ROCK Inhibitor Is Not Required for Embryoid Body Formation from Singularized Human Embryonic Stem Cells

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications

A large-scale proteomic analysis of human embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>Much of our current knowledge of the molecular expression profile of human embryonic stem cells (hESCs) is based on transcriptional approaches. These analyses are only partly predictive of protein expression however, and do not shed light on post-translational regulation, leaving a large gap in our knowledge of the biology of pluripotent stem cells.</p> <p>Results</p> <p>Here we describe the use of two large-scale western blot assays to identify over 600 proteins expressed in undifferentiated hESCs, and highlight over 40 examples of multiple gel mobility variants, which are suspected protein isoforms and/or post-translational modifications. Twenty-two phosphorylation events in cell signaling molecules, as well as potential new markers of undifferentiated hESCs were also identified. We confirmed the expression of a subset of the identified proteins by immunofluorescence and correlated the expression of transcript and protein for key molecules in active signaling pathways in hESCs. These analyses also indicated that hESCs exhibit several features of polarized epithelia, including expression of tight junction proteins.</p> <p>Conclusion</p> <p>Our approach complements proteomic and transcriptional analysis to provide unique information on human pluripotent stem cells, and is a framework for the continued analyses of self-renewal.</p

Efficient Generation of Functional Dopaminergic Neurons from Human Induced Pluripotent Stem Cells Under Defined Conditions

Human induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells represent a promising unlimited cell source for generating patient-specific cells for biomedical research and personalized medicine. As a first step, critical to clinical applications, we attempted to develop defined culture conditions to expand and differentiate human iPSCs into functional progeny such as dopaminergic neurons for treating or modeling Parkinson's disease (PD). We used a completely defined (xeno-free) system that we previously developed for efficient generation of authentic dopaminergic neurons from human embryonic stem cells (hESCs), and applied it to iPSCs. First, we adapted two human iPSC lines derived from different somatic cell types for the defined expansion medium and showed that the iPSCs grew similarly as hESCs in the same medium regarding pluripotency and genomic stability. Second, by using these two independent adapted iPSC lines, we showed that the process of differentiation into committed neural stem cells (NSCs) and subsequently into dopaminergic neurons was also similar to hESCs. Importantly, iPSC-derived dopaminergic neurons were functional as they survived and improved behavioral deficits in 6-hydroxydopamine-leasioned rats after transplantation. In addition, iPSC-derived NSCs and neurons could be efficiently transduced by a baculoviral vector delivering episomal DNA for future gene function study and disease modeling using iPSCs. We also performed genome-wide microarray comparisons between iPSCs and hESCs, and we derived NSC and dopaminergic neurons. Our data revealed overall similarity and visible differences at a molecular level. Efficient generation of functional dopaminergic neurons under defined conditions will facilitate research and applications using PD patient-specific iPSCs. Stem Cells 2010;28:1893–190

Identification by Automated Screening of a Small Molecule that Selectively Eliminates Neural Stem Cells Derived from hESCs but Not Dopamine Neurons

BACKGROUND:We have previously described fundamental differences in the biology of stem cells as compared to other dividing cell populations. We reasoned therefore that a differential screen using US Food and Drug Administration (FDA)-approved compounds may identify either selective survival factors or specific toxins and may be useful for the therapeutically-driven manufacturing of cells in vitro and possibly in vivo. METHODOLOGY/PRINCIPAL FINDINGS:In this study we report on optimized methods for feeder-free culture of hESCs and hESC-derived neural stem cells (NSCs) to facilitate automated screening. We show that we are able to measure ATP as an indicator of metabolic activity in an automated screening assay. With this optimized platform we screened a collection of FDA-approved drugs to identify compounds that have differential toxicity to hESCs and their neural derivatives. Nine compounds were identified to be specifically toxic for NSCs to a greater extent than for hESCs. Six of these initial hits were retested and verified by large-scale cell culture to determine dose-responsive NSC toxicity. One of the compounds retested, amiodarone HCL, was further tested for possible effects on postmitotic neurons, a likely target for transplant therapy. Amiodarone HCL was found to be selectively toxic to NSCs but not to differentiated neurons or glial cells. Treated and untreated NSCs and neurons were then interrogated with global gene expression analysis to explore the mechanisms of action of amiodarone HCl. The gene expression analysis suggests that activation of cell-type specific cationic channels may underlie the toxicity of the drug. CONCLUSIONS/SIGNIFICANCE:In conclusion, we have developed a screening strategy that allows us to rapidly identify clinically approved drugs for use in a Chemistry, Manufacture and Control protocol that can be safely used to deplete unwanted contaminating precursor cells from a differentiated cell product. Our results also suggest that such a strategy is rich in the potential of identifying lineage specific reagents and provides additional evidence for the utility of stem cells in screening and discovery paradigms

Induction of early neural precursors and derivation of tripotent neural stem cells from human pluripotent stem cells under xeno-free conditions

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) can differentiate into many cell types and are important for regenerative medicine; however, further work is needed to reliably differentiate hESC and hiPSC into neural-restricted multipotent derivatives or specialized cell types under conditions that are free from animal products. Toward this goal, we tested the transition of hESC and hiPSC lines onto xeno-free (XF) / feeder-free conditions and evaluated XF substrate preference, pluripotency, and karyotype. Critically, XF transitioned H9 hESC, Shef4 hESC, and iPS6-9 retained pluripotency (Oct-4 and NANOG), proliferation (MKI67 and PCNA), and normal karyotype. Subsequently, XF transitioned hESC and hiPSC were induced with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to generate neuralized spheres containing primitive neural precursors, which could differentiate into astrocytes and neurons, but not oligoprogenitors. Further neuralization of spheres via LIF supplementation and attachment selection on CELLstart substrate generated adherent human neural stem cells (hNSC) with normal karyotype and high proliferation potential under XF conditions. Interestingly, adherent hNSC derived from H9, Shef4, and iPS6-9 differentiated into significant numbers of O4+ oligoprogenitors (~20-30%) with robust proliferation; however, very few GalC+ cells were observed (~2-4%), indicative of early oligodendrocytic lineage commitment. Overall, these data demonstrate the transition of multiple hESC and hiPSC lines onto XF substrate and media conditions, and a reproducible neuralization method that generated neural derivatives with multipotent cell fate potential and normal karyotype

Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors

Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis, and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here, we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus, a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated cassette exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus, this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate recombinase-mediated cassette exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome

The PDZ Domain as a Complex Adaptive System

Specific protein associations define the wiring of protein interaction networks and thus control the organization and functioning of the cell as a whole. Peptide recognition by PDZ and other protein interaction domains represents one of the best-studied classes of specific protein associations. However, a mechanistic understanding of the relationship between selectivity and promiscuity commonly observed in the interactions mediated by peptide recognition modules as well as its functional meaning remain elusive. To address these questions in a comprehensive manner, two large populations of artificial and natural peptide ligands of six archetypal PDZ domains from the synaptic proteins PSD95 and SAP97 were generated by target-assisted iterative screening (TAIS) of combinatorial peptide libraries and by synthesis of proteomic fragments, correspondingly. A comparative statistical analysis of affinity-ranked artificial and natural ligands yielded a comprehensive picture of known and novel PDZ ligand specificity determinants, revealing a hitherto unappreciated combination of specificity and adaptive plasticity inherent to PDZ domain recognition. We propose a reconceptualization of the PDZ domain in terms of a complex adaptive system representing a flexible compromise between the rigid order of exquisite specificity and the chaos of unselective promiscuity, which has evolved to mediate two mutually contradictory properties required of such higher order sub-cellular organizations as synapses, cell junctions, and others – organizational structure and organizational plasticity/adaptability. The generalization of this reconceptualization in regard to other protein interaction modules and specific protein associations is consistent with the image of the cell as a complex adaptive macromolecular system as opposed to clockwork

The Intracellular Threonine of Amyloid Precursor Protein That Is Essential for Docking of Pin1 Is Dispensable for Developmental Function

Background: Processing of Ab-precursor protein (APP) plays an important role in Alzheimer’s Disease (AD) pathogenesis. Thr residue at amino acid 668 of the APP intracellular domain (AID) is highly conserved. When phosphorylated, this residue generates a binding site for Pin1. The interaction of APP with Pin1 has been involved in AD pathogenesis. Methodology/Principal Findings: To dissect the functions of this sequence in vivo, we created an APP knock-in allele, in which Thr 668 is replaced by an Ala (T 668 A). Doubly deficient APP/APP-like protein 2 (APLP2) mice present postnatal lethality and neuromuscular synapse defects. Previous work has shown that the APP intracellular domain is necessary for preventing early lethality and neuromuscular junctions (NMJ) defects. Crossing the T 668 A allele into the APLP2 knockout background showed that mutation of Thr 668 does not cause a defective phenotype. Notably, the T 668 A mutant APP is able to bind Mint1. Conclusions/Significance: Our results argue against an important role of the Thr 668 residue in the essential function of APP in developmental regulation. Furthermore, they indicate that phosphorylation at this residue is not functionally involved i

Comparative transcriptome profiling of amyloid precursor protein family members in the adult cortex

<p>Abstract</p> <p>Background</p> <p>The β-amyloid precursor protein (APP) and the related β-amyloid precursor-like proteins (APLPs) undergo complex proteolytic processing giving rise to several fragments. Whereas it is well established that Aβ accumulation is a central trigger for Alzheimer's disease, the physiological role of APP family members and their diverse proteolytic products is still largely unknown. The secreted APPsα ectodomain has been shown to be involved in neuroprotection and synaptic plasticity. The γ-secretase-generated APP intracellular domain (AICD) functions as a transcriptional regulator in heterologous reporter assays although its role for endogenous gene regulation has remained controversial.</p> <p>Results</p> <p>To gain further insight into the molecular changes associated with knockout phenotypes and to elucidate the physiological functions of APP family members including their proposed role as transcriptional regulators, we performed DNA microarray transcriptome profiling of prefrontal cortex of adult wild-type (WT), APP knockout (APP^-/-), APLP2 knockout (APLP2^-/-) and APPsα knockin mice (APP^α/α) expressing solely the secreted APPsα ectodomain. Biological pathways affected by the lack of APP family members included neurogenesis, transcription, and kinase activity. Comparative analysis of transcriptome changes between mutant and wild-type mice, followed by qPCR validation, identified co-regulated gene sets. Interestingly, these included heat shock proteins and plasticity-related genes that were both down-regulated in knockout cortices. In contrast, we failed to detect significant differences in expression of previously proposed AICD target genes including <it>Bace1</it>, <it>Kai1</it>, <it>Gsk3b</it>, <it>p53</it>, <it>Tip60</it>, and <it>Vglut2</it>. Only <it>Egfr </it>was slightly up-regulated in APLP2^-/-mice. Comparison of APP^-/-and APP^α/αwith wild-type mice revealed a high proportion of co-regulated genes indicating an important role of the C-terminus for cellular signaling. Finally, comparison of APLP2^-/-on different genetic backgrounds revealed that background-related transcriptome changes may dominate over changes due to the knockout of a single gene.</p> <p>Conclusion</p> <p>Shared transcriptome profiles corroborated closely related physiological functions of APP family members in the adult central nervous system. As expression of proposed AICD target genes was not altered in adult cortex, this may indicate that these genes are not affected by lack of APP under resting conditions or only in a small subset of cells.</p