Summary

トランスポゾンのシステムを用いたマウスの羊水細胞から多能性幹細胞の作製

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

出生前診断は、遺伝性疾患( すなわち染色体異常、単成または複成/多因子疾患)と先天性奇形( すなわち先天性横隔膜ヘルニア、嚢胞性肺病変、臍帯ヘルニア、胃壁破裂)を評価するための重要な臨床的ツールです。羊水(AF)細胞( すなわち 、羊水穿刺およびamnioreduction)または帝王切開1、2妊娠中期の間に定期的にスケジュールされた手順から取得するのは簡単です。出生前または新生児の患者からのAF細胞の利用可能性は、再生医療のためのこのソースを使用する可能性を提供し、いくつかの研究は、AF 3、4、5、6から単離された幹細胞集団を使用して、異なる組織の損傷または疾患を治療する可能性を検討しました、7、8、9、10、11、12。簡単に罹患した患者からのAF細胞を得る可能性は、疾患はしばしば静止している時間ウィンドウ内で、再プログラミングのために、この細胞源を使用するというアイデアに道を開きます。実際、AF細胞由来の人工多能性幹(iPS)細胞は、出産前の適切な患者特有の治療を製造するために、 インビトロでの薬物試験のために、または組織工学的アプローチのための目的の細胞に分化することができます。多くの研究は、既に細胞型13、14、15、16、17 <広範囲に再プログラムと区別されるAF細胞の能力を実証しています/ SUP>、18、19、20、21、22、23、24、25、26、27。

4転写因子(Oct4の、Sox2の、CMYCおよびKlf4の)の強制発現を介して、再プログラミングされた体細胞の高橋と山中28によって発見されて以来、進展が再プログラミングの分野で行われています。異なる方法を考慮すると、我々は、ウイルスおよび非ウイルス性のアプローチを区別することができます。最初は、部分的に再プログラムされた細胞株の結果とのリスクの両方で、高効率レトロウイルス導入遺伝子の通常不完全なサイレンシングを有するウイルスベクター(レトロウイルスおよびレンチウイルス)の使用を想定し挿入突然変異29、30、31。 すなわちプラスミド、ベクター、mRNA、タンパク質、トランスポゾン:非ウイルス法は、異なる戦略を使用しています。トランスジェニック配列を含まiPS細胞の誘導は、漏出性導入遺伝子発現および挿入突然変異の潜在的に有害な影響を回避することを目指しています。上記の全ての非ウイルス戦略の中で、のpiggyBac(PB)トランスポゾン/トランスポザーゼシステムは挿入または切除イベント32を触媒する導入遺伝子およびトランスポザーゼ酵素の一過性発現に隣接する唯一の逆方向末端反復を必要とします。 iPS細胞の生成のための他の方法を介してトランスポゾンを使用する利点は、レトロウイルスベクターの同じ効率を示す非ウイルスベクターアプローチとベクトルフリーiPS細胞を得る可能性があります。これはreprogrのための統合されたトランスポゾンエンコーディングのトレースレス切除することにより可能ですiPS細胞33トランスポザーゼの新たな一過性の発現以下の要因をamming。 PBは、異なる細胞型34、35、36、37に効率的であることを考えると、ウイルスベクターに対する臨床的アプローチに適している、および生体異物を使用し、現在のウイルス産生プロトコルに反して異種非含有のiPS細胞の産生を可能にします条件は、このシステムは、マウスAFからiPS細胞を得るために使用されます。

ここでは、マウスのAF細胞(IPS-AF細胞)38からの多能性のiPSクローンの生産を表示するには、すでに発表された研究を、以下の詳細なプロトコルを提案します。

Protocol

すべての手順は、イタリアの法律に従いました。マウスAFサンプルをGFPと呼ばれるC57BL / 6-Tgの(UBC-GFP)30Scha / Jマウスからの13.5日後交尾(DPC)で妊娠したマウスから採取しました。 1.トランスポゾン生産注:トランスポゾン発現ベクターは、標準的なクローニング手順を用いて作製しました。マウスAF細胞のトランスフェクション用のプラスミドDNAを?…

Representative Results

再プログラミングの能力を評価するために、マウスAF細胞をGFPマウスの胎児から採取しました。細胞は、PB-(ドキシサイクリン誘導方法でmCherryを蛍光タンパク質に連結山中因子(Oct4の、Sox2の、CMYCおよびKlf4の)を発現円形トランスポゾンプラスミドPB-tetO2-IRES-OKMSでトランスフェクション、およびテトラサイクリントランスを逆にしました。 CAG-のrtTA)トランスポザー…

Discussion

多能性の誘導を得るために選択された方法は、長期移植に関して細胞臨床的安全性に関連します。今日では、再プログラミングに適したいくつかの方法があります。非組込み方法のうち、センダイウイルス(センダイウイルス)ベクターは、感染した細胞40の核内に統合することなく、タンパク質を大量に生産することができ、iPS細胞を得るための戦略であり得るRNAウイル?…

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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