Omdannelse af humane induceret pluripotente stamceller (iPSCs) til funktionel spinal og kraniel motor neuroner brug PiggyBac vektorer
Summary
Denne protokol giver mulighed for hurtig og effektiv omdannelse af inducerede pluripotente stamceller til motoriske neuroner med en spinal eller kranie identitet, ved Ektopisk udtryk af transkription faktorer fra inducerble piggybac vektorer.
Abstract
Vi beskriver her en metode til at opnå funktionelle spinal og kraniel motoriske neuroner fra menneskeskabte pluripotente stamceller (iPSCs). Direkte omdannelse til motor neuron er opnået ved Ektopisk udtryk af alternative moduler af transkription faktorer, nemlig Ngn2, Isl1 og Lhx3 (NIL) eller Ngn2, Isl1 og Phox2a (NIP). NIL og NIP specificere henholdsvis spinal og kraniel motoriske neuron identitet. Vores protokol starter med dannelsen af modificerede iPSC linjer, hvor NIL eller NIP er stabilt integreret i genomet via en piggyBac transposon vektor. Ekspressionen af transgener induceres derefter af doxycyclin og fører i 5 dage til omdannelsen af iPSCs til MN-progenitorer. Efterfølgende modning, i 7 dage, fører til homogene populationer af spinal eller craniale MNs. Vores metode rummer flere fordele i forhold til tidligere protokoller: den er ekstremt hurtig og forenklet; Det kræver ikke virus infektion eller yderligere MN-isolation. Det gør det muligt at generere forskellige MN subpopulationer (spinal og Cranial) med en bemærkelsesværdig grad af modning, som påvist ved evnen til at brand tog af action potentialer. Desuden kan et stort antal motoriske neuroner opnås uden rensning fra blandede populationer. iPSC-afledte spinal og kraniel motor neuroner kan bruges til in vitro modellering af amyotrofisk lateral sklerose og andre neurodegenerative sygdomme i motor neuron. Homogene motoriske neuron populationer kan repræsentere en vigtig ressource for celletype specifikke Drug screeninger.
Introduction
Motorisk neuron (MN) degeneration spiller en årsagssammenhæng rolle i menneskelige sygdomme som Amyotrofisk lateral sklerose (ALS) og spinal muskuløs atrofi (SMA). Etablering af egnede in vitro-celle modelsystemer, der oversigter kompleksiteten af det menneskelige MN, er et vigtigt skridt i retning af udvikling af nye terapeutiske tilgange. Induceret pluripotente stamceller (ipscs), som er udstyret med bemærkelsesværdige plurilineage differentiering egenskaber, er nu blevet afledt af en række patienter ramt af motoriske neuron sygdomme1,2. Yderligere iPSC linjer transporterer patogene mutationer forbundet til MN sygdomme er blevet genereret af genredigering, begyndende fra kontrol “sund” pluripotente stamceller3. Disse linjer repræsenterer nyttige værktøjer til in vitro-sygdoms modellering og lægemiddel screening, forudsat at der findes passende metoder til differentiering af iPSC i MNs. Rationalet bag udviklingen af denne metode er at give forskersamfundet, der er interesseret i MN-sygdomme, en hurtig og effektiv differentierings protokol, der giver anledning til modne funktionelle MNs. Den første fordel ved denne metode er dens tidsramme for udførelse. Et andet relevant punkt af styrke kommer fra fjernelsen af ethvert rensningstrin. Endelig kan protokollen bruges til at generere to forskellige populationer af motoriske neuroner.
Muligheden for at generere forskellige undertyper af MNs er særlig relevant for modellering af MN-sygdomme. Ikke alle MN-undertyper er lige så sårbare i ALS og SMA, og symptomdebut i forskellige motor enheder påvirker i høj grad prognosen. I ALS, spinal debut med symptomer, der starter i øvre og nedre lemmer fører til døden i omkring 3-5 år4. Omvendt, bulbar debut, begyndende med degeneration af kranielle MNs, har en værste prognose. Desuden er andelen af bulbar debut signifikant højere hos patienter med mutationer i RNA-bindende proteiner FUS og TDP-43 end hos personer med SOD1 mutationer5. Næsten alle alternative MN-differentierings protokoller er baseret på aktiviteten af retinoinsyre (RA), som giver en spinal karakter til at differentiere ipscs6,7,8. Dette begrænser muligheden for at studere iboende faktorer, som kan være beskyttende i specifikke MN undertyper9,10.
I overensstemmelse med en tidligere arbejde i mus embryonale stamceller11, har vi for nylig vist, at i human ipscs ektopic udtryk for Ngn2, Isl1 og Lhx3 (Nil) inducerer en spinal MN identitet, mens Ngn2 og Isl1 plus Phox2a (NIP) angive kranie mns12. Vi har derfor udviklet en effektiv protokol, der fører til produktion af menneskelige MNs udstyret med funktionelle egenskaber i en 12 dages turnaround. Formålet med denne metode er at opnå, inden for en kort tidsramme og uden behov for rensning (f. eks. ved FACS), cellepopulationer stærkt beriget for MNs med spinal eller kraniel identitet.
Protocol
Representative Results
Discussion
Denne protokol gør det muligt effektivt at konvertere humane iPSCs til spinal og kraniel motor neuroner takket være ektopic udtryk af Lineage-specifikke transkription faktorer. Disse transgener er inducerbare af doxycyclin og stabilt integreret i genomet takket være en transposon-baseret vektor med piggyBac. I en blandet population, en eller flere eksemplarer af piggyBac vektor vil blive tilfældigt integreret i genomet af de enkelte celler, øge risikoen for genomintegritet ændringer. Desuden kan en progressiv udvæ…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne ønsker at takke Imaging Facility på Center for Life Nano Science, Istituto Italiano di tecnologia, for støtte og teknisk rådgivning. Vi er taknemmelige for medlemmer af Center for Life Nano Science for nyttige diskussioner. Dette arbejde blev delvist understøttet af et tilskud fra AriSLA (pilot tilskud 2016 “StressFUS”) til AR.
Materials
| 5-Bapta | Sigma-Aldrich | A4926-1G | chemicals for electrophysiological solutions |
| Accutase | Sigma-Aldrich | A6964-100ML | Cell dissociation reagent |
| anti-CHAT | EMD Millipore | AB144P | Anti-Choline Acetyltransferase. Primary antibody used in immunostaining assays. RRID: AB_2079751; Lot number: 2971003 |
| anti-goat Alexa Fluor 488 | Thermo Fisher Scientific | A11055 | Secondary antibody used for immonofluorescence assays. RRID: AB_2534102; Lot number: 1915848 |
| anti-mouse Alexa Fluor 647 | Thermo Fisher Scientific | A31571 | Secondary antibody used for immonofluorescence assays. RRID: AB_162542; Lot number: 1757130 |
| anti-Oct4 | BD Biosciences | 611202 | Primary antibody used in immunostaining assays. RRID: AB_398736; Lot number: 5233722 |
| anti-Phox2b | Santa Cruz Biotechnology, Inc. | sc-376997 | Primary antibody used in immunostaining assays. Lot number: E0117 |
| anti-rabbit Alexa Fluor 594 | Immunological Sciences | IS-20152-1 | Secondary antibody used for immonofluorescence assays |
| anti-TUJ1 | Sigma-Aldrich | T2200 | Primary antibody used in immunostaining assays. RRID: AB_262133 |
| B27 | Miltenyi Biotec | 130-093-566 | Serum free supplement for neuronal cell maintenance |
| Bambanker | Nippon Genetics | NGE-BB02 | Cell freezing medium, used here for motor neuron progenitors |
| BDNF | PreproTech | 450-02 | Brain-Derived Neurotrophic Factor |
| Blasticidin | Sigma-Aldrich | 203350 | Nucleoside antibiotic that inhibits protein synthesis in prokaryotes and eukaryotes |
| BSA | Sigma-Aldrich | A2153 | Bovine Serum Albumin. Blocking agent to prevent non-specific binding of antibodies in immunostaining assays |
| CaCl2 | Sigma-Aldrich | C3881 | chemicals for electrophysiological solutions |
| Clampex 10 software | Molecular Devices | Clampex 10 | Membrane currents recording system |
| Corning Matrigel hESC-qualified Matrix | Corning | 354277 | Reconstituted basement membrane preparation from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. Used for adhesion of iPSC to plastic and glass supports |
| CRYOSTEM ACF FREEZING MEDIA | Biological Industries | 05-710-1E | Freezing medium for human iPSCs |
| D-Glucose | Sigma-Aldrich | G5146 | chemicals for electrophysiological solutions |
| DAPI powder | Roche | 10236276001 | 4′,6-diamidino-2-phenylindole. Fluorescent stain that binds to adenine–thymine rich regions in DNA used for nuclei staining in immonofluorescence assays |
| DAPT | AdipoGen | AG-CR1-0016-M005 | Gamma secretase inhibitor |
| Dispase | Gibco | 17105-041 | Reagent for gentle dissociation of human iPSCs |
| DMEM/F12 | Sigma-Aldrich | D6421-500ML | Basal medium for cell culture |
| Doxycycline | Sigma-Aldrich | D9891-1G | Used to induce expression of transgenes from epB-Bsd-TT-NIL and epB-Bsd-TT-NIP vectors |
| DS2U | WiCell | UWWC1-DS2U | Commercial human iPSC line |
| E.Z.N.A Total RNA Kit | Omega bio-tek | R6834-02 | Kit for total extraction of RNA from cultured eukaryotic cells |
| GDNF | PreproTech | 450-10 | Glial-Derived Neurotrophic Factor |
| Gibco Episomal hiPSC Line | Thermo Fisher Scientific | A18945 | Commercial human iPSC line |
| Glutamax | Thermo Fisher Scientific | 35050038 | An alternative to L-glutamine with increased stability. Improves cell health. |
| Hepes | Sigma-Aldrich | H4034 | chemicals for electrophysiological solutions |
| iScript Reverse Transcription Supermix for RT-qPCR | Bio-Rad | 1708841 | Kit for gene expression analysis using real-time qPCR |
| iTaqTM Universal SYBR Green Supermix | Bio-Rad | 172-5121 | Ready-to-use reaction master mix optimized for dye-based quantitative PCR (qPCR) on any real-time PCR instrument |
| K-Gluconate | Sigma-Aldrich | G4500 | chemicals for electrophysiological solutions |
| KCl | Sigma-Aldrich | P9333 | chemicals for electrophysiological solutions |
| L-ascorbic acid | LKT Laboratories | A7210 | Used in cell culture as an antioxidant |
| Laminin | Sigma-Aldrich | 11243217001 | Promotes attachment and growth of neural cells in vitro |
| Laser scanning confocal microscope | Olympus | iX83 FluoView1200 | Confocal microscope for acquisition of immunostaining images |
| Mg-ATP | Sigma-Aldrich | A9187 | chemicals for electrophysiological solutions |
| MgCl2 | Sigma-Aldrich | M8266 | chemicals for electrophysiological solutions |
| Mounting Medium | Ibidi | 50001 | Mounting solution used for confocal microscopy and immunofluorescence assays |
| Multiclamp patch-clamp amplifier | Molecular Devices | 700B | Membrane currents recording system |
| Na-GTP | Sigma-Aldrich | G8877 | chemicals for electrophysiological solutions |
| NaCl | Sigma-Aldrich | 71376 | chemicals for electrophysiological solutions |
| NEAA | Thermo Fisher Scientific | 11140035 | Non-Essential Amino Acids. Used as a supplement for cell culture medium, to increase cell growth and viability. |
| Neon 100 μL Kit | Thermo Fisher Scientific | MPK10096 | Cell electroporation kit |
| Neon Transfection System | Thermo Fisher Scientific | MPK5000 | Cell electroporation system |
| Neurobasal Medium | Thermo Fisher Scientific | 21103049 | Basal medium designed for long-term maintenance and maturation of neuronal cell populations without the need for an astrocyte feeder layer |
| NutriStem-XF/FF | Biological Industries | 05-100-1A | Human iPSC culture medium |
| Paraformaldehyde | Electron Microscopy Sciences | 157-8 | Used for cell fixation in immunostaining assays |
| PBS | Sigma-Aldrich | D8662-500ML | Dulbecco s Phosphate Buffer Saline w Calcium w Magnesium |
| PBS Ca2+/Mg2+ free | Sigma-Aldrich | D8537-500ML | Dulbecco s Phosphate Buffer Saline w/o Calcium w/o Magnesium |
| Penicillin/Streptomycin | Sigma-Aldrich | P4333-100ML | Penicillin/Streptomicin solution used to prevent cell culture contamination from bacteria. |
| poly-ornithine | Sigma-Aldrich | P4957 | Promotes attachment and growth of neural cells in vitro |
| SU5402 | Sigma-Aldrich | SML0443-5MG | Selective inhibitor of vascular endothelial growth factor receptor 2 (VEGFR-2) |
| Triton X-100 | Sigma-Aldrich | T8787 | 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenyl ether. Used for cell permeabilization in immunostaining assays |
| Upright microscope | Olympus | BX51VI | Microscope for electrophysiological recording equipped with CoolSnap Myo camera |
| Y-27632 (ROCK inhibitor) | Enzo Life Sciences | ALX-270-333-M005 | Cell-permeable selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK). Increases iPSC survival |
| μ-Slide 8 Well | Ibidi | 80826 | Support for high–end microscopic analysis of fixed cells |
References
- Dimos, J. T., et al. Induced Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into Motor Neurons. Science (New York, NY). 321 (5893), 1218 (2008).
- Ebert, A. D., et al. Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature. 457 (7227), 277-280 (2009).
- Lenzi, J., et al. ALS mutant FUS proteins are recruited into stress granules in induced Pluripotent Stem Cells (iPSCs) derived motoneurons. Disease models & mechanisms. 8 (7), 755-766 (2015).
- Wijesekera, L. C., Leigh, P. N. Amyotrophic lateral sclerosis. Orphanet journal of rare diseases. 4 (3), 1-22 (2009).
- Yan, J., et al. Frameshift and novel mutations in FUS in familial amyotrophic lateral sclerosis and ALS/dementia. Neurology. 75 (9), 807-814 (2010).
- Boulting, G. L., et al. A functionally characterized test set of human induced pluripotent stem cells. Nature biotechnology. 29 (3), 279-286 (2011).
- Amoroso, M. W., et al. Accelerated high-yield generation of limb-innervating motor neurons from human stem cells. The Journal of neuroscience: the official journal of the Society for Neuroscience. 33 (2), 574-586 (2013).
- Maury, Y., et al. Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes. Nature biotechnology. 33 (1), 89-96 (2015).
- Allodi, I., et al. Differential neuronal vulnerability identifies IGF-2 as a protective factor in ALS. Scientific reports. 6, 25960 (2016).
- Kaplan, A., et al. Neuronal matrix metalloproteinase-9 is a determinant of selective neurodegeneration. Neuron. 81 (2), 333-348 (2014).
- Mazzoni, E. O., et al. Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity. Nature neuroscience. 16 (9), 1219-1227 (2013).
- De Santis, R., Garone, M. G., Pagani, F., de Turris, V., Di Angelantonio, S., Rosa, A. Direct conversion of human pluripotent stem cells into cranial motor neurons using a piggyBac vector. Stem Cell Research. 29, 189-196 (2018).
- Yusa, K., Zhou, L., Li, M. A., Bradley, A., Craig, N. L. A hyperactive piggyBac transposase for mammalian applications. Proceedings of the National Academy of Sciences of the United States of America. 108 (4), 1531-1536 (2011).
- Sances, S., et al. Modeling ALS with motor neurons derived from human induced pluripotent stem cells. Nature Neuroscience. 16 (4), 542-553 (2016).
- Miller, J. D., et al. Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell stem cell. 13 (6), 691-705 (2013).
- Lenzi, J., et al. Differentiation of control and ALS mutant human iPSCs into functional skeletal muscle cells, a tool for the study of neuromuscolar diseases. Stem Cell Research. 17 (1), 140-147 (2016).
- Zhang, Y., et al. Rapid Single-Step Induction of Functional Neurons from Human Pluripotent Stem Cells. Neuron. 78 (5), 785-798 (2013).
- Theka, I., et al. Rapid generation of functional dopaminergic neurons from human induced pluripotent stem cells through a single-step procedure using cell lineage transcription factors. Stem cells translational medicine. 2 (6), 473-479 (2013).
- Pawlowski, M., et al. Inducible and Deterministic Forward Programming of Human Pluripotent Stem Cells into Neurons, Skeletal Myocytes, and Oligodendrocytes. Stem Cell Reports. 8 (4), 803-812 (2017).
- Li, X., et al. Fast Generation of Functional Subtype Astrocytes from Human Pluripotent Stem Cells. Stem Cell Reports. 11 (4), 998-1008 (2018).
- Nehme, R., et al. Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission. Cell reports. 23 (8), 2509-2523 (2018).
