Produktionen av pluripotenta stamceller från mus fostervatten celler med användning av en transposon System
Summary
In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.
Abstract
Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.
Introduction
Fosterdiagnostik är ett viktigt kliniskt verktyg för att utvärdera genetiska sjukdomar (dvs kromosomavvikelser, monogena eller polygenetisk / multifaktoriella sjukdomar) och medfödda missbildningar (dvs. medfödd diafragmabråck, cystisk lunglesioner, exomphalos, gastroschis). Fostervatten (AF) celler är enkelt att få från rutinmässigt schemalagda förfaranden under andra trimestern av graviditeten (dvs. fostervattenprov och amnioreduction) eller kejsarsnitt 1, 2. Tillgängligheten av AF-celler från prenatal eller neonatala patienter ger möjligheten att använda denna källa för regenerativ medicin, och flera forskare undersökt möjligheten att behandla olika vävnads skador eller sjukdomar med användning av en stamcellspopulation isolerades från AF 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Möjligheten att enkelt få AF celler från sjuka patienter, i ett tidsfönster där sjukdomen är ofta stillastående, öppnar vägen till idén att använda denna cellkälla för omprogrammering ändamål. I själva verket kan inducerade pluripotenta stamceller (IPS) celler från AF-celler differentieras i cellerna av intresse för in vitro-drogtestning eller för vävnadstekniska metoder, i syfte att förbereda en lämplig patientspecifik behandling före förlossningen. Många studier har redan visat förmågan hos AF celler som skall omprogrammeras och differentieras till ett brett spektrum av celltyper 13, 14, 15, 16, 17 </ sup>, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27.
Sedan upptäckten av Takahashi och Yamanaka 28 omprogrammeras somatiska celler genom tvångs uttryck av fyra transkriptionsfaktorer (Oct4, Sox2, cMyc och Klf4) har framsteg gjorts när det gäller omprogrammering. Med tanke på de olika metoder, kan vi skilja mellan virala och icke-virala metoder. Det första förväntar sig att användningen av virala vektorer (retrovirus och lentivirus), som har hög effektivitet men vanligtvis ofullständigt tystande av den retrovirala transgenen, med både en följd av en delvis omprogrammeras cellinje och risken förinsättningsmutagenes 29, 30, 31. Den icke-virala metod använder olika strategier: dvs plasmider, vektorer, mRNA, protein, transposoner. Härledningen av iPS-celler fria från transgena sekvenser syftar till att kringgå de potentiellt skadliga effekterna av läckande transgenexpression och insertionsmutationer. Bland alla de ovan nämnda icke-virala strategier kräver piggyBac (PB) transposon / transposas systemet endast de inverterade terminala upprepningar som flankerar en transgen och transient uttryck av nämnda transposas enzym att katalysera inser eller excision händelser 32. Fördelen med att använda transposoner jämfört med andra metoder för IPS cell generation är möjligheten att erhålla vektorfria iPS-celler med en icke-viral vektor tillvägagångssätt som visar samma effektivitet av retrovirala vektorer. Detta är möjligt genom spårlösa excision av den integrerade transposonen kodning för reprogrAmming faktorer efter en ny transient uttryck av transposaset i iPS-celler 33. Med tanke på att PB är effektiv i olika celltyper 34, 35, 36, 37, är mer lämplig för ett kliniskt synsätt med avseende på virala vektorer, och möjliggör för framställningen av xeno-fria iPS-celler i motsats till nuvarande virusproduktionsprotokoll som använder xenobiotisk betingelser, är detta system användas för att erhålla iPS-celler från murin AF.
Här föreslår vi ett detaljerat protokoll följer redan publicerat arbete som visar framställningen av pluripotenta iPS kloner från mus AF-celler (iPS-AF-celler) 38.
Protocol
Representative Results
Discussion
Den metod som valts för att erhålla induktion av pluripotens är relevant för cell klinisk säkerhet med avseende på lång sikt transplantation. Numera finns det flera metoder som är lämpliga för omprogrammering. Bland de icke-integrerande metoder är Sendai virus (SeV) vektor ett RNA-virus som kan producera stora mängder protein utan integreras i kärnan av de infekterade cellerna 40 och kan vara en strategi för att få iPS-celler. SeV-vektorer kan vara en attraktiv kandidat för alstri…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by CARIPARO Foundation Grant number 13/04 and Fondazione Istituto di Ricerca Pediatrica Città della Speranza Grant number 10/02. Martina Piccoli, Chiara Franzin and Michela Pozzobon are funded by Fondazione Istituto di Ricerca Pediatrica Città della Speranza. Enrica Bertin is funded by CARIPARO Foundation Grant number 13/04. Paolo De Coppi is funded by Great Ormond Street Hospital Children’s Charity.
Materials
| 100 mm Bacterial-grade Petri Dishes | BD Falcon | 351029 | For in vitro differentiation |
| 2-mercaptoethanol | Sigma | M6250 | For mouse AF, iPS-AF cells and differentiation medium |
| Alexa568-conjugated goat anti-mouse IgM | Thermo Fisher Scientific | A21043 | Secondary antibody (immunofluorescence) |
| Alexa594-conjugated chicken anti-goat IgG | Thermo Fisher Scientific | A21468 | Secondary antibody (immunofluorescence) |
| Alexa594-conjugated chicken anti-rabbit IgG | Thermo Fisher Scientific | A21442 | Secondary antibody (immunofluorescence) |
| Alexa594-conjugated goat anti-mouse IgG | Thermo Fisher Scientific | A11005 | Secondary antibody (immunofluorescence) |
| Alkaline Phosphatase kit | Sigma | 85L1 | Alkaline Phosphatase staining |
| Ampicillin | Sigma | A0166 | For bacterial selection |
| Bovine Serum Albumin | Sigma | A7906 | BSA, for blocking solution. Diluted in PBS 1X |
| Chloroform | Sigma | C2432 | For RNA extraction |
| DH5α cells | Thermo Fisher Scientific | 18265-017 | Bacteria for cloning procedure |
| Dulbecco's Modified Eagle Medium (DMEM) | Thermo Fisher Scientific | 41965039 | For MEF, mouse AF, iPS-AF cells and differentiation medium |
| Doxycycline | Sigma | D9891 | For exogenous factors expression |
| Microcentrifuge tubes (1.5 mL) | Sarstedt | 72.706 | For PB production |
| ES FBS | Thermo Fisher Scientific | 10439024 | For mouse AF, iPS-AF cells and differentiation medium |
| FBS | Thermo Fisher Scientific | 10270106 | For MEF medium |
| Fine point forceps | F.S.T | Dumont #5 | AF isolation |
| Gelatin | J.T.Baker | 131 | Used 0.1%, diluted in PBS 1X |
| Glycine | Bio-Rad | 161-0718 | For blocking solution. Diluted in PBS 1X |
| Haematoxylin QS | Vector Laboratories | H3404 | Nuclei detection |
| HE | Bio-Optica | 04-061010 | Histological analysis of teratoma |
| Hoechst | Thermo Fisher Scientific | H3570 | Nuclei detection |
| Horse Serum | Thermo Fisher Scientific | 16050-122 | For blocking solution |
| HRP-conjugated goat anti-mouse IgG | SantaCruz | sc2005 | Secondary antibody (immunoperoxidase) |
| ImmPACT NovaRED | Vector Laboratories | SK4805 | Peroxidase substrate |
| Insulin syringe with needle (25G) | Terumo | SS+01H25161 | Amniocentesis procedure |
| Klf4 | SantaCruz | sc-20691 | Rabbit polyclonal IgG |
| L-glutamine | Thermo Fisher Scientific | 25030 | For mouse AF, iPS-AF cells and differentiation medium |
| LB broth (Lennox) | Sigma | L3022 | For bacterial growth |
| LIF | Sigma | L5158 | For mouse AF and iPS-AF cells medium |
| Matrigel | BD | 354234 | For in vitro differentiation. Diluted 1:10 in DMEM |
| Methanol | Sigma | 32213 | Peroxidase blocking |
| MULTIWELL 24 well plate | BD Falcon | 353047 | For in vitro differentiation |
| MULTIWELL 6 well plate | BD Falcon | 353046 | For MEF, mouse AF and iPS-AF cells culture |
| Nanog | ReproCELL | RCAB0002P-F | Rabbit polyclonal IgG |
| Non-essential amino acids | Sigma | M7145 | For mouse AF, iPS-AF cells and differentiation medium |
| Normal Goat Serum | Vector Laboratories | S2000 | For blocking solution. Diluted in PBS 1X |
| NP-40 | Sigma | 12087-87-0 | For cell permeabilization. Diluted in PBS 1X |
| Oct4 | SantaCruz | sc-5279 | Mouse monoclonal IgG2b |
| Oligo (dT) | Thermo Fisher Scientific | 18418012 | For RT-PCR |
| Paraformaldehyde (solution) | Sigma | 441244 | PFA, fixative, diluted in PBS |
| PBS 10X | Thermo Fisher Scientific | 14200-067 | D-PBS, free of Ca2+/Mg2+. Diluted with sterile water to obtain PBS 1X |
| Penicillin – Streptomycin | Thermo Fisher Scientific | 15070063 | For MEF, mouse AF, iPS-AF cells and differentiation medium |
| Petri Dish (150mm) | BD Falcon | 353025 | For MEF culture, tissue culture |
| PiggyBac transposase expression plasmid | Provided by professor Andras Nagy laboratory | – | mPBase |
| PiggyBac-tetO2-IRES-OKMS transposon plasmid | Provided by professor Andras Nagy laboratory | – | PB-tetO2-IRES-OKMS |
| QIAprep Spin Maxiprep Kit | Qiagen | 12663 | For plasmids purification |
| QIAprep Spin Miniprep Kit | Qiagen | 27106 | For plasmids purification |
| Reverse tetracycline transactivator transposon plasmid | Provided by professor Andras Nagy laboratory | – | rtTA |
| RNeasy Mini Kit | Qiagen | 74134 | For RNA extraction |
| Sox2 | SantaCruz | sc-17320 | Goat polyclonal IgG |
| SSEA1 | Abcam | ab16285 | Mouse monoclonal IgM |
| SuperScript II Reverse Transcriptase | Thermo Fisher Scientific | 18064-014 | For RT-PCR |
| T | Abcam | ab20680 | Rabbit polyclonal IgG |
| Taq DNA Polymerase | Thermo Fisher Scientific | 10342020 | PCR |
| Trypsin | Thermo Fisher Scientific | 25300-054 | Cell culture passaging |
| Triton X-100 | Bio-Rad | 161-047 | For cell permeabilization, diluted in PBS 1X |
| TRIzol Reagent | Thermo Fisher Scientific | 15596-026 | For RNA extraction |
| Tubb3 | Promega | G712A | Mouse monoclonal IgG1 |
| TWEEN-20 | Sigma | P1379 | For cell permeabilization, diluted in PBS 1X |
| αfp | R&D Systems | MAB1368 | Mouse Monoclonal IgG1 |
| αSMA | Abcam | ab7817 | Mouse Monoclonal IgG2a |
| Transfection Reagent (FuGENE HD) | Promega | E2311 | For AF cells transfection |
| Stereomicroscope | Nikon | SM2645 | To perform amniocentesis |
| 200 ul tips | Sarstedt | 70.760012 | To pick bacteria colonies |
| Scissor | F.S.T | 14094-11 stainless 25U | To perform amniocentesis |
| Ethanol | Sigma | 2860 | To clean the abdominal wall of the pregnant dam |
| Tissue culture petri dish (150 mm) | BD Falcon | 353025 | For MEF expansion |
| Mitomycin C | Sigma | M4287-2MG | For MEF inactivation |
| MULTIWELL 96 well plate | BD Falcon | 353071 | For iPS-AF culture |
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