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The CD64 receptor has been described as an interesting bacterial infection biomarker. Its expression has not been studied in previously healthy children admitted to pediatric critical care unit (PICU). Our objective was firstly to describe the CD64 expression and secondly study its diagnostic accuracy to discriminate bacterial versus viral infection in this children. We made a prospective double-blind observational study (March 2016-February 2018). A flow cytometry (FC) was done from peripheral blood at PICU admission. We studied the percentage of CD64+ neutrophils and the CD64 mean fluorescence intensity (MFI) on neutrophils (nCD64) and monocytes (mCD64). Statistical analyses were performed with non-parametric tests (p < 0.05). Twenty children in the bacterial infection group (BIG) and 25 in the viral infection group (VIG). Children in BIG showed higher values of CD64+ neutrophils (p = 0.000), nCD64 (p = 0.001), and mCD64 (p = 0.003). In addition, CD64+ neutrophils and nCD64 expression have positive correlation with procalcitonin and C reactive protein. The nCD64 area under the curve (AUC) was 0.83 (p = 0.000). The %CD64+ neutrophils showed an AUC of 0.828 (p = 0.000). The mCD64 AUC was 0.83 (p = 0.003). The nCD64 and %CD64+ neutrophils also showed higher combined values of sensitivity (74%) and specificity (90%) than all classical biomarkers.In our series CD64 expression allows to discriminate between bacterial and viral infection at PICU admission. Future studies should confirm this and be focused in the study of CD64 correlation with clinical data and its utility as an evolution biomarker in critical care children.

The selective delivery of therapeutic and imaging agents to tumoral cells has been postulated as one of the most important challenges in the nanomedicine field. Meta-iodobenzilguanidine (MIBG) is widely used for the diagnosis of neuroblastoma (NB) due to its strong affinity for the norepinephrine transporter (NET), usually overexpressed on the membrane of malignant cells. Herein, a family of novel Y-shaped scaffolds has been synthesized, which have structural analogues of MIBG covalently attached at each end of the Y-structure. The cellular uptake capacity of these double-targeting ligands has been evaluated in vitro and in vivo, yielding one specific Y-shaped structure that is able to be engulfed by the malignant cells, and accumulates in the tumoral tissue, at significantly higher levels than the structure containing only one single targeting agent. This Y-shaped ligand can provide a powerful tool for the current treatment and diagnosis of this disease., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The CD64 receptor has been described as a biomarker of bacterial infection. We speculated that CD64 surface expression on monocytes and granulocytes of children with severe acute bronchiolitis (SAB) could be altered in cases of probable bacterial infection (PBI) determined using classical biomarkers (procalcitonin and C-reactive protein, leukocyte count, and radiographic findings). A prospective observational pilot study was conducted from October 2015 to February 2016 in children admitted for pediatric critical care. A blood sample was taken in the first 24 hours of admission, and CD64 was measured by flow cytometry. The values obtained were analyzed and correlated with traditional biomarkers of PBI. Thirty-two children were included; a correlation was found between CD64 expression and the PBI criteria. CD64 surface expression was higher in children with PBI (area under the receiver operating characteristic curve of 0.73; P = 0.042) and the percentage of CD64 + granulocytes was higher in children with PBI. This is the first study to describe CD64 surface expression on monocytes and granulocytes in SAB, finding CD64 values to be higher in children with PBI. Larger clinical studies are needed to elucidate the real accuracy of CD64 as a biomarker of bacterial infection., (©2018 Society for Leukocyte Biology.)

Aim: To develop a NB animal model which makes possible studies related to tumor immunity., Materials and Methods: Two types of NB cells were used. Cell line 36769 was derived from TH-MYCN+ mouse in which overexpression of the MYCN gene is governed by rat tyrosine hydroxylase promotor. Cell line 4040 was derived from TH-MYCN/ALK mice, which in addition express an activating mutation of ALK gene. For each cell type, 1x10 6 neurospheres were implanted in 129/SVJ mice (with the same genetic background as donors, n=8), via orthotopic injection in the left suprarenal gland by intraperitoneal approach, through a transverse supraumbilical laparotomy. Daily postsurgical clinical follow-up of the animals was done until they were sacrificed at four weeks. The tumor presence was macroscopically confirmed. The tumoral sample was excised and was processed for cellular immunity and molecular tolerance mediator's studies. The existence of metastasis was investigated by flow cytometry in the spleen, bone marrow and peripheral blood., Results: 1) Orthotopic Neuroblastoma was generated in all the transplanted mice. 2) The tumors were infiltrated by several immune subpopulations, with effector, regulatory and suppressor inmunophenotype. This was similar to the inmunophenotype described in human NB. Furthermore, the molecular mediators of the environment point to a state of protumoral tolerance., Conclusion: The orthotopic implantation of NB neurospheres in syngeneic mice has allowed us to generate a NB model in which it has been possible to study the tumor immunity.

We report here our clinical experience of a program of compassionate use of Celyvir--autologous marrow-derived mesenchymal stem cells (MSCs) carrying an oncolytic adenovirus--for treating children with advanced metastatic neuroblastoma. Children received weekly doses of Celyvir with no concomitant treatments. The tolerance was excellent, with very mild and self-limited viral-related symptoms. Patients could be distinguished based on their response to therapy: those who had a clinical response (either complete, partial or stabilization) and those who did not respond. We found differences between patients who responded versus those who did not when analyzing their respective MSCs, at the expression levels of adhesion molecules (CCR1, CXCR1 and CXCR4) and in migration capacities in transwell assays, and in immune-related molecules (IFNγ, HLA-DR). These results suggest interpatient differences in the homing and immune modulation capacities of the therapy administered. In addition, the pretherapy immune T cell status and the T effector response were markedly different between responders and non-responders. We conclude that multidoses of Celyvir have an excellent safety profile in children with metastatic neuroblastoma. Intrinsic patients' and MSCs' factors appear to be related to clinical outcome., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Oncolytic virotherapy is gaining interest in the clinic as a new weapon against cancer. In vivo administration of oncolytic viruses showed important limitations that decrease their effectiveness very significantly: the antiviral immune response causes the elimination of the therapeutic effect, and the poor natural ability of oncolytic viruses to infect micrometastatic lesions significantly minimizes the effective dose of virus. This review will focus on updating the technical and scientific foundations of one of the strategies developed to overcome these limitations, ie, using cells as vehicles for oncolytic viruses. Among many candidates, a special type of adult stem cell, mesenchymal stem cells (MSCs), have already been used in the clinic as cell vehicles for oncolytic viruses, partly due to the fact that these cells are actively being evaluated for other indications. MSC carrier cells are used as Trojan horses loaded with oncoviruses, are administered systemically, and release their cargos at the right places. MSCs are equipped with an array of molecules involved in cell arrest in the capillaries (integrins and selectins), migration toward specific parenchymal locations within tissues (chemokine receptors), and invasion and degradation of the extracellular matrix (proteases). In addition to anatomical targeting capacity, MSCs have a well-recognized role in modulating immune responses by affecting cells of the innate (antigen-presenting cells, natural killer cells) and adaptive immune system (effector and regulatory lymphocytes). Therefore, carrier MSCs may also modulate the immune responses taking place after therapy, ie, the antiviral and the antitumor immune responses.

We studied whether chemokines may have a role in relapses in childhood acute lymphoblastic leukemia (ALL). We compared the levels of chemokine receptors in marrow samples from 82 children with ALL at diagnosis versus 15 at relapses, and quantified the levels of chemokines in central system fluid (CSF) samples. The functional role of specific chemokines was studied in vitro and in vivo. The expression of some chemokine receptors was upregulated upon leukemic relapse, both in B- and in T-ALL, and in cases of medullary and extramedullary involvement. CXCL10 induced chemotaxis in leukemic cell lines and in primary leukemic cells, depending upon the levels of CXCR3 expression. CXCL10 specifically diminished chemotherapy-induced apoptosis on ALL cells expressing CXCR3, partially inhibiting caspase activation and maintaining the levels of the antiapoptotic protein Bcl-2. Finally, immunodeficient mice engrafted with CXCR3-expressing human leukemic cells showed decreased infiltration of marrow, spleen, and CNS after receiving a CXCR3-antagonist molecule. CXCR3 signaling in ALL may have a dual function: chemotactic for the localisation of leukemic blasts in specific niches, and it may also confer resistance to chemotherapy, enhancing the chances for relapses., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The genetic programs that maintain hematopoiesis during steady state in physiologic conditions are different from those activated during stress. Here, we show that hematopoietic stem cells (HSCs) with deficiencies in components of the alternative NFκB pathway (the NFκB inducing kinase, NIK, and the downstream molecule NFκB2) had a defect in response to stressors such as supraphysiological doses of cytokines, chemotherapy, and hematopoietic transplantation. NIK-deficient mice had peripheral blood and bone marrow leukocyte numbers within normal ranges (except for the already reported defects in B-cell maturation); however, HSCs showed significantly slower expansion capacity in in vitro cultures compared to wild-type HSCs. This was due to a delayed cell cycle and increased apoptosis. In vivo experiments showed that NIK-deficient HSCs did not recover at the same pace as controls when challenged with myeloablative chemotherapy. Finally, NIK-deficient HSCs showed a significantly decreased competitive repopulation capacity in vivo. Using HSCs from mice deficient in one of two downstream targets of NIK, that is, either NFκB2 or c-Rel, only NFκB2 deficiency recapitulated the defects detected with NIK-deficient HSCs. Our results underscore the role of NIK and the alternative NFκB pathway for the recovery of normal levels of hematopoiesis after stress., (© 2015 AlphaMed Press.)

Mesenchymal stem cells (MSCs) are key components of the bone marrow microenvironment which contribute to the maintenance of the hematopoietic stem cell niche and exert immunoregulatory functions in innate and adaptive immunity. We analyze the immunobiology of MSCs derived from acute lymphoblastic leukemia (ALL) patients and their impact on NK cell function. In contrast to the inhibitory effects on the immune response exerted by MSCs from healthy donors (Healthy-MSCs), we demonstrate that MSCs derived from low/intermediate risk ALL patients at diagnosis (ALL-MSCs) promote an efficient NK cell response including cytokine production, phenotypic activation and most importantly, cytotoxicity. Longitudinal studies indicate that these immunostimulatory effects of ALL-MSCs are progressively attenuated. Healthy-MSCs adopt ALL-MSC-like immunomodulatory features when exposed to leukemia cells, acquiring the ability to stimulate NK cell antitumor function. The mechanisms underlying to these functional changes of ALL-MSCs include reduced production of soluble inhibitory factors, differential expression of costimulatory and coinhibitory molecules, increased expression of specific TLRs and Notch pathway activation. Collectively our findings indicate that, in response to leukemia cells, ALL-MSCs could mediate a host beneficial immunomodulatory effect by stimulating the antitumor innate immune response., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Novel targeting agents against neuroblastoma based on the meta-iodobenzylguanidine (MIBG) moiety were synthesized and biologically evaluated for nanocarrier vectorization. These compounds have been anchored on the surface of drug loaded mesoporous silica nanocarriers, resulting in the improved cellular uptake in tumoral cells. Neuroblastoma (NB) is the most frequent extracranial pediatric tumor. Advanced forms of the disease (metastatic and/or refractory) have a dismal prognosis despite the combination of chemotherapy, radiotherapy, surgery and bone narrow transplants. These treatments carry severe side effects and, in some cases, compromise the life of the patient. MIBG has been widely applied in the medical diagnosis of NB due to its affinity for tumor cells through the norepinephrine transporter (NET), which is expressed in 90% of NB tumors. The exclusive accumulation of MIBG in neuroblastoma has been widely studied; however, its properties have been never exploited as a targeting agent in nanocarrier drug delivery systems. Several structural analogues of MIBG have been prepared and attached on the surface of nanocarriers. Their selective internalization has been tested against human neuroblastoma cells, which show, in the best case, cellular uptake four times higher than that of the naked nanosystem. Furthermore, in vivo experiments showed preferential and selective accumulation and retention of the targeted nanosystem comparing with the naked and only PEGylated counterpart systems. This novel nanosystem could be easily applicable to all kinds of drug delivery nanocarriers, providing a universal tool for neuroblastoma chemotherapies that is superior to classical approaches through a novel nanosystem exclusively designed to target this terrible malignancy.

Purpose: Traditional inflammatory biomarkers are insufficient for the evaluation of bronchiolitis severity. Recent investigations have shown that the receptor for advanced glycation end product (RAGE) and its soluble isoforms (sRAGE) play a critical role in the pathogenesis of lung injury. Main objective was to assess the serum levels of sRAGE of children with severe bronchiolitis admitted to the pediatric intensive care unit (PICU). Secondary objective was to study sRAGE correlation with the evolution and traditional biomarkers., Methods: Prospective, observational and descriptive study, 43 healthy controls and 37 patients (December 2011-February 2012) were enrolled. sRAGE levels were assessed and compared. In patients, the relation between sRAGE levels and clinical evolution, respiratory assistance, white blood cell count, absolute neutrophils count, serum C-reactive protein, and serum procalcitonin was analyzed., Results: A statistical difference was found in the mean value of sRAGE at PICU admission between patients and controls (1,215.7 ± 535 vs 849 ± 579 pg/ml). Also a significant inverse correlation was found between sRAGE and the Wood-Downes Score at admission (p = 0.02)., Conclusions: Serum sRAGE could be elevated in children with bronchiolitis. Larger clinical studies are necessary to elucidate its role as a bronchiolitis inflammatory and/or lung injury biomarker.

Natural killer (NK) cells are critical for innate tumor immunity due to their specialized ability to recognize and kill neoplastically transformed cells. However, NK cells require a specific set of cytokine-mediated signals to achieve optimal effector function. Th1-associated cytokines promote effector functions that are inhibited by the prototypic Th2 cytokine IL4 and the TGFβ superfamily members TGFβ1 and activin-A. Interestingly, the largest subgroup of the TGFβ superfamily are the bone morphogenetic proteins (BMP), but the effects of BMP signaling on NK cell effector functions have not been evaluated. Here, we demonstrate that blood-circulating NK cells express type I and II BMP receptors, BMP-2 and BMP-6 ligands, and phosphorylated isoforms of Smad-1/-5/-8, which mediate BMP family member signaling. In opposition to the inhibitory effects of TGFβ1 or activin-A, autocrine BMP signaling was supportive to NK cell function. Mechanistic investigations in cytokine and TLR-L-activated NK cells revealed that BMP signaling optimized IFNγ and global cytokine and chemokine production, phenotypic activation and proliferation, and autologous dendritic cell activation and target cytotoxicity. Collectively, our findings identify a novel auto-activatory pathway that is essential for optimal NK cell effector function, one that might be therapeutically manipulated to help eradicate tumors. Cancer Res; 74(18); 5019-31. ©2014 AACR., (©2014 American Association for Cancer Research.)

An enzymatically activatable prodrug of doxorubicin was covalently coupled, using click-chemistry, to the hydrophobic core of poly(ethylene glycol)- b -poly[N-(2-hydroxypropyl)-methacrylamide-lactate] micelles. The release and cytotoxic activity of the prodrug was evaluated in vitro in A549 non-small-cell lung cancer cells after adding β-glucuronidase, an enzyme which is present intracellularly in lysosomes and extracellularly in necrotic areas of tumor lesions. The prodrug-containing micelles alone and in combination with standard and β-glucuronidase-producing oncolytic vaccinia viruses were also evaluated in vivo, in mice bearing A549 xenograft tumors. When combined with the oncolytic viruses, the micelles completely blocked tumor growth. Moreover, a significantly better antitumor efficacy as compared to virus treatment alone was observed when β-glucuronidase virus treated tumor-bearing mice received the prodrug-containing micelles. These findings show that combining tumor-targeted drug delivery systems with oncolytic vaccinia viruses holds potential for improving anticancer therapy.

The relevance of tumor microenvironment for the development and progression of tumor cells in hematological malignancies has been extensively reported. Identification of factors involved in the information exchange between the malignant cells and the bone marrow mesenchymal stem cells (BM-MSCs) and the knowledge on their functioning may provide important information to eliminate leukemic cells from protective BM niches. We evaluated changes in BM-MSCs obtained from children with acute lymphoblastic leukemia (ALL) at different times in the course of disease. Whereas ALL-MSCs did not exhibit phenotypic changes compared to BM-derived MSCs isolated from healthy donors, they exhibited increased adipogenic capacity. In addition, the viability of healthy CD34+ hematopoietic progenitors was significantly reduced when co-cultured with ALL-MSCs. ALL-MSCs grow less efficiently, although gradually recover normal growth with treatment. Accordingly, proliferation is particularly low in MSCs obtained at diagnosis and in the first days of treatment (+15 days), recovering to control levels after 35 days of treatment. Correlating these results with bone morphogenetic protein 4 (BMP4) production, a molecule demonstrated to affect MSC biology, we found higher production of BMP4 in ALL-MSCs derived from patients over the course of disease but not in those free of leukemia. However, no significant differences in the expression of different members of the BMP4 signaling pathway were observed. Furthermore, an inverse correlation between high levels of BMP4 production in the cultures and MSC proliferation was found, as observed in MSCs derived from patients at diagnosis that produce high BMP4 levels. In addition, co-culturing ALL-MSC with the REH leukemia cell line, but not CD34+ hematopoietic progenitors, powerfully enhanced BMP4 production, suggesting an intimate crosstalk among ALL-MSCs isolated from BM colonized by ALL cells that presumably also occurs in situ conditions. Our data may support the participation of BMP4 in BM niche, but the mechanism remains to be elucidated.

Neuroblastoma (NB) is one of the most common pediatric solid tumors and, like most human cancers, is characterized by a broad variety of genomic alterations. Although mesenchymal stem cells (MSCs) are known to interact with cancer cells, the relationship between MSCs and metastatic NB cancer cells in bone marrow (BM) is unknown. To obtain genetic evidence about this interaction, we isolated ΒΜ-derived MSCs from children with NB and compared their global expression patterns with MSCs obtained from normal pediatric donors, using the Agilent 44K microarrays. Significance analysis of microarray results with a false discovery rate (FDR) <5% identified 496 differentially expressed genes showing either a 2-fold upregulation or downregulation between both groups of samples. Comparison of gene ontology categories of differentially expressed genes revealed the upregulation of genes categorized as 'neurological system process', 'cell adhesion', 'apoptosis', 'cell surface receptor linked signal transduction', 'intrinsic to membrane' and 'extracellular region'. Among the downregulated genes, several immunology-related terms were the most abundant. These findings provide preliminary genetic evidence of the interaction between MSCs and NB cancer cells in ΒΜ as well as identify relevant biological processes potentially altered in MSCs in response to NB.

The MLL-AF4 fusion gene is a hallmark genomic aberration in high-risk acute lymphoblastic leukemia in infants. Although it is well established that MLL-AF4 arises prenatally during human development, its effects on hematopoietic development in utero remain unexplored. We have created a human-specific cellular system to study early hemato-endothelial development in MLL-AF4-expressing human embryonic stem cells (hESCs). Functional studies, clonal analysis and gene expression profiling reveal that expression of MLL-AF4 in hESCs has a phenotypic, functional and gene expression impact. MLL-AF4 acts as a global transcriptional activator and a positive regulator of homeobox gene expression in hESCs. Functionally, MLL-AF4 enhances the specification of hemogenic precursors from hESCs but strongly impairs further hematopoietic commitment in favor of an endothelial cell fate. MLL-AF4 hESCs are transcriptionally primed to differentiate towards hemogenic precursors prone to endothelial maturation, as reflected by the marked upregulation of master genes associated to vascular-endothelial functions and early hematopoiesis. Furthermore, we report that MLL-AF4 expression is not sufficient to transform hESC-derived hematopoietic cells. This work illustrates how hESCs may provide unique insights into human development and further our understanding of how leukemic fusion genes, known to arise prenatally, regulate human embryonic hematopoietic specification.

Human sarcomas have been modeled in mice by expression of specific fusion genes in mesenchymal stem cells (MSCs). However, sarcoma models based on human MSCs are still missing. We attempted to develop a model of liposarcoma by expressing FUS (FUsed in Sarcoma; also termed TLS, Translocated in LipoSarcoma)-CHOP (C/EBP HOmologous Protein; also termed DDIT3, DNA Damage-Inducible Transcript 3), a hallmark mixoid liposarcoma-associated fusion oncogene, in wild-type and p53-deficient mouse and human adipose-derived mesenchymal stem/stromal cells (ASCs). FUS-CHOP induced liposarcoma-like tumors when expressed in p53(-/-) but not in wild-type (wt) mouse ASCs (mASCs). In the absence of FUS-CHOP, p53(-/-) mASCs forms leiomyosarcoma, indicating that the expression of FUS-CHOP redirects the tumor genesis/phenotype. FUS-CHOP expression in wt mASCs does not initiate sarcomagenesis, indicating that p53 deficiency is required to induce FUS-CHOP-mediated liposarcoma in fat-derived mASCs. In a human setting, p53-deficient human ASCs (hASCs) displayed a higher in vitro growth rate and a more extended lifespan than wt hASCs. However, FUS-CHOP expression did not induce further changes in culture homeostasis nor initiated liposarcoma in either wt or p53-depleted hASCs. These results indicate that FUS-CHOP expression in a p53-deficient background is sufficient to initiate liposarcoma in mouse but not in hASCs, suggesting the need of additional cooperating mutations in hASCs. A microarray gene expression profiling has shed light into the potential deregulated pathways in liposarcoma formation from p53-deficient mASCs expressing FUS-CHOP, which might also function as potential cooperating mutations in the transformation process from hASCs., (Copyright © 2010 AlphaMed Press.)

Lineage-specific differentiation potential varies among different human pluripotent stem cell (hPSC) lines, becoming therefore highly desirable to prospectively know which hPSC lines exhibit the highest differentiation potential for a certain lineage. We have compared the hematopoietic potential of 14 human embryonic stem cell (hESC)/induced pluripotent stem cell (iPSC) lines. The emergence of hemogenic progenitors, primitive and mature blood cells, and colony-forming unit (CFU) potential was analyzed at different time points. Significant differences in the propensity to differentiate toward blood were observed among hPSCs: some hPSCs exhibited good blood differentiation potential, whereas others barely displayed blood-differentiation capacity. Correlation studies revealed that the CFU potential robustly correlates with hemogenic progenitors and primitive but not mature blood cells. Developmental progression of mesoendodermal and hematopoietic transcription factors expression revealed no correlation with either hematopoietic initiation or maturation efficiency. Microarray studies showed distinct gene expression profile between hPSCs with good versus poor hematopoietic potential. Although neuroectoderm-associated genes were downregulated in hPSCs prone to hematopoietic differentiation many members of the Nodal/Activin signaling were upregulated, suggesting that this signaling predicts those hPSC lines with good blood-differentiation potential. The association between Nodal/Activin signaling and the hematopoietic differentiation potential was confirmed using loss- and gain-of-function functional assays. Our data reinforce the value of prospective comparative studies aimed at determining the lineage-specific differentiation potential among different hPSCs and indicate that Nodal/Activin signaling seems to predict those hPSC lines prone to hematopoietic specification.

MLL-AF4 fusion is a hallmark genetic abnormality in infant B-acute lymphoblastic leukemia (B-ALL) known to arise in utero. The cellular origin of leukemic fusion genes during human development is difficult to ascertain. The bone marrow (BM) microenvironment plays an important role in the pathogenesis of several hematological malignances. BM mesenchymal stem cells (BM-MSC) from 38 children diagnosed with cytogenetically different acute leukemias were screened for leukemic fusion genes. Fusion genes were absent in BM-MSCs of childhood leukemias carrying TEL-AML1, BCR-ABL, AML1-ETO, MLL-AF9, MLL-AF10, MLL-ENL or hyperdiploidy. However, MLL-AF4 was detected and expressed in BM-MSCs from all cases of MLL-AF4(+) B-ALL. Unlike leukemic blasts, MLL-AF4(+) BM-MSCs did not display monoclonal Ig gene rearrangements. Endogenous or ectopic expression of MLL-AF4 exerted no effect on MSC culture homeostasis. These findings suggest that MSCs may be in part tumor-related, highlighting an unrecognized role of the BM milieu on the pathogenesis of MLL-AF4(+) B-ALL. MLL-AF4 itself is not sufficient for MSC transformation and the expression of MLL-AF4 in MSCs is compatible with a mesenchymal phenotype, suggesting a differential impact in the hematopoietic system and mesenchyme. The absence of monoclonal rearrangements in MLL-AF4(+) BM-MSCs precludes the possibility of cellular plasticity or de-differentiation of B-ALL blasts and suggests that MLL-AF4 might arise in a population of prehematopoietic precursors.

MLL rearrangements are hallmark genetic abnormalities in infant leukemia known to arise in utero. They can be induced during human prenatal development upon exposure to etoposide. We also hypothesize that chronic exposure to etoposide might render cells more susceptible to other genomic insults. Here, for the first time, human embryonic stem cells (hESCs) were used as a model to test the effects of etoposide on human early embryonic development. We addressed whether: (i) low doses of etoposide promote MLL rearrangements in hESCs and hESCs-derived hematopoietic cells; (ii) MLL rearrangements are sufficient to confer hESCs with a selective growth advantage and (iii) continuous exposure to low doses of etoposide induces hESCs to acquire other chromosomal abnormalities. In contrast to cord blood-derived CD34(+) and hESC-derived hematopoietic cells, exposure of undifferentiated hESCs to a single low dose of etoposide induced a pronounced cell death. Etoposide induced MLL rearrangements in hESCs and their hematopoietic derivatives. After long-term culture, the proportion of hESCs harboring MLL rearrangements diminished and neither cell cycle variations nor genomic abnormalities were observed in the etoposide-treated hESCs, suggesting that MLL rearrangements are insufficient to confer hESCs with a selective proliferation/survival advantage. However, continuous exposure to etoposide induced MLL breaks and primed hESCs to acquire other major karyotypic abnormalities. These data show that chronic exposure of developmentally early stem cells to etoposide induces MLL rearrangements and make hESCs more prone to acquire other chromosomal abnormalities than postnatal CD34(+) cells, linking embryonic genotoxic exposure to genomic instability.

A paracrine regulation was recently proposed in human embryonic stem cells (hESCs) grown in mouse embryonic fibroblast (MEF)-conditioned media (MEF-CM), where hESCs spontaneously differentiate into autologous fibroblast-like cells to maintain culture homeostasis by producing TGF-beta and insulin-like growth factor-II (IGF-II) in response to basic fibroblast growth factor (bFGF). Although the importance of TGF-beta family members in the maintenance of pluripotency of hESCs is widely established, very little is known about the role of IGF-II. In order to ease hESC culture conditions and to reduce xenogenic components, we sought (i) to determine whether hESCs can be maintained stable and pluripotent using CM from human foreskin fibroblasts (HFFs) and human mesenchymal stem cells (hMSCs) rather than MEF-CM, and (ii) to analyze whether the cooperation of bFGF with TGF-beta and IGF-II to maintain hESCs in MEF-CM may be extrapolated to hESCs maintained in allogeneic mesenchymal stem cell (MSC)-CM and HFF-CM. We found that MSCs and HFFs express all FGF receptors (FGFR1-4) and specifically produce TGF-beta in response to bFGF. However, HFFs but not MSCs secrete IGF-II. Despite the absence of IGF-II in MSC-CM, hESC pluripotency and culture homeostasis were successfully maintained in MSC-CM for over 37 passages. Human ESCs derived on MSCs and hESCs maintained in MSC-CM retained hESC morphology, euploidy, expression of surface markers and transcription factors linked to pluripotency and displayed in vitro and in vivo multilineage developmental potential, suggesting that IGF-II may be dispensable for hESC pluripotency. In fact, IGF-II blocking had no effect on the homeostasis of hESC cultures maintained either on HFF-CM or on MSC-CM. These data indicate that hESCs are successfully maintained feeder-free with IGF-II-lacking MSC-CM, and that the previously proposed paracrine mechanism by which bFGF cooperates with TGF-beta and IGF-II in the maintenance of hESCs in MEF-CM may not be fully extrapolated to hESCs maintained in CM from human MSCs.

MicroRNAs (miRNAs) play a central role in the regulation of multiple biological processes including the maintenance of stem cell self-renewal and pluripotency. Recently, the miRNA cluster miR302-367 was shown to be differentially expressed in embryonic stem cells (ESCs). Unfortunately, very little is known about the genomic structure of miRNA-encoding genes and their transcriptional units. Here, we have characterized the structure of the gene coding for the human miR302-367 cluster. We identify the transcriptional start and functional core promoter region which specifically drives the expression of this miRNA cluster. The promoter activity depends on the ontogeny and hierarchical cellular stage. It is functional during embryonic development, but it is turned off later in development. From a hierarchical standpoint, its activity decays upon differentiation of ESCs, suggesting that its activity is restricted to the ESC compartment and that the ESC-specific expression of the miR302-367 cluster is fully conferred by its core promoter transcriptional activity. Furthermore, algorithmic prediction of transcription factor binding sites and knockdown studies suggest that ESC-associated transcription factors, including Nanog, Oct3/4, Sox2, and Rex1 may be upstream regulators of miR302-367 promoter. This study represents the first identification, characterization, and functional validation of a human miRNA promoter in stem cells. This study opens up new avenues to further investigate the upstream transcriptional regulation of the miR302-367 cluster and to dissect how these miRNAs integrate in the complex molecular network conferring stem cell properties to ESCs.

Translating a graded morphogen distribution into tight response borders is central to all developmental processes. Yet, the molecular mechanisms generating such behavior are poorly understood. During patterning of the Drosophila embryonic ventral ectoderm, a graded mitogen-activated protein kinase (MAPK) activation is converted into an all-or-none degradation switch of the Yan transcriptional repressor. Replacing the cardinal phosphorylated amino acid of Yan by a phosphomimetic residue allowed its degradation in a MAPK-independent manner, consistent with Yan phosphorylation being the critical event in generating the switch. Several alternative threshold mechanisms that could, in principle, be realized by this phosphorylation, including first order, cooperativity, positive feedback and zero-order ultrasensitivity, were analyzed. We found that they can be distinguished by their kinetics and steady-state responses to Yan overexpression. In agreement with the predictions for zero-order kinetics, an increase in Yan levels did not shift the degradation border, but significantly elevated the time required to reach steady state. We propose that a reversible loop of Yan phosphorylation implements a zero-order ultrasensitivity-like threshold mechanism, with the capacity to form sharp thresholds that are independent of the level of Yan.

Splitting and apparent splicing of ribosomal RNA, both previously unknown in vertebrates, were found in rodents of the genus Ctenomys. Instead of being formed by a single molecule of 4.4 kb, 28S rRNA is split in two molecules of 2.6 and 1.8 kb. A hidden break, mapping within a 106 bp 'intron' located in the D6 divergent region, is expressed in mature ribosomes of liver, lung, heart and spleen, as well as in primary fibroblast cultures. Testis-specific processing eliminates the intron and concomitantly the break site, producing non-split 28S rRNA molecules exclusively in this organ. The intron is flanked by two 9 bp direct repeats, revealing the acquisition by insertion of a novel rRNA processing strategy in the evolution of higher organisms.