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Pluripotent stem cells can be genetically labeled to facilitate differentiation studies. In this paper,we describe a gene-targeting protocol to knock in a GFP cassette into key gene loci in human pluripotent stem cells (hPSCs),and then use the genetically tagged hPSCs to guide in vitro differentiation,immunocytochemical and electrophysiological profiling and in vivo characterization after cell transplantation. The Olig transcription factors have key roles in the transcription regulatory pathways for the genesis of motor neurons (MNs) and oligodendrocytes (OLs). We have generated OLIG2-GFP hPSC reporter lines that reliably mark MNs and OLs for monitoring their sequential differentiation from hPSCs. The expression of the GFP reporter recapitulates the endogenous expression of OLIG genes. The in vitro characterization of fluorescence-activated cell sorting-purified cells is consistent with cells of the MN or OL lineages,depending on the stages at which they are collected. This protocol is efficient and reliable and usually takes 5-7 months to complete. The genetic tagging-differentiation methodology used herein provides a general framework for similar work for differentiation of hPSCs into other lineages. 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

Neurotoxicity testing still relies on ethically debated,expensive and time consuming in vivo experiments,which are unsuitable for high-throughput toxicity screening. There is thus a clear need for a rapid in vitro screening strategy that is preferably based on human-derived neurons to circumvent interspecies translation. Recent availability of commercially obtainable human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes holds great promise in assisting the transition from the current standard of rat primary cortical cultures to an animal-free alternative. We therefore composed several hiPSC-derived neuronal models with different ratios of excitatory and inhibitory neurons in the presence or absence of astrocytes. Using immunofluorescent stainings and multi-well micro-electrode array (mwMEA) recordings we demonstrate that these models form functional neuronal networks that become spontaneously active. The differences in development of spontaneous neuronal activity and bursting behavior as well as spiking patterns between our models confirm the importance of the presence of astrocytes. Preliminary neurotoxicity assessment demonstrates that these cultures can be modulated with known seizurogenic compounds,such as picrotoxin (PTX) and endosulfan,and the neurotoxicant methylmercury (MeHg). However,the chemical-induced effects on different parameters for neuronal activity,such as mean spike rate (MSR) and mean burst rate (MBR),may depend on the ratio of inhibitory and excitatory neurons. Our results thus indicate that hiPSC-derived neuronal models must be carefully designed and characterized prior to large-scale use in neurotoxicity screening. View Publication

Induced pluripotent stem cells (iPSCs) offer an unlimited resource of cells to be used for the study of underlying molecular biology of disease,therapeutic drug screening,and transplant-based regenerative medicine. However,methods for the directed differentiation of skeletal muscle for these purposes remain scarce and incomplete. Here,we present a novel,small molecule-based protocol for the generation of multinucleated skeletal myotubes using eight independent iPSC lines. Through combinatorial inhibition of phosphoinositide 3-kinase (PI3K) and glycogen synthase kinase 3β (GSK3β) with addition of bone morphogenic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2),we report up to 64% conversion of iPSCs into the myogenic program by day 36 as indicated by MYOG+ cell populations. These cells began to exhibit spontaneous contractions as early as 34 days in vitro in the presence of a serum-free medium formulation. We used this protocol to obtain iPSC-derived muscle cells from frontotemporal dementia (FTD) patients harboring C9orf72 hexanucleotide repeat expansions (rGGGGCC),sporadic FTD,and unaffected controls. iPSCs derived from rGGGGCC carriers contained RNA foci but did not vary in differentiation efficiency when compared to unaffected controls nor display mislocalized TDP-43 after as many as 120 days in vitro. This study presents a rapid,efficient,and transgene-free method for generating multinucleated skeletal myotubes from iPSCs and a resource for further modeling the role of skeletal muscle in amyotrophic lateral sclerosis and other motor neuron diseases. SIGNIFICANCE Protocols to produce skeletal myotubes for disease modeling or therapy are scarce and incomplete. The present study efficiently generates functional skeletal myotubes from human induced pluripotent stem cells using a small molecule-based approach. Using this strategy,terminal myogenic induction of up to 64% in 36 days and spontaneously contractile myotubes within 34 days were achieved. Myotubes derived from patients carrying the C9orf72 repeat expansion show no change in differentiation efficiency and normal TDP-43 localization after as many as 120 days in vitro when compared to unaffected controls. This study provides an efficient,novel protocol for the generation of skeletal myotubes from human induced pluripotent stem cells that may serve as a valuable tool in drug discovery and modeling of musculoskeletal and neuromuscular diseases. View Publication

Human embryonic stem cells (hESCs) are emerging as an attractive alternative source for cell replacement therapy since the cells can be expanded in culture indefinitely and differentiated into any cell types in the body. In order to optimize cell-to-cell interaction,cell proliferation and differentiation into specific lineages as well as tissue organization,it is important to provide a microenvironment for the hESCs which mimics the stem cell niche. One approach is to provide a three-dimensional (3D) environment such as encapsulation. We present an approach to culture and differentiate hESCs into midbrain dopamine (mdDA) neurons in a 3D microenvironment using alginate microcapsules for the first time. A detailed gene and protein expression analysis during neuronal differentiation showed an increased gene and protein expression of various specific DA neuronal markers,particularly tyrosine hydroxylase (TH) by textgreater100 folds after 2weeks and at least 50% higher expression after 4weeks respectively,compared to cells differentiated under conventional two-dimensional (2D) platform. The encapsulated TH+ cells co-expressed mdDA neuronal markers,forkhead box protein A-2 (FOXA2) and pituitary homeobox-3 (PITX3) after 4weeks and secreted approximately 60pg/ml/106 cells higher DA level when induced. We propose that the 3D platform facilitated an early onset of DA neuronal generation compared to that with conventional 2D system which also secretes more DA under potassium-induction. It is a very useful model to study the proliferation and directed differentiation of hESCs to various lineages,particularly to mdDA neurons. This 3D system also allows the separation of feeder cells from hESCs during the process of differentiation and also has potential for immune-isolation during transplantation studies. ?? 2013 Elsevier B.V. View Publication

Pluripotent stem cells can become any cell type found in the body. Accordingly,one of the major challenges when working with pluripotent stem cells is producing a highly homogenous population of differentiated cells,which can then be used for downstream applications such as cell therapies or drug screening. The transcription factor Ascl1 plays a key role in neural development and previous work has shown that Ascl1 overexpression using viral vectors can reprogram fibroblasts directly into neurons. Here we report on how a recombinant version of the Ascl1 protein functionalized with intracellular protein delivery technology (Ascl1-IPTD) can be used to rapidly differentiate human induced pluripotent stem cells (hiPSCs) into neurons. We first evaluated a range of Ascl1-IPTD concentrations to determine the most effective amount for generating neurons from hiPSCs cultured in serum free media. Next,we looked at the frequency of Ascl1-IPTD supplementation in the media on differentiation and found that one time supplementation is sufficient enough to trigger the neural differentiation process. Ascl1-IPTD was efficiently taken up by the hiPSCs and enabled rapid differentiation into TUJ1-positive and NeuN-positive populations with neuronal morphology after 8 days. After 12 days of culture,hiPSC-derived neurons produced by Ascl1-IPTD treatment exhibited greater neurite length and higher numbers of branch points compared to neurons derived using a standard neural progenitor differentiation protocol. This work validates Ascl1-IPTD as a powerful tool for engineering neural tissue from pluripotent stem cells. View Publication

The chaperone GRP78/Dna K is conserved throughout evolution down to prokaryotes. The GRP78 inhibitor OSU-03012 (AR-12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes. Similar data with the drug combination were obtained for: HSP70,HSP90,GRP94,GRP58,HSP27,HSP40 and HSP60. OSU-03012/sildenafil treatment killed brain cancer stem cells and decreased the expression of: NPC1 and TIM1; LAMP1; and NTCP1,receptors for Ebola/Marburg/Hepatitis A,Lassa fever,and Hepatitis B viruses,respectively. Pre-treatment with OSU-03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce. Similar data were obtained using Chikungunya,Mumps,Measles,Rubella,RSV,CMV,and Influenza viruses. OSU-03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi-drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression. The PDE5 inhibitors sildenafil or tadalafil enhanced OSU-03012 killing in N. gonorrhoeae and MRSE and low marginally toxic doses of OSU-03012 could restore bacterial sensitivity in N. gonorrhoeae to multiple antibiotics. Thus,Dna K and bacterial phosphodiesterases are novel antibiotic targets,and inhibition of GRP78 is of therapeutic utility for cancer and also for bacterial and viral infections. View Publication