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Human pluripotent stem cells hold promising potential in many therapeutics applications including regenerative medicine and drug discovery. Over the past three decades,embryonic stem cell research has illustrated that embryonic stem cells possess two important and distinct properties: the ability to continuously self-renew and the ability to differentiate into all specialized cell types. In this article,we will discuss the continuing evolution of human pluripotent stem cell culture by examining requirements needed for the maintenance of self-renewal in vitro. We will also elaborate on the future direction of the field toward generating a robust and completely defined culture system,which has brought forth collaborations amongst biologists and engineers. As human pluripotent stem cell research progresses towards identifying solutions for debilitating diseases,it will be critical to establish a defined,reproducible and scalable culture system to meet the requirements of these clinical applications. View Publication

This unit describes the generation of tetracycline-inducible short hairpin RNA interference (shRNAi) human embryonic stem cell (hESC) lines. Using this vector-based approach enables stable and long-term expression of target hairpins under the control of doxycycline/tetracycline. Target degradation can be controlled in both a dose- and time-dependent manner that can even be switched off,depending upon the particular requirements of the study. View Publication

The midbody is a singular organelle formed between daughter cells during cytokinesis and required for their final separation. Midbodies persist in cells long after division as midbody derivatives (MB(d)s),but their fate is unclear. Here we show that MB(d)s are inherited asymmetrically by the daughter cell with the older centrosome. They selectively accumulate in stem cells,induced pluripotent stem cells and potential cancer 'stem cells' in vivo and in vitro. MB(d) loss accompanies stem-cell differentiation,and involves autophagic degradation mediated by binding of the autophagic receptor NBR1 to the midbody protein CEP55. Differentiating cells and normal dividing cells do not accumulate MB(d)s and possess high autophagic activity. Stem cells and cancer cells accumulate MB(d)s by evading autophagosome encapsulation and exhibit low autophagic activity. MB(d) enrichment enhances reprogramming to induced pluripotent stem cells and increases the in vitro tumorigenicity of cancer cells. These results indicate unexpected roles for MB(d)s in stem cells and cancer 'stem cells'. View Publication

The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected,patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression,avoiding the risk of insertional mutagenesis by therapeutic vectors,and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However,gene targeting in human pluripotent cells has remained challenging and inefficient. Recently,engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs,raising the prospect of using this technology to correct disease causing mutations. Here,we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug-resistance genes. By testing various conditions,we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient,transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications. View Publication

Pluripotency,the ability of a cell to differentiate and give rise to all embryonic lineages,defines a small number of mammalian cell types such as embryonic stem (ES) cells. While it has been generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes,accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluripotency of ES cells,as well as maintaining the identity of differentiated cell types. In order to better understand the role of epigenetic mechanisms in pluripotency,we have examined the dynamics of chromatin modifications genome-wide in human ES cells (hESCs) undergoing differentiation into a mesendodermal lineage. We found that chromatin modifications at promoters remain largely invariant during differentiation,except at a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression,suggesting a hierarchy in cell fate commitment over most differentially expressed genes. We also mapped over 50 000 potential enhancers,and observed much greater dynamics in chromatin modifications,especially H3K4me1 and H3K27ac,which correlate with expression of their potential target genes. Further analysis of these enhancers revealed potentially key transcriptional regulators of pluripotency and a chromatin signature indicative of a poised state that may confer developmental competence in hESCs. Our results provide new evidence supporting the role of chromatin modifications in defining enhancers and pluripotency. View Publication

Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology,focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS),CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin,pFAK [Y(397)],and HSP90α/β and p130CAS,and analysed for reactivity,intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences,and subcellular localisation analysis revealed that in pathological MSCs,paxillin,pFAK [Y(397)],and HSP90α/β formed nuclear molecular complexes. Increased expression of paxillin,pFAK [Y(397)],and HSP90α/β and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further,because FAK is an HSP90α/β client protein,these results suggest the utility of HSP90α/β inhibition as a target for adjuvant therapy for myelodysplasia. View Publication

Microbial growth in damp indoor environments has been correlated with risks to human health. This study was aimed to determine the cytotoxicity of 1-octen-3-ol (mushroom alcohol")� View Publication

Human embryonic stem cells (hESCs) and their differentiated progeny allow for investigation of important changes/events during normal embryonic development. Currently most of the research is focused on proteinacous changes occurring as a result of differentiation of stem cells and little is known about changes in cell surface glycosylation patterns. Identification of cell lineage specific glycans can help in understanding their role in maintenance,proliferation and differentiation. Furthermore,these glycans can serve as markers for isolation of homogenous populations of cells. Using a panel of eight biotinylated lectins,the glycan expression of hESCs,hESCs-derived human neural progenitors (hNP) cells,and hESCs-derived mesenchymal progenitor (hMP) cells was investigated. Our goal was to identify glycans that are unique for hNP cells and use the corresponding lectins for cell isolation. Flow cytometry and immunocytochemistry were used to determine expression and localization of glycans,respectively,in each cell type. These results show that the glycan expression changes upon differentiation of hESCs and is different for neural and mesenchymal lineage. For example,binding of PHA-L lectin is low in hESCs (14±4.4%) but significantly higher in differentiated hNP cells (99±0.4%) and hMP cells (90±3%). Three lectins: VVA,DBA and LTL have low binding in hESCs and hMP cells,but significantly higher binding in hNP cells. Finally,VVA lectin binding was used to isolate hNP cells from a mixed population of hESCs,hNP cells and hMP cells. This is the first report that compares glycan expression across these human stem cell lineages and identifies significant differences. Also,this is the first study that uses VVA lectin for isolation for human neural progenitor cells. View Publication

An important risk in the clinical application of human pluripotent stem cells (hPSCs),including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs),is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs,designated anti-stage-specific embryonic antigen (SSEA)-5,which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells,we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9,CD30,CD50,CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures. View Publication

Cellular decisions of self-renewal or differentiation arise from integration and reciprocal titration of numerous regulatory networks. Notch and Wnt/β-catenin signalling often intersect in stem and progenitor cells and regulate each other transcriptionally. The biological outcome of signalling through each pathway often depends on the context and timing as cells progress through stages of differentiation. Here,we show that membrane-bound Notch physically associates with unphosphorylated (active) β-catenin in stem and colon cancer cells and negatively regulates post-translational accumulation of active β-catenin protein. Notch-dependent regulation of β-catenin protein did not require ligand-dependent membrane cleavage of Notch or the glycogen synthase kinase-3β-dependent activity of the β-catenin destruction complex. It did,however,require the endocytic adaptor protein Numb and lysosomal activity. This study reveals a previously unrecognized function of Notch in negatively titrating active β-catenin protein levels in stem and progenitor cells. View Publication

Numerous matrices for the growth of human embryonic stem cells (hESC) in vitro have been described. However,their exact composition is typically unknown. Information on the components of these matrices will aid in the development of a fully defined growth surface for hESCs. These matrices typically consist of mixture of proteins present in a wide range of abundance making their characterization challenging. In this study,we performed the proteomic analysis of five previously uncharacterized matrices: CellStart,Human Basement Membrane Extract (Human BME),StemXVivo,Bridge Human Extracellular Matrix (BridgeECM),and mouse embryonic fibroblast conditioned matrix (MEF-CMTX). Based on a proteomics protocol optimized using lysates from HeLa cells,we undertook the analysis of the five complex extracellular matrix (ECM) samples using a combination of strong anion and cation exchange chromatography and SDS-PAGE. For each of these matrices,we identify numerous proteins,indicating their complex nature. We also compared these results with a similar proteomics analysis of the growth matrix,Matrigel™. From these analyses,we observed that fibronectin is a primary component of nearly all hESC supportive matrices. This observation led to the investigation of the suitability of fibronectin as a defined ECM for the growth of hESCs. We found that fibronectin promotes the maintenance of pluripotent H9 and CA1 hESCs in an undifferentiated state using mTeSR1 medium. This finding validates the utility of characterizing matrices used for hESC growth in revealing ECM components required for culturing hESCs in a universally applicable defined system. View Publication

Efficient derivation and isolation of hematopoietic stem cells (HSCs) from human pluripotent stem cell (hPSC) populations remains a major goal in the field of developmental hematopoiesis. These enticing pluripotent stem cells (comprising both human embryonic stem cells and induced pluripotent stem cells) have been successfully used to generate a wide array of hematopoietic cells in vitro,from primitive hematoendothelial precursors to mature myeloid,erythroid,and lymphoid lineage cells. However,to date,PSC-derived cells have demonstrated only limited potential for long-term multilineage hematopoietic engraftment in vivo - the test by which putative HSCs are defined. Successful generation and characterization of HSCs from hPSCs not only requires an efficient in vitro differentiation system that provides insight into the developmental fate of hPSC-derived cells,but also necessitates an in vivo engraftment model that allows identification of specific mechanisms that hinder or promote hematopoietic engraftment. In this chapter,we will describe a method that utilizes firefly luciferase-expressing hPSCs and bioluminescent imaging to noninvasively track the survival,proliferation,and migration of transplanted hPSC-derived cells. Combined with lineage and functional analyses of engrafted cells,this system is a useful tool to gain insight into the in vivo potential of hematopoietic cells generated from hPSCs. View Publication