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Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless,clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally,derivation of hiPSCs should be rapid and efficient in order to minimize manipulations,reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here,we provide an optimized,fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity,purity,stability and safety at a GMP facility and cryopreserved. To our knowledge,as a proof of principle,these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes. View Publication

The process of X chromosome inactivation (XCI) during reprogramming to produce human induced pluripotent stem cells (iPSCs),as well as during the extensive programming that occurs in human preimplantation development,is not well-understood. Indeed,studies of XCI during reprogramming to iPSCs report cells with two active X chromosomes and/or cells with one inactive X chromosome. Here,we examine expression of the long noncoding RNA,XIST,in single cells of human embryos through the oocyte-to-embryo transition and in new mRNA reprogrammed iPSCs. We show that XIST is first expressed beginning at the 4-cell stage,coincident with the onset of embryonic genome activation in an asynchronous manner. Additionally,we report that mRNA reprogramming produces iPSCs that initially express XIST transcript; however,expression is rapidly lost with culture. Loss of XIST and H3K27me3 enrichment at the inactive X chromosome at late passage results in X chromosome expression changes. Our data may contribute to applications in disease modeling and potential translational applications of female stem cells. View Publication

Pluripotent stem cell (PSC) usage in heart regenerative medicine requires producing enriched cardiomyocytes (CMs) with mature phenotypes in a defined medium. However,current methods are typically performed in 2D environments that produce immature CMs. Here we report a simple,growth factor-free 3D culture system to rapidly and efficiently generate 85.07 ± 1.8% of spontaneously contractile cardiac spheres (scCDSs) using 3D-cultured human and monkey PSC-spheres. Along with small molecule-based 3D induction,this protocol produces CDSs of up to 95.7% CMs at a yield of up to 237 CMs for every input pluripotent cell,is effective for human and monkey PSCs,and maintains 81.03 ± 12.43% of CDSs in spontaneous contractibility for over three months. These CDSs displayed CM ultrastructure,calcium transient,appropriate pharmacological responses and CM gene expression profiles specific for maturity. Furthermore,3D-derived CMs displayed more mature phenotypes than those from a parallel 2D-culture. The system is compatible to large-scaly produce CMs for disease study,cell therapy and pharmaceutics screening. View Publication

Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy,reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative,but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC,comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated,through enrichment analysis,their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally,we report an unprecedented coverage of MSC CD markers,as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC. View Publication

The use of induced pluripotent stem cells (iPSCs) as a source of cells for cell-based therapy in regenerative medicine is hampered by the limited efficiency and safety of the reprogramming procedure and the low efficiency of iPSC differentiation to specialized cell types. Evidence suggests that iPSCs retain an epigenetic memory of their parental cells with a possible influence on their differentiation capacity in vitro. We reprogramme three cell types,namely human umbilical cord vein endothelial cells (HUVECs),endothelial progenitor cells (EPCs) and human dermal fibroblasts (HDFs),to iPSCs and compare their hematoendothelial differentiation capacity. HUVECs and EPCs were at least two-fold more efficient in iPSC reprogramming than HDFs. Both HUVEC- and EPC-derived iPSCs exhibited high potentiality toward endothelial cell differentiation compared with HDF-derived iPSCs. However,only HUVEC-derived iPSCs showed efficient differentiation to hematopoietic stem/progenitor cells. Examination of DNA methylation at promoters of hematopoietic and endothelial genes revealed evidence for the existence of epigenetic memory at the endothelial genes but not the hematopoietic genes in iPSCs derived from HUVECs and EPCs indicating that epigenetic memory involves an endothelial differentiation bias. Our findings suggest that endothelial cells and EPCs are better sources for iPSC derivation regarding their reprogramming efficiency and that the somatic cell type used for iPSC generation toward specific cell lineage differentiation is of importance. View Publication

Human B cells can be cultured ex vivo for a few weeks,following stimulation of the CD40 cell surface molecule in the presence of recombinant cytokines such as interleukin-4 (IL-4). However,attempts to produce polyclonal antigen-specific human antibodies by in vitro culture of human B cells obtained from immunized donors have not been successful. It has been shown in mice that lipopolysaccharide (LPS) is a potent mitogen for B cells and plays an important role in the generation of antigen-specific antibody responses. Although it has long been believed that LPS has no direct effect on human B cells,recent data indicating that IL-4-activated human B cells are induced to express Toll-like receptor-4,the main LPS receptor,prompted us to study the effects of LPS on the proliferation and antibody secretion of human B cells. Our results showed that LPS caused a reduction in the expansion of CD40-activated human B cells,accompanied by an increase in antigen-specific antibody secretion. This result suggested that some,but not all,B cells were able to differentiate into antibody-secreting cells in response to LPS. This increased differentiation could be explained by the observation that LPS-stimulated human B cells were induced to secrete higher amounts of IL-6,a pleiotropic cytokine well-known for its B-cell differentiation activity. In vivo,the effect of LPS on cytokine secretion by B cells may not only enhance B-cell differentiation but also help to sustain a local ongoing immune response to invading Gram-negative bacteria,until all pathogens have been cleared from the organism. View Publication

We describe the generation of a fusion cytokine consisting of GM-CSF in tandem with N-terminal-truncated MCP-1 (6-76),hereafter GMME1. Treatment of activated T cells with recombinant GMME1 protein leads to proinflammatory cytokine reduction and apoptosis via a CCR2-restricted pathway. Similarly,cell death is triggered in macrophages cultured with GMME1,while an inhibition of Ab production from plasma cells is observed. Treatment of CD4 T cells derived from experimental autoimmune encephalomyelitis mice with GMME1 leads to p38 hyperphosphorylation,inhibition of p44/42,AKT and STAT3 phosphorylation,and caspase-3 activation. GMME1 administration to experimental autoimmune encephalomyelitis mice suppresses symptomatic disease and correlates with decreased levels of inflammatory cytokines including IL-17,MOG-specific Ab titers,and blockade of CD4 and CD8 T cell infiltration in spinal cords. We propose that GMME1 defines a new class of agents for the treatment of autoimmune ailments by selectively targeting lymphomyeloid cells expressing CCR2. View Publication

The concept of cancer stem cells (CSC) embodies two aspects: the stem cell as the initial target of the oncogenic process and the existence of two populations of cells in cancers: the CSC and derived cells. The second is discussed in this review. CSC are defined as cells having three properties: a selectively endowed tumorigenic capacity,an ability to recreate the full repertoire of cancer cells of the parent tumor and the expression of a distinctive repertoire of surface biomarkers. In operational terms,the CSC are among all cancer cells those able to initiate a xenotransplant. Other explicit or implicit assumptions exist,including the concept of CSC as a single unique infrequent population of cells. To avoid such assumptions,we propose to use the operational term tumor-propagating cells (TPC); indeed,the cells that initiate transplants did not initiate the cancer. The experimental evidence supporting the explicit definition is analyzed. Cancers indeed contain a fraction of cells mainly responsible for the tumor development. However,there is evidence that these cells do not represent one homogenous population. Moreover,there is no evidence that the derived cells result from an asymmetric,qualitative and irreversible process. A more general model is proposed of which the CSC model could be one extreme case. We propose that the TPC are multiple evolutionary selected cancer cells with the most competitive properties [maintained by (epi-)genetic mechanisms],at least partially reversible,quantitative rather than qualitative and resulting from a stochastic rather than deterministic process. View Publication

BACKGROUND: To maintain pluripotency of human embryonic stem (huES) cells in feeder-free culture it has been necessary to provide a Matrigel substratum,which is a complex of poorly defined extracellular matrices and growth factors derived from mouse Engelbreth-Holm-Swarm sarcoma cells. Culture of stem cells under ill-defined conditions can inhibit the effectiveness of maintaining cells in a pluripotent state and reduce reproducibility of differentiation protocols. Moreover recent batches of Matrigel have been found to be contaminated with the single stranded RNA virus,Lactate Dehydrogenase Elevating Virus (LDEV),raising concerns regarding the safety of using stem cells that have been cultured on Matrigel in a therapeutic setting. To circumvent such concerns,we attempted to identify a recombinant matrix that could be used as an alternative to Matrigel for the culture of human pluripotent stem cells. huES and human induced pluripotent stem (hiPS) cells were grown on plates coated with a fusion protein consisting of E-cadherin and the IgG Fc domain using mTeSR1 medium.backslashnbackslashnRESULTS: Cells grown under these conditions maintained similar morphology and growth rate to those grown on Matrigel and retained all pluripotent stem cell features,including an ability to differentiate into multiple cell lineages in teratoma assays. We,therefore,present a culture system that maintains the pluripotency of huES and hiPS cells under completely defined conditions.backslashnbackslashnCONCLUSIONS: We propose that this system should facilitate growth of stem cells using good manufacturing practices (GMP),which will be necessary for the clinical use of pluripotent stem cells and their derivatives. View Publication

The contribution of mammary epithelial cells (MEC) to human immunodeficiency virus type 1 (HIV-1) in breast milk remains largely unknown. While breast milk contains CD4(+) cells throughout the breast-feeding period,it is not known whether MEC directly support HIV-1 infection or facilitate infection of CD4(+) cells in the breast compartment. This study evaluated primary human MEC for direct infection with HIV-1 and for indirect transfer of infection to CD4(+) target cells. Primary human MEC were isolated and assessed for expression of HIV-1 receptors. MEC were exposed to CCR5-,CXCR4- and dual-tropic strains of HIV-1 and evaluated for viral reverse transcription and integration and productive viral infection. MEC were also tested for the ability to transfer HIV to CD4(+) target cells and to activate resting CD4(+) T cells. Our results demonstrate that MEC express HIV-1 receptor proteins CD4,CCR5,CXCR4,and galactosyl ceramide (GalCer). While no evidence for direct infection of MEC was found,HIV-1 virions were observed in MEC endosomal compartments. Coculture of HIV-exposed MEC resulted in productive infection of activated CD4(+) T cells. In addition,MEC secretions increased HIV-1 replication and proliferation of infected target cells. Overall,our results indicate that MEC are capable of endosomal uptake of HIV-1 and can facilitate virus infection and replication in CD4(+) target cells. These findings suggest that MEC may serve as a viral reservoir for HIV-1 and may enhance infection of CD4(+) T lymphocytes in vivo. View Publication

Although many previous investigations have studied how mercury compounds cause cell death,sub-cytotoxic levels may affect mechanisms essential for the proper development of the nervous system. The present study investigates whether low doses of methylmercury (MeHg) and mercury chloride (HgCl2) can modulate the activity of JAK/STAT signaling,a pathway that promotes gliogenesis. We report that sub-cytotoxic doses of MeHg enhance ciliary neurotrophic factor (CNTF) evoked STAT3 phosphorylation in human SH-SY5Y neuroblastoma and mouse cortical neural progenitor cells (NPCs). This effect is specific for MeHg,since HgCl2 fails to enhance JAK/STAT signaling. Exposing NPCs to these low doses of MeHg (30-300nM) enhances CNTF-induced expression of STAT3-target genes such as glial fibrillary acidic protein (GFAP) and suppressors of cytokine signaling 3 (SOCS3),and increases the proportion of cells expressing GFAP following 2 days of differentiation. Higher,near-cytotoxic concentrations of MeHg and HgCl2 inhibit STAT3 phosphorylation and lead to increased production of superoxide. Lower concentrations of MeHg effective in enhancing JAK/STAT signaling (30nM) do not result in a detectable increase in superoxide nor increased expression of the oxidant-responsive genes,heme oxygenase 1,heat shock protein A5 and sirtuin 1. These findings suggest that low concentrations of MeHg inappropriately enhance STAT3 phosphorylation and glial differentiation,and that the mechanism causing this enhancement is distinct from the reactive oxygen species-associated cell death observed at higher concentrations of MeHg and HgCl2. View Publication

Human adipose-derived stem cells (ADSCs) can release exosomes; however,their specific functions remain elusive. In this study,we verified that exosomes derived from osteogenically differentiated ADSCs can promote osteogenic differentiation of ADSCs. Furthermore,in order to investigate the importance of exosomal microRNAs (miRNAs) in osteogenic differentiation of ADSCs,we used microarray assays to analyze the expression profiles of exosomal miRNAs derived from undifferentiated as well as osteogenically differentiated ADSCs; 201 miRNAs were upregulated and 33 miRNAs were downregulated between the two types of exosomes. Additionally,bioinformatic analyses,which included gene ontology analyses,pathway analysis,and miRNA-mRNA-network investigations,were performed. The results of these analyses revealed that the differentially expressed exosomal miRNAs participate in multiple biological processes,such as gene expression,synthesis of biomolecules,cell development,differentiation,and signal transduction,among others. Moreover,we found that these differentially expressed exosomal miRNAs connect osteogenic differentiation to processes such as axon guidance,MAPK signaling,and Wnt signaling. To the best of our knowledge,this is the first study to identify and characterize exosomal miRNAs derived from osteogenically differentiated ADSCs. This study confirms that alterations in the expression of exosomal miRNAs can promote osteogenic differentiation of ADSCs,which also provides the foundation for further research on the regulatory functions of exosomal miRNAs in the context of ADSC osteogenesis. View Publication