative strategies to repair tendon defects without the undesirable side effects associated with the current options are needed. With this in mind, a tissue engineering approach has gained considerable attention as a promising strategy. Here we investigated a synthetic three-dimensional (3D) microenvironment able to interact with stem cells and inducing, via coupled biochemical and physical signals, their early commitment toward the tenogenic lineage. This multiphase 3D construct consisted of a braided hyaluronate elastic band merged with human bone marrow mesenchymal stem cells (hBMSCs) and poly-lactic-co-glycolic acid microcarriers loaded with human growth differentiation factor 5 (hGDF-5) by means of fibrin hydrogel. The multiphase structure allowed hBMSC culture under cyclic strain within a microenvironment where a controlled amount of hGDF-5 was regularly delivered. The cooperative biochemical and physical stimuli induced significantly increased expression of tenogenic markers, such as collagen type I and III, decorin, scleraxis, and tenascin-C, within only 3 days of dynamic hBMSC culture. This approach opens exciting perspectives for future development of engineered tendon tissue substitutes.

Tissue engineering is now increasingly focusing on cell-based treatments as promising tools to improve tendon repair. However, many crucial aspects of tendon biology remain to be understood before adopting the best experimental approach for cell-tissue engineering.The role played by Ascorbic Acid (AA) alone and in combination with thyroid hormone TIn thisWe showed that the highest concentration of AA (100 and 500 µg/ml) caused cytotoxicity to human tenocytes. Moreover, it was shown that AA reduces NO synthesis. These results show that AA is a cell proliferation inducer that triggers tenocyte growth, while it reduces NO synthesis.

The in vitro generation of hematopoietic stem cells (HSCs) and mature hematopoietic cells from hemangioblast derived from embryonic stem (ES) or induced pluripotent stem (iPS) cells promises to provide an alternative source of cells that could replace total bone marrow cells or HSC-enriched fractions. This mini-review deals with innovation related to hemangioblast-based methods for blood cells production as disclosed in recent patent literature and current barriers to clinical translation are discussed.

The use of high-dose ascorbate (ASC) for the treatment of human cancer has been attempted several decades ago and has been recently revived by several in vitro and in vivo studies in solid tumors. We tested the cytotoxic effects of ASC, alone or in combination with arsenic trioxide (ATO) in acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL). Leukemic cell lines and primary blasts from AML and APL patients were treated with graded concentrations of ASC, alone or in association with standard concentration (1 μM) of ATO. The ASC/ATO combination killed myeloid blasts, including leukemic CD34+ cells, while sparing CD34+ progenitors obtained from normal cord blood and bone marrow. Actually, approximately one-third (11/36) of primary AML cases were highly sensitive to the ASC/ATO combination. The mechanism of cell killing appeared to be related to increased oxidative stress and overproduction of ROS in a non-quantitative fashion, which resulted in induction of apoptosis. These effects were reverted by the addition of the antioxidant N-Acetyl-Cysteine (NAC). In the APL NB4 model, ASC induced direct degradation of the PML and PML/RARA proteins via caspase activation, while the transcriptional repressor DAXX was recruited in re-constituted PML nuclear bodies. Our findings encourage the design of pilot studies to explore the potential clinical benefit of ASC alone or in combination with ATO in advanced AML and APL.

Tendinopathies have a multifactorial etiology driven by extrinsic and intrinsic factors. Recent studies have elucidated the importance of thyroid hormones in the alteration of tendons homeostasis and in the failure of tendon healing after injury. The effects of thyroid hormones are mediated by receptors (TR)-α and -β that seem to be ubiquitous. In particular, T3 and T4 play an antiapoptotic role on tenocytes, causing an increase in vital tenocytes isolated from tendons in vitro and a reduction of apoptotic ones; they are also able to influence extra cellular matrix proteins secretion in vitro from tenocytes, enhancing collagen production. From a clinical point of view, disorders of thyroid function have been investigated only for rotator cuff calcific tendinopathy and tears. In this complex scenario, further research is needed to clarify the role of thyroid hormones on the onset of tendinopathies.

In an attempt to investigate whether the genetic defect in the HEXA and HEXB genes (which causes the absence of the lysosomal β-N-acetyl-hexosaminidase), are related to the wide inflammation in GM2 gangliosidoses (Tay-Sachs and Sandhoff disease), we have chosen the dendritic cells (DCs) as a study model. Using the RNA interference approach, we generated an in vitro model of HEXs knock-down immunogenic DCs (i-DCs) from CD34(+)-haemopoietic stem cells (CD34(+)-HSCs), thus mimicking the Tay-Sachs (HEXA-/-) and Sandhoff (HEXB-/-) cells. We showed that the absence of β-N-acetyl-hexosaminidase activity does not alter the differentiation of i-DCs from HSCs, but it is critical for the activation of CD4(+)T cells because knock-down of HEXA or HEXB gene causes a loss of function of i-DCs. Notably, the silencing of the HEXA gene had a stronger immune inhibitory effect, thereby indicating a major involvement of β-N-acetyl-hexosaminidase A isoenzyme within this mechanism.

A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast, we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34, lineage markers, CD45 and CD133 (CD34−Lin−CD45−CD133− cells), endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34−Lin−CD45−CD133− cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo, CD34−Lin−CD45−CD133− cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34−Lin−CD45−CD133− cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials, characterized by the expression of c-Kit and CXCR4 as well as EphB4, EphB2, and ephrinB2. Further molecular and functional characterization of CD34−Lin−CD45−CD133− cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life.

Background Tendinopathies negatively affect the quality of life of millions of people, but we still do not know the factors involved in the development of tendon conditions. Sources of data Published articles in English in PubMed and Google Scholar up to June 2015 about hormonal influence on tendinopathies onset. One hundred and two papers were included following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Areas of agreement In vitro and in vivo, tenocytes showed changes in their morphology and in their functional properties according to hormonal imbalances. Areas of controversy Genetic pattern, sex, age and comorbidities can influence the hormonal effect on tendons. Growing points The increasing prevalence of metabolic disorders prompts to investigate the possible connection between metabolic problems and musculoskeletal diseases. Areas timely for developing research The influence of hormones on tendon structure and metabolism needs to be further investigated. If found to be significant, multidisciplinary preventive and therapeutic strategies should then be developed.

Tendinopathies have a multifactorial etiology driven by extrinsic and intrinsic factors. Recent studies have elucidated the importance of thyroid hormones in the alteration of tendons homeostasis and in the failure of tendon healing after injury. The effects of thyroid hormones are mediated by receptors (TR)-α and –β that seem to be ubiquitous. In particular, T3 and T4 play an antiapoptotic role on tenocytes, causing an increase in vital tenocytes isolated from tendons in vitro and a reduction of apoptotic ones; they are also able to influence extra cellular matrix proteins secretion in vitro from tenocytes, enhancing collagen production. From a clinical point of view, disorders of thyroid function have been investigated only for rotator cuff calcific tendinopathy and tears. In this complex scenario, further research is needed to clarify the role of thyroid hormones on the onset of tendinopathies.

The effect of high doses of intravenous (sodium) ascorbate (ASC) in the treatment of cancer has been controversial although there is growing evidence that ASC in high (pharmacologic) concentrations induces dose-dependent pro-apoptotic death of tumor cells, in vitro. Very few data are available on the role of ASC in the treatment of acute myeloid leukemia (AML). Ascorbate behaves as an antioxidant at low (physiologic), and as pro-oxidant at pharmacologic, concentrations, and this may account for the differences reported in different experimental settings, when human myeloid cell lines, such as HL60, were treated with ASC. Considering the myeloid origin of HL60 cells, and previous literature reports showing that some cell lines belonging to the myeloid lineage could be sensitive to the pro-apoptotic effects of high concentrations of ASC, we investigated in more details the effects of high doses (0.5 to 7 mM) of ASC in vitro, on a variety of human myeloid cell lines including the following: HL60, U937, NB4, NB4-R4 (retinoic acid [RA]-resistant), NB4/AsR (ATO-resistant) acute promyelocytic leukemia (APL)-derived cell lines, and K562 as well as on normal CD34+ progenitors derived from human cord blood. Our results indicate that all analyzed cell lines including all-trans retinoic acid (ATRA)- and arsenic trioxide (ATO)-resistant ones are highly sensitive to the cytotoxic, pro-oxidant effects of high doses of ASC, with an average 50 % lethal concentration (LC50) of 3 mM, depending on cell type, ASC concentration, and time of exposure. Conversely, high doses of ASC neither did exert significant cytotoxic effects nor impaired the differentiation potential in cord blood (CB) CD34+ normal cells. Since plasma ASC concentrations within the millimolar (mM) range can be easily and safely reached by intravenous administration, we conclude that phase I/II clinical trials using high doses of ASC should be designed for patients with advanced/refractory AML and APL.

we previously demonstrated the presence of high levels of thyroid hormones (THs) receptors isoforms in healthy tendons, their protective action during tenocyte apoptosis, and the capability to enhance tenocyte proliferation in vitro. In the present study we tested the ability of THs to influence ECM protein tenocyte secretion in an in vitro system.primary tenocyte-like cells were cultivated for 1, 7 and 14 days in the presence of T3 or T4 individually or in combination with ascorbic acid (AA).THs (T3 or T4) in synergism with AA increase significantly the total collagen production after 14 days. THs in synergism with AA increase significantly the expression of collagen I,biglycan and COMP, after some days.THs play a role on the extra cellular matrix of tendons, enhancing in vitro the production of several proteins such as collagen I, biglycan and COMP. THs receptors are active on human tenocytes, and can play a role in tendon ailments.

Background: we previously demonstrated the presence of high levels of thyroid hormones (THs) receptors isoforms in healthy tendons, their protective action during tenocyte apoptosis, and the capability to enhance tenocyte proliferation in vitro. In the present study we tested the ability of THs to influence ECM protein tenocyte secretion in an in vitro system. Methods: primary tenocyte-like cells were cultivated for 1, 7 and 14 days in the presence of T3 or T4 individually or in combination with ascorbic acid (AA). Results: THs (T3 or T4) in synergism with AA increase significantly the total collagen production after 14 days. THs in synergism with AA increase significantly the expression of collagen I,biglycan and COMP, after some days. Conclusion: THs play a role on the extra cellular matrix of tendons, enhancing in vitro the production of several proteins such as collagen I, biglycan and COMP. THs receptors are active on human tenocytes, and can play a role in tendon ailments.

Identification of human hematopoietic stem cells and analysis of molecular mechanisms regulating their function require biological assays that permit differentiation in all hematopoietic lineages simultaneously. In this study, we established conditions that permit the joint expression of the B-lymphoid and myeloid potential from cord blood-derived CD34+CD38lowCD19−/CD10− primitive progenitors that lack B-specific markers and transcripts. When cocultured during 6 weeks with the murine stromal cells MS-5 in the absence of exogenous human cytokines, CD34+CD38lowCD19−CD10− cells generated a high number of CD19+ B cells. Virtually all of these cells expressed a CD34−CD10+CD19+cIgM− phenotype of late pro-B cells and transcripts of Pax-5, λ-like, and μ chain were detected. We further show that 7% of CD34+CD38lowCD19− cells from cord blood, when grown individually with MS-5 cells, generated both CD19+ and CD11b+ cells after 6 weeks. Efficient B-cell differentiation was also observed in vivo after transplantation of human cord blood-derived unfractionated mononuclear cells or CD34+CD19+CD10− cells into immune-deficient mice. In contrast to the in vitro situation, all stages of B-cell differentiation were observed in vivo, including pro-B, pre-B, and sIgM+ B cells. Interestingly, human progenitors with the ability to differentiate along both B-lymphoid and granulocytic pathways were also detected among human CD34+CD38low cells in the marrow of chimeric mice 6 to 7 weeks after transplantation. Both in vitro and in vivo systems will offer an invaluable tool to further identify the lymphoid and myeloid potentialities of primitive progenitor cells isolated from fetal as well as adult human hematopoietic tissues and characterize stromal-derived signals that regulate their function.

The purpose of this study was to determine the efficacy of platelet-rich plasma (PRP) 1-injection during an Achilles tendon rat tear model. 80 male adult imbreded rats (Wistar Kyoto), underwent under surgical tendon rupture. 40 Animal (PRP group rats) were given a local injection with 0,25 mL of PRP, and 40 animal (control group) were given the same quantity of control solution. The rats were sacrified at 1, 2, 4 and 6 weeks (each time point, 20 rats of the each group) after surgical tear and tendon tissue was analysed by macroscopic aspect, histology, immunostaining and Real Time (RT)-PCR to evaluate tissue repair. PRP improved tendon remodelling by better coordination of the reconstructive process with earlier formation of tendon-like continuity only in the first week after surgery. However, after 2,4 and 6 weeks, Achilles tendons in the PRP group had no difference compared to the control group. Immunostaining and RT-PCR did not show any difference between PRP treated and untreated group. Based on these findings a single injection of PRP appear not useful for Achilles rat tendon tear.

Correction to: Cell Death and Disease (2013) 4, e705; doi:10.1038/cddis.2013.229; published online 4 July 2013

Basic fibroblast growth factor or fibroblast growth factor-2 (FGF) has been shown to affect myeloid cell proliferation and hypothesized to stimulate primitive hematopoietic cells. We sought to evaluate the effect of FGF on hematopoietic stem cells and to determine if FGF mediated its effects on progenitor cells directly or through the induction of other cytokines. To address the direct effects of FGF, we investigated whether FGF induced production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, IL-6, granulocyte colony- stimulating factor, or granulocyte-macrophage colony-stimulating factor by two types of accessory cells, bone marrow (BM) fibroblasts and macrophages. We further evaluated whether antibodies to FGF-induced cytokines affected colony formation. To determine if FGF was capable of stimulating multipotent progenitors, we assessed the output of different colony types after stimulation of BM mononuclear cells (BMMC) or CD34+ BMMC and compared the effects of FGF with the stem cell active cytokine, kit ligand (KL). In addition, a subset of CD34+ BMMC with characteristics of hematopoietic stem cells was isolated by functional selection and their response to FGF was evaluated using proliferation, colony-forming, and single-cell polymerase chain reaction (PCR) assays. We determined that FGF had a stimulatory effect on the production of a single cytokine, IL-6, but that the effects of FGF on colony formation were not attributable to that induction. FGF was more restricted in its in vitro effects on BM progenitors than KL was, having no effect on erythroid colony formation. FGF did not stimulate stem cells and FGF receptors were not detected on stem cells as evaluated by single-cell reverse transcription PCR. In contrast, FGF receptor gene expression was detected in myeloid progenitor populations. These data support a directly mediated effect for FGF that appears to be restricted to lineage-committed myeloid progenitor cells. FGF does not appear to modulate the human hematopoietic stem cell.

The molecular mechanism responsible for Ewing's Sarcoma (ES) remains largely unknown. MicroRNAs (miRNAs), a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs) by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis.

The identity of biochemical players which underpin the commitment of CD34(+) hematopoietic stem cells to immunogenic or tolerogenic dendritic cells is largely unknown. To explore this issue, we employed a previously established cell-based system amenable to shift dendritic cell differentiation from the immunogenic into the tolerogenic pathway upon supplementation with a conventional cytokine cocktail containing thrombopoietin (TPO) and IL-16. We show that stringent regulation of cathepsins S and D, two proteases involved in antigen presentation, is crucial to engage cell commitment to either route. In response to TPO+IL-16-dependent signaling, both cathepsins undergo earlier maturation and down-regulation. Additionally, cystatin C orchestrates cathepsin S expression through a tight but reversible interaction that, based on a screen of adult stem cells from disparate origins, CD14(+) cells, primary fibroblasts and the MCF7 cell line, appears unique to CD34(+) stem cells from peripheral and cord blood. As shown by CD4(+) T cell proliferation in mixed-lymphocyte reactions, cell commitment to either pathway is disrupted upon cathepsin knockdown by RNAi. Surprisingly, similar effects were also observed upon gene overexpression, which prompts atypically accelerated maturation of cathepsins S and D in cells of the immunogenic pathway, similar to the tolerogenic route. Furthermore, RNAi studies revealed that cystatin C is a proteolytic target of cathepsin D and has a direct, causal impact on cell differentiation. Together, these findings uncover a novel biochemical cluster that is subject to time-controlled and rigorously balanced expression to mediate specific stem cell commitment at the crossroads towards tolerance or immunity.

The design of bioactive scaffold materials able to guide cellular processes involved in new-tissue genesis is key determinant in bone tissue engineering. The aim of this study was the design and characterization of novel multi-phase biomaterials to be processed for the fabrication of 3D porous scaffolds able to provide a temporary biocompatible substrate for mesenchymal stem cells (MSCs) adhesion, proliferation and osteogenic differentiation. The biomaterials were prepared by blending poly(epsilon-caprolactone) (PCL) with thermoplastic zein (TZ), a thermoplastic material obtained by de novo thermoplasticization of zein. Furthermore, to bioactivate the scaffolds, microparticles of osteoconductive hydroxyapatite (HA) were dispersed within the organic phases. Results demonstrated that materials and formulations strongly affected the micro-structural properties and hydrophilicity of the scaffolds and, therefore, had a pivotal role in guiding cell/scaffold interaction. In particular, if compared to neat PCL, PCL-HA composite and PCL/TZ blend, the three-phase PCL/TZ-HA showed improved MSCs adhesion, proliferation and osteogenic differentiation capability, thus demonstrating potential for bone regeneration.

This report describes our experience with various techniques for the freezing of platelet-rich plasma, removed from the final product after leukapheresis procedures performed on 14 hematological patients. A total of 194 platelet units were frozen for subsequent autologous transfusion, by the following four methods: (1) 6% dimethyl sulfoxide (DMSO); (2) a combination of 5% DMSO/6% hydroxyethyl starch; (3) 3% glycerol; (4) 5% glycerol/4% glucose. Each technique was evaluated by measuring the percentage of platelet recovery, malondialdehyde (MDA) production, and lactate dehydrogenase release. To investigate the safety and therapeutic effectiveness of the previously frozen platelets, in vivo comparison of four platelet freezing methods was made in 8 thrombocytopenic patients, using corrected platelet increment (CCI), determined at 24 h. Our in vitro results indicate that the cryopreservation with 6% DMSO, without controlled cooling rate, provides significantly (p less than 0.05) greater platelet recovery (75%) as compared to other systems. The decrease of MDA production and the increase in plasma lactate measured after the thawing process was less in the DMSO-frozen units than in the other platelet units. When platelets, cryopreserved by this method, were subsequently transfused into patients, a significantly better CCI (greater than 5,000/microliters) was obtained. In our series, 6 patients were entirely supported with frozen autologous platelets. It appears from this study that a better understanding of the physical and biochemical events occurring during the freezing process will improve platelet cryopreservation, allowing a more systematic use of frozen platelets in the support of thrombocytopenic patients.

We previously reported that interleukin (IL)-16 can induce CD34(+) hematopoietic cells to proliferate and differentiate in vitro into phenotypically and functionally mature dendritic cells. In this study, we investigated the effects of IL-16 on the expansion of CD34(+) cells from human cord blood (CB). CD34(+) CB cells were cultured for 14 days in medium containing a basal cocktail (BC) containing stem cell factor, Flt-3 ligand, thrombopoietin, IL-6, and IL-3 with and without IL-16 as a control. Interleukin-16 added to BC significantly enhanced the expansion of CD34(+) cells (66.47 +/- 1.46-fold vs. 36.23 +/- 1.67-fold), as well as CD34(+)CD38(-) early stem cells (106.67 +/- 2.34-fold vs. 63.42 +/- 1.89-fold) and progenitor cells [colony-forming unit (CFU) -mixed -(GEMM)] and multilineage-committed progenitors [burst-forming unit (BFU-E), CFU-granulocyte, macrophage (-GM), CFU-megakaryocyte (-MK)]. Interleukin-16 also significantly increased long-term culture-initiating cells (160.8 +/- 3.45-fold vs. 83 +/- 2.89-fold with BC alone). Moreover, CD34(+) cells expanded with IL-16 maintained the capacity to differentiate into the lymphoid-B and -NK lineage. The addition of IL-16 to BC increased the migratory capacity of expanded CD34(+) cells compared to BC alone, leaving the expression of CXCR4 unaffected, and decreased the percentage of CD34(+)CD4(+) cells. We showed that IL-16 released endogenously affected the ex vivo expansion of CD34(+) cells. Overall, this study suggests that IL-16 may have a new role in promoting the expansion of hematopoietic stem cells and may represent a new tool for the expansion of CD34(+) cells for clinical applications.

BACKGROUND: Increased expression of Eph receptors and their ephrin ligands has been implicated in promoting angiogenesis and tumour progression in several malignancies. Here the expression of mRNA for ephrin-B and EphB receptors in rhabdomyosarcoma (RMS) cell lines and primary tumours was investigated. MATERIALS AND METHODS: Expression of mRNA for ephrin-B and EphB receptors in RMS cell lines and primary tumours was measured by real-time RT-PCR and compared with the expression in normal striated muscle. RESULTS: A dysregulation of both ligands and receptors was found in all cell lines. In embryonal tumours, overexpression of ephrin-B1 correlated with overexpression of EphB1 (r = 0.97, p < 0.01) and EphB3 (r = 0.94, p < 0.05); overexpression of ephrin-B2 correlated with overexpression of EphB1 (r = 0.94, p < 0.05), EphB2 (r = 0.88, p < 0.01) and EphB4 (r = 0.76, p < 0.01). In alveolar tumours, no similar correlations were found. A correlation between EphB2 and EphB4 receptors was demonstrated in both tumour types, being positive in embryonal cases (r = 0.81, p < 0.01) and negative in alveolar (r = -1.00, p < 0.01). CONCLUSION: A global up-regulation of ephrin-B and EphB receptors in RMS tumours was found. The correlation between EphB2 and EphB4 receptors suggests a possible role for ephrin-B and EphB receptors in RMS development

High levels of PDGFR expression in primary rhabdomyosarcoma (RMS) have been associated with disease progression. To date however, there are no reports on the activity of imatinib mesylate, a selective PDGFR inhibitor, in RMS preclinical models. A panel of 5 RMS cell lines was used to investigate the expression of PDGFRalpha and PDGFRbeta, c-Kit and the multidrug transporter ABCG2 (also inhibited by imatinib). In vitro and in vivo experiments were performed using RD (embryonal) and RH30 (alveolar) cell lines to determine the efficacy of imatinib as single agent and in combination with topotecan (TPT). PDGFRbeta was significantly expressed in all cell lines, with the highest levels in RD, while PDGFR alpha and ABCG2 were significantly expressed only in RH30 and RMZ-RC2. c-Kit was not detected. PDGFRbeta signaling was active in RD but not in RH30, whilst PDGFRalpha signaling was not active in either cell lines. Significant ABCG2-mediated extrusion of Hoechst 33342 was demonstrated in RH30 but not in RD, and was inhibited by imatinib and the specific ABCG2 inhibitor Ko143. In vitro, imatinib was not active as a single agent at therapeutic concentrations, but significantly potentiated TPT antitumor activity in both cell lines. In vivo experiments using tumor xenografts confirmed the synergistic interaction in both cell lines. These results suggest that at least 2 different mechanisms--inhibition of ABCG2 and/or PDGFRbeta--are involved in the synergistic interaction between imatinib and TPT, and support the use of this combination for the treatment of high-risk RMS patients.

The effects of interleukin 16 (IL-16) on dendritic cell (DC) generation from human CD34+ progenitor cells are not known. Here, we show that IL-16 added to a basal cocktail comprised of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, Flt-3 ligand (Flt3L), and tumor necrosis factor α (TNF-α) does induce the CD34+ hematopoietic cells to proliferate in vitro and to differentiate into phenotypically and functionally mature DCs. IL-16 exerts this function more efficiently than stem cell factor (SCF) as a control, thrombopoietin (TPO), or IL-16 plus TPO. Moreover, we show that the combination of IL-16 plus TPO induces the generation of tolerogenic DCs, able to induce an anergic state in T cells that persists when T cells are rechallenged with immunogenic DCs. An altered pattern of cytokine production, a reduced expression of the C-type lectin DC-SIGN, and an increased surface expression of the inhibitory molecules immunoglobulin-like transcript 2 (ILT-2), ILT-3, and ILT-4 may all contribute to confer the tolerogenic properties of these DCs. Generation of tolerogenic DCs may aid the exploration of new therapeutic strategies to promote tolerance to autoantigens and prevent disease development. (Blood. 2004;104:4020-4028)

Identification of human hematopoietic stem cells and analysis of molecular mechanisms regulating their function require biological assays that permit differentiation in all hematopoietic lineages simultaneously. In this study, we established conditions that permit the joint expression of the B-lymphoid and myeloid potential from cord blood-derived CD34+CD38lowCD19−/CD10− primitive progenitors that lack B-specific markers and transcripts. When cocultured during 6 weeks with the murine stromal cells MS-5 in the absence of exogenous human cytokines, CD34+CD38lowCD19−CD10− cells generated a high number of CD19+ B cells. Virtually all of these cells expressed a CD34−CD10+CD19+cIgM− phenotype of late pro-B cells and transcripts of Pax-5, λ-like, and μ chain were detected. We further show that 7% of CD34+CD38lowCD19− cells from cord blood, when grown individually with MS-5 cells, generated both CD19+ and CD11b+ cells after 6 weeks. Efficient B-cell differentiation was also observed in vivo after transplantation of human cord blood-derived unfractionated mononuclear cells or CD34+CD19+CD10− cells into immune-deficient mice. In contrast to the in vitro situation, all stages of B-cell differentiation were observed in vivo, including pro-B, pre-B, and sIgM+ B cells. Interestingly, human progenitors with the ability to differentiate along both B-lymphoid and granulocytic pathways were also detected among human CD34+CD38low cells in the marrow of chimeric mice 6 to 7 weeks after transplantation. Both in vitro and in vivo systems will offer an invaluable tool to further identify the lymphoid and myeloid potentialities of primitive progenitor cells isolated from fetal as well as adult human hematopoietic tissues and characterize stromal-derived signals that regulate their function.

Hematopoietic cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Human bone marrow cells that were responsive to the early-acting cytokines Kit ligand and interleukin-3 were forced to a metabolic death. The subfraction remaining represented 1 in 10(5) bone marrow mononuclear cells, were determined to be quiescent by cell cycle analysis, and had a stem cell immunophenotype. The cells were highly enriched for long-term culture-initiating cells, were capable of secondary colony formation, and produced both myeloid and lymphoid progeny. Thus, this technically simple strategy led to the efficient purification of cells with characteristics of hematopoietic stem cells.

A key mechanism responsible for processing of peptide-MHC class II complexes in mature Dendritic Cells (DCs) is the generalized activation of lysosomal function. Mechanisms underlie these developmental changes are controversial. Thus, it is unclear whether immature DCs can present self antigens, and which are the checkpoints that regulate antigen presentation in immature and mature DCs. Here we generated in-vitro human DCs from peripheral blood CD34+ hematopoietic stem cells (HSCs), by adding to the medium culture Flt-3, GM-CSF, IL-4, and TNF-a (cytokine cocktail, CC) at 37°C for 14 days, and analysed the lysosomal glycohydrolases production and function. Lysosomal enzymes, b-N-Acetyl-Hexosaminidase, a-Mannosidase, b-Galactosidase and b-Glucoronidase are highly increased in a wide range in DCs (14 days of culture) with respect to the CD34+HSCs. All the glycohydrolases activities measured at 3 and 7 days in-vitro culture, were similar and four times more than CD34+HSCs (day 0) respectively. Interestingly, no activities increase were observed, even when SCF, an early acting cytokine, promoting cellular proliferation, were added to the CC medium, indicating that this phenomenon is independent from the proliferation process. Moreover, LPS treatment, to induce DCs maturation, slightly enhance the specific activities of all enzymes that we tested as respect to the untreated cells. and support the evidence that the lysosomal glycohydrolases activation is up-stream to DCs maturation process. Furthermore, for the first time, this date indicated that lysosomal glycohydrolases are regulated during the stem cell differentiation process. Understanding the key mechanism leading this phenomenon is critical for therapeutic application in immunologic or neoplastic disease.