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Human macrophages are specialised hosts for HIV-1,dengue virus,Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens,because available myeloid cell lines are,by definition,not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined,feeder- and serum-free culture conditions and produces very consistent,pure,high yields across both human Embryonic Stem Cell (hESC) and multiple human induced Pluripotent Stem Cell (hiPSC) lines over time periods of up to one year. Cumulatively,up to ∼3×10(7) monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14(+),CD16(low),CD163(+)). Differentiation with M-CSF produces macrophages that are highly phagocytic,HIV-1-infectable,and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate,as they recognise foreign nucleic acids; the lentivector system described here overcomes this,as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells,surmounting issues of transgene silencing. Overall,the method we describe here is an efficient,effective,scalable system for the reproducible production and genetic modification of human macrophages,facilitating the interrogation of human macrophage biology. View Publication

Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro,but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal,we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray,and tested their ability to perform mature hepatocyte functions (albumin and urea secretion,cytochrome activity). By these measures,ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. View Publication

Human embryonic stem cells (hESCs) are promising in regenerative medicine (RM) due to their differentiation plasticity and proliferation potential. However,a major challenge in RM is the generation of a vascular system to support nutrient flow to newly synthesized tissues. Here we refined an existing method to generate tight vessels by differentiating hESCs in CD34(+) vascular progenitor cells using chemically defined media and growth conditions. We selectively purified these cells from CD34(-) outgrowth populations also formed. To analyze these differentiation processes,we compared the proteomes of the hESCs with those of the CD34(+) and CD34(-) populations using high resolution mass spectrometry,label-free quantification,and multivariate analysis. Eighteen protein markers validate the differentiated phenotypes in immunological assays; nine of these were also detected by proteomics and show statistically significant differential abundance. Another 225 proteins show differential abundance between the three cell types. Sixty-three of these have known functions in CD34(+) and CD34(-) cells. CD34(+) cells synthesize proteins implicated in endothelial cell differentiation and smooth muscle formation,which support the bipotent phenotype of these progenitor cells. CD34(-) cells are more heterogeneous synthesizing muscular/osteogenic/chondrogenic/adipogenic lineage markers. The remaining textgreater150 differentially abundant proteins in CD34(+) or CD34(-) cells raise testable hypotheses for future studies to probe vascular morphogenesis. View Publication

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Electrospun scaffolds with varied stiffness promote distinct colony morphology of human induced pluripotent stem cells,which affects their subsequent differentiation. On soft scaffolds,induced pluripotent stem cells develop 3D colonies due to the pliability of the electrospun fibrous networks,leading to greater differentiation tendency to ectodermal lineage. View Publication

PURPOSE To determine the safety,survival,and functionality of human embryonic stem cell-derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats. METHODS Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n = 46) received vitronectin-coated parylene membrane without cells (rMSPM+VN),group 2 (n = 59) received rCPCB-RPE1 implants,and group 3 (n = 13) served as the control group. Animals that are selected based on optical coherence tomography screening were subjected to visual function assays using optokinetic (OKN) testing and superior colliculus (SC) electrophysiology. At approximately 25 weeks of age (21 weeks after surgery),the eyes were examined histologically for cell survival,phagocytosis,and local toxicity. RESULTS Eighty-seven percent of the rCPCB-RPE1-implanted animals showed hESC-RPE survivability. Significant numbers of outer nuclear layer cells were rescued in both group 1 (rMSPM+VN) and group 2 (rCPCB-RPE1) animals. A significantly higher ratio of rod photoreceptor cells to cone photoreceptor cells was found in the rCPCB-RPE1-implanted group. Animals with rCPCB-RPE1 implant showed hESC-RPE cells containing rhodopsin-positive particles in immunohistochemistry,suggesting phagocytic function. Superior colliculus mapping data demonstrated that a significantly higher number of SC sites responded to light stimulus at a lower luminance threshold level in the rCPCB-RPE1-implanted group. Optokinetic data suggested both implantation groups showed improved visual acuity. CONCLUSIONS These results demonstrate the safety,survival,and functionality of the hESC-RPE monolayer transplantation in an RPE dysfunction rat model. View Publication

Monoclonal antibodies offer high specificity and this makes it an important tool for molecular biology,biochemistry and medicine. Typically,monoclonal antibodies are generated by fusing mouse spleen cells that have been immunized with the desired antigen with myeloma cells to create immortalized hybridomas. Here,we describe the generation of monoclonal antibodies that are specific to Chikungunya virus using ClonaCell-HY system. View Publication

The lipophilic statin lovastatin decreases cholesterol synthesis and is a safe and effective treatment for the prevention of cardiovascular diseases. Growing evidence points at antitumor potential of lovastatin. Therefore,understanding the molecular mechanism of lovastatin function in different cell types is critical to effective therapy design. In this study,we investigated the effects of lovastatin on the differentiation potential of human embryonic stem (hES) cells (H9 cell line). Multiparameter flow cytometric assay was used to detect changes in the expression of transcription factors characteristic of hES cells. We found that lovastatin treatment delayed NANOG downregulation during ectodermal and endodermal differentiation. Likewise,expression of ectodermal (SOX1 and OTX2) and endodermal (GATA4 and FOXA2) markers was higher in treated cells. Exposure of hES cells to lovastatin led to a minor decrease in the expression of SSEA-3 and a significant reduction in CD133 expression. Treated cells also formed fewer embryoid bodies than control cells. By analyzing hES with and without CD133,we discovered that CD133 expression is required for proper formation of embryoid bodies. In conclusion,lovastatin reduced the heterogeneity of hES cells and impaired their differentiation potential. View Publication

Stem cell-induced hepatocytes (SC-iHeps) have been suggested as a valuable model for evaluating drug toxicology. Here,human-induced pluripotent stem cells (QIA7) and embryonic stem cells (WA01) were differentiated into hepatocytes,and the hepatotoxic effects of acetaminophen (AAP) and aflatoxin B1 (AFB1) were compared with primary hepatocytes (p-Heps) and HepG2. In a cytotoxicity assay,the IC50 of SC-iHeps was similar to that in p-Heps and HepG2 in the AAP groups but different from that in p-Heps of the AFB1 groups. In a multi-parameter assay,phenotypic changes in mitochondrial membrane potential,calcium influx and oxidative stress were similar between QIA7-iHeps and p-Heps following AAP and AFB1 treatment but relatively low in WA01-iHeps and HepG2. Most hepatic functional markers (hepatocyte-specific genes,albumin/urea secretion,and the CYP450 enzyme activity) were decreased in a dose-dependent manner following AAP and AFB1 treatment in SC-iHeps and p-Heps but not in HepG2. Regarding CYP450 inhibition,the cell viability of SC-iHeps and p-Heps was increased by ketoconazole,a CYP3A4 inhibitor. Collectively,SC-iHeps and p-Heps showed similar cytotoxicity and hepatocyte functional effects for AAP and AFB1 compared with HepG2. Therefore,SC-iHeps have phenotypic characteristics and sensitivity to cytotoxic chemicals that are more similar to p-Heps than to HepG2 cells. View Publication

Recent advances in developmental biology and stem cell technology have led to the engineering of functional organs in a dish. However,the limited size of these organoids and absence of a large circulatory system poses limits to its clinical translation. To overcome these issues,decellularized whole kidney scaffolds with native microstructure and extracellular matrix (ECM) are employed for kidney bioengineering,using human-induced pluripotent-stem-cell-derived renal progenitor cells and endothelial cells. To demonstrate ECM-guided cellular assembly,the present work is focused on generating the functional unit of the kidney,the glomerulus. In the repopulated organ,the presence of endothelial cells broadly upregulates the expression level of genes related to renal development. When the cellularized native scaffolds are implanted in SCID mice,glomeruli assembly can be achieved by co-culture of the renal progenitors and endothelial cells. These individual glomerular units are shown to be functional in the context of the whole organ using a simulated bio-reactor set-up with urea and creatinine excretion and albumin reabsorption. Our results indicate that the repopulation of decellularized native kidney using clinically relevant,expandable patient-specific renal progenitors and endothelial cells may be a viable approach for the generation of a functional whole kidney. View Publication

As known from model organisms,such as frog,fish,mouse and chicken,the anterior-posterior patterning of the definitive endoderm (DE) into distinct domains is controlled by a variety of signaling interactions between the DE and its surrounding mesoderm. This includes Wnt/FGFs and BMPs in the posterior half and all-trans-retinoic acid,TGF-$$-ligands,Wnt- and BMP-inhibitors in the anterior half of the DE sheet. However,it is currently unclear how these embryonic tissue interactions can be translated into a defined differentiation protocol for human embryonic stem cells. Activin A has been proposed to direct DE into a SOX2-positive foregut-like cell type. Due to the pleiotropic nature of SOX2 in pluripotency and developing cells of the foregut we purified DE-cells by magnetic cell sorting and tested the effects of anteriorizing and posteriorizing factors on pure endoderm. We show in contrast to previous studies that the generation of the foregut marked by SOX2/FOXA2 double-positive cells does not depend on activin A/TGF-$$-signaling but is mediated by the inhibition of Wnt- and BMP-signaling. Retinoic acid can posteriorize and at the same time dorsalize the foregut towards a PDX1-positive pancreatic duodenal cell type whereas active Wnt/beta-catenin signaling synergistically with FGF-2,BMP-4 and RA induces the formation of CDX2-positive posterior endoderm. Thus,these results provide new insights into the mechanisms behind cell specification of human DE derived from pluripotent stem cells. This article is protected by copyright. All rights reserved. View Publication

Mechanisms determining intrinsic differentiation bias inherent to human pluripotent stem cells (hPSCs) toward cardiogenic fate remain elusive. We evaluated the interplay between ErbB4 and EGFR in determining cardiac differentiation in vitro as these receptor tyrosine kinases (RTKs) are key to heart and brain development in vivo. Our results demonstrate that during cardiac differentiation,cell fate biases exist in hPSCs due to cardiac/neuroectoderm divergence post cardiac mesoderm stage. Stage-specific up-regulation of EGFR in concert with persistent Wnt3a signaling post cardiac mesoderm favors commitment towards neural progenitor cells (NPCs). Inhibition of EGFR abrogates these effects with enhanced (textgreater2-fold) cardiac differentiation efficiencies by increasing proliferation of Nkx2-5 expressing cardiac progenitors while reducing proliferation of Sox2 expressing NPCs. Forced overexpression of ErbB4 rescued cardiac commitment by augmenting Wnt11 signaling. Convergence between EGFR/ErbB4 and canonical/non-canonical Wnt signaling determines cardiogenic fate in hPSCs. This article is protected by copyright. All rights reserved. View Publication