Summary

De productie van pluripotente stamcellen van Mouse Vruchtwater cellen met behulp van een transposon System

Published: February 28, 2017
doi:

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

Prenatale diagnostiek is een belangrijk klinisch hulpmiddel om genetische ziekten (dwz chromosomale afwijkingen, monogenetische of polygenetische / multifactoriële aandoeningen) en aangeboren afwijkingen (dwz hernia diafragmatica, cystic long laesies, exomphalos, gastroschisis) te evalueren. Vruchtwater (AF) cellen zijn eenvoudig te verkrijgen van routinematig geplande procedures tijdens het tweede trimester van de zwangerschap (dwz vruchtwaterpunctie en amnioreduction) of keizersneden 1, 2. De beschikbaarheid van AF cellen van prenatale en neonatale patiënten geeft de mogelijkheid om deze bron regeneratieve medicijnen en verscheidene onderzoekers onderzochten de mogelijkheid om verschillende weefselbeschadigingen of ziekten met een stamcelpopulatie geïsoleerd uit AF 3, 4, 5, 6 behandel, 7, 8, 9, 10, 11, 12. Zich gemakkelijk verkrijgen AF cellen van zieke patiënten in een tijdvenster waarin de ziekte is vaak stilstaat, opent de weg naar het idee van deze cel bron voor herprogrammering doeleinden. Inderdaad zou geïnduceerde pluripotente stamcellen (iPS) cellen afkomstig van AF cellen worden gedifferentieerd in de cellen van belang in vitro drug tests of weefselengineering, teneinde een adequate patiënt-specifieke therapie bereiden voor de bevalling. Vele studies hebben reeds werd aangetoond dat AF cellen te herprogrammeren en onderverdeeld in een groot aantal celtypes 13, 14, 15, 16, 17 </ sup>, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27.

Sinds de ontdekking van Takahashi en Yamanaka 28 van reprogrammed somatische cellen door de geforceerde expressie van vier transcriptiefactoren (Oct4, Sox2, cMYC en Klf4), is er vooruitgang geboekt op het gebied van de herprogrammering. Gezien de verschillende werkwijzen kunnen onderscheiden tussen virale en niet-virale benaderingen. De eerste verwacht dat het gebruik van virale vectoren (retrovirussen en lentivirussen), die een hoge efficiëntie, maar meestal onvolledige onderdrukking van de retrovirale transgen met zowel het gevolg van een gedeeltelijk geherprogrammeerd cellijn en het risico van zijninsertiemutagenese 29, 30, 31. De niet-virale werkwijze gebruikt verschillende strategieën: bijvoorbeeld plasmiden, vectoren, mRNA, eiwit, transposons. De afleiding van iPS-cellen vrij van transgene sequenties heeft tot doel de mogelijke schadelijke effecten van lekkende transgenexpressie en insertiemutagenese omzeilen. Van alle hierboven vermelde niet-virale strategie, de piggyBac (PB) transposon / transposase vereist alleen de omgekeerde terminale herhalingen flankeren een transgen en tijdelijke expressie van het transposase enzym katalyseren invoeging of excisie gebeurtenissen 32. Het voordeel bij het gebruik van transposons boven andere werkwijzen voor iPS celgeneratie is de mogelijkheid om vectorvrije iPS cellen met een niet-virale vector benadering dezelfde efficiëntie van retrovirale vectoren toont. Dit kan door trace-less excisie van het transposon geïntegreerde codering voor de reprogramming factoren naar aanleiding van een nieuwe tijdelijke expressie van de transposase in de iPS-cellen 33. Aangezien PB efficiënt in verschillende celtypes 34, 35, 36, 37, is meer geschikt voor een klinische benadering met betrekking tot virale vectoren, en maakt de productie van xeno-vrije iPS cellen strijd met de huidige virusproductie protocollen die xenobiotische gebruiken voorwaarden, wordt dit systeem gebruikt om iPS cellen te verkrijgen uit muizen AF.

Hier stellen we een gedetailleerd protocol volgende reeds gepubliceerde werk om de productie van pluripotente iPS klonen van muizen AF-cellen (iPS-AF-cellen) 38 te tonen.

Protocol

Alle procedures waren in overeenstemming met de Italiaanse wetgeving. Murine AF monsters werden geoogst van zwangere muizen op 13,5 dagen na coïtum (DPC) van C57BL / 6-Tg (UBC-GFP) 30Scha / J muizen geroepen GFP. 1. Transposon Production OPMERKING: Transposon expressievectoren werden gegenereerd onder toepassing van standaard kloneringswerkwijzen. Het plasmide DNA voor transfectie muis AF-cellen werd bereid onder toepassing van commerciële kits. Meng…

Representative Results

De capaciteit van herprogrammering evalueren, werden muizen AF cellen verzameld uit foetussen van GFP muizen. Cellen werden getransfecteerd met de cirkelvormige transposon plasmide PB-tetO2-IRES-OKMS, waarbij de Yamanaka factoren (Oct4, Sox2, cMYC en Klf4) gerelateerd aan de mCherry fluorescerend eiwit in een doxycycline-induceerbare wijze tot expressie brengt, en omgekeerde tetracycline transactivator (PB- CAG-rtTA) plasmiden met het transposase expressieplasmide (mPBase). Muis AF-celle…

Discussion

De voor het induceren van pluripotentie verkrijgen werkwijze is relevant voor mobiele klinische veiligheid met betrekking tot langdurige transplantatie. Tegenwoordig zijn er verscheidene werkwijzen geschikt voor de herprogrammering. Onder de niet-integrerende methoden, het Sendai virus (SeV) vector een RNA virus dat grote hoeveelheden eiwitten kan produceren zonder integratie in de kern van de geïnfecteerde cellen 40 en een strategie om iPS cellen te verkrijgen zijn. SeV vectoren zou een aantrek…

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
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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Cite This Article
Bertin, E., Piccoli, M., Franzin, C., Nagy, A., Mileikovsky, M., De Coppi, P., Pozzobon, M. The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System. J. Vis. Exp. (120), e54598, doi:10.3791/54598 (2017).

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