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Stover A.E.,CHOC Research Institute | Schwartz P.H.,Childrens Hospital of Orange County Research Institute
Methods in Molecular Biology | Year: 2011

Embryoid body (EB) formation is a traditional method of inducing differentiation of pluripotent stem cells (PSCs). It is a routine in vitro test of pluripotency as well as the first stage in many differentiation protocols targeted toward the production of a specific lineage or cellular population, as in neural differentiation (see Chapters 29 and 30). The induction of differentiation via EB formation is fairly straightforward. However, depending on the specific PSC culture conditions - substrate, feeders, medium, and eventual cell type of interest - various methods are applied in order to most routinely obtain healthy EB cultures. © 2011 Springer Science+Business Media, LLC.

Stover A.E.,CHOC Research Institute | Schwartz P.H.,Childrens Hospital of Orange County Research Institute
Methods in Molecular Biology | Year: 2011

This protocol describes the culture of human pluripotent stem cells (PSCs) under feeder-free conditions in a commercially available, chemically defined, growth medium, using Matrigel as a substrate and the enzyme solution Accutase for single-cell passaging. This system is strikingly different from traditional PSC culture, where the cells are co-cultured with feeder cells and in medium containing serum replacement. PSCs cultured in this new system have a different morphology than those cultured on feeder cells but retain their characteristic pluripotency. This feeder-free PSC culture system is conceptually similar to feeder-free systems that use mouse embryonic fibroblast (MEF)-conditioned medium (MEF-CM) and Matrigel substratum. Instead of MEF-CM, a very complex and undefined medium, this new system uses StemPro SFM, a chemically defined medium that permits enzymatic passaging with Accutase to disaggregate the colonies into single cells. Accutase passaging has been used in conjunction with Stempro in our hands for 20+ passages without detectable karyotypic abnormalities. We will also review techniques for adapting cultures previously grown on MEFs, routine passaging of the cells, and cryopreservation. © 2011 Springer Science+Business Media, LLC.

Marei H.E.S.,Mansoura University | Althani A.,Qatar University | Afifi N.,Qatar University | Michetti F.,University Cattolica Del ore | And 6 more authors.
PLoS ONE | Year: 2011

Neural stem cells (NSC) with self-renewal and multipotent properties serve as an ideal cell source for transplantation to treat neurodegenerative insults such as Parkinson's disease. We used Agilent's and Illumina Whole Human Genome Oligonucleotide Microarray to compare the genomic profiles of human embryonic NSC at a single time point in culture, and a multicellular tissue from postmortem adult substantia nigra (SN) which are rich in dopaminergic (DA) neurons. We identified 13525 up-regulated genes in both cell types of which 3737 (27.6%) genes were up-regulated in the hENSC, 4116 (30.4%) genes were up-regulated in the human substantia nigra dopaminergic cells, and 5672 (41.93%) were significantly up-regulated in both cell population. Careful analysis of the data that emerged using DAVID has permitted us to distinguish several genes and pathways that are involved in dopaminergic (DA) differentiation, and to identify the crucial signaling pathways that direct the process of differentiation. The set of genes expressed more highly at hENSC is enriched in molecules known or predicted to be involved in the M phase of the mitotic cell cycle. On the other hand, the genes enriched in SN cells include a different set of functional categories, namely synaptic transmission, central nervous system development, structural constituents of the myelin sheath, the internode region of axons, myelination, cell projection, cell somata, ion transport, and the voltage-gated ion channel complex. Our results were also compared with data from various databases, and between different types of arrays, Agilent versus Illumina. This approach has allowed us to confirm the consistency of our obtained results for a large number of genes that delineate the phenotypical differences of embryonic NSCs, and SN cells. © 2011 Marei et al.

Duan Y.,CAS Wuhan Institute of Virology | Miao L.,CAS Wuhan Institute of Virology | Ye H.,CAS Wuhan Institute of Virology | Yang C.,CAS Wuhan Institute of Virology | And 5 more authors.
Acta Biochimica et Biophysica Sinica | Year: 2012

Immunofluorescence assay (IFA) is one of the most frequently used methods in the biological sciences and clinic diagnosis, but it is expensive and time-consuming. To overcome these limitations, we developed a faster and more cost-effective IFA (f-IFA) by modifying the standard IFA, and applied this method to track the progression of human cytomegalovirus (HCMV) infection in different cells. The f-IFA that we developed not only saves time, but also dramatically reduces the quantity of antibody (Ab), which will facilitate the application of IFA in clinic diagnosis. f-IFA requires only 15 min for blocking, 10 min incubation for each primary and secondary Abs, followed by 1 min extensive wash after each incubation. Only 25 μl of diluted Ab solution was needed for each coverslip at the primary and secondary Ab incubation steps. In addition, all steps were performed at room temperature. This f-IFA has been applied successfully to follow virion entry (pp65) and expression of viral genes (IE1, UL44, and pp65) in order to track the details of HCMV infection process. We found that ∼0.5 HCMV-infected T98G cells formed multiple-micronuclei (IE1 and nucleus staining) and had virus shedding (pp65 staining) by f-IFA, which could not be detected by the traditional IFA. Our results indicated that f-IFA is a sensitive, convenient, fast, and cost-effective method for investigating the details of virus infection progress, especially HCMV infection. The faster and cost-effective feature with higher sensitivity and specificity implies that f-IFA has potential applications in clinical diagnosis. © The Author 2012. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Nethercott H.E.,CHOC Research Institute | Brick D.J.,Childrens Hospital of Orange County Research Institute | Schwartz P.H.,Childrens Hospital of Orange County Research Institute
Methods in Molecular Biology | Year: 2011

This chapter provides a method for reprogramming human dermal fibroblasts into induced pluripotent stem cells (iPSCs) using three lentiviruses containing cDNAs for OCT4 and SOX2, KLF4 and C-MYC, and NANOG and LIN28, respectively. Lentiviral vectors are based on the human immunodeficiency virus (HIV) and provide an effective means for the delivery, integration, and expression of exogenous genes in mammalian cells. Lentiviruses are attractive gene delivery vehicles as they are able to infect both proliferating and nonproliferating cells. Lentiviruses stably integrate into the genome without incurring cellular toxicity and can maintain sustained transgene expression during prolonged host cell proliferation and differentiation. In this protocol, we describe how to prepare lentiviruses, stably transduce human fibroblasts, and identify bona fide iPSC colonies based on morphological similarity to human embryonic stem cell (ESC) colonies and live-cell immunological staining using cell-surface markers of human PSCs such as Tra-1-60 and Tra-1-81. © 2011 Springer Science+Business Media, LLC.

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