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Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases,yet current therapeutic treatments are inadequate due to a complex disease pathogenesis. The plant polyphenol apigenin has been shown to have anti-inflammatory and neuroprotective properties in a number of cell and animal models; however a comprehensive assessment has not been performed in a human model of AD. Here we have used a human induced pluripotent stem cell (iPSC) model of familial and sporadic AD,in addition to healthy controls,to assess the neuroprotective activity of apigenin. The iPSC-derived AD neurons demonstrated a hyper-excitable calcium signalling phenotype,elevated levels of nitrite,increased cytotoxicity and apoptosis,reduced neurite length and increased susceptibility to inflammatory stress challenge from activated murine microglia,in comparison to control neurons. We identified that apigenin has potent anti-inflammatory properties with the ability to protect neurites and cell viability by promoting a global down-regulation of cytokine and nitric oxide (NO) release in inflammatory cells. In addition,we show that apigenin is able to protect iPSC-derived AD neurons via multiple means by reducing the frequency of spontaneous Ca(2+) signals and significantly reducing caspase-3/7 mediated apoptosis. These data demonstrate the broad neuroprotective action of apigenin against AD pathogenesis in a human disease model. View Publication

The emerging models of human embryonic stem cell (hESC) self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen,we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the absence of growth factors. Gene expression analyses and functional assays demonstrated an endothelial identity of this balloon-like structure,while cell surface marker analyses revealed a VE-cadherin+CD31+CD34+KDR+CD43???putative endothelial progenitor population. Furthermore,molecular marker labeling and morphological examinations characterized several other distinct DiI-Ac-LDL+multi-cellular modules and a VEGFR3+sprouting structure in the balloon cultures that likely represented intermediate structures of balloon-formation. View Publication

Williams syndrome is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with Williams syndrome lack precisely the same set of genes,with breakpoints in chromosome band 7q11.23 (refs 1-5). The contribution of specific genes to the neuroanatomical and functional alterations,leading to behavioural pathologies in humans,remains largely unexplored. Here we investigate neural progenitor cells and cortical neurons derived from Williams syndrome and typically developing induced pluripotent stem cells. Neural progenitor cells in Williams syndrome have an increased doubling time and apoptosis compared with typically developing neural progenitor cells. Using an individual with atypical Williams syndrome,we narrowed this cellular phenotype to a single gene candidate,frizzled 9 (FZD9). At the neuronal stage,layer V/VI cortical neurons derived from Williams syndrome were characterized by longer total dendrites,increased numbers of spines and synapses,aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in neurons from Williams syndrome were validated after Golgi staining of post-mortem layer V/VI cortical neurons. This model of human induced pluripotent stem cells fills the current knowledge gap in the cellular biology of Williams syndrome and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain. View Publication

With the advent of induced pluripotent stem cells and directed differentiation techniques,it is now feasible to derive individual-specific cardiac cells for human heart tissue engineering. Here we report the generation of functional engineered human cardiac patches using human induced pluripotent stem cells-derived cardiac cells and decellularized natural heart ECM as scaffolds. The engineered human cardiac patches can be tailored to any desired size and shape and exhibited normal contractile and electrical physiology in vitro. Further,when patching on the infarct area,these patches improved heart function of rats with acute myocardial infarction in vivo. These engineered human cardiac patches can be of great value for normal and disease-specific heart tissue engineering,drug screening,and meet the demands for individual-specific heart tissues for personalized regenerative therapy of myocardial damages in the future. View Publication

Various scalable three-dimensional culture systems for regenerative medicine using human induced pluripotent stem cells (hiPSCs) have been developed to date. However,stable production of hiPSCs with homogeneous qualities still remains a challenge. Here,we describe a novel and simple embryoid body (EB) formation system using unique microfabricated culture vessels. Furthermore,this culture system is useful for high throughput EB formation and rapid generation of differentiated cells such as neural stem cells (NSCs) from hiPSCs. The period of NSC differentiation was significantly shortened under high EB density culture conditions. Simultaneous mass production of a pure population of NSCs was possible within 4 days. These results indicate that the novel culture system might not only become a unique tool to obtain new insights into developmental biology based on human stem cells,but also provide an important tractable platform for efficient and stable production of NSCs for clinical applications. View Publication

Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse,but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30-50%). After cell division,RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome. View Publication

Consistent and robust manufacturing is essential for the translation of cell therapies,and the utilisation automation throughout the manufacturing process may allow for improvements in quality control,scalability,reproducibility and economics of the process. The aim of this study was to measure and establish the comparability between alternative process steps for the culture of hiPSCs. Consequently,the effects of manual centrifugation and automated non-centrifugation process steps,performed using TAP Biosystems' CompacT SelecT automated cell culture platform,upon the culture of a human induced pluripotent stem cell (hiPSC) line (VAX001024c07) were compared. This study,has demonstrated that comparable morphologies and cell diameters were observed in hiPSCs cultured using either manual or automated process steps. However,non-centrifugation hiPSC populations exhibited greater cell yields,greater aggregate rates,increased pluripotency marker expression,and decreased differentiation marker expression compared to centrifugation hiPSCs. A trend for decreased variability in cell yield was also observed after the utilisation of the automated process step. This study also highlights the detrimental effect of the cryopreservation and thawing processes upon the growth and characteristics of hiPSC cultures,and demonstrates that automated hiPSC manufacturing protocols can be successfully transferred between independent laboratories. View Publication

The differentiation efficiency of human embryonic stem cells (hESCs) into heart muscle cells (cardiomyocytes) is highly sensitive to culture conditions. To elucidate the regulatory mechanisms involved,we investigated hESCs grown on three distinct culture platforms: feeder-free Matrigel,mouse embryonic fibroblast feeders,and Matrigel replated on feeders. At the outset,we profiled and quantified their differentiation efficiency,transcriptome,transcription factor binding sites and DNA-methylation. Subsequent genome-wide analyses allowed us to reconstruct the relevant interactome,thereby forming the regulatory basis for implicating the contrasting differentiation efficiency of the culture conditions. We hypothesized that the parental expressions of FOXC1,FOXD1 and FOXQ1 transcription factors (TFs) are correlative with eventual cardiomyogenic outcome. Through WNT induction of the FOX TFs,we observed the co-activation of WNT3 and EOMES which are potent inducers of mesoderm differentiation. The result strengthened our hypothesis on the regulatory role of the FOX TFs in enhancing mesoderm differentiation capacity of hESCs. Importantly,the final proportions of cells expressing cardiac markers were directly correlated to the strength of FOX inductions within 72 hours after initiation of differentiation across different cell lines and protocols. Thus,we affirmed the relationship between early FOX TF expressions and cardiomyogenesis efficiency. View Publication

Gamma-aminobutyric acid (GABA)-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here,we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs) into GABAergic neurons (iGNs) with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6-8 weeks. Furthermore,in vitro,iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs). Upon transplantation into immunodeficient mice,human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together,our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons. View Publication

Human embryonic stem cells (hESCs) can differentiate into all somatic lineages including stratified squamous epithelia. Thus,efficient methods are required to direct hESC differentiation to obtain a pure subpopulation for tissue engineering. The study aimed to assess the effects of retinoic acid (RA),bone morphogenetic protein-4 (BMP4),and ascorbic acid (AA) on the differentiation of hESCs into keratinocyte progenitors in vitro. The first media contained AA and BMP4; the second contained RA,AA,and BMP4; the third was commercial-defined keratinocyte serum-free medium,which was used to differentiate H9 hESCs (direct approach) or embryoid bodies (EBs) (indirect approach) into keratinocyte progenitors. Real-time RT-PCR,immunofluorescence,and flow-cytometry were used to characterize the differentiated cells. Cells induced by AA + BMP4 + RA showed the typical epithelial morphology,while cells induced by AA + BMP4 showed multiple appearances. CK14 and p63 messenger RNA (mRNA) expressions in the AA + BMP4 + RA-treated cells were higher than those of the AA + BMP4-treated cells (CK14: 22.4-fold; p63: 84.7-fold). Epithelial marker CK18 mRNA expressions at 14 d of differentiation and keratinocyte marker CK14 and transcription factor p63 mRNA expressions at 35 d of differentiation were higher in cells differentiated from hESCs compared with those differentiated from EBs (CK18 10.51 ± 3.26 vs. 6.67 ± 1.28; CK14 9.27 ± 3.61 vs. 5.32 ± 1.86; p63 0.73 ± 0.06 vs. 0.44 ± 0.12,all P textless 0.05) After hESC induction by AA+BMP4+RA,CK14 mRNA expression was upregulated after day 21,peaking by 35 d of differentiation. Combined RA,BMP4,and AA could effectively induce differentiation of hESCs into keratinocyte progenitors in vitro. These keratinocytes could be used for oral mucosa and skin tissue engineering. View Publication

PURPOSE Proliferative vitreoretinopathy (PVR) is a blinding disorder that develops after a retinal tear or detachment. Activation of the retinal pigmented epithelium (RPE) is implicated in PVR; however,the mechanisms leading to enhanced RPE proliferation,migration,and contraction remain largely unknown. This study utilized an in vitro model of PVR to investigate the role of acetylation in RPE activation and its contribution to the progression of this disease. METHODS ARPE-19 cells,primary cultures of porcine RPE,and induced pluripotent stem cell-derived RPE (iPS-RPE) were utilized for cellular and molecular analyses. Cells treated with transforming growth factor beta 2 (TGF$$2; 10 ng/mL) alone or in the presence of the broad-spectrum histone deacetylase (HDAC) inhibitor,trichostatin A (TSA; 0.1 $$M),were assessed for contraction and migration through collagen contraction and scratch assays,respectively. Western blotting and immunofluorescence analysis were performed to assess $$-smooth muscle actin ($$-SMA) and $$-catenin expression after TGF$$2 treatment alone or in combination with TSA. RESULTS TGF$$2 significantly increased RPE cell contraction in collagen matrix and this effect was inhibited in the presence of TSA (0.1 $$M). In agreement with these data,immunofluorescence analysis of TSA-treated iPS-RPE wounded monolayers revealed decreased $$-SMA as compared with control. Scratch assays to assess wound healing revealed TSA inhibited TGF$$2-mediated iPS-RPE cell migration. CONCLUSIONS Our findings indicate a role of acetylation in RPE activation. Specifically,the HDAC inhibitor TSA decreased RPE cell proliferation and TGF$$2-mediated cell contraction and migration. Further investigation of pharmacological compounds that modulate acetylation may hold promise as therapeutic agents for PVR. View Publication

ALS is a rapidly progressive,devastating neurodegenerative illness of adults that produces disabling weakness and spasticity arising from death of lower and upper motor neurons. No meaningful therapies exist to slow ALS progression,and molecular insights into pathogenesis and progression are sorely needed. In that context,we used high-depth,next generation RNA sequencing (RNAseq,Illumina) to define gene network abnormalities in RNA samples depleted of rRNA and isolated from cervical spinal cord sections of 7 ALS and 8 CTL samples. We aligned textgreater50 million 2X150 bp paired-end sequences/sample to the hg19 human genome and applied three different algorithms (Cuffdiff2,DEseq2,EdgeR) for identification of differentially expressed genes (DEG's). Ingenuity Pathways Analysis (IPA) and Weighted Gene Co-expression Network Analysis (WGCNA) identified inflammatory processes as significantly elevated in our ALS samples,with tumor necrosis factor (TNF) found to be a major pathway regulator (IPA) and TNF$$-induced protein 2 (TNFAIP2) as a major network hub" gene (WGCNA). Using the oPOSSUM algorithm� View Publication