Omprogrammere primære fostervann og membran celler Pluripotency Xeno-gratis forhold
Summary
Denne protokollen beskriver omprogrammering av primære amniotic væske og membran mesenchymal stamceller til indusert pluripotent stamceller med en ikke-integrere episomal tilnærming i fullt kjemisk definerte forhold. Prosedyrer for utvinning, kultur, omprogrammering og karakterisering av resulterende indusert pluripotent stamceller strenge metoder er detaljerte.
Abstract
Autologous cellen-basert behandling fikk et skritt nærmere virkeligheten med innføringen av indusert pluripotent stamceller. Fetal stamceller, som fostervann og membran mesenchymal stamceller, representerer en unik udifferensierte celler med løfte vev engineering og omprogrammere til iPSC for fremtidige pediatric intervensjoner og stilk cellen bank. Protokollen presenteres her beskriver en optimalisert prosedyre for å trekke ut og dyrking primære amniotic væske og membran mesenchymal stamceller og generere episomal indusert pluripotent stamceller fra disse cellene i fullt kjemisk definerte kultur forhold menneskelige rekombinant vitronectin og E8 medium. Karakterisering av de nye linjene ved å bruke strenge metoder-flowcytometri, AC confocal imaging, teratoma dannelse og transcriptional profilering-er også beskrevet. De nyopprettede linjene uttrykke markører av embryonale stamceller-Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4-samtidig er negativt for SSEA-1 markøren. Stamcelleforskningen linjer danner teratomer i scid-beige mus i 6-8 uker og i teratomer inneholder vev representant for alle tre bakterie lag. Transcriptional profilering av linjene ved sending av globale uttrykk microarray data til en bioinformatic pluripotency vurdering algoritme anses alle linjer pluripotent og derfor denne tilnærmingen er et attraktivt alternativ til dyreforsøk. De nye iPSC linjene kan lett brukes i nedstrøms forsøk med optimalisering av differensiering og vev engineering.
Introduction
Teknologien for indusert pluripotent stamceller (iPSC) bringer om potensielle celle erstatning terapi, sykdom og utviklingsmessige modellering, og narkotika og toksikologisk screening1,2,3. Erstatning terapi kan konseptuelt oppnås ved celle injeksjon, in vitro differensiert vev (som cardiac patcher) implantasjon, eller guidet fornyelse ved hjelp av vev engineering. Fostervann (AFSC) og membran stamceller (AMSC) er en utmerket kilde til celler for disse intervensjonene enten direkte4,5,6,7 eller som en start celle befolkningen omprogrammere i pluripotency8,9,10,11,12.
Tidlig tilnærminger brukt udefinert kultur systemer eller omprogrammering metoder som krever innebære genomisk integrering av konstruerer9,10,11,12. En nyere studie ansatt en xeno-fri medium, selv om en mindre definerte kjelleren membran vedlegg matrise (BMM) ble brukt, generere iPSC fra amniotic væske epitelceller. Imidlertid var teratoma formasjon analysen ikke inkludert i studien en rekke in vitro og molekylære data. Amniotic væske epitelceller ble funnet for å ha en omtrent 8-fold høyere reprogramming effektivitet sammenlignet med neonatal fibroblaster13. I en annen studie fant også mesenchymal stamceller fra fostervann omprogrammeres til iPSC med en mye høyere effektivitet12.
Pluripotent stamceller kan differensieres til vev representant for alle 3 bakterie lag og dermed har den bredeste muligheten. Pediatriske pasienter kan dra nytte av høsting, omprogrammering og vev utvikling av sine autologous amniotic væske stamceller prenatally og amniotic membran stamceller øye. Videre kan på relativt lavt nivå av (lavere enn voksne stamceller14,15) fetal stamceller teoretisk hjelpe i adressering observert oppbevaring av epigenetic bias fra kildecellene i iPSC16.
Presenterer her vi en protokoll for å omprogrammere fostervann og membran stamceller til pluripotency i kjemisk definert xeno-fri E8 medium på rekombinant vitronectin17 (VTN) bruker episomal plasmider18. Den største fordelen med amniotic væske og membran celler som cellene omprogrammere ligger i tilgjengelighet pre og øye og dermed denne tilnærmingen vil hovedsakelig nytte forskning i pediatric tissue engineering.
Protocol
Representative Results
Discussion
Den innledende fasen av iPSC generasjon fra fetal stamceller innebærer utvinning av kildecellene fra fosterets vev, kultur, utvidelse og innføring av episomal reprogramming plasmider. Denne fasen er etterfulgt av en kultur periode på rundt 14-18 dager før første fullt omprogrammeres koloniene kan utvides. Sluttfasen er modning iPSC kloner. Første utvinning av amniotic membran stamceller oppnås ved hjelp av en kombinert mekanisk og enzymatiske fordøyelsen av amnion. Vi fant som en inkubering periode av 30 min resu…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbeidet ble støttet av Fonds Medizinische Forschung Universitetet i Zurich, Forschungskredit Universitetet i Zurich, The SCIEX NMSlm under stipend 10.216 og 12.176, den sveitsiske Society of Cardiology, The Swiss National Science Foundation under Grant [320030-122273] og [310030-143992], 7th Framework Programme, livet Valve, Europakommisjonen under Grant [242008], Olga Mayenfisch Foundation, EMDO stiftelsen, oppstart stipendet 2012 universitetssykehus Zürich, og intern finansiering av Mitchell Cancer Institute.
Materials
| Tumor Dissociation Kit, human | Miltenyi Biotec | 130-095-929 | tissue dissociation system, reagent kit, includes tissue dissociation tubes and tissue dissociation enzymes |
| gentleMACS Dissociator | Miltenyi Biotec | 130-093-235 | tissue dissociation system, dissociator |
| Thermo Scientific™ Shandon™ Disposable Scalpel No. 10, Sterile, Individually Wrapped, 5.75 (14.6cm) | Thermo-Fisher | 3120032 | |
| 70 µm cell strainers | Corning | 10054-456 | |
| RPMI 1640 medium | Thermo-Fisher | 32404014 | |
| rocking platform | VWR | 40000-300 | |
| 50 ml centrifuge tubes | Thermo-Fisher | 339652 | |
| 15 ml centrifuge tubes | Thermo-Fisher | 339650 | |
| EBM-2 basal medium | Lonza | CC-3156 | basal medium for AFMC medium |
| FGF 2 Human (expressed in E. coli, non-glycosylated) | Prospec Bio | CYT-218 | bFGF, supplement for AFMC medium |
| EGF Human, Pichia | Prospec Bio | CYT-332 | EGF, supplement for AFMC medium |
| LR3 Insulin Like Growth Factor-1 Human Recombinant | Prospec Bio | CYT-022 | IGF, supplement for AFMC medium |
| Fetal Bovine Serum, embryonic stem cell-qualified | Thermo-Fisher | 10439024 | FBS |
| Antibiotic-Antimycotic (100X) | Thermo-Fisher | 15240062 | for primary AFSC/AMSC, for routine AFSC/AMSC it should not be necessary, do not use in medium for transfected cells! |
| Accutase cell detachment solution | StemCell Technologies | 07920 | cell detachment enzyme |
| CryoStor™ CS10 | StemCell Technologies | 07930 | complete freezing medium |
| PBS, pH 7.4 | Thermo-Fisher Scientific | 10010023 | |
| EndoFree Plasmid Maxi Kit (10) | Qiagen | 12362 | for plasmid isolation |
| pEP4 E02S EN2K | Addgene | 20925 | EN2K, reprogramming factors Oct4+Sox2, Nanog+Klf4 |
| pEP4 E02S ET2K | Addgene | 20927 | ET2K, reprogramming factors Oct4+Sox2, SV40LT+Klf4 |
| pCEP4-M2L | Addgene | 20926 | M2L, reprogramming factors c-Myc+LIN28 |
| NanoDrop 2000c UV-Vis Spectrophotometer | Thermo-Fisher | ND-2000C | spectrophotometer |
| Neon® Transfection System | Thermo-Fisher | MPK5000 | transfection system, components: Neon pipette – transfection pipette Neon device – transfection device |
| Neon® Transfection System 10 µL Kit | Thermo-Fisher | MPK1025 | consumables kit for the Neon Transfection System, it contains: Neon tip – transfection tip Neon tube – transfection tube buffer R – resuspension buffer buffer E – electrolytic buffer |
| Stemolecule™ Sodium Butyrate | StemGent | 04-0005 | small molecule enhancer of reprogramming |
| TeSR-E8 | StemCell Technologies | 05940 | E8 medium |
| Vitronectin XF™ | StemCell Technologies | 07180 | VTN, stock concentration 250 µg/ml, used for coating at 1 µg/cm2 in vitronectin dilution (CellAdhere) buffer |
| CellAdhere™ Dilution Buffer | StemCell Technologies | 07183 | vitronectin dilution buffer |
| UltraPure™ 0.5M EDTA, pH 8.0 | Thermo-Fisher | 15575020 | dilute with PBS to 0.5 mM before use |
| EVOS® FL Imaging System | Thermo-Fisher Scientific | AMF4300 | LCD imaging microscope system |
| CKX53 Inverted Microscope | Olympus | phase contrast cell culture microscope | |
| Pierce™ 16% Formaldehyde (w/v), Methanol-free | Thermo-Fisher | 28908 | dilute to 4% with PBS before use, diluted can be stored at 2-8 °C for 1 week |
| Perm Buffer III | BD Biosciences | 558050 | permeabilization buffer, chill to -20 °C before use |
| Mouse IgG1, κ Isotype Control, Alexa Fluor® 488 | BD Biosciences | 557782 | isotype control for Oct3/4A, Nanog |
| Mouse IgG1, κ Isotype Control, Alexa Fluor® 647 | BD Biosciences | 557783 | isotype control for Sox2 |
| Mouse anti-human Oct3/4 (Human Isoform A), Alexa Fluor® 488 | BD Biosciences | 561628 | |
| Mouse anti-human Nanog, Alexa Fluor® 488 | BD Biosciences | 560791 | |
| Mouse anti-human Sox-2, Alexa Fluor® 647 | BD Biosciences | 562139 | |
| Mouse IgGM, κ Isotype Control, Alexa Fluor® 488 | BD Biosciences | 401617 | isotype control for TRA-1-60 |
| Mouse IgGM, κ Isotype Control, Alexa Fluor® 647 | BD Biosciences | 401618 | isotype control for TRA-1-81 |
| Mouse anti-human TRA-1-60, Alexa Fluor® 488 | BD Biosciences | 330613 | |
| Mouse anti-human TRA-1-81, Alexa Fluor® 647 | BD Biosciences | 330705 | |
| Mouse IgG1, κ Isotype Control, Alexa Fluor® 488 | BD Biosciences | 400129 | isotype control for SSEA-1 |
| Mouse IgG3, κ Isotype Control, Alexa Fluor® 647 | BD Biosciences | 401321 | isotype control for SSEA-4 |
| Mouse anti-human SSEA-1, Alexa Fluor® 488 | BD Biosciences | 323010 | |
| Mouse anti-human SSEA-4, Alexa Fluor® 647 | BD Biosciences | 330407 | |
| Affinipure F(ab')2 Fragment Goat Anti-Mouse IgG+IgM, Alexa Fluor® 488 | Jackson Immunoresearch | 115-606-068 | use at a dilution of 1:600 or further optimize |
| Affinipure F(ab')2 Fragment Goat Anti-Mouse IgG+IgM, Alexa Fluor® 647 | Jackson Immunoresearch | 115-546-068 | use at a dilution of 1:600 or further optimize |
| DAPI | Thermo-Fisher Scientific | D21490 | stock solution 10 mM, further dilute to 1:12.000 for a working solution |
| Corning® Matrigel® Growth Factor Reduced, Phenol Red-Free | Corning | 356231 | basement membrane matrix (BMM) |
| scid-beige mice, female | Taconic | CBSCBG-F | |
| RNeasy Plus Mini Kit (50) | Qiagen | 74134 | RNA isolation kit |
| T-25 flasks, tissue culture-treated | Thermo-Fisher | 156367 | |
| T-75 flasks, tissue culture-treated | Thermo-Fisher | 156499 | |
| Nunc™ tissue-culture dish | Thermo-Fisher | 12-567-650 | 10 cm tissue culture dish |
| 6-well plates, tissue-culture treated | Thermo-Fisher | 140675 | |
| Neubauer counting chamber (hemacytometer) | VWR | 15170-173 | |
| Mr. Frosty™ Freezing Container | Thermo-Fisher | 5100-0001 | freezing container |
| FACS tubes, Round Bottom Polystyrene Test Tube, 5ml | Corning | 352058 | 5 ml polystyrene tubes |
| Eppendorf tubes, 1.5 ml | Thermo-Fisher | 05-402-96 | 1.5 ml microcentrifuge tubes |
| PCR tubes, 200 µl | Thermo-Fisher | 14-222-262 | |
| pipette tips, 100 to 1250 µl | Thermo-Fisher | 02-707-407 | narrow-bore 1 mL tips |
| pipette tips, 5 to 300 µl | Thermo-Fisher | 02-707-410 | |
| pipette tips, 0.1 to 10 µl | Thermo-Fisher | 02-707-437 | |
| wide-bore pipette tips, 1000 µl | VWR | 89049-166 | wide-bore 1 mL tips |
| glass Pasteur pipettes | Thermo-Fisher | 13-678-20A | |
| ethanol, 200 proof | Thermo-Fisher | 04-355-451 | |
| vortex mixer | VWR | 10153-842 | |
| chambered coverglass, 8-well, 1.5mm borosilicate glass | Thermo-Fisher | 155409 | glass-bottom confocal-grade cultureware |
| 22G needles | VWR | 82002-366 | |
| insulin syringes | Thermo-Fisher | 22-253-260 | |
| Formalin solution, neutral buffered, 10% | Sigma-Aldrich | HT501128-4L | fixation of explanted teratomas |
| Illumina HT-12 v4 Expression BeachChip | Illumina | BD-103-0204 | expression microarray, supported by PluriTest, discontinued by manufacturer |
| PrimeView Human Genome U219 Array Plate | Thermo-Fisher | 901605 | expression microarray (formerly Affymetrix brand), soon to be supported by PluriTest |
| GeneChip™ Human Genome U133 Plus 2.0 Array | Thermo-Fisher | 902482 | expression microarray (formerly Affymetrix brand), supported by CellNet, soon to be supported by PluriTest |
| PluriTest® | Coriell Institute | www.pluritest.org, free service for bioinformatic assessment of pluripotency, accepts microarray data – *.idat files from HT-12 v4 platform, soon to support U133, U219 microarray and RNA sequencing data | |
| CellNet | Johns Hopkins University | cellnet.hms.harvard.edu, free service for bioinformatic identification of cell type, including plutipotent stem cells, based on U133 microarray data – *.cel files, soon to support RNA sequencing data |
References
- Takahashi, K., Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126 (4), 663-676 (2006).
- Yu, J., et al. Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Science. 318 (5858), 1917-1920 (2007).
- Trounson, A., DeWitt, N. D. Pluripotent stem cells progressing to the clinic. Nat. Rev. Mol. Cell Biol. 17 (3), 194-200 (2016).
- Schmidt, D., et al. Prenatally fabricated autologous human living heart valves based on amniotic fluid derived progenitor cells as single cell source. Circulation. 116 (11 Suppl), I64-I70 (2007).
- Weber, B., Zeisberger, S. M., Hoerstrup, S. P. Prenatally harvested cells for cardiovascular tissue engineering: Fabrication of autologous implants prior to birth. Placenta. 32, S316-S319 (2011).
- Weber, B., et al. Prenatally engineered autologous amniotic fluid stem cell-based heart valves in the fetal circulation. Biomaterials. 33 (16), 4031-4043 (2012).
- Kehl, D., Weber, B., Hoerstrup, S. P. Bioengineered living cardiac and venous valve replacements: current status and future prospects. Cardiovasc. Pathol. 25 (4), 300-305 (2016).
- Slamecka, J., et al. Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions. Cell Cycle. 15 (2), 234-249 (2016).
- Jiang, G., et al. Human Transgene-Free Amniotic-Fluid-Derived Induced Pluripotent Stem Cells for Autologous Cell Therapy. Stem Cells Dev. 23 (21), 2613-2625 (2014).
- Pipino, C., et al. Trisomy 21 mid-trimester amniotic fluid induced pluripotent stem cells maintain genetic signatures during reprogramming: implications for disease modeling and cryobanking. Cell. Reprogram. 16 (5), 331-344 (2014).
- Cai, J., et al. Generation of human induced pluripotent stem cells from umbilical cord matrix and amniotic membrane mesenchymal cells. J Biol. Chem. 285 (15), 11227-11234 (2010).
- Ge, X., et al. Human Amniotic Mesenchymal Stem Cell-Derived Induced Pluripotent Stem Cells May Generate a Universal Source of Cardiac Cells. Stem Cells Dev. 21 (15), 2798-2808 (2012).
- Drozd, A. M., Walczak, M. P., Piaskowski, S., Stoczynska-Fidelus, E., Rieske, P., Grzela, D. P. Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP/EBNA-1 episomal reprogramming system. Stem Cell Res. Ther. 6 (1), (2015).
- Kang, N. -. H., et al. Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells. Cancer Gene Ther. 19 (8), 517-522 (2012).
- Moschidou, D., et al. Valproic Acid Confers Functional Pluripotency to Human Amniotic Fluid Stem Cells in a Transgene-free Approach. Mol. Ther. 20 (10), 1953-1967 (2012).
- Kim, K., et al. Epigenetic memory in induced pluripotent stem cells. Nature. 467 (7313), 285-290 (2010).
- Chen, G., et al. Chemically defined conditions for human iPSC derivation and culture. Nature Methods. 8 (5), 424-429 (2011).
- Yu, J., et al. Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences. Science. 324 (5928), 797-801 (2009).
- Martí, M., et al. Characterization of pluripotent stem cells. Nat. Protoc. 8 (2), 223-253 (2013).
- Chan, E. M., et al. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat. Biotechnol. 27 (11), 1033-1037 (2009).
- Adewumi, O., et al. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat. Biotechnol. 25 (7), 803-816 (2007).
- Müller, F. -. J., et al. A bioinformatic assay for pluripotency in human cells. Nature Methods. 8 (4), 315-317 (2011).
- Cahan, P., Li, H., Morris, S. A., Lummertz da Rocha, E., Daley, G. Q., Collins, J. J. CellNet: Network Biology Applied to Stem Cell Engineering. Cell. 158 (4), 903-915 (2014).
- Schopperle, W. M., DeWolf, W. C. The TRA-1-60 and TRA-1-81 Human Pluripotent Stem Cell Markers Are Expressed on Podocalyxin in Embryonal Carcinoma. STEM CELLS. 25 (3), 723-730 (2007).
- Ohnishi, K., et al. Premature Termination of Reprogramming In Vivo Leads to Cancer Development through Altered Epigenetic Regulation. Cell. 156 (4), 663-677 (2014).
- Schlaeger, T. M., et al. A comparison of non-integrating reprogramming methods. Nature Biotechnology. 33 (1), 58-63 (2014).
- Müller, F. -. J., Goldmann, J., Löser, P., Loring, J. F. A call to standardize teratoma assays used to define human pluripotent cell lines. Cell Stem Cell. 6 (5), 412-414 (2010).
- Beers, J., et al. Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat. Protoc. 7 (11), 2029-2040 (2012).
