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NOVATO, CA, United States

Momcilovic O.,Buck Institute for Research on Aging | Liu Q.,Buck Institute for Research on Aging | Liu Q.,Chinese Academy of Agricultural Sciences | Swistowski A.,Buck Institute for Research on Aging | And 6 more authors.
Stem Cells and Development | Year: 2014

Recent advances in human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) biology enable generation of dopaminergic neurons for potential therapy and drug screening. However, our current understanding of molecular and cellular signaling that controls human dopaminergic development and function is limited. Here, we report on a whole genome analysis of gene expression during dopaminergic differentiation of human ESC/iPSC using Illumina bead microarrays. We generated a transcriptome data set containing the expression levels of 28,688 unique transcripts by profiling five lines (three ESC and two iPSC lines) at four stages of differentiation: (1) undifferentiated ESC/iPSC, (2) neural stem cells, (3) dopaminergic precursors, and (4) dopaminergic neurons. This data set provides comprehensive information about genes expressed at each stage of differentiation. Our data indicate that distinct pathways are activated during neural and dopaminergic neuronal differentiation. For example, WNT, sonic hedgehog (SHH), and cAMP signaling pathways were found over-represented in dopaminergic populations by gene enrichment and pathway analysis, and their role was confirmed by perturbation analyses using RNAi (small interfering RNA of SHH and WNT) or small molecule [dibutyryl cyclic AMP (dcAMP)]. In summary, whole genome profiling of dopaminergic differentiation enables systematic analysis of genes/pathways, networks, and cellular/molecular processes that control cell fate decisions. Such analyses will serve as the foundation for better understanding of dopaminergic development, function, and development of future stem cell-based therapies. © Copyright 2014, Mary Ann Liebert, Inc. 2014.

Shaltouki A.,Buck Institute | Sivapatham R.,Buck Institute | Pei Y.,Buck Institute | Gerencser A.A.,Buck Institute | And 4 more authors.
Stem Cell Reports | Year: 2015

In this study, we used patient-specific and isogenic PARK2-induced pluripotent stem cells (iPSCs) to show that mutations in PARK2 alter neuronal proliferation. The percentage of TH+ neurons was decreased in Parkinson's disease (PD) patient-derived neurons carrying various mutations in PARK2 compared with an age-matched control subject. This reduction was accompanied by alterations in mitochondrial:cell volume fraction (mitochondrial volume fraction). The same phenotype was confirmed in isogenic PARK2 null lines. The mitochondrial phenotype was also seen in non-midbrain neurons differentiated from the PARK2 null line, as was the functional phenotype of reduced proliferation in culture. Whole genome expression profiling at various stages of differentiation confirmed the mitochondrial phenotype and identified pathways altered by PARK2 dysfunction that include PD-related genes. Our results are consistent with current model of PARK2 function where damaged mitochondria are targeted for degradation via a PARK2/PINK1-mediated mechanism. © 2015 The Authors.

Peng J.,Buck Institute for Age Research | Liu Q.,Buck Institute for Age Research | Rao M.S.,NxCell | Zeng X.,Buck Institute for Age Research | Zeng X.,Xcell Science, Inc.
Cytotherapy | Year: 2014

Background aims: We have previously reported a Good Manufacturing Practice (GMP)-compatible process for generating authentic dopaminergic neurons in defined media from human pluripotent stem cells and determined the time point at which dopaminergic precursors/neurons (day 14 after neuronal stem cell [NSC] stage) can be frozen, shipped and thawed without compromising their viability and ability to mature in vitro. One important issue we wished to address is whether dopaminergic precursors/neurons manufactured by our GMP-compatible process can be cryopreserved and engrafted in animal Parkinson disease (PD) models. Methods: In this study, we evaluated the efficacy of freshly prepared and cryopreserved dopaminergic neurons in the 6-hydroxydopamine-lesioned rat PD model. Results: We showed functional recovery up to 6 months post-transplantation in rats transplanted with our cells, whether freshly prepared or cryopreserved. In contrast, no motor improvement was observed in two control groups receiving either medium or cells at a slightly earlier stage (day 10 after NSC stage). Histologic analysis at the end point of the study (6 months post-transplantation) showed robust long-term survival of donor-derived tyrosine hydroxylase (TH)+ dopaminergic neurons in rats transplanted with day 14 dopaminergic neurons. Moreover, TH+ fibers emanated from the graft core into the surrounding host striatum. Consistent with the behavioral analysis, no or few TH+ neurons were detected in animals receiving day 10 cells, although human cells were present in the graft. Importantly, no tumors were detected in any grafted rats, but long-term tumorigenic studies will need to determine the safety of our products. Conclusions: Dopaminergic neurons manufactured by a GMP-compatible process from human ESC survived and engrafted efficiently in the 6-OHDA PD rat model. © 2014 International Society for Cellular Therapy.

Swistowski A.,Buck Institute for Age Research | Swistowski A.,Xcell Science, Inc. | Zeng X.,Buck Institute for Age Research
Current Protocols in Stem Cell Biology | Year: 2012

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are potentially an unlimited cell source for cell replacement therapy and personalized medicine. Before hESC- and iPSC-based therapy can be moved from bench to bedside, however, it is essential to establish protocols for generating therapeutically relevant cells, like dopaminergic neurons in defined conditions that are suitable for scalable good manufacturing practice (GMP)-compliant protocols. Here, the derivation and differentiation of functional dopaminergic neurons from hESCs and iPSCs under xeno-free defined conditions are described. These protocols have been validated in multiple hESC and iPSC lines. © 2012 by John Wiley & Sons, Inc.

Efthymiou A.,U.S. National Institutes of Health | Shaltouki A.,Buck Institute for Research on Aging | Steiner J.P.,U.S. National Institutes of Health | Jha B.,U.S. National Institutes of Health | And 5 more authors.
Journal of Biomolecular Screening | Year: 2014

Rapid and effective drug discovery for neurodegenerative disease is currently impeded by an inability to source primary neural cells for high-throughput and phenotypic screens. This limitation can be addressed through the use of pluripotent stem cells (PSCs), which can be derived from patient-specific samples and differentiated to neural cells for use in identifying novel compounds for the treatment of neurodegenerative diseases. We have developed an efficient protocol to culture pure populations of neurons, as confirmed by gene expression analysis, in the 96-well format necessary for screens. These differentiated neurons were subjected to viability assays to illustrate their potential in future high-throughput screens. We have also shown that organelles such as nuclei and mitochondria could be live-labeled and visualized through fluorescence, suggesting that we should be able to monitor subcellular phenotypic changes. Neurons derived from a green fluorescent protein-expressing reporter line of PSCs were live-imaged to assess markers of neuronal maturation such as neurite length and co-cultured with astrocytes to demonstrate further maturation. These studies confirm that PSC-derived neurons can be used effectively in viability and functional assays and pave the way for high-throughput screens on neurons derived from patients with neurodegenerative disorders. © 2013 Society for Laboratory Automation and Screening.

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