JoVE Journal > Developmental Biology
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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발생학

트랜스포존 시스템을 사용하여 마우스 양수 세포로부터 다 능성 줄기 세포의 제조

Published: February 28, 2017
doi:
10.3791/54598
, , , , , ,
1Stem Cell and Regenerative Medicine Laboratory,Fondazione Istituto di Ricerca Pediatrica Citta della Speranza, 2Lunenfeld-Tanenbaum Research Institute,Mount Sinai Hospital, 3Stem Cells and Regenerative Medicine Section, Developmental Biology and Cancer Programme,UCL Institute of Child Health and Great Ormond Street Hospital

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

산전 진단은 유전 질환 (즉, 염색체 이상, monogenetic 또는 인성 / polygenetic 질환)과 선천성 기형 (즉, 선천성 횡격막 탈장, 낭포 성 폐 병변, exomphalos, 배벽 갈림 증)을 평가하는 중요한 임상 도구입니다. 양수 (AF) 세포는 임신의 두 번째 임신 중 정기적으로 예약 절차에서 획득하는 간단한 (즉, 양수 천자 및 amnioreduction) 또는 제왕 절개 섹션 1, 2입니다. 태아 또는 신생아 환자 AF 세포의 이용은 재생 의학이 소스를 사용할 수있는 가능성을 제공하고, 여러 연구자들은 AF 3, 4, 5, 6에서 분리 된 줄기 세포 집단을 사용하여 서로 다른 조직 손상 또는 질환을 치료하는 가능성을 조사7, 8, 9, 10, 11, 12. 쉽게 질병들은 종종 고정 된 시간 윈도우에서 질병 환자 AF 셀을 획득 할 가능성은 프로그래밍 목적이 셀 소스를 사용하는 아이디어에 방법을 연다. 실제로, AF 세포로부터 유래 된 유도 다 능성 줄기 (IPS) 세포는 출산 전에 적절한 환자 별 치료를 준비하기 위해, 시험관 내 약물 검사 또는 조직 공학 방법에 대한 관심의 세포 분화 될 수있다. 많은 연구가 이미 AF 세포의 능력은 세포 형태 13, 14, 15, 16, 17 <넓은 범위로 재 프로그램과 차별화 될 입증/ SUP> 18, 19, 20, 21, 22, 23, 24, 25, 26, 27.

네 개의 전사 인자 (인 Oct4, Sox2이, cMyc 및 Klf4)의 강제 표현을 통해 다카하시 및 재 프로그램 체세포의 야마나카 (28)에 의해 발견 이후, 진행 프로그래밍의 분야에서했다. 다른 방법을 고려하여, 우리는 바이러스 및 비 – 바이러스 성 방법을 구별 할 수있다. 첫번째 부분 재 프로그램 세포주의 결과와의 위험 모두 고효율이지만 레트로 바이러스 유전자 보통 불완전 침묵을 가질 바이러스 벡터 (레트로 바이러스 및 렌티 바이러스)의 사용을 예상삽입 성 돌연변이 29, 30, 31. 즉, 플라스미드 벡터, mRNA의 단백질, 트랜스포존 : 비 – 바이러스 성 방법은 다른 전략을 사용한다. 유전자 서열의 무료 만능 줄기 세포의 유도는 새는 유전자 발현 및 삽입 성 돌연변이 유발의 잠재적으로 해로운 영향을 회피하는 것을 목표로하고있다. 위에서 언급 한 모든 비 바이러스 전략 중, 피기 백 (PB) 트랜스포존 / 트랜스 시스템은 삽입 또는 절제 이벤트 (32)을 촉매하는 유전자와 트랜스 효소의 일시적인 발현을 측면에만 역전 된 말단 반복을 필요로한다. 만능 줄기 세포의 생성을위한 다른 방법을 통해 트랜스포존을 사용의 장점은 레트로 바이러스 벡터의 동일 효율을 나타내는 비 – 바이러스 벡터 방식 벡터 프리 만능 줄기 세포를 얻을 수 있다는 것이다. 이것은 reprogr위한 통합 트랜스포존 인코딩 트레이스 이하로 절단 할 수있다만능 줄기 세포 (33)의 트랜스의 새로운 일시적 발현 다음과 같은 요소를 amming. PB가 다른 세포 유형 34, 35, 36, 37 효율적임을 감안할 때, 바이러스 벡터에 대한 임상 적 접근 방식에 더 적합하며, 생체이 물을 사용하여 현재의 바이러스 생산 프로토콜 달리 이종없는 만능 줄기 세포의 생산을 허용 조건은,이 시스템은 뮤린 AF에서 만능 줄기 세포를 수득하기 위해 사용된다.

여기에서 우리는 마우스 AF 세포 (IPS-AF 세포) (38)로부터 만능 만능 줄기 클론의 생산을 보여주기 위해 이미 게시 작업을 다음과 같은 세부적인 프로토콜을 제안한다.

Protocol

모든 절차 이탈리아 법에 따라했다. 쥐의 AF 샘플은 GFP라는 C57BL / 6-Tg는 (UBC-GFP) 30Scha / J 마우스에서 13.5 일 후 교미 (DPC)에서 임신 한 쥐에서 수확했다. 1. 트랜스포존 생산 주 : 트랜스포존 발현 벡터는 표준 클로닝 방법을 사용하여 생성 하였다. 마우스 AF 세포 형질 전환 용 플라스미드 DNA 상업용 키트를 사용하여 제조 하였다. 1.5 ml의 마이크로 원…

Representative Results

프로그래밍의 능력을 평가하기 위해 마우스 AF 세포를 GFP 마우스의 태아로부터 수집 하였다. 세포는 솔더 (Soler) (a 독시사이클린 – 유도 방식으로 mCherry 형광 단백질에 연결된 야마나카 인자 (인 Oct4, Sox2이, cMyc 및 Klf4)를 나타내고, 원형 트랜스포존 플라스미드 PB-tetO2-IRES-OKMS, 형질 및 테트라 사이클린 transactivator 역방향했다 CAG-rtTA)은 트랜스의 발현 플라스미드 (mPBase)와 함?…

Discussion

능성의 유도를 얻기 위해 선택된 방법은 장기 이식에 대한 임상 세포의 안전을 위해 적합하다. 요즘 프로그래밍에 적합한 여러 가지 방법이 있습니다. 비 통합 방법 중에서, 센다이 바이러스 (SEV) 벡터는 감염된 세포의 핵 (40)로 통합하지 않고 대량의 단백질을 생산할 수 및 만능 줄기 세포를 수득하기위한 전략 될 수있는 RNA 바이러스이다. SEV 벡터는 병진 급 IPS 세포의 생성을위?…

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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Tags

Mouse Amniotic Fluid CellsPluripotent Stem CellsTransposon SystemReprogrammingFetal TherapyCell IsolationCell CultureMEF Feeder Cells
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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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