Scientific Resources

BACKGROUND The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases,the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. METHODS AND RESULTS Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug,which elicits lethargy,anorexia,weight loss,and peritoneal fibrosis,all of which confound the interpretation of autophagy. Given this,we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms,reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation,we studied animals with diminished autophagic activity resulting from haploinsufficiency for Beclin 1. Beclin 1(+/-) mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely,animals overexpressing Beclin 1 manifested an amplified cardiotoxic response. CONCLUSIONS Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity. View Publication

Cell replacement therapy with human pluripotent stem cell-derived neurons has the potential to ameliorate neurodegenerative dysfunction and central nervous system injuries,but reprogrammed neurons are dissociated and spatially disorganized during transplantation,rendering poor cell survival,functionality and engraftment in vivo. Here,we present the design of three-dimensional (3D) microtopographic scaffolds,using tunable electrospun microfibrous polymeric substrates that promote in situ stem cell neuronal reprogramming,neural network establishment and support neuronal engraftment into the brain. Scaffold-supported,reprogrammed neuronal networks were successfully grafted into organotypic hippocampal brain slices,showing an ∼3.5-fold improvement in neurite outgrowth and increased action potential firing relative to injected isolated cells. Transplantation of scaffold-supported neuronal networks into mouse brain striatum improved survival ∼38-fold at the injection site relative to injected isolated cells,and allowed delivery of multiple neuronal subtypes. Thus,3D microscale biomaterials represent a promising platform for the transplantation of therapeutic human neurons with broad neuro-regenerative relevance. View Publication

Alterations in DNA damage response and repair have been observed in Huntington's disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis,while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX,indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus,increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown. View Publication

Diploidy is a fundamental genetic feature in mammals,in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species,but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes,leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics,such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover,we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts,they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation,alongside reduction in absolute gene expression levels and cell size. Surprisingly,we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state,but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo,despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development. View Publication

Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However,primary hepatocyte scarcity,cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies,HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology,such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study,the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions,cytochrome P450 drug metabolism and serum protein secretion,in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug,primarily metabolised by Cyp2D6. In addition to metabolic capacity,fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker,alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype. View Publication

Transplantation of ex vivo expanded corneal limbal stem cells (LSCs) has been the main treatment for limbal stem cell deficiency,although the shortage of donor corneal tissues remains a major concern for its wide application. Due to the development of tissue engineering,embryonic stem cells (ESCs)-derived corneal epithelial-like cells (ESC-CECs) become a new direction for this issue. However,the immunogenicity of ESC-CECs is a critical matter to be solved. In the present study,we explored the immunological properties of ESC-CECs,which were differentiated from ESCs. The results showed that ESC-CECs had a similar character and function with LSCs both in vitro and in vivo. In ESC-CECs,a large number of genes related with immune response were down-regulated. The expressions of MHC-I,MHC-II,and co-stimulatory molecules were low,but the expression of HLA-G was high. The ESC-CECs were less responsible for T cell proliferation and NK cell lysis in vitro,and there was less immune cell infiltration after transplantation in vivo compared with LSCs. Moreover,the immunological properties were not affected by interferon-$$. All these results indicated a low immunogenicity of ESC-CECs,and they can be promising in clinical use. View Publication

Remyelination is the goal of potential cell transplantation therapies for demyelinating diseases and other central nervous system injuries. Transplantation of oligodendrocyte precursor cells (OPCs) can result in remyelination in the central nervous system,and induced pluripotent stem cells (iPSCs) are envisioned to be an autograft cell source of transplantation therapy for many cell types. However,it remains time-consuming and difficult to generate OPCs from iPSCs. Clonal sphere preparations are reliable cell culture methods for purifying select populations of proliferating cells. To make clonal neurospheres from human embryonic stem cell (ESC)/iPSC colonies,we have found that a monolayer differentiation phase helps to increase the numbers of neural precursor cells. Indeed,we have compared a direct isolation of neural stem cells from human ESC/iPSC colonies (protocol 1) with monolayer neural differentiation,followed by clonal neural stem cell sphere preparations (protocol 2). The two-step method combining monolayer neuralization,followed by clonal sphere preparations,is more useful than direct sphere preparations in generating mature human oligodendrocytes. The initial monolayer culture stage appears to bias cells toward the oligodendrocyte lineage. This method of deriving oligodendrocyte lineage spheres from iPSCs represents a novel strategy for generating OPCs. View Publication

Cockayne syndrome (CS) is a severe neurodevelopmental disorder characterized by growth abnormalities,premature aging,and photosensitivity. Mutation of Cockayne syndrome B (CSB) affects neuronal gene expression and differentiation,so we attempted to bypass its function by expressing downstream target genes. Intriguingly,ectopic expression of Synaptotagmin 9 (SYT9),a key component of the machinery controlling neurotrophin release,bypasses the need for CSB in neuritogenesis. Importantly,brain-derived neurotrophic factor (BDNF),a neurotrophin implicated in neuronal differentiation and synaptic modulation,and pharmacological mimics such as 7,8-dihydroxyflavone and amitriptyline can compensate for CSB deficiency in cell models of neuronal differentiation as well. SYT9 and BDNF are downregulated in CS patient brain tissue,further indicating that sub-optimal neurotrophin signaling underlies neurological defects in CS. In addition to shedding light on cellular mechanisms underlying CS and pointing to future avenues for pharmacological intervention,these data suggest an important role for SYT9 in neuronal differentiation. View Publication

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function,developmental pathways,and disease mechanisms. Here,we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi,in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain,can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors,cardiomyocytes,and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn),CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types,dissect developmental pathways,and model disease. View Publication

Manipulation of genes in human embryonic stem cells (hESCs) is imperative for their highly potential applications; however,the transduction efficiency remains very low. Although existing evidence revealed the type,size,and zeta potential of vector affect gene transfection efficiency in cells,the systematic study in hESCs is scarce. In this study,using poly(amidoamine) (PAMAM) dendrimers ended with amine,hydroxyl,or carboxyl as model,we tested the influences of size and surface group as well as cytotoxicity and endocytosis on hESC gene transfection. We found that in culture medium of mTeSR the particle sizes of G5,G7,G4.5COOH,and G5OH were around 5 nm and G1 had a smaller size of 3.14 nm. G5 and G7 had a slight and significant positive zeta potential,respectively,whereas G1 was slightly negative,and G4.5COOH and G5OH were significantly negative. We demonstrated that only amine-terminated dendrimers accomplished gene transfection in hESCs,which is greater than that from Lipofectamine 2000 transfection. Ten micromolar G5 had the greatest efficiency and was better than 1000 μM G1. Only a low concentration (0.5 and 1 μM) of G7 realized gene delivery. Amine-ended dendrimers,especially with higher generations,were detrimental to the growth and pluripotent maintenance of hESCs. In contrast,similarly sized hydroxyl- and carboxyl-terminated dendrimers exerted much lower cytotoxicity,in which carboxyl-terminated dendrimer maintained pluripotency of hESCs. We also confirmed the endocytosis into and significant exocytosis from hESCs using FITC-labeled G5 dendrimer. These results suggested that careful considerations of size,concentration,and zeta potential,particularly the identity and position of groups,as well as minimized exocytosis in the design of a vector for hESC gene delivery are necessary,which helps to better design an effective vector in hESC gene transduction. View Publication

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung,liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells,hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2,SOX17,HNF1B,members of the GATA family,and the surface receptor CXCR4. However,differentiation protocols are rarely 100% efficient. Here,we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations. View Publication

Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4high cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation,efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved,while retaining their pluripotency. When added during the reprogramming process,CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus,CD30-LV may serve as novel tool for the selective gene transfer into pluripotent stem cells with broad applications in basic and therapeutic research. View Publication