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Routine commercial and clinical applications of human pluripotent stem cells (hPSCs) and their progenies will require increasing cell quantities that cannot be provided by conventional adherent culture technologies. Here we describe a straightforward culture protocol for the expansion of undifferentiated human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) in suspension culture. This culture technique was successfully tested on two hiPSC clones,three hESC lines and on a nonhuman primate ESC line. It is based on a defined medium and single-cell inoculation,but it does not require culture preadaptation,use of microcarriers or any other matrices. Over a time course of 4-7 d,hPSCs can be expanded up to sixfold. Preparation of a high-density culture and its subsequent translation to scalable stirred suspension in Erlenmeyer flasks and stirred spinner flasks are also feasible. Importantly,hPSCs maintain pluripotency and karyotype stability for more than ten passages. View Publication

AIM: Dendritic cell (DC)-based vaccines are designed to exploit the intrinsic capacity of these highly effective antigen presenting cells to prime and boost antigen-specific T-cell immune responses. Successful development of DC-based vaccines will be dependent on the ability to utilize and harness the full potential of these potent immune stimulatory cells. Recent advances to generate DCs derived from human embryonic stem cells (hESCs) that are suitable for clinical use represent an alternative strategy from conventional approaches of using patient-specific DCs. Although the differentiation of hESC-derived DCs in serum-free defined conditions has been established,the stimulatory potential of these hESC-derived DCs have not been fully evaluated. METHODS: hESC-derived DCs were differentiated in serum-free defined culture conditions. The delivery of antigen into hESC-derived DCs was investigated using mRNA transfection and replication-deficient adenoviral vector transduction. hESC-derived DCs modified with antigen were evaluated for their capacity to stimulate antigen-specific T-cell responses with known HLA matching. Since IL-12 is a key cytokine that drives T-cell function,further enhancement of DC potency was evaluated by transfecting mRNA encoding the IL-12p70 protein into hESC-derived DCs. RESULTS: The transfection of mRNA into hESC-derived DCs was effective for heterologous protein expression. The efficiency of adenoviral vector transduction into hESC-derived DCs was poor. These mRNA-transfected DCs were capable of stimulating human telomerase reverse transcriptase antigen-specific T cells composed of varying degrees of HLA matching. In addition,we observed the transfection of mRNA encoding IL-12p70 enhanced the T-cell stimulation potency of hESC-derived DCs. CONCLUSION: These data provide support for the development and modification of hESC-derived DCs with mRNA as a potential strategy for the induction of T-cell-mediated immunity. View Publication

Human induced pluripotent stem cells (iPSCs) are a potential source of hepatocytes for liver transplantation to treat end-stage liver disease. In vitro differentiation of human iPSCs into hepatic cells has been achieved using a multi- stage differentiation protocol,but whether these cells are functional and capable of engrafting and regenerating diseased liver tissue is not clear. We show that human iPSC-derived hepatic cells at various differentiation stages can engraft the liver in a mouse transplantation model. Using the same differentiation and transplantation protocols,we also assessed the ability of human iPSCs derived from each of the three developmental germ layer tissues (that is,ectoderm,mesoderm,and endoderm) to regenerate mouse liver. These iPSC lines,with similar but distinct global DNA methylation patterns,differentiated into multistage hepatic cells with an efficiency similar to that of human embryonic stem cells. Human hepatic cells at various differentiation stages derived from iPSC lines of different origins successfully repopulated the liver tissue of mice with liver cirrhosis. They also secreted human-specific liver proteins into mouse blood at concentrations comparable to that of proteins secreted by human primary hepato- cytes. Our results demonstrate the engraftment and liver regenerative capabilities of human iPSC-derived multi- stage hepatic cells in vivo and suggest that human iPSCs of distinct origins and regardless of their parental epigenetic memory can efficiently differentiate along the hepatic lineage. View Publication

Transient lymphopenia is a hallmark of measles virus (MV)-induced immunosuppression. To address to what extent replenishment of the peripheral lymphocyte compartment from bone marrow (BM) progenitor/stem cells might be affected,we analyzed the interaction of wild-type MV with hematopoietic stem and progenitor cells (HS/PCs) and stroma cells in vitro. Infection of human CD34(+) HS/PCs or stroma cells with wild-type MV is highly inefficient yet noncytolytic. It occurs independently of CD150 in stroma cells but also in HS/PCs,where infection is established in CD34(+) CD150(-) and CD34(+) CD150(+) (in humans representing HS/PC oligopotent precursors) subsets. Stroma cells and HS/PCs can mutually transmit MV and may thereby create a possible niche for continuous viral exchange in the BM. Infected lymphocytes homing to this compartment may serve as sources for HS/PC or stroma cell infection,as reflected by highly efficient transmission of MV from both populations in cocultures with MV-infected B or T cells. Though MV exposure does not detectably affect the viability,expansion,and colony-forming activity of either CD150(+) or CD150(-) HS/PCs in vitro,it efficiently interferes with short- but not long-term hematopoietic reconstitution in NOD/SCID mice. Altogether,these findings support the hypothesis that MV accession of the BM compartment by infected lymphocytes may contribute to peripheral blood mononuclear cell lymphopenia at the level of BM suppression. View Publication

Directed hepatocyte differentiation from human induced pluripotent stem cells (iPSCs) potentially provides a unique platform for modeling liver genetic diseases and performing drug-toxicity screening in vitro. Wilson's disease is a genetic disease caused by mutations in the ATP7B gene,whose product is a liver transporter protein responsible for coordinated copper export into bile and blood. Interestingly,the spectrum of ATP7B mutations is vast and can influence clinical presentation (a variable spectrum of hepatic and neural manifestations),though the reason is not well understood. We describe the generation of iPSCs from a Chinese patient with Wilson's disease that bears the R778L Chinese hotspot mutation in the ATP7B gene. These iPSCs were pluripotent and could be readily differentiated into hepatocyte-like cells that displayed abnormal cytoplasmic localization of mutated ATP7B and defective copper transport. Moreover,gene correction using a self-inactivating lentiviral vector that expresses codon optimized-ATP7B or treatment with the chaperone drug curcumin could reverse the functional defect in vitro. Hence,our work describes an attractive model for studying the pathogenesis of Wilson's disease that is valuable for screening compounds or gene therapy approaches aimed to correct the abnormality. In the future,once relevant safety concerns (including the stability of the mature liver-like phenotype) and technical issues for the transplantation procedure are solved,hepatocyte-like cells from similarly genetically corrected iPSCs could be an option for autologous transplantation in Wilson's disease. View Publication

RATIONALE: Cardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis,the key process underlying excitation-contraction coupling. OBJECTIVE: We investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs). METHODS AND RESULTS: We differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes,spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore,in contrast with hESC-derived cardiomyocytes,ryanodine did not reduce the amplitudes,maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition,spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2),sacroplasmic recticulum calcium-ATPase (SERCA),junction (Jun) and triadin (TRDN),were significantly lower in hiPSC than in hESCs. CONCLUSIONS: The results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts. View Publication

The mitochondrial contribution to the maintenance of human embryonic stem cell (hESC) pluripotency and culture homeostasis remains poorly understood. Here,we sought to determine whether hESC adaptation to different feeder-free culture conditions is linked to a mitochondrial adaptation. The expression of ESC pluripotency factors and parameters of mitochondrial contribution including mitochondrial membrane potential,mtDNA content,and the expression of master mitochondrial genes implicated in replication,transcription,and biogenesis were determined in 8 hESC lines maintained in 2 distinct human feeders-conditioned media (CM): human foreskin fibroblast-CM (HFF-CM) and mesenchymal stem cell-CM (MSC-CM). We show a robust parallel trend between the expression of ESC pluripotency factors and the mitochondrial contribution depending on the culture conditions employed to maintain the hESCs,with those in MSC-CM consistently displaying increased levels of pluripotency markers associated to an enhanced mitochondrial contribution. The differences in the mitochondrial status between hESCs maintained in MSC-CM versus HFF-CM respond to coordinated changes in mitochondrial gene expression and biogenesis. Importantly,the culture conditions determine the mitochondrial distribution within the stage-specific embryonic antigen 3 positive (SSEA3(+)) and negative (SSEA3(-)) isolated cell subsets. hESC colonies in MSC-CM display an intrinsic" high mitochondrial status which may suffice to support undifferentiated growth� View Publication

By providing access to affected neurons,human induced pluripotent stem cells (iPSc) offer a unique opportunity to model human neurodegenerative diseases. We generated human iPSc from the skin fibroblasts of children with mucopolysaccharidosis type IIIB. In this fatal lysosomal storage disease,defective α-N-acetylglucosaminidase interrupts the degradation of heparan sulfate (HS) proteoglycans and induces cell disorders predominating in the central nervous system,causing relentless progression toward severe mental retardation. Partially digested proteoglycans,which affect fibroblast growth factor signaling,accumulated in patient cells. They impaired isolation of emerging iPSc unless exogenous supply of the missing enzyme cleared storage and restored cell proliferation. After several passages,patient iPSc starved of an exogenous enzyme continued to proliferate in the presence of fibroblast growth factor despite HS accumulation. Survival and neural differentiation of patient iPSc were comparable with unaffected controls. Whereas cell pathology was modest in floating neurosphere cultures,undifferentiated patient iPSc and their neuronal progeny expressed cell disorders consisting of storage vesicles and severe disorganization of Golgi ribbons associated with modified expression of the Golgi matrix protein GM130. Gene expression profiling in neural stem cells pointed to alterations of extracellular matrix constituents and cell-matrix interactions,whereas genes associated with lysosome or Golgi apparatus functions were downregulated. Taken together,these results suggest defective responses of patient undifferentiated stem cells and neurons to environmental cues,which possibly affect Golgi organization,cell migration and neuritogenesis. This could have potential consequences on post-natal neurological development,once HS proteoglycan accumulation becomes prominent in the affected child brain. View Publication

Elucidating the in vitro differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose,a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study,we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover,single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new,robust,and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation. 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

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

Histone deacetylases (HDAC) are key enzymes in the epigenetic control of gene expression. Recently,inhibitors of class I and class II HDAC have been successfully employed for the treatment of different inflammatory diseases such as rheumatoid arthritis,colitis,airway inflammation and asthma. So far,little is known so far about a similar therapeutic effect of inhibitors specifically directed against sirtuins,the class III HDAC. In this study,we investigated the expression and localization of endogenous sirtuins in primary human dermal microvascular endothelial cells (HDMEC),a cell type playing a key role in the development and maintenance of skin inflammation. We then examined the biological activity of sirtinol,a specific sirtuin inhibitor,in HDMEC response to pro-inflammatory cytokines. We found that,even though sirtinol treatment alone affected only long-term cell proliferation,it diminishes HDMEC inflammatory responses to tumor necrosis factor (TNF)α and interleukin (IL)-1β. In fact,sirtinol significantly reduced membrane expression of adhesion molecules in TNFã- or IL-1β-stimulated cells,as well as the amount of CXCL10 and CCL2 released by HDMEC following TNFα treatment. Notably,sirtinol drastically decreased monocyte adhesion on activated HDMEC. Using selective inhibitors for Sirt1 and Sirt2,we showed a predominant involvement of Sirt1 inhibition in the modulation of adhesion molecule expression and monocyte adhesion on activated HDMEC. Finally,we demonstrated the in vivo expression of Sirt1 in the dermal vessels of normal and psoriatic skin. Altogether,these findings indicated that sirtuins may represent a promising therapeutic target for the treatment of inflammatory skin diseases characterized by a prominent microvessel involvement. View Publication