את ייצור תאי גזע פלוריפוטנטיים מתאי מי שפירים עכבר באמצעות מערכת transposon
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
אבחון טרום לידתי הוא כלי קליני חשוב להעריך מחלות גנטיות (סטיות כרומוזומליות כלומר, monogenetic או polygenetic / מחל מולטיפקטוריאלית) ו מומים מולדים (כלומר מולד בקע סרעפתי, נגעי ריאות פיברוזיס, exomphalos, gastroschisis). מי שפיר (AF) תאים הם פשוט להשיג מנהלים מתוכננים באופן שגרתי במהלך השליש השני של הריון (כלומר מי שפיר ו amnioreduction) או ניתוחים קיסריים 1, 2. הזמינות של תאי AF מחולים טרום לידתי או בילוד מספק את האפשרות של שימוש מקור זה עבור רפואה רגנרטיבית, וכמה החוקרים בדקו את האפשרות לטיפול נזקים ברקמות שונות או מחלות באמצעות האוכלוסייה בתאי גזע שבודדו 3 AF, 4, 5, 67, 8, 9, 10, 11, 12. האפשרות של קבלה בקלות תאי AF מחולים חולים, בחלון הזמן בו המחלה היא לעתים קרובות נייחת, פותחת את הדרך אל הרעיון של שימוש מקור התא הזה למטרות תכנות מחדש. ואכן, גזע מושר (iPS) תאים שמקורם בתאי AF יכולים להיות מובחנים בתאים של ריבית עבור בדיקות סמים במבחנה או גישות הנדסת רקמות, כדי להכין טיפול למטופל ספציפי נאות לפני הלידה. מחקרים רבים כבר הוכיחו את יכולתם של תאי AF כדי לתכנת מחדש בדיל לתוך מגוון רחב של סוגי תאי 13, 14, 15, 16, 17 </ sup>, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27.
מאז גילויו על ידי טקהאשי יאמאנאקה 28 תאים סומטיים לתכנות מחדש דרך הביטוי הכפוי של ארבעה גורמי שעתוק (Oct4, Sox2, cMyc ו Klf4), הושג התקדמות בתחום של תכנות מחדש. בהתחשב בשיטות שונות, אנו יכולים להבחין בין גישות ויראלי ולא ויראלי. הראשון מצפה השימוש של וקטורים ויראליים (רטרווירוסים ו lentiviruses), אשר יש יעילות גבוהה, אך בדרך כלל השתקה שלמה של transgene retroviral, הוא עם התוצאה של שורת תאים לתכנות מחדש באופן חלקי את הסיכוןinsertional mutagenesis 29, 30, 31. השיטה הלא-ויראלי משתמשת באסטרטגיות שונות: פלסמידים כלומר, וקטורים, mRNA, חלבון, transposons. על הגזרה של תאי iPS ללא רצפים מהונדסים שואפת לעקוף את השפעות מזיקי ביטוי transgene דולף mutagenesis insertional. בין כל האסטרטגיות הלא-ויראלי הנ"ל, את PiggyBac (PB) מערכת transposon / transposase דורשת חוזרת מסוף ההפוך רק חובק transgene ו ביטוי חולף של אנזים transposase לזרז אירועי כניסה או כריתה 32. היתרון בשימוש transposons על פני שיטות אחרות לייצור תאי שב"ס הוא האפשרות לקבל תאי iPS ללא וקטור עם גישת וקטור שאינו ויראלי שמראה אותה היעילות של וקטורי retroviral. זה אפשרי על ידי כריתת עקבות-פחות של קידוד transposon המשולב עבור reprogramming גורמים הבאים ביטוי חולף חדש של transposase בתאי שב"ס 33. בהתחשב בכך PB הוא יעיל בסוגי תאים שונים 34, 35, 36, 37, הוא מתאים יותר עבור גישה קלינית ביחס וקטורים ויראליים, ומאפשר את ייצור תאי iPS ללא קסנו בניגוד פרוטוקולי ייצור ויראלי הנוכחיים משתמשות xenobiotic תנאים, מערכת זו משמשת להשיג תאי שב"ס מן בעכברים AF.
כאן אנו מציעים פרוטוקול מפורט לאחר עבודה שכבר פורסם להראות לייצור שיבוטי שב"ס פלוריפוטנטיים מתאי AF עכבר (תאי iPS-AF) 38.
Protocol
Representative Results
Discussion
השיטה שנבחרה כדי להשיג את האינדוקציה של pluripotency רלוונטית לבטיחות קלינית תא ביחס השתלה לטווח ארוכה. כיום, קיימות מספר שיטות מתאימות תכנות מחדש. בין השיטות הלא-אינטגרטיבי יותר, ויראלי סנדאי (SEV) הווקטור הוא וירוס RNA אשר יכול לייצר כמויות גדולות של חלבון ללא להשתלב בגרעין …
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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