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The monocyte population in blood is considered a possible source of endothelial precursors. Because endothelial-specific receptor tyrosine kinases act as regulators of endothelial cell function, we investigated whether expression of the vascular endothelial growth factor receptor-2 (VEGFR-2) on monocytes is important for their endothelial-like functional capacity. Peripheral-blood monocytes expressing vascular endothelial growth factor receptor-2 (VEGFR-2), or CD14+/VEGFR-2+, were isolated, and their phenotypic, morphologic, and functional capacities were compared with those of monocytes negative for this marker (CD14+/VEGFR-2-). CD14+/VEGFR-2+ cells constituted approximately 2% +/- 0.5% of the total population of monocytes and 0.08% +/- 0.04% of mononuclear cells in blood. CD14+/VEGFR-2+ cells exhibited the potential to differentiate in vitro into cells with endothelial characteristics. The cells were efficiently transduced by a lentiviral vector driving expression of the green fluorescence protein (GFP). Transplantation of GFP-transduced cells into balloon-injured femoral arteries of nude mice significantly contributed to efficient reendothelialization. CD14+/VEGFR-2- did not exhibit any of these characteristics. These data demonstrate that the expression of VEGFR-2 on peripheral blood monocytes is essential for their endothelial-like functional capacity and support the notion of a common precursor for monocytic and endothelial cell lineage. Our results help clarify which subpopulations may restore damaged endothelium and may participate in the maintenance of vascular homeostasis.

Optimization of pluripotent stem cell expansion and differentiation is facilitated by biological tools that permit non-invasive and dynamic monitoring of pluripotency, and the ability to select for an undifferentiated input cell population. Here we report on the generation and characterisation of clonal human embryonic stem (HES3, H9) and human induced pluripotent stem cell lines (UQEW01i-epifibC11) that have been stably modified with an artificial EOS(C3+) promoter driving expression of EGFP and puromycin resistance-conferring proteins. We show that EGFP expression faithfully reports on the pluripotency status of the cells in these lines and that antibiotic selection allows for an efficient elimination of differentiated cells from the cultures. We demonstrate that the extinction of the expression of the pluripotency reporter during differentiation closely correlates with the decrease in expression of conventional pluripotency markers, such as OCT4 (POU5F1), TRA-1-60 and SSEA4 when screening across conditions with various levels of pluripotency-maintaining or differentiation-inducing signals. We further illustrate the utility of these lines for real-time monitoring of pluripotency in embryoid bodies and microfluidic bioreactors.

Neutrophils are the most abundant WBCs and have an essential role in the clearance of pathogens. Tight regulation of neutrophil numbers and their recruitment to sites of inflammation is critical in maintaining a balanced immune response. In various inflammatory conditions, such as rheumatoid arthritis, vasculitis, cystic fibrosis, and inflammatory bowel disease, increased serum G-CSF correlates with neutrophilia and enhanced neutrophil infiltration into inflamed tissues. We describe a fully human therapeutic anti-G-CSFR antibody (CSL324) that is safe and well tolerated when administered via i.v. infusion to cynomolgus macaques. CSL324 was effective in controlling G-CSF-mediated neutrophilia when administered either before or after G-CSF. A single ascending-dose study showed CSL324 did not alter steady-state neutrophil numbers, even at doses sufficient to completely prevent G-CSF-mediated neutrophilia. Weekly infusions of CSL324 (%10 mg/kg) for 3 wk completely neutralized G-CSF-mediated pSTAT3 phosphorylation without neutropenia. Moreover, repeat dosing up to 100 mg/kg for 12 wk did not result in neutropenia at any point, including the 12-wk follow-up after the last infusion. In addition, CSL324 had no observable effect on basic neutrophil functions, such as phagocytosis and oxidative burst. These data suggest that targeting G-CSFR may provide a safe and effective means of controlling G-CSF-mediated neutrophilia as observed in various inflammatory diseases.

Spinocerebellar ataxia type 2 (SCA2) is caused by triple nucleotidebackslashnrepeat (CAG) expansion in the coding region of the ATAXN2 gene onbackslashnchromosome 12, which produces an elongated, toxic polyglutamine tract,backslashnleading to Purkinje cell loss. There is currently no effective therapy.backslashnOne of the main obstacles that hampers therapeutic development is lackbackslashnof an ideal disease model. In this study, we have generated andbackslashncharacterized SCA2-induced pluripotent stem (iPS) cell lines as an inbackslashnvitro cell model. Dermal fibroblasts (FBs) were harvested from primarybackslashncultures of skin explants obtained from a SCA2 subject and a healthybackslashnsubject. For reprogramming, hOct4, hSox2, hKlf4, and hc-Myc werebackslashntransduced to passage-3 FBs by retroviral infection. Both SCA2 iPS andbackslashncontrol iPS cells were successfully generated and showed typical stembackslashncell growth patterns with normal karyotype. All iPS cell lines expressedbackslashnstem cell markers and differentiated in vitro into cells from threebackslashnembryonic germ layers. Upon in vitro neural differentiation, SCA2 iPSbackslashncells showed abnormality in neural rosette formation but successfullybackslashndifferentiated into neural stem cells (NSCs) and subsequent neuralbackslashncells. SCA2 and normal FBs showed a comparable level of ataxin-2backslashnexpression; whereas SCA2 NSCs showed less ataxin-2 expression thanbackslashnnormal NSCs and SCA2 FBs. Within the neural lineage, neurons had thebackslashnmost abundant expression of ataxin-2. Time-lapsed neural growth assaybackslashnindicated terminally differentiated SCA2 neural cells were short-livedbackslashncompared with control neural cells. The expanded CAG repeats of SCA2backslashnwere stable throughout reprogramming and neural differentiation. Inbackslashnconclusion, we have established the first disease-specific human SCA2backslashniPS cell line. These mutant iPS cells have the potential for neuralbackslashndifferentiation. These differentiated neural cells harboring mutationsbackslashnare invaluable for the study of SCA2 pathogenesis and therapeutic drugbackslashndevelopment.

Gaucher disease (GD) is caused by insufficient activity of acid $\$-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD, induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs, NPCs, and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition, GD2 neurons showed increased $\$-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons, but intriguingly, those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes, sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings, providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge, this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease.

Mast cells are unique hematopoietic cells that are richly distributed in the skin and mucosal surfaces of the respiratory and gastrointestinal tract. They play a key role in allergic inflammation by releasing a cocktail of granular constituents, including histamine, serine proteases, and various eicosanoids and cytokines. As such, a number of drugs target either inhibition of mast cell degranulation or the products of degranulation. To identify potential novel drugs and mechanisms in mast cell biology, assays were developed to identify inhibitors of mast cell degranulation and activation in a phenotypic screen. Due to the challenges associated with obtaining primary mast cells, cord blood-derived mononuclear cells were reproducibly differentiated to mast cells and assays developed to monitor tryptase release and prostaglandin D2 generation. The tryptase assay was particularly sensitive, requiring only 500 cells per data point, which permitted a set of approximately 12,000 compounds to be screened robustly and cost-effectively. Active compounds were tested for concomitant inhibition of prostaglandin D2 generation. This study demonstrates the robustness and effectiveness of this approach in the identification of potential novel compounds and mechanisms targeting mast cell-driven inflammation, to enable innovative drug discovery efforts to be prosecuted.

Human induced pluripotent stem cells have the potential to become an unlimited cell source for cell replacement therapy. The realization of this potential, however, depends on the availability of culture methods that are robust, scalable, and use chemically defined materials. Despite significant advances in hiPSC technologies, the expansion of hiPSCs relies upon the use of animal-derived extracellular matrix extracts, such as Matrigel, which raises safety concerns over the use of these products. In this work, we investigated the feasibility of expanding and differentiating hiPSCs on a chemically defined, xeno-free synthetic peptide substrate, i.e. Corning Synthemax(®) Surface. We demonstrated that the Synthemax Surface supports the attachment, spreading, and proliferation of hiPSCs, as well as hiPSCs' lineage-specific differentiation. hiPSCs colonies grown on Synthemax Surfaces exhibit less spread and more compact morphology compared to cells grown on Matrigel™. The cytoskeleton characterization of hiPSCs grown on the Synthemax Surface revealed formation of denser actin filaments in the cell-cell interface. The down-regulation of vinculin and up-regulation of zyxin expression were also observed in hiPSCs grown on the Synthemax Surface. Further examination of cell-ECM interaction revealed that hiPSCs grown on the Synthemax Surface primarily utilize α(v)β(5) integrins to mediate attachment to the substrate, whereas multiple integrins are involved in cell attachment to Matrigel. Finally, hiPSCs can be maintained undifferentiated on the Synthemax Surface for more than ten passages. These studies provide a novel approach for expansion of hiPSCs using synthetic peptide engineered surface as a substrate to avoid a potential risk of contamination and lot-to-lot variability with animal derived materials.

BACKGROUND: Due to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions. METHODOLOGY/PRINCIPAL FINDINGS: HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions. CONCLUSIONS/SIGNIFICANCE: The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types, identification and isolation of stem cell subpopulations, and generation of single cell clones. Finally, these results demonstrate an additional application of ROCK inhibition to hESC research.

We have recently shown that more than 90% of long-term culture initiating cells (LTC-IC) mobilized in the peripheral blood (PB) of normal individuals express HLA-DR and CD38 antigens and can sustain hematopoiesis for only 5 weeks. However, 10% of LTC-IC in mobilized PB are CD34+ HLA-DR- and CD34+ CD38- and can sustain hematopoiesis for at least 8 weeks. We now examine the ex vivo expansion potential of CD34+ HLA-DR+ cells (rich in mature LTC-IC) and CD34+ HLA-DR- cells (rich in primitive LTC-IC) in granulocyte colony-stimulating factor (G-CSF) mobilized PB progenitor cells (PBPC). Cells were cultured in contact with M2-10B4 cells (contact) or in transwells above M2-10B4 (noncontact) without and with interleukin-3 (IL-3) and macrophage inflammatory protein (MIP-1alpha) for 2 and 5 weeks. Progeny were evaluated for the presence of colony-forming cells (CFC) and LTC-IC. When CD34+ HLA-DR+ PB cells were cultured in contact cultures without cytokines, a threefold expansion of CFC was seen at 2 weeks, but an 80% decrease in CFC was seen at week 5. Further, the recovery of LTC-IC at week 2 was only 17% and 1% at week 5. This confirms our previous observation that although CD34+ HLA-DR+ mobilized PB cells can initiate long-term cultures, they are relatively mature and cannot sustain long-term hematopoiesis. In contrast, when CD34+ HLA-DR- mobilized PB cells were cultured in contact cultures without cytokines, CFC expansion persisted until week 5 and 49% and 11% of LTC-IC were recovered at week 2 and 5, respectively. As we have shown for steady state bone marrow (BM) progenitors, recovery of LTC-IC was threefold higher when CD34+ HLA-DR- PBPC were cultured in noncontact rather than contact cultures, and improved further when IL-3 and MIP-1alpha were added to noncontact cultures (96 +/- 2% maintained at week 5). We conclude that although G-CSF mobilizes a large population of mature" CD34+ HLA-DR+ LTC-IC with a limited proliferative capacity�

Malignant cells have a higher nicotinamide adenine dinucleotide (NAD(+)) turnover rate than normal cells, making this biosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD(+) synthesis, Nampt, in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 triggered cytotoxicity in MM cell lines and patient MM cells, but not normal donor as well as MM patients PBMCs. Importantly, FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6, IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD(+) stores. Interestingly, cytotoxicity of FK866 triggered autophagy, but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity. Finally, FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model, associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Our data therefore define a key role of Nampt in MM biology, providing the basis for a novel targeted therapeutic approach.

Adipogenesis plays a key role in the pathogenesis of obesity. It begins with the commitment of mesenchymal stem cells (MSCs) to the adipocyte lineage, followed by terminal differentiation of preadipocytes to mature adipocytes. A critical, but poorly understood, component of adipogenesis involves proliferation of MSCs and preadipocytes. The present study was undertaken to examine the hypothesis that bone morphogenetic protein-3 (BMP-3) promotes adipogenesis using C3H10T1/2 MSCs and 3T3-L1 preadipocytes as in vitro model systems. We demonstrated that although it did not promote the commitment of MSCs to the adipocyte lineage or the differentiation of preadipocytes to adipocytes, BMP-3-stimulated proliferation by threefold in both cell types. Owing to a lack of information on MSC proliferation, we then delineated the molecular mechanisms underlying BMP-3-stimulated MSC proliferation. We showed that BMP-3 activated the transforming growth factor-beta (TGF-beta)/activin but not ERK1/2, p38 MAPK, or JNK signaling pathways in C3H10T1/2 cells. Furthermore, the TGF-beta/activin receptor kinase inhibitor SB-431542 blocked BMP-3-stimulated proliferation. Importantly, siRNA-mediated knockdown of the key TGF-beta/activin signaling pathway components, ActRIIB, ALK4, or Smad2, abrogated the mitogenic effects of BMP-3 on MSCs. Together, these results demonstrate that BMP-3 stimulates MSC proliferation via the TGF-beta/activin signaling pathway, thus revealing a novel role for this divergent and poorly understood member of the TGF-beta superfamily in regulating MSC proliferation.

Stem cell differentiation is regulated by complex repertoires of signaling ligands which often use multivalent interactions, where multiple ligands tethered to one entity interact with multiple cellular receptors to yield oligomeric complexes. One such ligand is Sonic hedgehog (Shh), whose posttranslational lipid modifications and assembly into multimers enhance its biological potency, potentially through receptor clustering. Investigations of Shh typically utilize recombinant, monomeric protein, and thus the impact of multivalency on ligand potency is unexplored. Among its many activities, Shh is required for ventralization of the midbrain and forebrain and is therefore critical for the development of midbrain dopaminergic (mDA) and forebrain gamma-aminobutyric acid (GABA) inhibitory neurons. We have designed multivalent biomaterials presenting Shh in defined spatial arrangements and investigated the role of Shh valency in ventral specification of human embryonic stem cells (hESCs) into these therapeutically relevant cell types. Multivalent Shh conjugates with optimal valencies, compared to the monomeric Shh, increased the percentages of neurons belonging to mDA or forebrain GABAergic fates from 33% to 60% or 52% to 86%, respectively. Thus, multivalent Shh bioconjugates can enhance neuronal lineage commitment of pluripotent stem cells and thereby facilitate efficient derivation of neurons that could be used to treat Parkinson's and epilepsy patients.