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Microglia perform key homeostatic functions to protect the central nervous system (CNS). However,in many brain disorders their protective functions are abrogated,contributing to disease progression. Therefore,studies of microglial function are critical to developing treatments for brain disorders. Different in vitro microglia models have been established,including primary human and rodent cells,induced pluripotent stem cell (iPSC)-derived models,and immortalised cell lines. However,a direct comparative analysis of the phenotypic and functional characteristics of these models has not been undertaken. Accurate modelling of human microglia in vitro is critical for ensuring the translatability of results from the bench to the brain. Therefore,our study aimed to characterise and compare commonly utilised in vitro microglia models. We assessed four established microglia models: primary human microglia,human iPSC-derived microglia,the human microglial clone 3 (HMC3) cell line,and primary mouse microglia,with primary human brain pericytes acting as a negative control. Primary human microglia,iPSC-derived microglia,and mouse microglia stained positive for myeloid-cell markers (Iba1,CD45 and PU.1),while HMC3 cells only stained positive for mural-cell markers (PDGFRβ and NG2). Distinct secretomes were observed in all cell models in response to inflammatory treatment,with iPSC-derived microglia showing the most significant inflammatory secretions. Notably,nitric oxide was only secreted by mouse microglia. Although all cell types exhibited phagocytic capacity,primary human microglia and iPSC-derived microglia displayed significantly higher levels of phagocytosis. Overall,comparative analysis revealed notable differences between human microglia,iPSC-derived microglia,HMC3 cells and mouse microglia. Such differences should be considered when using these models to study human brain diseases. Experimental findings obtained from mouse models or cell lines should ultimately be cross validated to ensure the translatability of results to the human condition. View Publication

We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells,but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions. View Publication

Cryopreservation is an essential technique to preserve stem cells,semipermanently sustaining their potentials. There are two main approaches of cryopreservation for human pluripotent stem cells (hPSCs). The first is the vitrification,which involves instantaneous freeze and thaw of hPSCs. The second is the conventional slow-cooling method and a rapid thaw. Both cryopreservation protocols have been standardized and optimized to yield high survivability of hPSCs. View Publication

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However,it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here,we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days,at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis,thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional,morphological and functional signatures of differentiated neurons,with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons,suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types. View Publication

Human embryonic stem cells (hESCs) share an identical genome with lineage-committed cells,yet possess the remarkable properties of self-renewal and pluripotency. The diverse cellular properties in different cells have been attributed to their distinct epigenomes,but how much epigenomes differ remains unclear. Here,we report that epigenomic landscapes in hESCs and lineage-committed cells are drastically different. By comparing the chromatin-modification profiles and DNA methylomes in hESCs and primary fibroblasts,we find that nearly one-third of the genome differs in chromatin structure. Most changes arise from dramatic redistributions of repressive H3K9me3 and H3K27me3 marks,which form blocks that significantly expand in fibroblasts. A large number of potential regulatory sequences also exhibit a high degree of dynamics in chromatin modifications and DNA methylation. Additionally,we observe novel,context-dependent relationships between DNA methylation and chromatin modifications. Our results provide new insights into epigenetic mechanisms underlying properties of pluripotency and cell fate commitment. textcopyright 2010 Elsevier Inc. View Publication

BACKGROUND: Constitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications,including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking. METHODOLOGY/PRINCIPAL FINDINGS: We have quantitatively compared promoter activities of five commonly used constitutive promoters,including the human β-actin promoter (ACTB),cytomegalovirus (CMV),elongation factor-1α,(EF1α),phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene transfer was used to ensure stable integration of promoter-eGFP constructs into the hESCs genome. Promoter activities were quantitatively compared in long term culture of undifferentiated hESCs and in their differentiated progenies. CONCLUSION/SIGNIFICANCE: The ACTB,EF1α and PGK promoters showed stable activities during long term culture of undifferentiated hESCs. The ACTB promoter was superior by maintaining expression in 75-80% of the cells after 50 days in culture. During embryoid body (EB) differentiation,promoter activities of all five promoters decreased. Although the EF1α promoter was downregulated in approximately 50% of the cells,it was the most stable promoter during differentiation. Gene expression analysis of differentiated eGFP+ and eGFP- cells indicate that promoter activities might be restricted to specific cell lineages,suggesting the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs. View Publication

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24(low/�?�)). Since then,CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties,including aldehyde dehydrogenase (ALDH) activity,have also been used to isolate CSCs from malignant tissues. Recently,we and others identified CSCs from pancreatic adenocarcinoma based on ALDH activity and the expression of the cell surface antigens CD44 and CD24,and CD133. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic,but ALDH+ cells are relatively more invasive. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human xenografts. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,a fluorescent substrate of ALDH. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells. View Publication