En guide til generering og brug hiPSC Afledte NPC'ere for Studiet af neurologiske sygdomme
Summary
This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.
Abstract
Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.
Introduction
Genekspressionsstudier af neuroner differentierede in vitro fra humane inducerede pluripotente stamceller (hiPSCs) af os 1 og andre 2,3 angiver, at hiPSC neuroner ligne føtalt snarere end voksne hjernevæv. På nuværende tidspunkt kan hiPSC-baserede modeller være mere passende for studiet af disposition til, i stedet for sene træk, neurologisk sygdom. Vi har tidligere rapporteret, at en betydelig del af genet underskrift af skizofreni hiPSC afledt neuroner er konserveret i skizofreni hiPSC afledt neurale progenitorceller (NPCs), hvilket indikerer, at NPC kan være en nyttig celletype til undersøgelse af de molekylære veje bidrager til skizofreni 1 . Vi og andre har rapporteret afvigende migration, øget oxidativt stress og reaktive ilt arter, følsomhed over for sub-tærskel miljømæssige belastninger og nedsat mitokondriefunktion i skizofreni hiPSC NPC'ere 1,4-6, samt nedsat neuronal ctilslutningsmuligheder og synaptiske funktion i skizofreni hiPSC neuroner 5,7-10. Hvis de molekylære faktorer, der bidrager til afvigende migration og / eller oxidativt stress i skizofreni hiPSC NPC'ere ligger til grund også den reducerede neuronal tilslutningsmuligheder i skizofreni hiPSC-afledte neuroner, kan NPC'ere være en robust og yderst replikativ neurale population med til at undersøge de mekanismer, der er ansvarlige for sygdom. Endvidere, fordi man hurtigt kan frembringe et stort antal celler og behøver ikke vente uger eller måneder for neuronal modning, NPC-baserede assays er egnede til undersøgelse af større patientkohorter og er mere modtagelig for high throughput screening. Vi mener, at hiPSC NPC'ere kan tjene som målestok for de udviklingsmæssige veje potentielt bidrager til sygdommen patogenese, som det allerede er påvist i sygdomme så forskellige som skizofreni 1 og Huntingtons sygdom 11.
For at differentiere NPC'ere fra hiPSCs, indledende neurale iduktion opnås ved dual-SMAD hæmning (0,1 mM LDN193189 og 10 mM SB431542) 12. Ved at antagonisering BMP og TGF signalering med disse små molekyler, er endoderm og mesoderm specifikation blokeret, accelererende neuronal differentiering og fører til dannelsen af synlige neurale rosetter inden for en uge plating. Neural mønsterdannelse forekommer tidligt i denne proces, formodentlig i løbet af neural roset-dannelse og umiddelbart derefter. I mangel af andre signaler, disse primitive neurale celler antager en forreste forhjerne-lignende skæbne 13. Umiddelbart efter neurale roset formation, og løbende i hele NPC ekspansion, forhjernen NPC'ere dyrkes med FGF2 8,14. De har dobbelt afstamning potentiale og kan differentieres til neurale populationer af 70-80% III-tubulin-positive neuroner og 20-30% glial fibrillært surt protein (GFAP) -positive astrocytter (figur 1). Størstedelen af forhjernen hiPSC neuroner VGLUT1-positiveOg er således formentlig glutamaterge, selvom ca. 30% af neuroner er GAD67-positive (GABAergic) 8.
NPC rutinemæssigt passeret mere end ti gange in vitro, samtidig med at konsistente differentiering profiler og uden akkumulering karyotype abnormiteter. Grupper har rapporteret overførselsteknikker NPC'ere mere end 40 gange 15 dog finder vi, at ud over ti passager, viser NPC'ere øget tilbøjelighed til astrocyt differentiering. NPC'ere godt tåle flere fryse-tøs op og kan skiftet til vokse som neurosfærer ved blot at dyrke i ikke-klæbende plader. NPC effektivt transduceres af virale vektorer, der muliggør hurtig evaluering af de molekylære og cellulære konsekvenser af genetisk forstyrrelse, og let kan udvides til opnåelse tilstrækkeligt materiale til biokemiske undersøgelser. Endvidere, fordi virale vektorer tillader robust over-ekspression og / eller knockdown af sygdomstilstande relevante gener, enten kontrol eller patient afledt NEURAL-celler, kan man anvende denne platform til at teste virkningen af genetisk baggrund på disse manipulationer. Selv om det ikke er egnet til synaptisk eller aktivitet assays kræver modne neuroner, kan NPC'ere være et praktisk alternativ for mange enkle molekylære eller biokemiske analyser af patient-afledte neurale celler.
Protocol
Representative Results
Discussion
Vi har beskrevet metoder, som at differentiere hiPSCs til NPC'ere, en neural celletype, hvor en betydelig del af genet underskrift hiPSC-afledte neuroner er bevaret, og som kan fungere som en målestok for de udviklingsmæssige veje potentielt bidrager til sygdommen patogenese 8, 11. Derudover som vi har beskrevet, NPC'ere er en robust replikativ og nemt transducerede neurale befolkning, som vi mener kan være egnet til molekylære og biokemiske studier af sygdom disposition.
<p class="jove_conten…Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Kristen Brennand is a New York Stem Cell Foundation – Robertson Investigator. The Brennand Laboratory is supported by a Brain and Behavior Young Investigator Grant, National Institute of Health (NIH) grant R01 MH101454 and the New York Stem Cell Foundation.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments |
| DMEM/F12 | Life Technologies | #11330 | for HES media |
| DMEM/F12 | Life Technologies | #10565 | for neural media |
| KO-Serum Replacement | Life Technologies | #10828 | Needs to be lot tested |
| Glutamax | Life Technologies | #35050 | |
| NEAA | Life Technologies | #11140 | |
| 2‐mercaptoethanol (55mM 1000x) | Life Technologies | #21985-023 | |
| N2 | Life Technologies | #17502-048 | Needs to be lot tested |
| B27-RA | Life Technologies | #12587-010 | Needs to be lot tested |
| FGF2 | Life Technologies | #13256-029 | Resuspend in PBS + 1% BSA |
| LDN193189 | Stemgent | #04-0074 | |
| SB431542 | Stemgent | #04-0010 | |
| BDNF | Peprotech | #450-02 | Resuspend in PBS + 0.1% BSA |
| GDNF | Peprotech | #450-10 | Resuspend in PBS + 0.1% BSA |
| Dibutyryl cyclic-AMP | Sigma | #D0627 | Resuspend in PBS + 0.1% BSA |
| L-ascorbic acid | Sigma | #A0278 | Resuspend in H20 |
| STEMdiff Neural Rosette Selection Reagent | Stemcell Technologies | #05832 | |
| Accutase | Innovative Cell Technologies | AT-104 | |
| Collagenase IV | Life Technologies | #17104019 | |
| CF1 mEFs | Millipore | #PMEF-CF | |
| Poly-L-Ornithine | Sigma | P3655 | |
| Laminin, Natural Mouse 1mg | Life Technologies | #23017-015 | |
| BD Matrigel | BD | #354230 | Resuspend on ice in cold DMEM at 10mg/ml, use 1mg per two 6-well plate equivalent |
| Tissue culture treated 6-well plates | Corning | 3506 | |
| Ultra low attachment 6-well plates | Corning | 3471 | |
| goat anti-Sox2 | Santa Cruz | sc17320 | use at 1:200 |
| mouse anti-human Nestin | Millipore | MAB5326 | use at 1:200 |
| rabbit anti-βIII-tubulin | Covance | PRB435P | use at 1:500 |
| mouse anti-βIII-tubulin | Covance | MMS435P | use at 1:500 |
| mouse anti-MAP2AB | Sigma | M1406 | use at 1:200 |
| Plate centrifuge | Beckman Coulter | Beckman Coulter Allegra X-14 (with SX4750 plate carrier) |
Riferimenti
- Brennand, K., et al. Phenotypic differences in hiPSC NPCs derived from patients with schizophrenia. Mol Psychiatry. , (2014).
- Nicholas, C. R., et al. Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development. Cell Stem Cell. 12, 573-586 (2013).
- Mariani, J., et al. Modeling human cortical development in vitro using induced pluripotent stem cells. Proc Natl Acad Sci U S A. 109, 12770-12775 (2012).
- Hashimoto-Torii, K., et al. Roles of heat shock factor 1 in neuronal response to fetal environmental risks and its relevance to brain disorders. Neuron. 82, 560-572 (2014).
- Robicsek, O., et al. Abnormal neuronal differentiation and mitochondrial dysfunction in hair follicle-derived induced pluripotent stem cells of schizophrenia patients. Mol Psychiatry. 18 (10), 1067-1076 (2013).
- Paulsen, B. D., et al. Altered oxygen metabolism associated to neurogenesis of induced pluripotent stem cells derived from a schizophrenic patient. Cell Transplant. 21 (7), 1547-1559 (2012).
- Yu, D. X., et al. Modeling hippocampal neurogenesis using human pluripotent stem cells. Stem Cell Reports. 2, 295-310 (2014).
- Brennand, K. J., et al. Modelling schizophrenia using human induced pluripotent stem cells. Nature. 473 (7346), 221-225 (2011).
- Wen, Z., et al. Synaptic dysregulation in a human iPS cell model of mental disorders. Nature. , (2014).
- Zhang, L., Song, X., Mohri, Y., Qiao, L. Role pf Inflammation and Tumor Microenvironment in the Development of Gastrointestinal Cancers: What Induced Pluripotent Stem Cells Can Do. Current stem cell research & therapy. , (2014).
- An, M. C., et al. Genetic Correction of Huntington’s Disease Phenotypes in Induced Pluripotent Stem Cells. Cell Stem Cell. 11 (2), 253-263 (2012).
- Chambers, S. M., et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 27, 275-280 (2009).
- Pankratz, M. T., et al. Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage. Stem Cells. 25, 1511-1520 (2007).
- Marchetto, M. C., et al. A model for neural development and treatment of rett syndrome using human induced pluripotent stem cells. Cell. 143, 527-539 (2010).
- Sareen, D., et al. Chromosome 7 and 19 trisomy in cultured human neural progenitor cells. PLoS One. 4, e7630 (2009).
- Thomson, J. A., et al. Embryonic stem cell lines derived from human blastocysts. Science. 282, 1145-1147 (1998).
- Cowan, C. A., et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med. 350, 1353-1356 (2004).
- Ludwig, T. E., et al. Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol. 24, 185-187 (2006).
- Coufal, N. G., et al. L1 retrotransposition in human neural progenitor cells. Nature. 460, 1127-1131 (2009).
- Xia, G., et al. Generation of human-induced pluripotent stem cells to model spinocerebellar ataxia type 2 in vitro. Journal of molecular neuroscience : MN. 51, 237-248 (2013).
- Aasen, T., et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol. 26, 1276-1284 (2008).
- Delaloy, C., et al. MicroRNA-9 coordinates proliferation and migration of human embryonic stem cell-derived neural progenitors. Cell Stem Cell. 6, 323-335 (2010).
- Kriks, S., et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature. 480, 547-551 (2011).
- Maroof, A. M., et al. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell. 12, 559-572 (2013).
- Shi, Y., Kirwan, P., Smith, J., Robinson, H. P., Livesey, F. J. Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nat Neurosci. 15, 477-486 (2012).
- Espuny-Camacho, I., et al. Pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo. Neuron. 77, 440-456 (2013).
