Scientific Resources

Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease,as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells,their reprogramming and the subsequent verification of iPSC pluripotency are laborious,manual processes limiting the scale and reproducibility of this technology. Here we describe a modular,robotic platform for iPSC reprogramming enabling automated,high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality,stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines. View Publication

Substrate composition significantly impacts human pluripotent stem cell (hPSC) self-renewal and differentiation,but relatively little is known about the role of endogenously produced extracellular matrix (ECM) components in regulating hPSC fates. Here we identify $\$-5 laminin as a signature ECM component endogenously synthesized by undifferentiated hPSCs cultured on defined substrates. Inducible shRNA knockdown and Cas9-mediated disruption of the LAMA5 gene dramatically reduced hPSC self-renewal and increased apoptosis without affecting the expression of pluripotency markers. Increased self-renewal and survival was restored to wild-type levels by culturing the LAMA5-deficient cells on exogenous laminin-521. Furthermore,treatment of LAMA5-deficient cells with blebbistatin or a ROCK inhibitor partially restored self-renewal and diminished apoptosis. These results demonstrate that endogenous $\$-5 laminin promotes hPSC self-renewal in an autocrine and paracrine manner. This finding has implications for understanding how stem cells dynamically regulate their microenvironment to promote self-renewal and provides guidance for efforts to design substrates for stem cell bioprocessing. View Publication

UNLABELLED: Human bocavirus 1 (HBoV1) is a single-stranded DNA parvovirus that causes lower respiratory tract infections in young children worldwide. In this study,we identified novel splice acceptor and donor sites,namely,A1' and D1',in the large nonstructural protein (NS1)-encoding region of the HBoV1 precursor mRNA. The novel small NS proteins (NS2,NS3,and NS4) were confirmed to be expressed following transfection of an HBoV1 infectious proviral plasmid and viral infection of polarized human airway epithelium cultured at an air-liquid interface (HAE-ALI). We constructed mutant pIHBoV1 infectious plasmids which harbor silent mutations (sm) smA1' and smD1' at the A1' and D1' splice sites,respectively. The mutant infectious plasmids maintained production of HBoV1 progeny virions at levels less than five times lower than that of the wild-type plasmid. Importantly,the smA1' mutant virus that does not express NS3 and NS4 replicated in HAE-ALI as effectively as the wild-type virus; however,the smD1' mutant virus that does not express NS2 and NS4 underwent an abortive infection in HAE-ALI. Thus,our study identified three novel NS proteins,NS2,NS3,and NS4,and suggests an important function of the NS2 protein in HBoV1 replication in HAE-ALI. IMPORTANCE: Human bocavirus 1 infection causes respiratory diseases,including acute wheezing in infants,of which life-threatening cases have been reported. In vitro,human bocavirus 1 infects polarized human bronchial airway epithelium cultured at an air-liquid interface that mimics the environment of human lower respiratory airways. Viral nonstructural proteins are often important for virus replication and pathogenesis in infected tissues or cells. In this report,we identified three new nonstructural proteins of human bocavirus 1 that are expressed during infection of polarized human bronchial airway epithelium. Among them,we proved that one nonstructural protein is critical to the replication of the virus in polarized human bronchial airway epithelium. The creation of nonreplicating infectious HBoV1 mutants may have particular utility in vaccine development for this virus. View Publication

T-cell genome engineering holds great promise for cell-based therapies for cancer,HIV,primary immune deficiencies,and autoimmune diseases,but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types� View Publication

Type 2 diabetes mellitus (T2D) is a chronic metabolic disorder resulting from defects in both insulin secretion and insulin activity. The deficit and dysfunction of insulin secreting $\$-cells are signature symptoms of T2D. Additionally,in pancreatic $\$-cells,a small group of genes that are abundantly expressed in most other tissues is highly selectively repressed. Monocarboxylate transporter 1 (MCT1) is one of these genes. In this study,we identified an MCT1-suppressing microRNA (hsa-miR-495) and used this microRNA together with human embryonic stem cell (hESC) derived pancreatic endoderm (PE) cells transplanted into a high-fat diet induced T2D mouse model. Glucose metabolism significantly improved and other symptoms of T2D were attenuated after the procedure. Our findings support the potential for T2D treatment using the combination of microRNA and hESC differentiated PE cells. View Publication

Tissue engineering strategies,based on solid/porous scaffolds,suffer from several limitations,such as ineffective vascularization,poor cell distribution and organization within scaffold,in addition to low final cell density,among others. Therefore,the search for other tissue engineering approaches constitutes an active area of investigation. Decellularized matrices (DM) present major advantages compared to solid scaffolds,such as ideal chemical composition,the preservation of vascularization structure and perfect three-dimensional structure. In the present study,we aimed to characterize and investigate murine heart decellularized matrices as biomaterials for regular and whole organ tissue engineering. Heart decellularized matrices were characterized according to: 1. DNA content,through DNA quantificationo and PCR of isolated genomic DNA; 2. Histological structure,assessed after Hematoxylin and Eosin,as well as Masson's Trichrome stainings; 3. Surface nanostructure analysis,performed,using SEM. Those essays allowed us to conclude that DM was indeed decellularized,with preserved extracellular matrix structure. Following characterization,decellularized heart slices were seeded with induced Pluripotent Stem cells (iPS). As expected,but - to the best of our knowledge - never shown before,decellularization of murine heart matrices maintained matrix biocompatibility,as iPS cells rapidly attached to the surface of the material and proliferated. Strikingly though,heart DM presented a differentiation induction effect over those cells,which lost their pluripotency markers after 7 days of culture in the DM. Such loss of differentiation markers was observed,even though bFGF containing media mTSR was used during such period. Gene expression of iPS cells cultured on DM will be further analyzed,in order to assess the effects of culturing pluripotent stem cells in decellularized heart matrices. View Publication

Decabromodiphenyl ether (BDE-209) has been detected in human serum,semen,placenta,cord blood and milk worldwide. However,little is known regarding the potential effects on the early human embryonic development of BDE-209. In this study,human embryonic stem cell lines FY-hES-10 and FY-hES-26 were used to evaluate the potential effects and explore the toxification mechanisms using low-level BDE-209 exposure. Our data showed that BDE-209 exposure (1,10 and 100 nM) reduced the expression of pluripotent genes such as OCT4,SOX2 and NANOG and induced human embryonic stem cells (hESCs) apoptosis. The downregulation of BIRC5/BCL2 and upregulation of BAX were related to apoptosis of hESCs induced by BDE-209 exposure. A mechanism study showed that OCT4 down-regulation accompanied by OCT4 promoter hypermethylation and increasing miR-145/miR-335 levels,OCT4 inhibitors. Moreover,BDE-209 could increase the generation of intracellular reactive oxygen species (ROS) and decrease SOD2 expression. The ROS increase and OCT4 downregulation after BDE-209 exposure could be reversed partly by antioxidant N-acetylcysteine supplement. These findings showed that BDE-209 exposure could decrease pluripotent genes expression via epigenetic regulation and induce apoptosis through ROS generation in human embryonic stem cells in vitro. View Publication

Tissue morphogenesis and organ formation are the consequences of biochemical and biophysical cues that lead to cellular spatial patterning in development. To model such events in vitro,we use PEG-patterned substrates to geometrically confine human pluripotent stem cell colonies and spatially present mechanical stress. Modulation of the WNT/β-catenin pathway promotes spatial patterning via geometric confinement of the cell condensation process during epithelial-mesenchymal transition,forcing cells at the perimeter to express an OCT4+ annulus,which is coincident with a region of higher cell density and E-cadherin expression. The biochemical and biophysical cues synergistically induce self-organizing lineage specification and creation of a beating human cardiac microchamber confined by the pattern geometry. These highly defined human cardiac microchambers can be used to study aspects of embryonic spatial patterning,early cardiac development and drug-induced developmental toxicity. 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

Regulatory T cells (Tregs) are physiologically designed to prevent autoimmune disease and maintain self-tolerance. In tumour microenvironments,their presence is related to a poor prognosis,and they influence the therapeutic outcome due to their capacity to suppress the immune response by cell-cell contact and to release immunosuppressive cytokines. In this study,we demonstrate that Treg immunosuppressive activity can be modulated by the cross-linking between the CD45RA expressed by Tregs and the C-type lectin MGL. This specific interaction strongly decreases the immunosuppressive activity of Tregs,restoring the proliferative capacity of co-cultured T lymphocytes. This effect can be attributed to changes in CD45RA and TCR signalling through the inhibition of Lck and inactivation of Zap-70,an increase in the Foxp3 methylation status and,ultimately,the reduced production of suppressive cytokines. These results indicate a role of MGL as an immunomodulator within the tumour microenvironment interfering with Treg functions,suggesting its possible use in the design of anticancer vaccines. View Publication

Stem cells integrate inputs from multiple sources. Stem cell niches provide signals that promote stem cell maintenance,while differentiated daughter cells are known to provide feedback signals to regulate stem cell replication and differentiation. Recently,stem cells have been shown to regulate themselves using an autocrine mechanism. The existence of a 'stem cell niche' was first postulated by Schofield in 1978 to define local environments necessary for the maintenance of haematopoietic stem cells. Since then,an increasing body of work has focused on defining stem cell niches. Yet little is known about how progenitor cell and differentiated cell numbers and proportions are maintained. In the airway epithelium,basal cells function as stem/progenitor cells that can both self-renew and produce differentiated secretory cells and ciliated cells. Secretory cells also act as transit-amplifying cells that eventually differentiate into post-mitotic ciliated cells . Here we describe a mode of cell regulation in which adult mammalian stem/progenitor cells relay a forward signal to their own progeny. Surprisingly,this forward signal is shown to be necessary for daughter cell maintenance. Using a combination of cell ablation,lineage tracing and signalling pathway modulation,we show that airway basal stem/progenitor cells continuously supply a Notch ligand to their daughter secretory cells. Without these forward signals,the secretory progenitor cell pool fails to be maintained and secretory cells execute a terminal differentiation program and convert into ciliated cells. Thus,a parent stem/progenitor cell can serve as a functional daughter cell niche. View Publication

BACKGROUND Haemolytic infection lyses red blood cells,releasing haemoglobin (Hb) into the plasma. Although recent studies showed that immune cells recognize redox-active cytotoxic extracellular Hb (metHb) bound to pathogen-associated molecular patterns (PAMPs),currently available information is limited to experiments performed in defined conditions using single cell lines. Therefore,a systemic approach targeting primary whole blood cells is required to better understand the cellular immune defence against metHb and PAMPs,when under a haemolytic infection. METHODS We investigated how human white blood cells,including neutrophils,respond to metHb and lipoteichoic acid (LTA) by measuring reactive oxygen species (ROS),signalling mediators (ERK and p38),NF-κB,cytokines,elastase secretion and cell activation markers. FINDINGS metHb activates NF-κB in TLR2-expressing HEK293 cells but not in normal or TLR9-expressing HEK293 cells. Treatment of isolated neutrophils with metHb increased production of ROS and expressions of IL-8,TNFα,and CD11b,which were further enhanced by metHb + LTA complex. While LTA stimulated the survival of neutrophils,it caused apoptotic cell death when complexed with metHb. The activation of neutrophils by metHb + LTA was subdued by the presence of other types of white blood cells. INTERPRETATION metHb and metHb + LTA complex are ligands of TLR2,inducing an unconventional TLR signalling pathway. Neutrophils are a highly sensitive cell type to metHb + LTA complex. During a haemolytic infection,white blood cells in the vicinity crosstalk to modulate neutrophil TLR-signalling induced by metHb and LTA. View Publication