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An essential step for therapeutic and research applications of stem cells is the ability to differentiate them into specific cell types. Endodermal cell derivatives, including lung, liver, and pancreas, are of interest for regenerative medicine, but efforts to produce these cells have been met with only modest success. In a screen of 4000 compounds, two cell-permeable small molecules were indentified that direct differentiation of ESCs into the endodermal lineage. These compounds induce nearly 80% of ESCs to form definitive endoderm, a higher efficiency than that achieved by Activin A or Nodal, commonly used protein inducers of endoderm. The chemically induced endoderm expresses multiple endodermal markers, can participate in normal development when injected into developing embryos, and can form pancreatic progenitors. The application of small molecules to differentiate mouse and human ESCs into endoderm represents a step toward achieving a reproducible and efficient production of desired ESC derivatives.

Recent evidence indicates that mouse and human embryonic stem cells (ESCs) are fixed at different developmental stages, with the former positioned earlier. We show that a narrow concentration of the naturally occurring short-chain fatty acid, sodium butyrate, supports the extensive self-renewal of mouse and human ESCs, while promoting their convergence toward an intermediate stem cell state. In response to butyrate, human ESCs regress to an earlier developmental stage characterized by a gene expression profile resembling that of mouse ESCs, preventing precocious Xist expression while retaining the ability to form complex teratomas in vivo. Other histone deacetylase inhibitors (HDACi) also support human ESC self-renewal. Our results indicate that HDACi can promote ESC self-renewal across species, and demonstrate that ESCs can toggle between alternative states in response to environmental factors.

Proliferation in the nonpregnant human breast is highest in the luteal phase of the menstrual cycle when serum progesterone levels are high, and exposure to progesterone analogues in hormone replacement therapy is known to elevate breast cancer risk, yet the proliferative effects of progesterone in the human breast are poorly understood. In a model of normal human breast, we have shown that progesterone increased incorporation of 5-bromo-2'-deoxyuridine and increased cell numbers by activation of pathways involved in DNA replication licensing, including E2F transcription factors, chromatin licensing and DNA replication factor 1 (Cdt1), and the minichromosome maintenance proteins and by increased expression of proteins involved in kinetochore formation including Ras-related nuclear protein (Ran) and regulation of chromosome condensation 1 (RCC1). Progenitor cells competent to give rise to both myoepithelial and luminal epithelial cells were increased by progesterone, showing that progesterone influences epithelial cell lineage differentiation. Therefore, we have demonstrated that progesterone augments proliferation of normal human breast cells by both activating DNA replication licensing and kinetochore formation and increasing bipotent progenitor numbers.

The antigen recognition interface formed by T helper precursors (Thps) and antigen-presenting cells (APCs), called the immunological synapse (IS), includes receptors and signaling molecules necessary for Thp activation and differentiation. We have recently shown that recruitment of the interferon-gamma receptor (IFNGR) into the IS correlates with the capacity of Thps to differentiate into Th1 effector cells, an event regulated by signaling through the functionally opposing receptor to interleukin-4 (IL4R). Here, we show that, similar to IFN-gamma ligation, TCR stimuli induce the translocation of signal transducer and activator of transcription 1 (STAT1) to IFNGR1-rich regions of the membrane. Unexpectedly, STAT1 is preferentially expressed, is constitutively serine (727) phosphorylated in Thp, and is recruited to the IS and the nucleus upon TCR signaling. IL4R engagement controls this process by interfering with both STAT1 recruitment and nuclear translocation. We also show that in cells with deficient Th1 or constitutive Th2 differentiation, the IL4R is recruited to the IS. This observation suggest that the IL4R is retained outside the IS, similar to the exclusion of IFNGR from the IS during IL4R signaling. This study provides new mechanistic cues for the regulation of lineage commitment by mutual immobilization of functionally antagonistic membrane receptors.

Vandetanib is a novel, orally available inhibitor of different intracellular signaling pathways involved in tumor growth, progression, and angiogenesis: vascular endothelial growth factor receptor-2, epidermal growth factor receptor, and REarranged during Transfection tyrosine kinase activity. Phase I clinical trials have shown that vandetanib is well tolerated as a single agent at daily doses textless or =300 mg. In the phase II setting, negative results were observed with vandetanib in small cell lung cancer, metastatic breast cancer, and multiple myeloma. In contrast, three randomized phase II studies showed that vandetanib prolonged the progression-free survival (PFS) time of patients with non-small cell lung cancer (NSCLC) as a single agent when compared with gefitinib or when added to chemotherapy. Rash, diarrhea, hypertension, fatigue, and asymptomatic QTc prolongation were the most common adverse events. Antitumor activity was also observed in medullary thyroid cancer. Four randomized phase III clinical trials in NSCLC are exploring the efficacy of vandetanib in combination with docetaxel, the Zactima in cOmbination with Docetaxel In non-small cell lung Cancer (ZODIAC) trial, or with pemetrexed, the Zactima Efficacy with Alimta in Lung cancer (ZEAL) trial, or as a single agent, the Zactima Efficacy when Studied versus Tarceva (ZEST) and the Zactima Efficacy trial for NSCLC Patients with History of EGFR-TKI chemo-Resistance (ZEPHYR) trials. Based on a press release by the sponsor of these trials, the PFS time was longer with vandetanib in the ZODIAC and ZEAL trials; the ZEST trial was negative for its primary superiority analysis, but was successful according to a preplanned noninferiority analysis of PFS. Ongoing phase II and III clinical trials will better define the appropriate schedule, the optimal setting of evaluation, and the safety of long-term use of vandetanib.

Genetic evidence indicates that Wnt signaling is critically involved in bone homeostasis. In this study, we investigated the functions of canonical Wnts on differentiation of adult multipotent human mesenchymal stem cells (hMSCs) in vitro and in vivo. We observe differential sensitivities of hMSCs to Wnt inhibition of osteogenesis versus adipogenesis, which favors osteoblastic commitment under binary in vitro differentiation conditions. Wnt inhibition of osteogenesis is associated with decreased expression of osteoblastic transcription factors and inhibition of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation, which are involved in osteogenic differentiation. An hMSC subpopulation exhibits high endogenous Wnt signaling, the inhibition of which enhances osteogenic and adipogenic differentiation in vitro. In an in vivo bone formation model, high levels of Wnt signaling inhibit de novo bone formation by hMSCs. However, hMSCs with exogenous expression of Wnt1 but not stabilized beta-catenin markedly stimulate bone formation by naive hMSCs, arguing for an important role of a canonical Wnt gradient in hMSC osteogenesis in vivo.

The potential for directed differentiation of human-induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs), we found that once specified to a neural lineage, human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS-derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS-derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS-derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases.

Glioblastoma, the most malignant type of primary brain tumor, is one of the solid cancers where cancer stem cells have been isolated, and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here, we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serum-free medium with epidermal growth factor and fibroblast growth factor 2, varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem-like cells (GBM-SC), we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)-deleted mutants kill GBM-SCs as efficiently as wild-type HSV, the deletion of gamma34.5 significantly attenuated the vectors due to poor replication. However, this was significantly reversed by the additional deletion of alpha47. Infection with oHSV G47Delta (ICP6(-), gamma34.5(-), alpha47(-)) not only killed GBM-SCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly, despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs, intratumoral injection of G47Delta significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs, and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover, the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis.

Olive oil intake has been shown to induce significant levels of apoptosis in various cancer cells. These anti-cancer properties are thought to be mediated by phenolic compounds present in olive. These beneficial health effects of olive have been attributed, at least in part, to the presence of oleuropein and hydroxytyrosol. In this study, oleuropein and hydroxytyrosol, major phenolic compound of olive oil, was studied for its effects on growth in MCF-7 human breast cancer cells using assays for proliferation (MTT assay), cell viability (Guava ViaCount assay), cell apoptosis, cellcycle (flow cytometry). Oleuropein or hydroxytyrosol decreased cell viability, inhibited cell proliferation, and induced cell apoptosis in MCF-7 cells. Result of MTT assay showed that 200 mug/mL of oleuropein or 50 mug/mL of hydroxytyrosol remarkably reduced cell viability of MCF-7 cells. Oleuropein or hydroxytyrosol decrease of the number of MCF-7 cells by inhibiting the rate of cell proliferation and inducing cell apoptosis. Also hydroxytyrosol and oleuropein exhibited statistically significant block of G(1) to S phase transition manifested by the increase of cell number in G(0)/G(1) phase.

The cytidine deaminase AID (encoded by Aicda in mice and AICDA in humans) is critical for immunoglobulin class-switch recombination (CSR) and somatic hypermutation (SHM). Here we show that AID expression was induced by the HoxC4 homeodomain transcription factor, which bound to a highly conserved HoxC4-Oct site in the Aicda or AICDA promoter. This site functioned in synergy with a conserved binding site for the transcription factors Sp1, Sp3 and NF-kappaB. HoxC4 was 'preferentially' expressed in germinal center B cells and was upregulated by engagement of CD40 by CD154, as well as by lipopolysaccharide and interleukin 4. HoxC4 deficiency resulted in impaired CSR and SHM because of lower AID expression and not some other putative HoxC4-dependent activity. Enforced expression of AID in Hoxc4(-/-) B cells fully restored CSR. Thus, HoxC4 directly activates the Aicda promoter, thereby inducing AID expression, CSR and SHM.

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia.

Identification of rational therapeutic targets is an important strategy to improve the cure rate of diffuse large B-cell lymphoma (DLBCL). We previously showed that inhibition of the phosphodiesterase 4B (PDE4B) unleashes cyclic-AMP (cAMP) inhibitory effects toward the PI3K/AKT pathway and induces apoptosis. These data raised important considerations as to which upstream regulators mediate cAMP inhibition of PI3K/AKT, and how identifying this signaling route could be translated into clinical initiatives. We found that in normal and malignant B cells, cAMP potently inhibit the phosphorylation and activity of the tyrosine kinase SYK. Using genetic models of gain- and loss-of-function, we demonstrated the essential role for PDE4B in controlling these effects in DLBCL. Furthermore, we used a constitutively active SYK mutant to confirm its central role in transducing cAMP effects to PI3K/AKT. Importantly, given SYK credentials as a therapeutic target in B-cell tumors, we explored the role of PDE4B in these responses. In multiple DLBCL models, we found that genetically, hence specifically, inhibiting PDE4B expression significantly improved the efficacy of SYK inhibitors. Our data defined a hitherto unknown role for cAMP in negatively regulating SYK and indicate that combined inhibition of PDE4B and SYK should be actively pursued.