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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.

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.

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.

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.

Pei Y.,Buck Institute for Age Research | Sierra G.,Xcell Science, Inc. | Sivapatham R.,Buck Institute for Age Research | Swistowski A.,Xcell Science, Inc. | And 3 more authors.
Scientific Reports | Year: 2015

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript, we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore, we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages, further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays.

Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 714.36K | Year: 2016

DESCRIPTION provided by applicant Despite the fact that animal based neurotoxicology models have relatively low sensitivity and are burdened by high workload cost and animal ethics they have been the mainstay of evaluating neurotoxicology However toxicology today is looking for alternatives as it faces enormous challenges to the use of animals On one hand there is enormous societal pressure to reduce the use of animals and on the other hand the demand for testing is increasing More than chemicals are estimated in use without adequate toxicological information in the USA and Europe and the task of testing thousands of chemicals systematically with classical animal assays likely exceeds our present capabilities The recent advance in pluripotent stem cell PSC based technology and the ability to generate truly large numbers of allelically diverse cells and use uniform methods of differentiation into al the major types of cells in the nervous system offer a potential new tool for improved understanding of chemically induced toxicity This is especially useful for developmental neurotoxicity because neural cells differentiate early during development and this process is relatively easily recapitulated in vitro via rosette formation and isolation of neural stem cells NSC which can subsequently be differentiated into neurons and glia We have in a Phase I grant provided proof of concept for iPSC based neurotoxicity assays However several issues need to be addressed before such assays can be used routinely to test neurotoxicity For example there is a lack of neural reporters in referenced lines of both genders which will be invaluable for further assay development and refinement There is also a lack of datasets that can serve as baseline for toxicity assays and a lack of reference response to a reference set of compounds that can be used to calibrate the response of future lines and compare with the rodent data The objective of this Phase II application is to develop a neurotoxicity tool kit that addresses these issues and to commercialize it PUBLIC HEALTH RELEVANCE One emerging field in toxicology is the use of iPSC for neurotoxicology and developmental neurotoxicity If the proposed aims are achieved this project will provide the community a complete neurotoxicity tool kit with a database of baseline for toxicity assays and response to reference compounds This will help provide answers to a key question about the feasibility of using iPSC derived cells for neurotoxicity and to replace the in vivo animal tests

Zeng X.,Xcell Science, Inc. | Zeng X.,Buck Institute for Research on Aging | Hunsberger J.G.,U.S. National Institutes of Health | Simeonov A.,U.S. National Institutes of Health | And 4 more authors.
Stem Cells Translational Medicine | Year: 2014

Induced pluripotent stem cells (iPSCs) offer an opportunity to delve into the mechanisms underlying development while also affording the potential to take advantage of a number of naturally occurring mutations that contribute to either disease susceptibility or resistance. Just as with any new field, several models of screening are being explored, and innovators are working on the most efficient methods to overcome the inherent limitations of primary cell screens using iPSCs. In the present review, we provide a background regarding why iPSCs represent a paradigm shift for central nervous system (CNS) disease modeling. We describe the efforts in the field to develop more biologically relevant CNS disease models, which should provide screening assays useful for the pharmaceutical industry. We also provide some examples of successful uses for iPSC-based screens and suggest that additional development could revolutionize the field of drug discovery. The development and implementation of these advanced iPSC-based screens will create a more efficient disease-specific process underpinned by the biological mechanism in a patient- and disease-specific manner rather than by trial-and-error. Moreover, with careful and strategic planning, shared resources can be developed that will enable exponential advances in the field. This will undoubtedly lead tomore sensitive and accurate screens for early diagnosis and allow the identification of patient-specific therapies, thus, paving the way to personalized medicine. ©AlphaMed Press.

PubMed | Lonza Walkersville Inc., NxCell Inc, Institute of Stem Cells and Regenerative Medicine InSTEM, Buck Institute for Researching on Aging and Xcell Science, Inc.
Type: Journal Article | Journal: Stem cell reviews | Year: 2016

We have recently described manufacturing of human induced pluripotent stem cells (iPSC) master cell banks (MCB) generated by a clinically compliant process using cord blood as a starting material (Baghbaderani et al. in Stem Cell Reports, 5(4), 647-659, 2015). In this manuscript, we describe the detailed characterization of the two iPSC clones generated using this process, including whole genome sequencing (WGS), microarray, and comparative genomic hybridization (aCGH) single nucleotide polymorphism (SNP) analysis. We compare their profiles with a proposed calibration material and with a reporter subclone and lines made by a similar process from different donors. We believe that iPSCs are likely to be used to make multiple clinical products. We further believe that the lines used as input material will be used at different sites and, given their immortal status, will be used for many years or even decades. Therefore, it will be important to develop assays to monitor the state of the cells and their drift in culture. We suggest that a detailed characterization of the initial status of the cells, a comparison with some calibration material and the development of reporter sublcones will help determine which set of tests will be most useful in monitoring the cells and establishing criteria for discarding a line.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 224.99K | Year: 2013

7. Project Summary The recent advances in pluripotent stem cell (PSC) technology enable researchers to establish human tissue derived cellular platforms for toxicity testing that are capable of providing information on mechanism of action, while facilitating the incorporation of broad genetic diversity and clinically validated disease conditions. We and other investigators have shown that it is possible run neurotoxicity screens with primary cells derived from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) and obtain consistent and reliable results that offer advantages over screens run in cell lines or in rodent cells. The limiting factors in translating this technology into a commercial product have been an inability to establish large-scale reproducible protocols for generation and differentiation of PSCs and access to large collections of genetically diverse well-phenotyped cell lines. In this proposal, we aim to develop a fully automated platform for production of PSC-derived cells in a 96-well format utilizing a genetically diverse bank of cell lines. The large-scale approach will improve the statistical precision in the readouts and provide for parallel production not previously obtainable, which will translate into more predictive models. The product will be available to customers as a service for in-house screening of compounds or we can provide 96-well plates of iPSC-derived NSCs and DA neurons - vials of cells with instructions for plating and initial characterization will also be available. Plates will be generated from either a single cell line for initial screening of large numbers of compounds or independent cell lines from 96 healthy tissue donors representing the genetic diversity of the US population for hit validation. Analysis will include high-content image analysis of cell survival as well as transcriptional profiling. Our goal is to significantly reduce the costs of generating stem cell screening platforms and provide information that is more effective at predicting how human cells will respond to exposure to various toxicants during differentiation and at mature cell states. Our product will support the goals of the funding mechanisms, while also establishing a baseline of variation of transcription for neurallineages that will be critical when determining the significance of toxic effects of chemical compounds or developmental differences when analyzing diseased lines. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: In this application, we aim to develop a fully automated platform for production of PSC-derived neuronal cells in a 96-well format from one (initial screen) and 96 (validation) individuals representing the genetic diversity of the US population. The product will be available to customers as a service for in-house screening of compounds or we can provide 96-well plates of iPSC-derived NSCs, and DA neurons - vials of cells with instructions for plating and initial characterization will also be available. Analysis will include high-content imaging analysis of cell survival and transcriptional analysis.

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