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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. Source


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. Source


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 will describe the most common immunocytochemical method utilized in the stem cell field - using fluorescently tagged secondary antibodies to detect a primary antibody that is bound to an epitope on a molecule of interest. Secondary antibodies recognize the heavy chain of the primary antibody's isotype. Generally, these methods employ an incubation period of the sample with the primary antibody, a series of washes to remove unbound primary antibody, a secondary incubation period of the sample with the fluorescently conjugated secondary antibody, followed by washes and preparation for microscopy. © 2011 Springer Science+Business Media, LLC. Source


Lieber C.A.,CHOC Research Institute | Kabeer M.H.,Childrens Hospital of Orange County | Kabeer M.H.,University of California at Irvine
Journal of Pediatric Surgery | Year: 2010

Purpose: Raman spectroscopy has been successfully demonstrated as an effective tool for tissue characterization and diagnosis, but nearly all studies have interrogated adult tissues and diseases. In this study, we demonstrate the application of Raman spectroscopy and its background autofluorescence for pediatric Wilms' tumor diagnosis. Methods: Eight tumors were measured in this study, along with matched normal kidney tissue in 6 cases. Spectral comparisons were drawn, and diagnostic use was assessed using both the Raman spectral features as well as the inherent tissue fluorescence. Results: The fluorescent background spectra were able to discriminate normal kidney from Wilms' tumor with 81% sensitivity and 100% specificity. The Raman spectra obtained 93% sensitivity and 100% specificity. Conclusions: This pilot study shows that both autofluorescence and Raman spectra provide diagnostic use in discriminating Wilms' tumor from normal kidney. These techniques may be used individually or in tandem to develop a real-time intraoperative screening and diagnostic device. © 2010 Elsevier Inc. All rights reserved. Source


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. Source

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