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Mostafavi Abdolmaleky H, Alam R, Nohesara S, Deth RC, Zhou JR. iPSC-Derived Astrocytes and Neurons Replicate Brain Gene Expression, Epigenetic, Cell Morphology and Connectivity Alterations Found in Autism. Cells 2024; 13:1095. [PMID: 38994948 PMCID: PMC11240613 DOI: 10.3390/cells13131095] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2024] [Revised: 06/04/2024] [Accepted: 06/19/2024] [Indexed: 07/13/2024] Open
Abstract
Excessive inflammatory reactions and oxidative stress are well-recognized molecular findings in autism and these processes can affect or be affected by the epigenetic landscape. Nonetheless, adequate therapeutics are unavailable, as patient-specific brain molecular markers for individualized therapies remain challenging. METHODS We used iPSC-derived neurons and astrocytes of patients with autism vs. controls (5/group) to examine whether they replicate the postmortem brain expression/epigenetic alterations of autism. Additionally, DNA methylation of 10 postmortem brain samples (5/group) was analyzed for genes affected in PSC-derived cells. RESULTS We found hyperexpression of TGFB1, TGFB2, IL6 and IFI16 and decreased expression of HAP1, SIRT1, NURR1, RELN, GPX1, EN2, SLC1A2 and SLC1A3 in the astrocytes of patients with autism, along with DNA hypomethylation of TGFB2, IL6, TNFA and EN2 gene promoters and a decrease in HAP1 promoter 5-hydroxymethylation in the astrocytes of patients with autism. In neurons, HAP1 and IL6 expression trended alike. While HAP1 promoter was hypermethylated in neurons, IFI16 and SLC1A3 promoters were hypomethylated and TGFB2 exhibited increased promoter 5-hydroxymethlation. We also found a reduction in neuronal arborization, spine size, growth rate, and migration, but increased astrocyte size and a reduced growth rate in autism. In postmortem brain samples, we found DNA hypomethylation of TGFB2 and IFI16 promoter regions, but DNA hypermethylation of HAP1 and SLC1A2 promoters in autism. CONCLUSION Autism-associated expression/epigenetic alterations in iPSC-derived cells replicated those reported in the literature, making them appropriate surrogates to study disease pathogenesis or patient-specific therapeutics.
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Affiliation(s)
- Hamid Mostafavi Abdolmaleky
- Nutrition/Metabolism Laboratory, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA;
- Department of Medicine (Biomedical Genetics), Boston University Chobanian & Avedisian School of Medicine, Boston, MA 02118, USA;
| | - Reza Alam
- Nutrition/Metabolism Laboratory, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA;
| | - Shabnam Nohesara
- Department of Medicine (Biomedical Genetics), Boston University Chobanian & Avedisian School of Medicine, Boston, MA 02118, USA;
| | - Richard C. Deth
- Department of Pharmaceutical Sciences, Nova Southeastern University, Fort Lauderdale, FL 33328, USA;
| | - Jin-Rong Zhou
- Nutrition/Metabolism Laboratory, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA;
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Transition from Animal-Based to Human Induced Pluripotent Stem Cells (iPSCs)-Based Models of Neurodevelopmental Disorders: Opportunities and Challenges. Cells 2023; 12:cells12040538. [PMID: 36831205 PMCID: PMC9954744 DOI: 10.3390/cells12040538] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2022] [Revised: 01/25/2023] [Accepted: 02/02/2023] [Indexed: 02/11/2023] Open
Abstract
Neurodevelopmental disorders (NDDs) arise from the disruption of highly coordinated mechanisms underlying brain development, which results in impaired sensory, motor and/or cognitive functions. Although rodent models have offered very relevant insights to the field, the translation of findings to clinics, particularly regarding therapeutic approaches for these diseases, remains challenging. Part of the explanation for this failure may be the genetic differences-some targets not being conserved between species-and, most importantly, the differences in regulation of gene expression. This prompts the use of human-derived models to study NDDS. The generation of human induced pluripotent stem cells (hIPSCs) added a new suitable alternative to overcome species limitations, allowing for the study of human neuronal development while maintaining the genetic background of the donor patient. Several hIPSC models of NDDs already proved their worth by mimicking several pathological phenotypes found in humans. In this review, we highlight the utility of hIPSCs to pave new paths for NDD research and development of new therapeutic tools, summarize the challenges and advances of hIPSC-culture and neuronal differentiation protocols and discuss the best way to take advantage of these models, illustrating this with examples of success for some NDDs.
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3
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Xie Y, Ng NN, Safrina OS, Ramos CM, Ess KC, Schwartz PH, Smith MA, O'Dowd DK. Comparisons of dual isogenic human iPSC pairs identify functional alterations directly caused by an epilepsy associated SCN1A mutation. Neurobiol Dis 2019; 134:104627. [PMID: 31786370 DOI: 10.1016/j.nbd.2019.104627] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2019] [Revised: 09/05/2019] [Accepted: 09/26/2019] [Indexed: 02/06/2023] Open
Abstract
Over 1250 mutations in SCN1A, the Nav1.1 voltage-gated sodium channel gene, are associated with seizure disorders including GEFS+. To evaluate how a specific mutation, independent of genetic background, causes seizure activity we generated two pairs of isogenic human iPSC lines by CRISPR/Cas9 gene editing. One pair is a control line from an unaffected sibling, and the mutated control carrying the GEFS+ K1270T SCN1A mutation. The second pair is a GEFS+ patient line with the K1270T mutation, and the corrected patient line. By comparing the electrophysiological properties in inhibitory and excitatory iPSC-derived neurons from these pairs, we found the K1270T mutation causes cell type-specific alterations in sodium current density and evoked firing, resulting in hyperactive neural networks. We also identified differences associated with genetic background and interaction between the mutation and genetic background. Comparisons within and between dual pairs of isogenic iPSC-derived neuronal cultures provide a novel platform for evaluating cellular mechanisms underlying a disease phenotype and for developing patient-specific anti-seizure therapies.
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Affiliation(s)
- Yunyao Xie
- Department of Developmental and Cell Biology, University of California, Irvine, CA, United States of America
| | - Nathan N Ng
- Department of Developmental and Cell Biology, University of California, Irvine, CA, United States of America
| | - Olga S Safrina
- Department of Developmental and Cell Biology, University of California, Irvine, CA, United States of America
| | - Carmen M Ramos
- Department of Developmental and Cell Biology, University of California, Irvine, CA, United States of America
| | - Kevin C Ess
- Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, United States of America
| | - Philip H Schwartz
- Children's Hospital of Orange County Research Institute, Orange, CA, United States of America
| | - Martin A Smith
- Department of Anatomy and Neurobiology, University of California, Irvine, CA, United States of America
| | - Diane K O'Dowd
- Department of Developmental and Cell Biology, University of California, Irvine, CA, United States of America.
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Human Cytomegalovirus Compromises Development of Cerebral Organoids. J Virol 2019; 93:JVI.00957-19. [PMID: 31217239 DOI: 10.1128/jvi.00957-19] [Citation(s) in RCA: 54] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2019] [Accepted: 06/11/2019] [Indexed: 12/24/2022] Open
Abstract
Congenital human cytomegalovirus (HCMV) infection causes a broad spectrum of central and peripheral nervous system disorders, ranging from microcephaly to hearing loss. These ramifications mandate the study of virus-host interactions in neural cells. Neural progenitor cells are permissive for lytic infection. We infected two induced pluripotent stem cell (iPSC) lines and found these more primitive cells to be susceptible to infection but not permissive. Differentiation of infected iPSCs induced de novo expression of viral antigens. iPSCs can be cultured in three dimensions to generate cerebral organoids, closely mimicking in vivo development. Mock- or HCMV-infected iPSCs were subjected to a cerebral organoid generation protocol. HCMV IE1 protein was detected in virus-infected organoids at 52 days postinfection. Absent a significant effect on organoid size, infection induced regions of necrosis and the presence of large vacuoles and cysts. Perhaps more in parallel with the subtler manifestations of HCMV-induced birth defects, infection dramatically altered neurological development of organoids, decreasing the number of developing and fully formed cortical structure sites, with associated changes in the architectural organization and depth of lamination within these structures, and manifesting aberrant expression of the neural marker β-tubulin III. Our observations parallel published descriptions of infected clinical samples, which often contain only sparse antigen-positive foci yet display areas of focal necrosis and cellular loss, delayed maturation, and abnormal cortical lamination. The parallels between pathologies present in clinical specimens and the highly tractable three-dimensional (3D) organoid system demonstrate the utility of this system in modeling host-virus interactions and HCMV-induced birth defects.IMPORTANCE Human cytomegalovirus (HCMV) is a leading cause of central nervous system birth defects, ranging from microcephaly to hearing impairment. Recent literature has provided descriptions of delayed and abnormal maturation of developing cortical tissue in infected clinical specimens. We have found that infected induced pluripotent stem cells can be differentiated into three-dimensional, viral protein-expressing cerebral organoids. Virus-infected organoids displayed dramatic alterations in development compared to those of mock-infected controls. Development in these organoids closely paralleled observations in HCMV-infected clinical samples. Infection induced regions of necrosis, the presence of larger vacuoles and cysts, changes in the architectural organization of cortical structures, aberrant expression of the neural marker β-tubulin III, and an overall reduction in numbers of cortical structure sites. We found clear parallels between the pathologies of clinical specimens and virus-infected organoids, demonstrating the utility of this highly tractable system for future investigations of HCMV-induced birth defects.
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Harrill JA. Human-Derived Neurons and Neural Progenitor Cells in High Content Imaging Applications. Methods Mol Biol 2018; 1683:305-338. [PMID: 29082500 DOI: 10.1007/978-1-4939-7357-6_18] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/07/2023]
Abstract
Due to advances in the fields of stem cell biology and cellular engineering, a variety of commercially available human-derived neurons and neural progenitor cells (NPCs) are now available for use in research applications, including small molecule efficacy or toxicity screening. The use of human-derived neural cells is anticipated to address some of the uncertainties associated with the use of nonhuman culture models or transformed cell lines derived from human tissues. Many of the human-derived neurons and NPCs currently available from commercial sources recapitulate critical process of nervous system development including NPC proliferation, neurite outgrowth, synaptogenesis, and calcium signaling, each of which can be evaluated using high content image analysis (HCA). Human-derived neurons and NPCs are also amenable to culture in multiwell plate formats and thus may be adapted for use in HCA-based screening applications. This article reviews various types of HCA-based assays that have been used in conjunction with human-derived neurons and NPC cultures. This article also highlights instances where lower throughput analysis of neurodevelopmental processes has been performed and which demonstrate a potential for adaptation to higher-throughout imaging methods. Finally, a generic protocol for evaluating neurite outgrowth in human-derived neurons using a combination of immunocytochemistry and HCA is presented. The information provided in this article is intended to serve as a resource for cell model and assay selection for those interested in evaluating neurodevelopmental processes in human-derived cells.
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Affiliation(s)
- Joshua A Harrill
- Center for Toxicology and Environmental Health, LLC, 5120 Northshore Drive, Little Rock, AR, 72118, USA.
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6
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Xie Y, Schutte RJ, Ng NN, Ess KC, Schwartz PH, O'Dowd DK. Reproducible and efficient generation of functionally active neurons from human hiPSCs for preclinical disease modeling. Stem Cell Res 2017; 26:84-94. [PMID: 29272856 PMCID: PMC5899925 DOI: 10.1016/j.scr.2017.12.003] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/30/2017] [Revised: 10/22/2017] [Accepted: 12/07/2017] [Indexed: 02/08/2023] Open
Abstract
The use of human induced pluripotent stem cell (hiPSC)-derived neuronal cultures to study the mechanisms of neurological disorders is often limited by low efficiency and high variability in differentiation of functional neurons. Here we compare the functional properties of neurons in cultures prepared with two hiPSC differentiation protocols, both plated on astroglial feeder layers. Using a protocol with an expandable intermediate stage, only a small percentage of cells with neuronal morphology were excitable by 21-23days in culture. In contrast, a direct differentiation strategy of the same hiPSC line produced cultures in which the majority of neurons fired action potentials as early as 4-5days. By 35-38days over 80% of the neurons fired repetitively and many fired spontaneously. Spontaneous post-synaptic currents were observed in ~40% of the neurons at 4-5days and in ~80% by 21-23days. The majority (75%) received both glutamatergic and GABAergic spontaneous postsynaptic currents. The rate and degree of maturation of excitability and synaptic activity was similar between multiple independent platings from a single hiPSC line, and between two different control hiPSC lines. Cultures of rapidly functional neurons will facilitate identification of cellular mechanisms underlying genetically defined neurological disorders and development of novel therapeutics.
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Affiliation(s)
- Yunyao Xie
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, United States
| | - Ryan J Schutte
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, United States
| | - Nathan N Ng
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, United States
| | - Kevin C Ess
- Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, United States
| | - Philip H Schwartz
- Children's Hospital of Orange County Research Institute, Orange, CA, United States
| | - Diane K O'Dowd
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, United States.
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7
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Schutte RJ, Xie Y, Ng NN, Figueroa P, Pham AT, O'Dowd DK. Astrocyte-enriched feeder layers from cryopreserved cells support differentiation of spontaneously active networks of human iPSC-derived neurons. J Neurosci Methods 2017; 294:91-101. [PMID: 28746822 DOI: 10.1016/j.jneumeth.2017.07.019] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2016] [Revised: 07/19/2017] [Accepted: 07/19/2017] [Indexed: 01/08/2023]
Abstract
BACKGROUND Human induced pluripotent stem cell (hiPSC)-derived neuronal cultures are a useful tool for studying the mechanisms of neurological disorders and developing novel therapeutics. While plating hiPSC-derived neuronal progenitors onto glial feeder layers prepared from rodent cortex has been reported to promote functional differentiation of neuronal networks, this has not been examined in detail. NEW METHOD Here we describe a method of using cryopreserved cells from primary cultures for generation of mouse astrocyte-enriched, neuron-free feeder layers that grow from 10% to 100% confluence in 1 week. RESULTS Electrophysiological analysis demonstrated that compared to biochemical substrates alone, astrocyte-enriched feeder layers support more rapid differentiation of hiPSC-derived progenitors into excitable neurons that form spontaneously active networks in culture. There was a positive correlation between the degree of astroglial confluence at the time of progenitor plating and the average frequency of postsynaptic currents 3 weeks after plating. One disadvantage to plating on 100% confluent feeder layers was a high incidence of the astroglial layer with the overlying neurons detaching from the coverslips during transfer to the recording chamber. COMPARISON WITH EXISTING METHOD(S) Prevailing methods using primary glial feeder layers can result in possible contamination with rodent neurons and an unpredictable rate of growth. We provide a reliable method of generating mouse astroglial feeder layers from cryopreserved primary cultures to support differentiation of hiPSC-derived neurons. CONCLUSIONS The ability to make astrocyte-enriched feeder layers of defined confluence from cryopreserved primary cultures will facilitate the use of human stem cell derived neuronal cultures for disease modeling.
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Affiliation(s)
- Ryan J Schutte
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697, United States
| | - Yunyao Xie
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697, United States
| | - Nathan N Ng
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697, United States
| | - Priscilla Figueroa
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697, United States
| | - An T Pham
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697, United States
| | - Diane K O'Dowd
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697, United States.
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Stover AE, Herculian S, Banuelos MG, Navarro SL, Jenkins MP, Schwartz PH. Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research. J Vis Exp 2016. [PMID: 27341536 PMCID: PMC4927770 DOI: 10.3791/53685] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.
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Affiliation(s)
- Alexander E Stover
- National Human Neural Stem Cell Resource, Childrens Hospital of Orange County Research Institute
| | - Siranush Herculian
- National Human Neural Stem Cell Resource, Childrens Hospital of Orange County Research Institute
| | - Maria G Banuelos
- National Human Neural Stem Cell Resource, Childrens Hospital of Orange County Research Institute
| | - Samantha L Navarro
- National Human Neural Stem Cell Resource, Childrens Hospital of Orange County Research Institute
| | - Michael P Jenkins
- National Human Neural Stem Cell Resource, Childrens Hospital of Orange County Research Institute
| | - Philip H Schwartz
- National Human Neural Stem Cell Resource, Childrens Hospital of Orange County Research Institute;
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Schutte SS, Schutte RJ, Barragan EV, O'Dowd DK. Model systems for studying cellular mechanisms of SCN1A-related epilepsy. J Neurophysiol 2016; 115:1755-66. [PMID: 26843603 DOI: 10.1152/jn.00824.2015] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2015] [Accepted: 01/22/2016] [Indexed: 11/22/2022] Open
Abstract
Mutations in SCN1A, the gene encoding voltage-gated sodium channel NaV1.1, cause a spectrum of epilepsy disorders that range from genetic epilepsy with febrile seizures plus to catastrophic disorders such as Dravet syndrome. To date, more than 1,250 mutations in SCN1A have been linked to epilepsy. Distinct effects of individual SCN1A mutations on neuronal function are likely to contribute to variation in disease severity and response to treatment in patients. Several model systems have been used to explore seizure genesis in SCN1A epilepsies. In this article we review what has been learned about cellular mechanisms and potential new therapies from these model systems, with a particular emphasis on the novel model system of knock in Drosophila and a look toward the future with expanded use of patient-specific induced pluripotent stem cell-derived neurons.
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Affiliation(s)
- Soleil S Schutte
- Department of Developmental and Cell Biology and Department of Anatomy and Neurobiology, University of California, Irvine, California
| | - Ryan J Schutte
- Department of Developmental and Cell Biology and Department of Anatomy and Neurobiology, University of California, Irvine, California
| | - Eden V Barragan
- Department of Developmental and Cell Biology and Department of Anatomy and Neurobiology, University of California, Irvine, California
| | - Diane K O'Dowd
- Department of Developmental and Cell Biology and Department of Anatomy and Neurobiology, University of California, Irvine, California
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Kumari D, Bhattacharya A, Nadel J, Moulton K, Zeak NM, Glicksman A, Dobkin C, Brick DJ, Schwartz PH, Smith CB, Klann E, Usdin K. Identification of fragile X syndrome specific molecular markers in human fibroblasts: a useful model to test the efficacy of therapeutic drugs. Hum Mutat 2015; 35:1485-94. [PMID: 25224527 DOI: 10.1002/humu.22699] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2014] [Accepted: 09/04/2014] [Indexed: 11/06/2022]
Abstract
Fragile X syndrome (FXS) is the most frequent cause of inherited intellectual disability and autism. It is caused by the absence of the fragile X mental retardation 1 (FMR1) gene product, fragile X mental retardation protein (FMRP), an RNA-binding protein involved in the regulation of translation of a subset of brain mRNAs. In Fmr1 knockout mice, the absence of FMRP results in elevated protein synthesis in the brain as well as increased signaling of many translational regulators. Whether protein synthesis is also dysregulated in FXS patients is not firmly established. Here, we demonstrate that fibroblasts from FXS patients have significantly elevated rates of basal protein synthesis along with increased levels of phosphorylated mechanistic target of rapamycin (p-mTOR), phosphorylated extracellular signal regulated kinase 1/2, and phosphorylated p70 ribosomal S6 kinase 1 (p-S6K1). The treatment with small molecules that inhibit S6K1 and a known FMRP target, phosphoinositide 3-kinase (PI3K) catalytic subunit p110β, lowered the rates of protein synthesis in both control and patient fibroblasts. Our data thus demonstrate that fibroblasts from FXS patients may be a useful in vitro model to test the efficacy and toxicity of potential therapeutics prior to clinical trials, as well as for drug screening and designing personalized treatment approaches.
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Affiliation(s)
- Daman Kumari
- Laboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
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11
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Suter-Dick L, Alves PM, Blaauboer BJ, Bremm KD, Brito C, Coecke S, Flick B, Fowler P, Hescheler J, Ingelman-Sundberg M, Jennings P, Kelm JM, Manou I, Mistry P, Moretto A, Roth A, Stedman D, van de Water B, Beilmann M. Stem cell-derived systems in toxicology assessment. Stem Cells Dev 2015; 24:1284-96. [PMID: 25675366 DOI: 10.1089/scd.2014.0540] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Industrial sectors perform toxicological assessments of their potential products to ensure human safety and to fulfill regulatory requirements. These assessments often involve animal testing, but ethical, cost, and time concerns, together with a ban on it in specific sectors, make appropriate in vitro systems indispensable in toxicology. In this study, we summarize the outcome of an EPAA (European Partnership of Alternatives to Animal Testing)-organized workshop on the use of stem cell-derived (SCD) systems in toxicology, with a focus on industrial applications. SCD systems, in particular, induced pluripotent stem cell-derived, provide physiological cell culture systems of easy access and amenable to a variety of assays. They also present the opportunity to apply the vast repository of existing nonclinical data for the understanding of in vitro to in vivo translation. SCD systems from several toxicologically relevant tissues exist; they generally recapitulate many aspects of physiology and respond to toxicological and pharmacological interventions. However, focused research is necessary to accelerate implementation of SCD systems in an industrial setting and subsequent use of such systems by regulatory authorities. Research is required into the phenotypic characterization of the systems, since methods and protocols for generating terminally differentiated SCD cells are still lacking. Organotypical 3D culture systems in bioreactors and microscale tissue engineering technologies should be fostered, as they promote and maintain differentiation and support coculture systems. They need further development and validation for their successful implementation in toxicity testing in industry. Analytical measures also need to be implemented to enable compound exposure and metabolism measurements for in vitro to in vivo extrapolation. The future of SCD toxicological tests will combine advanced cell culture technologies and biokinetic measurements to support regulatory and research applications. However, scientific and technical hurdles must be overcome before SCD in vitro methods undergo appropriate validation and become accepted in the regulatory arena.
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Affiliation(s)
- Laura Suter-Dick
- 1University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Muttenz, Switzerland
| | - Paula M Alves
- 2iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.,3Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Bas J Blaauboer
- 4Division of Toxicology, Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, the Netherlands
| | - Klaus-Dieter Bremm
- 5Bayer Pharma AG, Global Drug Discovery-Global Early Development, Wuppertal, Germany
| | - Catarina Brito
- 2iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.,3Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Sandra Coecke
- 6European Commission Joint Research Centre, Institute for Health and Consumer Protection, EURL ECVAM, Ispra, Italy
| | - Burkhard Flick
- 7BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany
| | | | - Jürgen Hescheler
- 9Institut for Neurophysiology, University of Cologne, Cologne, Germany
| | | | - Paul Jennings
- 11Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria
| | | | - Irene Manou
- 13European Partnership for Alternative Approaches to Animal Testing (EPAA), B-Brussels, Belgium
| | - Pratibha Mistry
- 14Syngenta Ltd., Product Safety, Jealott's Hill International Research Station, Berkshire, United Kingdom
| | - Angelo Moretto
- 15Dipartimento di Scienze Biochimiche e Cliniche, Università degli Studi di Milano, Milano, Italy.,16Centro Internazionale per gli Antiparassitari e la Prevenzione Sanitaria, Luigi Sacco Hospital, Milano, Italy
| | - Adrian Roth
- 17F. Hoffmann-La Roche Ltd., Innovation Center Basel, Pharmaceutical Sciences, Basel, Switzerland
| | - Donald Stedman
- 18Pfizer Worldwide Research and Development, Cambridge, Massachusetts
| | - Bob van de Water
- 19Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, Leiden, the Netherlands
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12
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Mendez DC, Stover AE, Rangel AD, Brick DJ, Nethercott HE, Torres MA, Khalid O, Wong AM, Cooper JD, Jester JV, Monuki ES, McGuire C, Le SQ, Kan SH, Dickson PI, Schwartz PH. A novel, long-lived, and highly engraftable immunodeficient mouse model of mucopolysaccharidosis type I. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2015; 2:14068. [PMID: 26052536 PMCID: PMC4449030 DOI: 10.1038/mtm.2014.68] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/12/2014] [Revised: 12/04/2014] [Accepted: 12/14/2014] [Indexed: 02/06/2023]
Abstract
Mucopolysaccharidosis type I (MPS I) is an inherited α-L-iduronidase (IDUA, I) deficiency in which glycosaminoglycan (GAG) accumulation causes progressive multisystem organ dysfunction, neurological impairment, and death. Current MPS I mouse models, based on a NOD/SCID (NS) background, are short-lived, providing a very narrow window to assess the long-term efficacy of therapeutic interventions. They also develop thymic lymphomas, making the assessment of potential tumorigenicity of human stem cell transplantation problematic. We therefore developed a new MPS I model based on a NOD/SCID/Il2rγ (NSG) background. This model lives longer than 1 year and is tumor-free during that time. NSG MPS I (NSGI) mice exhibit the typical phenotypic features of MPS I including coarsened fur and facial features, reduced/abnormal gait, kyphosis, and corneal clouding. IDUA is undetectable in all tissues examined while GAG levels are dramatically higher in most tissues. NSGI brain shows a significant inflammatory response and prominent gliosis. Neurological MPS I manifestations are evidenced by impaired performance in behavioral tests. Human neural and hematopoietic stem cells were found to readily engraft, with human cells detectable for at least 1 year posttransplantation. This new MPS I model is thus suitable for preclinical testing of novel pluripotent stem cell-based therapy approaches.
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Affiliation(s)
- Daniel C Mendez
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, CHOC Children's Research Institute , Orange, California, USA
| | - Alexander E Stover
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, CHOC Children's Research Institute , Orange, California, USA
| | - Anthony D Rangel
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, CHOC Children's Research Institute , Orange, California, USA
| | - David J Brick
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, CHOC Children's Research Institute , Orange, California, USA
| | - Hubert E Nethercott
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, CHOC Children's Research Institute , Orange, California, USA
| | - Marissa A Torres
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, CHOC Children's Research Institute , Orange, California, USA
| | - Omar Khalid
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, CHOC Children's Research Institute , Orange, California, USA
| | - Andrew Ms Wong
- King's College, London, Institute of Psychiatry, Psychology & Neuroscience , London, UK
| | - Jonathan D Cooper
- King's College, London, Institute of Psychiatry, Psychology & Neuroscience , London, UK
| | - James V Jester
- Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine , Irvine, California, USA ; Department of Biomedical Engineering, Gavin Herbert Eye Institute, University of California, Irvine , Irvine, California, USA
| | - Edwin S Monuki
- Department of Pathology and Laboratory Medicine, Sue and Bill Gross Stem Cell Research Center, University of California, Irvine , Irvine, California, USA ; Department of Developmental and Cell Biology, Sue and Bill Gross Stem Cell Research Center, University of California, Irvine , Irvine, California, USA
| | - Cian McGuire
- Division of Medical Genetics, Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center , Torrance, California, USA
| | - Steven Q Le
- Division of Medical Genetics, Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center , Torrance, California, USA
| | - Shih-Hsin Kan
- Division of Medical Genetics, Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center , Torrance, California, USA
| | - Patricia I Dickson
- Division of Medical Genetics, Department of Pediatrics, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center , Torrance, California, USA
| | - Philip H Schwartz
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, CHOC Children's Research Institute , Orange, California, USA
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Crook JM, Wallace G, Tomaskovic-Crook E. The potential of induced pluripotent stem cells in models of neurological disorders: implications on future therapy. Expert Rev Neurother 2015; 15:295-304. [PMID: 25664599 DOI: 10.1586/14737175.2015.1013096] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
There is an urgent need for new and advanced approaches to modeling the pathological mechanisms of complex human neurological disorders. This is underscored by the decline in pharmaceutical research and development efficiency resulting in a relative decrease in new drug launches in the last several decades. Induced pluripotent stem cells represent a new tool to overcome many of the shortcomings of conventional methods, enabling live human neural cell modeling of complex conditions relating to aberrant neurodevelopment, such as schizophrenia, epilepsy and autism as well as age-associated neurodegeneration. This review considers the current status of induced pluripotent stem cell-based modeling of neurological disorders, canvassing proven and putative advantages, current constraints, and future prospects of next-generation culture systems for biomedical research and translation.
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Affiliation(s)
- Jeremy Micah Crook
- ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, AIIM Facility, Innovation Campus, University of Wollongong, Squires Way, Fairy Meadow, New South Wales 2519, Australia
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Brick DJ, Nethercott HE, Montesano S, Banuelos MG, Stover AE, Schutte SS, O'Dowd DK, Hagerman RJ, Ono M, Hessl DR, Tassone F, Schwartz PH. The Autism Spectrum Disorders Stem Cell Resource at Children's Hospital of Orange County: Implications for Disease Modeling and Drug Discovery. Stem Cells Transl Med 2014; 3:1275-86. [PMID: 25273538 PMCID: PMC4214842 DOI: 10.5966/sctm.2014-0073] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2014] [Accepted: 08/15/2014] [Indexed: 12/28/2022] Open
Abstract
The autism spectrum disorders (ASDs) comprise a set of neurodevelopmental disorders that are, at best, poorly understood but are the fastest growing developmental disorders in the United States. Because animal models of polygenic disorders such as the ASDs are difficult to validate, the derivation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming offers an alternative strategy for identifying the cellular mechanisms contributing to ASDs and the development of new treatment options. Access to statistically relevant numbers of ASD patient cell lines, however, is still a limiting factor for the field. We describe a new resource with more than 200 cell lines (fibroblasts, iPSC clones, neural stem cells, glia) from unaffected volunteers and patients with a wide range of clinical ASD diagnoses, including fragile X syndrome. We have shown that both normal and ASD-specific iPSCs can be differentiated toward a neural stem cell phenotype and terminally differentiated into action-potential firing neurons and glia. The ability to evaluate and compare data from a number of different cell lines will facilitate greater insight into the cause or causes and biology of the ASDs and will be extremely useful for uncovering new therapeutic and diagnostic targets. Some drug treatments have already shown promise in reversing the neurobiological abnormalities in iPSC-based models of ASD-associated diseases. The ASD Stem Cell Resource at the Children's Hospital of Orange County will continue expanding its collection and make all lines available on request with the goal of advancing the use of ASD patient cells as disease models by the scientific community.
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Affiliation(s)
- David J Brick
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California, USA; Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California, USA; Medical Investigation of Neurodevelopmental Disorders (MIND) Institute, Department of Pediatrics, and Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, California, USA
| | - Hubert E Nethercott
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California, USA; Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California, USA; Medical Investigation of Neurodevelopmental Disorders (MIND) Institute, Department of Pediatrics, and Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, California, USA
| | - Samantha Montesano
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California, USA; Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California, USA; Medical Investigation of Neurodevelopmental Disorders (MIND) Institute, Department of Pediatrics, and Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, California, USA
| | - Maria G Banuelos
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California, USA; Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California, USA; Medical Investigation of Neurodevelopmental Disorders (MIND) Institute, Department of Pediatrics, and Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, California, USA
| | - Alexander E Stover
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California, USA; Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California, USA; Medical Investigation of Neurodevelopmental Disorders (MIND) Institute, Department of Pediatrics, and Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, California, USA
| | - Soleil Sun Schutte
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California, USA; Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California, USA; Medical Investigation of Neurodevelopmental Disorders (MIND) Institute, Department of Pediatrics, and Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, California, USA
| | - Diane K O'Dowd
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California, USA; Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California, USA; Medical Investigation of Neurodevelopmental Disorders (MIND) Institute, Department of Pediatrics, and Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, California, USA
| | - Randi J Hagerman
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California, USA; Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California, USA; Medical Investigation of Neurodevelopmental Disorders (MIND) Institute, Department of Pediatrics, and Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, California, USA
| | - Michele Ono
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California, USA; Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California, USA; Medical Investigation of Neurodevelopmental Disorders (MIND) Institute, Department of Pediatrics, and Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, California, USA
| | - David R Hessl
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California, USA; Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California, USA; Medical Investigation of Neurodevelopmental Disorders (MIND) Institute, Department of Pediatrics, and Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, California, USA
| | - Flora Tassone
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California, USA; Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California, USA; Medical Investigation of Neurodevelopmental Disorders (MIND) Institute, Department of Pediatrics, and Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, California, USA
| | - Philip H Schwartz
- National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California, USA; Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California, USA; Medical Investigation of Neurodevelopmental Disorders (MIND) Institute, Department of Pediatrics, and Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, California, USA
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Hu B, Guo Y, Chen C, Li Q, Niu X, Guo S, Zhang A, Wang Y, Deng Z. Repression of SIRT1 promotes the differentiation of mouse induced pluripotent stem cells into neural stem cells. Cell Mol Neurobiol 2014; 34:905-12. [PMID: 24832395 PMCID: PMC11488914 DOI: 10.1007/s10571-014-0071-8] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2014] [Accepted: 04/27/2014] [Indexed: 12/13/2022]
Abstract
The use of transplanting functional neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) has increased for the treatment of brain diseases. As such, it is important to understand the molecular mechanisms that promote NSCs differentiation of iPSCs for future NSC-based therapies. Sirtuin 1 (SIRT1), a NAD(+)-dependent protein deacetylase, has attracted significant attention over the past decade due to its prominent role in processes including organ development, longevity, and cancer. However, it remains unclear whether SIRT1 plays a role in the differentiation of mouse iPSCs toward NSCs. In this study, we produced NSCs from mouse iPSCs using serum-free medium supplemented with retinoic acid. We then assessed changes in the expression of SIRT1 and microRNA-34a, which regulates SIRT1 expression. Moreover, we used a SIRT1 inhibitor to investigate the role of SIRT1 in NSCs differentiation of iPSCs. Data revealed that the expression of SIRT1 decreased, whereas miRNAs-34a increased, during this process. In addition, the inhibition of SIRT1 enhanced the generation of NSCs and mature neurocytes. This suggests that SIRT1 negatively regulated the differentiation of mouse iPSCs into NSCs, and that this process may be regulated by miRNA-34a.
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Affiliation(s)
- Bin Hu
- Institue of Orthopaedic Surgery, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai, 200233 China
| | - Ye Guo
- Graduate School of Nanchang University, Nanchang, 330006 China
- Department of Neurosurgery, Nanchang University Affiliated Second Hospital, Nanchang, 330006 China
| | - Chunyuan Chen
- Institue of Orthopaedic Surgery, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai, 200233 China
- Graduate School of Nanchang University, Nanchang, 330006 China
| | - Qing Li
- Institue of Orthopaedic Surgery, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai, 200233 China
| | - Xin Niu
- Institue of Orthopaedic Surgery, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai, 200233 China
| | - Shangchun Guo
- Institue of Orthopaedic Surgery, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai, 200233 China
| | - Aijun Zhang
- Department of Neurosurgery, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai, 200233 China
| | - Yang Wang
- Institue of Orthopaedic Surgery, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai, 200233 China
| | - Zhifeng Deng
- Department of Neurosurgery, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai, 200233 China
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Shu T, Pang M, Rong L, Zhou W, Wang J, Liu C, Wang X. Effects of Salvia miltiorrhiza on neural differentiation of induced pluripotent stem cells. JOURNAL OF ETHNOPHARMACOLOGY 2014; 153:233-241. [PMID: 24568774 DOI: 10.1016/j.jep.2014.02.028] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/11/2013] [Revised: 02/13/2014] [Accepted: 02/14/2014] [Indexed: 06/03/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE Salvia miltiorrhiza, a well-known traditional Chinese medicine, is commonly used to treat some neurological diseases because of its anti-oxidative, anti-inflammatory and anti-apoptotic properties. We investigate whether Salvia miltiorrhiza can improve the differentiation of induced pluripotent stem cells (iPSCs) into neurons in vitro, and promote iPSCs-derived neural stem cells survival, integrate, and differentiation after their transplantation to the ischemic brain tissues. MATERIALS AND METHODS Induced pluripotent stem cells were used to differentiate into neural stem cells, and further into neurons in induction medium with various concentrations of Salvia miltiorrhiza. The effects were assessed by immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting. iPSC-derived neural stem cells were transplanted into the brains of rats with middle cerebral artery occlusion, immunofluorescence staining was used to evaluate survival, integrate, and differentiation of grafted cells, the functional recovery of the animals was tested by the Longa scores and spontaneous motor activity. RESULTS Salvia miltiorrhiza (5μg/ml) significantly increased the gene and protein expression of Nestin compared with that in other groups. Microtubule-associated protein 2 (MAP2) expression in induction medium with 5μg/ml Salvia miltiorrhiza was significantly higher than that in the control group. After cells transplantation into the ischemic brain, more grafted MAP2(+) cells were found in Salvia miltiorrhiza-treated rats than others at 7 days. Salvia miltiorrhiza-treated rats showed the most remarkable functional recovery at 7 and 14 days. CONCLUSION Salvia miltiorrhiza induces differentiation of induced pluripotent stem cells to differentiate into neurons efficiently. The plant provides neuroprotection to implanted cells and improves functional recovery after their transplantation to the ischemic brain tissues.
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Affiliation(s)
- Tao Shu
- Department of Spine Surgery, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, China
| | - Mao Pang
- Department of Spine Surgery, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, China
| | - Limin Rong
- Department of Spine Surgery, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, China
| | - Wei Zhou
- Department of Spine Surgery, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, China
| | - Juan Wang
- Department of Gynecology, Baiyun Women׳s and Children׳s Hospital, Guangzhou, Guangdong 510000, China
| | - Chang Liu
- Department of Spine Surgery, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, China
| | - Xuan Wang
- Department of Spine Surgery, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510630, China
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Stanslowsky N, Haase A, Martin U, Naujock M, Leffler A, Dengler R, Wegner F. Functional differentiation of midbrain neurons from human cord blood-derived induced pluripotent stem cells. Stem Cell Res Ther 2014; 5:35. [PMID: 24636737 PMCID: PMC4055096 DOI: 10.1186/scrt423] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2013] [Accepted: 03/11/2014] [Indexed: 02/07/2023] Open
Abstract
Introduction Human induced pluripotent stem cells (hiPSCs) offer great promise for regenerative therapies or in vitro modelling of neurodegenerative disorders like Parkinson’s disease. Currently, widely used cell sources for the generation of hiPSCs are somatic cells obtained from aged individuals. However, a critical issue concerning the potential clinical use of these iPSCs is mutations that accumulate over lifetime and are transferred onto iPSCs during reprogramming which may influence the functionality of cells differentiated from them. The aim of our study was to establish a differentiation strategy to efficiently generate neurons including dopaminergic cells from human cord blood-derived iPSCs (hCBiPSCs) as a juvenescent cell source and prove their functional maturation in vitro. Methods The differentiation of hCBiPSCs was initiated by inhibition of transforming growth factor-β and bone morphogenetic protein signaling using the small molecules dorsomorphin and SB 431542 before final maturation was carried out. hCBiPSCs and differentiated neurons were characterized by immunocytochemistry and quantitative real time-polymerase chain reaction. Since functional investigations of hCBiPSC-derived neurons are indispensable prior to clinical applications, we performed detailed analysis of essential ion channel properties using whole-cell patch-clamp recordings and calcium imaging. Results A Sox1 and Pax6 positive neuronal progenitor cell population was efficiently induced from hCBiPSCs using a newly established differentiation protocol. Neuronal progenitor cells could be further maturated into dopaminergic neurons expressing tyrosine hydroxylase, the dopamine transporter and engrailed 1. Differentiated hCBiPSCs exhibited voltage-gated ion currents, were able to fire action potentials and displayed synaptic activity indicating synapse formation. Application of the neurotransmitters GABA, glutamate and acetylcholine induced depolarizing calcium signal changes in neuronal cells providing evidence for the excitatory effects of these ligand-gated ion channels during maturation in vitro. Conclusions This study demonstrates for the first time that hCBiPSCs can be used as a juvenescent cell source to generate a large number of functional neurons including dopaminergic cells which may serve for the development of novel regenerative treatment strategies.
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Li Y, Liu M, Yan Y, Yang ST. Neural differentiation from pluripotent stem cells: The role of natural and synthetic extracellular matrix. World J Stem Cells 2014; 6:11-23. [PMID: 24567784 PMCID: PMC3927010 DOI: 10.4252/wjsc.v6.i1.11] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/01/2013] [Revised: 10/23/2013] [Accepted: 11/02/2013] [Indexed: 02/06/2023] Open
Abstract
Neural cells differentiated from pluripotent stem cells (PSCs), including both embryonic stem cells and induced pluripotent stem cells, provide a powerful tool for drug screening, disease modeling and regenerative medicine. High-purity oligodendrocyte progenitor cells (OPCs) and neural progenitor cells (NPCs) have been derived from PSCs recently due to the advancements in understanding the developmental signaling pathways. Extracellular matrices (ECM) have been shown to play important roles in regulating the survival, proliferation, and differentiation of neural cells. To improve the function and maturation of the derived neural cells from PSCs, understanding the effects of ECM over the course of neural differentiation of PSCs is critical. During neural differentiation of PSCs, the cells are sensitive to the properties of natural or synthetic ECMs, including biochemical composition, biomechanical properties, and structural/topographical features. This review summarizes recent advances in neural differentiation of human PSCs into OPCs and NPCs, focusing on the role of ECM in modulating the composition and function of the differentiated cells. Especially, the importance of using three-dimensional ECM scaffolds to simulate the in vivo microenvironment for neural differentiation of PSCs is highlighted. Future perspectives including the immediate applications of PSC-derived neural cells in drug screening and disease modeling are also discussed.
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Affiliation(s)
- Yan Li
- Yan Li, Yuanwei Yan, Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL 32310, United States
| | - Meimei Liu
- Yan Li, Yuanwei Yan, Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL 32310, United States
| | - Yuanwei Yan
- Yan Li, Yuanwei Yan, Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL 32310, United States
| | - Shang-Tian Yang
- Yan Li, Yuanwei Yan, Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL 32310, United States
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