Summary

La production de cellules souches pluripotentes de souris amniotique Cellules Fluid Utilisation d'un système de transposon

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

Le diagnostic prénatal est un outil clinique important d'évaluer les maladies génétiques (ie des aberrations chromosomiques, des maladies monogéniques ou polygéniques / multifactorielle) et des malformations congénitales (hernie diaphragmatique congénitale ie, lésions pulmonaires kystiques, omphalocèle, gastroschisis). Cellules de liquide amniotique (AF) sont simples à obtenir des procédures régulièrement programmées au cours du deuxième trimestre de la grossesse (c. -à- amniocentèse et amnioreduction) ou césariennes 1, 2. La disponibilité des cellules AF de patients prénatales ou néonatales offre la possibilité d'utiliser cette source pour la médecine régénérative, et plusieurs chercheurs ont étudié la possibilité de traiter les dommages ou les maladies des tissus différents en utilisant une population de cellules souches isolées à partir AF 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. La possibilité d'obtenir facilement les cellules AF chez des patients malades, dans une fenêtre de temps dans lequel la maladie est souvent fixe, ouvre la voie à l'idée d'utiliser cette source de cellules à des fins de reprogrammation. En effet, souches pluripotentes induites (iPS) des cellules dérivées de cellules AF pourraient être différenciés dans les cellules d'intérêt pour les tests in vitro de médicaments ou pour les approches d'ingénierie tissulaire, afin de préparer une thérapie adéquate spécifique au patient avant l' accouchement. De nombreuses études ont déjà démontré la capacité des cellules AF à être reprogrammé et différenciées dans un large éventail de types de cellules 13, 14, 15, 16, 17 </ sup>, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27.

Depuis la découverte par Takahashi et Yamanaka 28 des cellules somatiques reprogrammées par l'expression forcée de quatre facteurs de transcription (Oct4, Sox2, cmyc et Klf4), des progrès ont été accomplis dans le domaine de la reprogrammation. Compte tenu des différentes méthodes, on peut distinguer entre les approches virales et non virales. Le premier attend l'utilisation de vecteurs viraux (rétrovirus et lentivirus), qui ont un rendement élevé, mais silencieux généralement incomplète du transgène rétroviral, à la fois la conséquence d'une lignée de cellules partiellement reprogrammée et le risque deinsertionnelle mutagenèse 29, 30, 31. La méthode non virale utilise des stratégies différentes: à savoir les plasmides, les vecteurs, les ARNm, les protéines, les transposons. La dérivation de cellules iPS libres de séquences transgéniques vise à contourner les effets potentiellement nocifs de l'expression du transgène qui fuit et la mutagenèse insertionnelle. Parmi toutes les stratégies non viraux mentionnés ci – dessus, le PiggyBac (PB) du système de transposon / transposase ne nécessite que les répétitions terminales inversées qui flanquent un transgène et de l' expression transitoire de l'enzyme transposase pour catalyser l' insertion ou l' excision d' événements de 32. L'avantage d'utiliser transposons sur les autres méthodes pour iPS génération de cellules est la possibilité d'obtenir des cellules sans vecteur-iPS avec une approche de vecteur non viral qui montre la même efficacité des vecteurs rétroviraux. Ceci est possible en trace-moins excision du codage de transposon intégré pour la reprogrammation facteurs suite à une nouvelle expression transitoire de la transposase dans les cellules iPS 33. Étant donné que le PB est efficace dans différents types de cellules 34, 35, 36, 37, est plus approprié pour une approche clinique en ce qui concerne les vecteurs viraux, et permet la production de cellules iPS libre xéno-contrairement aux protocoles de production viraux actuels qui utilisent des xénobiotiques conditions, ce système est utilisé pour obtenir des cellules iPS à partir AF murin.

Ici , nous proposons un protocole détaillé qui suit les travaux déjà publiés pour montrer la production de iPS pluripotentes clones à partir de cellules de souris AF (cellules iPS-AF) 38.

Protocol

Toutes les procédures étaient conformes à la loi italienne. échantillons murin AF ont été récoltées à partir de souris enceintes à 13,5 jours après le coït (dpc) à partir de C57BL / 6-Tg souris (UBC-GFP) 30Scha / J appelé GFP. 1. transposon production REMARQUE: transposon des vecteurs d'expression ont été générés en utilisant des procédures de clonage standard. L'ADN plasmidique pour la transfection des cellules de souris AF a été pré…

Representative Results

Pour évaluer la capacité de reprogrammation, les cellules AF souris ont été prélevés sur des foetus de souris GFP. Les cellules ont été transfectées avec le plasmide transposon circulaire PB-tetO2-IRES-OKMS, qui exprime les facteurs Yamanaka (Oct4, Sox2, cMyc et Klf4) liés à la protéine fluorescente mCherry d'une manière doxycycline inductible, et inverse la tetracycline transactivateur (PB- ACG rtTA) plasmides conjointement avec le plasmide d'expression de la transp…

Discussion

La méthode choisie pour obtenir l'induction de la pluripotence est pertinente pour la sécurité clinique cellule par rapport à la greffe à long terme. De nos jours, il existe plusieurs méthodes appropriées pour la reprogrammation. Parmi les méthodes non-intégration, le viral (SeV) vecteur Sendai est un virus à ARN qui peut produire de grandes quantités de protéines sans intégrer dans le noyau des cellules infectées 40 et pourrait être une stratégie pour obtenir des cellules iPS…

Divulgazioni

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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Citazione di questo articolo
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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