In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.
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.
Prænatal diagnose er et vigtigt klinisk redskab til at vurdere genetiske sygdomme (dvs. kromosomafvigelser, monogenetic eller polygenetic / multifaktoriel sygdomme) og medfødte misdannelser (dvs. medfødt diafragma brok, cystisk lungelæsioner, exomphalos, gastroschise). Fostervand (AF) celler er enkle at få fra rutinemæssigt planlagte procedurer i andet trimester af graviditeten (dvs. fostervandsprøve og amnioreduction) eller kejsersnit 1, 2. Tilgængeligheden af AF-celler fra prænatal eller neonatale patienter giver mulighed for at anvende denne kilde til regenerativ medicin, og flere forskere undersøgt muligheden for at behandle forskellige vævsskader eller sygdomme ved anvendelse af en stamcelle population isoleret fra AF 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Muligheden for nemt at få AF celler fra syge patienter, i et tidsvindue, hvor sygdommen er ofte stationær, baner vejen for tanken om at bruge denne celle kilde til omprogrammering formål. Faktisk kunne induceret pluripotente stamceller (IPS) celler afledt fra AF-celler differentieres i cellerne af interesse for in vitro-test af lægemidler eller væv tekniske tiltag, for at forberede en passende patient-specifik terapi før fødslen. Mange undersøgelser har allerede demonstreret evne AF celler, der skal omprogrammeres, og differentieret i en lang række celletyper 13, 14, 15, 16, 17 </ sup>, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27.
Siden opdagelsen af Takahashi og Yamanaka 28 omprogrammerede somatiske celler gennem den tvungne udtryk for fire transkriptionsfaktorer (Oct4, Sox2, cMyc og Klf4), er der gjort fremskridt med hensyn til omprogrammering. I betragtning af de forskellige metoder, kan vi skelne mellem virale og ikke-virale fremgangsmåder. Den første forventer brugen af virale vektorer (retrovirus og lentivira), som har høj effektivitet men normalt ufuldstændig inaktivering af den retrovirale transgen, med både en følge af en delvis omprogrammeret cellelinie og risikoen forinsertionsmutagenese 29, 30, 31. Den ikke-viral metode anvender forskellige strategier: dvs. plasmider, vektorer, mRNA, protein transposoner. Udledningen af iPS celler fri af transgene sekvenser har til formål at omgå de potentielt skadelige virkninger af utætte transgen ekspression og insertionsmutagenese. Blandt alle de ovennævnte ikke-virale strategier, den PiggyBac (PB) transposon / transposase systemet tager de inverterede terminale gentagelser flankerer et transgen og forbigående ekspression af transposasen enzymet at katalysere insertion eller excision events 32. Fordelen ved anvendelse transposoner i forhold til andre fremgangsmåder til iPS celle generation er muligheden for at opnå vektor-fri iPS-celler med en ikke-viral vektor tilgang, der viser den samme effektivitet af retrovirale vektorer. Dette er muligt ved spor-mindre udskæring af det integrerede transposon koder for reprogramming faktorer efter en ny kortvarig ekspression af transposasen i iPS celler 33. Eftersom PB er effektive i forskellige celletyper 34, 35, 36, 37, er mere egnet til en klinisk tilgang til virale vektorer, og muliggør fremstillingen af xeno-fri iPS celler strid med de gældende virusproduktion protokoller, der bruger xenobiotikum betingelser er dette system anvendes til at opnå iPS celler fra murine aF.
Her foreslår vi en detaljeret protokol efter allerede offentliggjorte arbejde for at vise produktionen af pluripotente iPS kloner fra muse AF celler (iPS-AF celler) 38.
Den valgte at opnå induktion af pluripotens metode er relevant for celle kliniske sikkerhed med hensyn til lang sigt transplantation. I dag er der flere metoder er egnede til omprogrammering. Blandt de ikke-integrative metoder, Sendai viral (SeV) vektor er et RNA-virus, der kan producere store mængder protein uden integration i kernen i de inficerede celler 40 og kunne være en strategi til opnåelse iPS-celler. SeV vektorer kunne være en attraktiv kandidat til generering af translationel-grad…
The authors have nothing to disclose.
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.
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 |