Omprogrammering primære fostervand og membran celler til Pluripotency i Xeno-fri betingelser
Summary
Denne protokol beskriver omprogrammering af primære fostervand væske og membran mesenkymale stamceller til induceret pluripotente stamceller ved hjælp af en ikke-integrere episomal tilgang i fuldt kemisk definerede betingelser. Procedurer for udvinding, kultur, omprogrammering og karakterisering af de resulterende inducerede pluripotente stamceller af strenge metoder er beskrevet.
Abstract
Autolog cellebaserede behandlinger fik et skridt tættere på virkeligheden med indførelsen af induceret pluripotente stamceller. Føtale stamceller, som fostervand og membran mesenkymale stamceller, repræsenterer en enestående type af udifferentierede celler med løftet i vævsmanipulering og for omprogrammering til iPSC for fremtidige pediatric interventioner og stamcelle bank. Protokollen præsenteres her beskriver en optimeret proceduren til ekstraktion og dyrkning af primære fostervand væske og membran mesenkymale stamceller og generere episomal induceret pluripotente stamceller fra disse celler som fuldt kemisk definerede kultur betingelser, der anvender humane rekombinante vitronectin og E8 medium. Karakterisering af de nye linjer ved at anvende strenge metoder – flowcytometri, Konfokal imaging, teratom dannelse og transcriptional profilering – er også beskrevet. De nyligt oprettede linjer express markører af embryonale stamceller-Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 – samtidig med at være negativ for SSEA-1 markør. Stamcellelinjer danner Teratomer i scid-beige mus i 6-8 uger og Teratomer indeholder væv repræsentativ for alle tre Kim lag. Transkriptionel profilering af linjerne ved at indsende globale udtryk microarray data til en bioinformatic pluripotency vurdering algoritmen anses for alle linjer pluripotente og derfor denne tilgang er et attraktivt alternativ til dyreforsøg. De nye iPSC linjer kan let anvendes downstream forsøg med optimering af differentiering og vævsmanipulering.
Introduction
Teknologien af induceret pluripotente stamceller (iPSC) bringer om potentielle celle udskiftning behandlingsformer, sygdom og udviklingsmæssige modellering, og narkotika og toksikologiske screening1,2,3. Udskiftning terapier kan konceptuelt opnås ved celle indsprøjtning, in vitro-opdelte væv (såsom hjerte patches) implantation eller guidede regenerering gennem vævsmanipulering. Fostervand (AFSC) og membran stamceller (AMSC) er en glimrende kilde til celler til disse interventioner enten direkte4,5,6,7 eller som en start celle population for omprogrammering i pluripotency8,9,10,11,12.
Tidlig tilgange bruges udefineret kultur systemer eller omprogrammering metoder, der kræver indebærer genomisk integration af konstruktioner9,10,11,12. En nyere undersøgelse ansat en xeno-frit medium, selv om en mindre definerede basalmembranen vedhæftet fil matrix (BMM) blev brugt til at generere iPSC fra fostervand væske epitelceller. Teratom dannelse analysen indgik ikke i undersøgelsen sammen med et væld af in vitro- og molekylære data. Fostervand væske epitelceller fandtes for at have en nogenlunde 8-fold højere omprogrammering effektivitet i forhold til neonatal fibroblaster13. I en anden undersøgelse fandtes mesenkymale stamceller fra fostervand også skal omprogrammeres til iPSC med en meget højere effektivitet12.
Pluripotente stamceller kan differentieres til væv repræsentant for alle 3 Kim lag og dermed har den bredeste potentiale. Pædiatriske patienter kunne drage fordel af den høst, omprogrammering, og vævsmanipulering af deres autolog fostervand væske stamceller prænatalt og fostervand membran stilk celler perinatal. Desuden kunne det forholdsvis lave niveau af differentiering af føtale stamceller (lavere end voksne stamceller14,15) teoretisk støtte i håndteringen af de observerede fastholdelse af epigenetiske bias fra kildecellerne i iPSC16.
Præsenterer her vi en protokol for omprogrammering fostervand og membran stamceller til pluripotency i kemisk definerede xeno-fri E8 medium rekombinante vitronectin17 (VTN) ved hjælp af episomal plasmider18. Den største fordel af fostervand væske og membran celler som en kilde til celler for omprogrammering ligger i deres tilgængelighed pre- og perinatal og dermed denne tilgang gavner hovedsagelig forskning i pediatric vævsmanipulering.
Protocol
Representative Results
Discussion
Den indledende fase af iPSC generation fra føtale stamceller indebærer udvinding af kildecellerne fra de føtale væv, deres kultur, ekspansion og indførelsen af de episomal omprogrammering plasmider. Denne fase efterfølges af en kultur omkring 14-18 dage før de første fuldt omprogrammeret kolonier kan udvides. Den sidste fase er modning af iPSC kloner. Den indledende ekstraktion af fostervand membran stamceller er opnået ved hjælp af en kombineret mekaniske og Enzymatisk nedbrydning af dyr. Vi fandt, at en inkub…
Declarações
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af Fonds Medizinische Forschung på universitetet i Zürich, Forschungskredit af universitetet i Zürich, The SCIEX NMSCh under stipendier 10.216 og 12.176, den schweiziske samfund kardiologi, The Swiss National Science Fundamentet under Grant [320030-122273] og [310030-143992], 7. rammeprogram, liv ventil, Europa-Kommissionen under tilskud [242008], Olga Mayenfisch Foundation, EMDO Foundation, starttilskud 2012 af Zürich Universitetshospital og interne finansiering af 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 |
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