Transcriptional, epigenetic and retroviral signatures identify regulatory regions involved in hematopoietic lineage commitment.

Genome-wide approaches allow investigating the molecular circuitry wiring the genetic and epigenetic programs of human somatic stem cells. Hematopoietic stem/progenitor cells (HSPC) give rise to the different blood cell types; however, the molecular basis of human hematopoietic lineage commitment is poorly characterized. Here, we define the transcriptional and epigenetic profile of human HSPC and early myeloid and erythroid progenitors by a combination of Cap Analysis of Gene Expression (CAGE), ChIP-seq and Moloney leukemia virus (MLV) integration site mapping. Most promoters and transcripts were shared by HSPC and committed progenitors, while enhancers and super-enhancers consistently changed upon differentiation, indicating that lineage commitment is essentially regulated by enhancer elements. A significant fraction of CAGE promoters differentially expressed upon commitment were novel, harbored a chromatin enhancer signature, and may identify promoters and transcribed enhancers driving cell commitment. MLV-targeted genomic regions co-mapped with cell-specific active enhancers and super-enhancers. Expression analyses, together with an enhancer functional assay, indicate that MLV integration can be used to identify bona fide developmentally regulated enhancers. Overall, this study provides an overview of transcriptional and epigenetic changes associated to HSPC lineage commitment, and a novel signature for regulatory elements involved in cell identity.

Neutrophil gelatinase-associated lipocalin increases HLA-G(+)/FoxP3(+) T-regulatory cell population in an in vitro model of PBMC.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is emerging as a mediator of various biological and pathological states. However, the specific biological role of this molecule remains unclear, as it serves as a biomarker for many conditions. The high sensitivity of NGAL as a biomarker coupled with relatively low specificity may hide important biological roles. Data point toward an acute compensatory, protective role for NGAL in response to adverse cellular stresses, including inflammatory and oxidative stress. The aim of this study was to understand whether NGAL modulates the T-cell response through regulation of the human leukocyte antigen G (HLA-G) complex, which is a mediator of tolerance. METHODOLOGY/PRINCIPAL FINDINGS: Peripheral blood mononuclear cells (PBMCs) were obtained from eight healthy donors and isolated by centrifugation on a Ficoll gradient. All donors gave informed consent. PBMCs were treated with four different concentrations of NGAL (40-320 ng/ml) in an iron-loaded or iron-free form. Changes in cell phenotype were analyzed by flow cytometry. NGAL stimulated expression of HLA-G on CD4+ T cells in a dose- and iron-dependent manner. Iron deficiency prevented NGAL-mediated effects, such that HLA-G expression was unaltered. Furthermore, NGAL treatment affected stimulation of regulatory T cells and in vitro expansion of CD4(+) CD25(+) FoxP3(+) cells. An NGAL neutralizing antibody limited HLA-G expression and significantly decreased the percentage of CD4(+) CD25(+) FoxP3(+) cells. CONCLUSIONS/SIGNIFICANCE: We provide in vitro evidence that NGAL is involved in cellular immunity. The potential role of NGAL as an immunomodulatory molecule is based on its ability to induce immune tolerance by upregulating HLA-G expression and expansion of T-regulatory cells in healthy donors. Future studies should further evaluate the role of NGAL in immunology and immunomodulation and its possible relationship to immunosuppressive therapy efficacy, tolerance induction in transplant patients, and other immunological disorders.

Human cadaver multipotent stromal/stem cells isolated from arteries stored in liquid nitrogen for 5 years.

INTRODUCTION: Regenerative medicine challenges researchers to find noncontroversial, safe and abundant stem cell sources. In this context, harvesting from asystolic donors could represent an innovative and unlimited reservoir of different stem cells. In this study, cadaveric vascular tissues were established as an alternative source of human cadaver mesenchymal stromal/stem cells (hC-MSCs). We reported the successful cell isolation from postmortem arterial segments stored in a tissue-banking facility for at least 5 years. METHODS: After thawing, hC-MSCs were isolated with a high efficiency (12×106) and characterized with flow cytometry, immunofluorescence, molecular and ultrastructural approaches. RESULTS: In early passages, hC-MSCs were clonogenic, highly proliferative and expressed mesenchymal (CD44, CD73, CD90, CD105, HLA-G), stemness (Stro-1, Oct-4, Notch-1), pericyte (CD146, PDGFR-ß, NG2) and neuronal (Nestin) markers; hematopoietic and vascular markers were negative. These cells had colony and spheroid-forming abilities, multipotency for their potential to differentiate in multiple mesengenic lineages and immunosuppressive activity to counteract proliferation of phytohemagglutinin-stimulated blood mononuclear cells. CONCLUSIONS: The efficient procurement of stem cells from cadaveric sources, as postmortem vascular tissues, demonstrates that such cells can survive to prolonged ischemic insult, anoxia, freezing and dehydration injuries, thus paving the way for a scientific revolution where cadaver stromal/stem cells could effectively treat patients demanding cell therapies.

Shiga toxin 1, as DNA repair inhibitor, synergistically potentiates the activity of the anticancer drug, mafosfamide, on raji cells.

Shiga toxin 1 (Stx1), produced by pathogenic Escherichia coli, targets a restricted subset of human cells, which possess the receptor globotriaosylceramide (Gb3Cer/CD77), causing hemolytic uremic syndrome. In spite of the high toxicity, Stx1 has been proposed in the treatment of Gb3Cer/CD77-expressing lymphoma. Here, we demonstrate in a Burkitt lymphoma cell model expressing this receptor, namely Raji cells, that Stx1, at quasi-non-toxic concentrations (0.05-0.1 pM), inhibits the repair of mafosfamide-induced DNA alkylating lesions, synergistically potentiating the cytotoxic activity of the anticancer drug. Conversely, human promyelocytic leukemia cells HL-60, which do not express Gb3Cer/CD77, were spared by the toxin as previously demonstrated for CD34+ human progenitor cells, and hence, in this cancer model, no additive nor synergistic effects were observed with the combined Stx1/mafosfamide treatment. Our findings suggest that Stx1 could be used to improve the mafosfamide-mediated purging of Gb3Cer/CD77+ tumor cells before autologous bone marrow transplantation.

A combination of temsirolimus, an allosteric mTOR inhibitor, with clofarabine as a new therapeutic option for patients with acute myeloid leukemia.

Signaling through the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream effectors, Akt and mechanistic target of rapamycin (mTOR), is aberrantly activated in acute myeloid leukemia (AML) patients, where it contributes to leukemic cell proliferation, survival, and drug-resistance. Thus, inhibiting mTOR signaling in AML blasts could enhance their sensitivity to cytotoxic agents. Preclinical data also suggest that allosteric mTOR inhibition with rapamycin impaired leukemia initiating cells (LICs) function. In this study, we assessed the therapeutic potential of a combination consisting of temsirolimus [an allosteric mTOR complex 1 (mTORC1) inhibitor] with clofarabine, a nucleoside analogue with potent inhibitory effects on both ribonucleotide reductase and DNA polymerase. The drug combination (CLO-TOR) displayed synergistic cytotoxic effects against a panel of AML cell lines and primary cells from AML patients. Treatment with CLO-TOR induced a G0/G1-phase cell cycle arrest, apoptosis, and autophagy. CLO-TOR was pro-apoptotic in an AML patient blast subset (CD34⁺/CD38⁻/CD123⁺), which is enriched in putative leukemia initiating cells (LICs). In summary, the CLO-TOR combination could represent a novel valuable treatment for AML patients, also in light of its efficacy against LICs.

Harnessing the PI3K/Akt/mTOR pathway in T-cell acute lymphoblastic leukemia: eliminating activity by targeting at different levels.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant hematological disorder arising in the thymus from T-cell progenitors. T-ALL mainly affects children and young adults, and remains fatal in 20% of adolescents and 50% of adults, despite progress in polychemotherapy protocols. Therefore, innovative targeted therapies are desperately needed for patients with a dismal prognosis. Aberrant activation of PI3K/Akt/mTOR signaling is a common event in T-ALL patients and portends a poor prognosis. Preclinical studies have highlighted that modulators of PI3K/Akt/mTOR signaling could have a therapeutic relevance in T-ALL. However, the best strategy for inhibiting this highly complex signal transduction pathway is still unclear, as the pharmaceutical companies have disclosed an impressive array of small molecules targeting this signaling network at different levels. Here, we demonstrate that a dual PI3K/PDK1 inhibitor, NVP-BAG956, displayed the most powerful cytotoxic affects against T-ALL cell lines and primary patients samples, when compared with a pan class I PI3K inhibitor (GDC-0941), an allosteric Akt inhibitor (MK-2206), an mTORC1 allosteric inhibitor (RAD-001), or an ATP-competitive mTORC1/mTORC2 inhibitor (KU63794). Moreover, we also document that combinations of some of the aforementioned drugs strongly synergized against T-ALL cells at concentrations well below their respective IC50. This observation indicates that vertical inhibition at different levels of the PI3K/Akt/mTOR network could be considered as a future innovative strategy for treating T-ALL patients.

Analysis of the effects of HIV-1 Tat on the survival and differentiation of vessel wall-derived mesenchymal stem cells.

HIV infection is an independent risk factor for atherosclerosis development and cardiovascular damage. As vessel wall mesenchymal stem cells (MSCs) are involved in the regulation of vessel structure homeostasis, we investigated the role of Tat, a key factor in HIV replication and pathogenesis, in MSC survival and differentiation. The survival of subconfluent MSCs was impaired when Tat was added at high concentrations (200-1,000 ng/ml), whereas lower Tat concentrations (1-100 ng/ml) did not promote apoptosis. Tat enhanced the differentiation of MSC toward adipogenesis by the transcription and activity upregulation of PPARγ. This Tat-related modulation of adipogenesis was tackled by treatment with antagonists of Tat-specific receptors such as SU5416 and RGD Fc. In contrast, Tat inhibited the differentiation of MSCs to endothelial cells by downregulating the expression of VEGF-induced endothelial markers such as Flt-1, KDR, and vWF. The treatment of MSCs with Tat-derived peptides corresponding to the cysteine-rich, basic, and RGD domains indicated that these Tat regions are involved in the inhibition of endothelial marker expression. The Tat-related impairment of MSC survival and differentiation might play an important role in vessel damage and formation of the atherosclerotic lesions observed in HIV-infected patients.

HIV-1 and recombinant gp120 affect the survival and differentiation of human vessel wall-derived mesenchymal stem cells.

BACKGROUND: HIV infection elicits the onset of a progressive immunodeficiency and also damages several other organs and tissues such as the CNS, kidney, heart, blood vessels, adipose tissue and bone. In particular, HIV infection has been related to an increased incidence of cardiovascular diseases and derangement in the structure of blood vessels in the absence of classical risk factors. The recent characterization of multipotent mesenchymal cells in the vascular wall, involved in regulating cellular homeostasis, suggests that these cells may be considered a target of HIV pathogenesis. This paper investigated the interaction between HIV-1 and vascular wall resident human mesenchymal stem cells (MSCs). RESULTS: MSCs were challenged with classical R5 and X4 HIV-1 laboratory strains demonstrating that these strains are able to enter and integrate their retro-transcribed proviral DNA in the host cell genome. Subsequent experiments indicated that HIV-1 strains and recombinant gp120 elicited a reliable increase in apoptosis in sub-confluent MSCs. Since vascular wall MSCs are multipotent cells that may be differentiated towards several cell lineages, we challenged HIV-1 strains and gp120 on MSCs differentiated to adipogenesis and endotheliogenesis. Our experiments showed that the adipogenesis is increased especially by upregulated PPARγ activity whereas the endothelial differentiation induced by VEGF treatment was impaired with a downregulation of endothelial markers such as vWF, Flt-1 and KDR expression. These viral effects in MSC survival and adipogenic or endothelial differentiation were tackled by CD4 blockade suggesting an important role of CD4/gp120 interaction in this context. CONCLUSIONS: The HIV-related derangement of MSC survival and differentiation may suggest a direct role of HIV infection and gp120 in impaired vessel homeostasis and in genesis of vessel damage observed in HIV-infected patients.

Preclinical testing of the Akt inhibitor triciribine in T-cell acute lymphoblastic leukemia.

Over the past 20 years, survival rates of T-cell acute lymphoblastic leukemia (T-ALL) patients have improved, mainly because of advances in polychemotherapy protocols. Despite these improvements, we still need novel and less toxic treatment strategies targeting aberrantly activated signaling networks which increase proliferation, survival, and drug resistance of T-ALL cells. One such network is represented by the phosphatidylinositol 3-kinase (PI3K)/Akt axis. PI3K inhibitors have displayed some promising effects in preclinical models of T-ALL. Here, we have analyzed the therapeutic potential of the Akt inhibitor, triciribine, in T-ALL cell lines. Triciribine caused cell cycle arrest and caspase-dependent apoptosis. Western blots demonstrated a dose-dependent dephosphorylation of Akt1/Akt2, and of mammalian target of rapamycin complex 1 downstream targets in response to triciribine. Triciribine induced autophagy, which could be interpreted as a defensive mechanism, because an autophagy inhibitor (chloroquine) increased triciribine-induced apoptosis. Triciribine synergized with vincristine, a chemotherapeutic drug employed for treating T-ALL patients, and targeted the side population of T-ALL cell lines, which might correspond to leukemia initiating cells. Our findings indicate that Akt inhibition, either alone or in combination with chemotherapeutic drugs, may serve as an efficient treatment towards T-ALL cells requiring upregulation of this signaling pathway for their proliferation and survival.

Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against T-cell acute lymphoblastic leukemia.

Recent findings have highlighted that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival, and drug resistance. These observations lend compelling weight to the application of PI3K/Akt/mTOR inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the novel dual PI3K/mTOR inhibitor NVP-BEZ235, an orally bioavailable imidazoquinoline derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. NVP-BEZ235 was cytotoxic to a panel of T-ALL cell lines as determined by MTT assays. NVP-BEZ235 treatment resulted in cell cycle arrest and apoptosis. Western blots showed a dose- and time-dependent dephosphorylation of Akt and mTORC1 downstream targets in response to NVP-BEZ235. Remarkably, NVP-BEZ235 targeted the side population of both T-ALL cell lines and patient lymphoblasts, which might correspond to leukemia-initiating cells, and synergized with chemotherapeutic agents (cyclophosphamide, cytarabine, dexamethasone) currently used for treating T-ALL patients. NVP-BEZ235 reduced chemoresistance to vincristine induced in Jurkat cells by coculturing with MS-5 stromal cells, which mimic the bone marrow microenvironment. NVP-BEZ235 was cytotoxic to T-ALL patient lymphoblasts displaying pathway activation, where the drug dephosphorylated eukaryotic initiation factor 4E-binding protein 1, at variance with rapamycin. Taken together, our findings indicate that longitudinal inhibition at two nodes of the PI3K/Akt/mTOR network with NVP-BEZ235, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment of those T-ALLs that have aberrant upregulation of this signaling pathway for their proliferation and survival.

Multidistrict human mesenchymal vascular cells: pluripotency and stemness characteristics.

BACKGROUND AIMS: The presence of ectopic tissues in the pathologic artery wall raises the issue of whether multipotent stem cells may reside in the vasculature itself. Recently mesenchymal stromal cells (MSC) have been isolated from different human vascular segments (VW MSC), belying the previous view that the vessel wall is a relatively quiescent tissue. METHODS: Resident multipotent cells were recovered from fresh arterial segments (aortic arches, thoracic and femoral arteries) collected in a tissue-banking facility and used to establish an in situ and in vitro study of the stemness features and multipotency of these multidistrict MSC populations. RESULTS: Notch-1+, Stro-1+, Sca-1+ and Oct-4+ cells were distributed along an arterial wall vasculogenic niche. Multidistrict VW MSC homogeneously expressed markers of stemness (Stro-1, Notch-1 and Oct-4) and MSC lineages (CD44, CD90, CD105, CD73, CD29 and CD166) whilst they were negative for hematopoietic and endothelial markers (CD34, CD45, CD31 and vWF). Each VW MSC population had characteristics of stem cells, i.e. a high efflux capability for Hoechst 33342 dye and the ability to form spheroids when grown in suspension and generate colonies when seeded at low density. Again, VW MSC cultured in induction media exhibited adipogenic, chondrogenic and leiomyogenic potential but less propensity to osteogenic differentiation, as documented by histochemical, immunohistochemical, molecular and electron microscopy analysis. CONCLUSIONS: Overall, these findings may enlighten the physiopathologic mechanisms of vascular wall diseases as well as having potential implications for cellular, genetic and tissue engineering approaches to treating vascular pathologies when these are unresponsive to medical and surgical therapies.

Isolation of stem cell populations with trophic and immunoregulatory functions from human intestinal tissues: potential for cell therapy in inflammatory bowel disease.

BACKGROUND AIMS: Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSC) are currently under evaluation in phase III clinical trials for inflammatory bowel disease and other intestinal disease manifestations. The therapeutic efficacy of these treatments may derive from a combination of the differentiation, trophic and immunomodulatory abilities of the transplanted cells. We investigated intestinal tissues as sources of MSC: such cells may support tissue-specific functions and hold advantages for engraftment and contribution in the gastrointestinal environment. METHODS: Intestinal specimens were collected, and the mucosa and submucosa mechanically separated and enzymatically digested. Mesenchymal stromal populations were isolated, expanded and characterized under conditions commonly used for MSC. The differentiation potential, trophic effect and immunomodulatory ability were investigated. Results We successfully isolated and extensively expanded populations showing the typical MSC profile: CD29+, CD44+, CD73+, CD105+ and CD166+, and CD14(-), CD34(-) and CD45(-). Intestinal mucosal (IM) MSC were also CD117+, while submucosal cultures (ISM MSC) showed CD34+ subsets. The cells differentiated toward osteogenic, adipogenic and angiogenic commitments. Intestinal-derived MSC were able to induce differentiation and organization of intestinal epithelial cells (Caco-2) in three-dimensional collagen cultures. Immunomodulatory activity was evidenced in co-cultures with normal heterologous phytohemagglutinin-stimulated peripheral blood mononuclear cells. Conclusions Multipotent MSC can be isolated from intestinal mucosal and submucosal tissues. IM MSC and ISM MSC are able to perform trophic and immunomodulatory functions. These findings could open a pathway for novel approaches to intestinal disease treatment.

Gravitational field-flow fractionation of human hemopoietic stem cells.

New cell sorting methodologies, which are simple, fast, non-invasive, and able to isolate homogeneous cell populations, are needed for applications ranging from gene expression analysis to cell-based therapy. In particular, in the forefront of stem cell isolation, progenitor cells have to be separated under mild experimental conditions from complex heterogeneous mixtures prepared from human tissues. Most of the methodologies now employed make use of immunological markers. However, it is widely acknowledged that specific markers for pluripotent stem cells are not as yet available, and cell labelling may interfere with the differentiation process. This work presents for the first time gravitational field-flow fractionation (GrFFF), as a tool for tag-less, direct selection of human hematopoietic stem and progenitor cells from cell samples obtained by peripheral blood aphaeresis. These cells are responsible to repopulate the hemopoietic system and they are used in transplantation therapies. Blood aphaeresis sample were injected into a GrFFF system and collected fractions were characterized by flow cytometry for CD34 and CD45 expression, and then tested for viability and multi-differentiation potential. The developed GrFFF method allowed obtaining high enrichment levels of viable, multi-potent hematopoietic stem cells in specific fraction and it showed to fulfil major requirements of analytical performance, such as selectivity and reproducibility of the fractionation process and high sample recovery.

Dual inhibition of class IA phosphatidylinositol 3-kinase and mammalian target of rapamycin as a new therapeutic option for T-cell acute lymphoblastic leukemia.

Recent investigations have documented that constitutively activated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it strongly influences growth and survival. These findings lend compelling weight for the application of PI3K/Akt/mTOR inhibitors in T-ALL. However, our knowledge of PI3K/Akt/mTOR signaling in T-ALL is limited and it is not clear whether it could be an effective target for innovative therapeutic strategies. Here, we have analyzed the therapeutic potential of the dual PI3K/mTOR inhibitor PI-103, a small synthetic molecule of the pyridofuropyrimidine class, on both T-ALL cell lines and patient samples, which displayed constitutive activation of PI3K/Akt/mTOR signaling. PI-103 inhibited the growth of T-ALL cells, including 170-kDa P-glycoprotein overexpressing cells. PI-103 cytotoxicity was independent of p53 gene status. PI-103 was more potent than inhibitors that are selective only for PI3K (Wortmannin, LY294002) or for mTOR (rapamycin). PI-103 induced G(0)-G(1) phase cell cycle arrest and apoptosis, which was characterized by activation of caspase-3 and caspase-9. PI-103 caused Akt dephosphorylation, accompanied by dephosphorylation of the Akt downstream target, glycogen synthase kinase-3beta. Also, mTOR downstream targets were dephosphorylated in response to PI-103, including p70S6 kinase, ribosomal S6 protein, and 4E-BP1. PI-103 strongly synergized with vincristine. These findings indicate that multitargeted therapy toward PI3K and mTOR alone or with existing drugs may serve as an efficient treatment toward T-ALL cells, which require up-regulation of PI3K/Akt/mTOR signaling for their survival and growth.

A novel stem cell tag-less sorting method.

Growing interest in stem cell research has led to the development of a number of new methods for isolation. The lack of homogeneity in stem cell preparation blurs standardization, which however is recommended for successful applications. Among stem cells, mesenchymal stem cells (MSCs) are promising candidates for cell therapy applications. This paper presents a fractionation protocol based on a tag-less, flow-assisted method of purifying, distinguishing and sorting MSCs. The protocol entails a suspension of cells in a transport fluid being injected into a ribbon-like capillary device by continuous flow. In a relatively short time (about 30 min) sorted cells are collected. The protocol has been applied to the improvement of MSC isolation, with a specific view to reducing cell manipulation operations, keeping instrumental simplicity and increasing analytical information for cell characterization. Applications such as MSC purification from epithelial contaminants, MSC characterization from various human sources and sorting of MSC subpopulations with high differentiation potential are described. The low cost, full biocompatibility and scale-up potential of the protocol presented could make the procedure attractive for stem cell selection.