Bir Transpozon Sistemi Kullanılarak Fare Amniyotik Sıvı Hücreleri pluripotent kök hücrelerin üretimi
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
Prenatal tanı genetik hastalıkların (örneğin kromozomal anormallikler, monogenetik veya çok faktörlü / polijenetik hastalıklar) ve konjenital anomali (yani konjenital diyafragma hernisi, kistik akciğer lezyonları, ekzomfalos, Gastroşizis) değerlendirmek için önemli bir klinik araçtır. Amniyotik sıvı (AF) hücreleri gebeliğin ikinci üç aylık döneminde rutin planlanan prosedürlerden elde etmek kolay (yani amniyosentez ve amniyoredüksiyon yapıldığında) ya da sezaryen bölümler 1, 2. Prenatal veya neonatal hastalarda AF hücrelerinin mevcudiyeti rejeneratif tıp için bu kaynak kullanma imkanı sağlar, ve birkaç araştırmacı AF 3, 4, 5, 6 izole bir kök hücre popülasyonu kullanarak farklı doku hasarları veya hastalıkları tedavi imkanı araştırıldı, 7, 8, 9, 10, 11, 12. Kolayca Hastalık genellikle hareketsiz olduğu bir zaman penceresinde, hastalıklı hastaların AF hücreleri elde etme ihtimali, yeniden programlama amacıyla bu hücre kaynağı kullanma düşüncesine yol açar. Gerçekten de, AF hücrelerinden türetilen uyarılmış pluripotent kök (IPS) hücreleri doğum öncesi uygun bir hastaya özgü tedavi hazırlamak amacıyla, in vitro ilaç testleri için veya doku mühendisliği yaklaşımlar ilgi hücrelerinde ayırt edilebilir. Birçok çalışma, daha önce AF hücrelerinin yeteneği hücre tipleri 13, 14, 15, 16, 17 <geniş bir aralığı içine yeniden programlanması ve ayırt edilmesi göstermiştir/ sup>, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27.
Dört transkripsiyon faktörleri (Oct4, Sox2, Myc'nin ve Klf4) zorla ifade ile Takahashi ve reprogrammed somatik hücre Yamanaka 28 ile keşfinden bu yana, ilerleme yeniden programlama alanında yapılmıştır. farklı yöntemler göz önüne alındığında, biz viral ve viral olmayan yaklaşımlar ayırt edebilirsiniz. İlk kısmen yeniden programlanan hücre hattının sonucu ve risk hem de yüksek verim ama retroviral transgen genellikle eksik susturulması, sahip viral vektörler (retrovirüsler ve lentiviruses), kullanımını bekliyoraraya sokma mutajenez 29, 30, 31. Yani plazmidler, vektörler, mRNA, protein, transpozonlar: viral olmayan yöntem farklı stratejiler kullanır. Transgenik dizilerin ücretsiz iPS hücrelerinin türetilmesi sızdıran transgen ifade ve girmeyle mutagenez potansiyel zararlı etkileri engelleyecek amaçlamaktadır. Yukarıda açıklanan tüm viral olmayan stratejiler arasında, piggyBac (PB) transpozon / transposase sistemi ekleme veya eksizyon olayları 32 katalize bir transgen ve transposase enzimin geçici ifadesini yan sadece ters terminali tekrarlar gerektirir. iPS hücresi üretimi için diğer yöntemlere göre transpozonlar kullanılarak avantajı retroviral vektörler aynı etkinliğini gösteren bir viral olmayan vektör yaklaşımla vektörü içermeyen iPS hücrelerini elde etme imkanı vardır. Bu reprogr için entegre transposon kodlama iz az eksizyon ile mümkündüriPS hücrelerinin 33 transpozazın yeni, geçici ifade, aşağıdaki faktörler amming. PB farklı hücre tipleri, 34, 35, 36, 37 etkili olduğu göz önüne alındığında, viral vektörler ile ilgili klinik yaklaşımın için daha uygundur, ve ksenobiyotik kullanarak mevcut virüs üretim protokolleri aksine kseno içermeyen iPS hücrelerinin üretimi sağlar Koşullar, bu sistem, kemirgen AF iPS hücreleri elde etmek için kullanılır.
Burada fare AF hücrelerinin (iPS-AF hücreleri) 38 dan pluripotent iPS klonların üretimini göstermek için zaten yayınlanmış çalışmaları takip ayrıntılı bir protokol öneriyoruz.
Protocol
Representative Results
Discussion
pluripotensin indüksiyon elde etmek için seçilen yöntem, uzun vadeli transplantasyonu ile ilgili hücre, klinik güvenliği için geçerlidir. Günümüzde, yeniden programlama için uygun çeşitli yöntemler vardır. Bütünleştirici olmayan yöntemler arasında, Sendai viral (SeV) vektörü bulaşmış hücreler 40 çekirdeği içine entegre olmayan proteinin büyük miktarlarda üretmek ve iPS hücreleri elde etmek için bir strateji olabilir bir RNA virüsüdür. SeV vektörleri ötelem…
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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