Generation der Integration freie Menschen induzierten pluripotenten Stammzellen Mit Haarstamm Keratinozyten
Summary
This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.
Abstract
Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.
Introduction
Die Entdeckung der menschlichen induzierten pluripotenten Stammzellen (hiPSCs) hat das Gebiet der regenerativen Medizin revolutioniert und bietet eine praktikable Methode zur Erzeugung von patientenspezifischen Stammzellen 1-3. hiPSCs wurden erfolgreich aus verschiedenen Zelltypen, einschließlich Fibroblasten, 4,5, 6,7 hämatopoetischen Zellen, Nieren-Epithelzellen aus Urin 8 und Keratinozyten 9,10 erzeugt. Bisher Haut-Fibroblasten und blutbildenden Zellen stellen die am häufigsten verwendeten Zellquellen zur Erzeugung von patientenspezifischen iPSCs. Dies mag auf die Tatsache zurückzuführen, dass Hautbiopsien und Blutentnahme sind Routine medizinischen Verfahren und großen Biobanken von Patienten Blut oder Hautproben wurden in vielen Ländern etabliert.
Im Gegensatz zu den Blutzellen und Hautfibroblasten, die invasive Extraktionsmethoden erfordern, Keratinozyten stellen eine leicht zugängliche Zelltyp für hiPSC Generation. Keratinocytes sind Keratin-reiche Epithelzellen, die das Äußere epidermalen Barriere der Haut zu bilden und werden auch in Nägel und Haare 11 gefunden. Insbesondere kann auf die Keratinozyten der äußeren Haarwurzelhülle (ORS) Haarfollikel von einer externen Zellschicht, die den Haarschaft zusammen mit dem inneren Wurzelscheide (IRS) Zellen (12, Figur 1) abgedeckt ermittelt werden. Wie Haar Sammlung ist ein einfaches Verfahren, das nicht die Unterstützung des medizinischen Personals erfordert, bietet es eine Chance für die Patienten zu sammeln und zu ihren eigenen Haarproben an Laboratorien, die die Sammlung von Patientenproben für hiPSC Generation erheblich erleichtern würde. Epidermiskeratinocyten auch eine höhere Wirksamkeit und eine schnellere Anpassung Umprogrammierung Kinetik im Vergleich zu Fibroblasten, zusätzlich zu den Vorteilen der Verwendung von Keratinozyten, wie die Ausgangszellen für die Erzeugung hiPSC 9,13. Weiterhin kann hiPSCs auch mit anderen Zellpopulationen in der Haarfollikel erzeugt werden,einschließlich der dermalen Papillenzellen an der Basis des Haarfollikels 14,15 entfernt.
Frühere Berichte des iPSC Generation mit Haar-abgeleiteten Zellen verwenden häufig retroviralen oder lentiviralen-basierte Verfahren der Reprogrammierung 9,14,15. Allerdings sind diese virale Verfahren einzuführen unerwünschte genomische Integration ausländischer Transgenen während Neuprogrammierung. Im Vergleich dazu ist die Verwendung von episomalen Vektoren, eine Möglichkeit, die nicht-virale Verfahren zum Umprogrammieren Integration freien iPSCs 4 zu erzeugen. Wir haben bereits eine einfache, kostengünstige und nicht-virale Verfahren zur effizienten umprogrammieren Keratinozyten in hiPSCs mit episomalen Vektoren 13 entwickelt. Hier stellen wir ein detailliertes Protokoll für die Erzeugung von Keratinozyten stamm hiPSCs, einschließlich der Ableitung von Keratinozyten aus gezupften Haare, Erweiterung und Wartung der Keratinozyten und anschließender Neuprogrammierung zu hiPSCs generieren.
Protocol
Representative Results
Discussion
Erzeugung von patientenspezifischer hiPSCs bietet einen einzigartigen Ansatz für die Untersuchung der Pathogenese in den erkrankten Zelltypen in vitro, und stellt auch eine Plattform zum Wirkstoff-Screening, neue Moleküle, die die Krankheitsphänotypen retten kann identifizieren. Diese Krankheit Modellansatz mit hiPSCs vielversprechende Ergebnisse für eine Vielzahl von Krankheiten, einschließlich Long-QT-Syndrom, Huntington-Krankheit, Parkinson-Krankheit und amyotrophe Lateralsklerose 22 ergab. M…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors wish to thank Harene Ranjithakumaran and Stacey Jackson for technical support. This work was supported in part by grants from the National Health and Medical Research Council (R.C.B. Wong, A. Pébay), the University of Melbourne (R.C.B. Wong), Retina Australia (R.C.B. Wong, S.S.C. Hung, A. Pébay) and the Ophthalmic Research Institute of Australia (R.C.B. Wong, S.S.C. Hung, A. Pébay); Australian Research Council Future Fellowship (A. Pébay, FT140100047), Cranbourne Foundation Fellowship (R.C.B. Wong); intramural funding from the National Institutes for Health (R.C.B. Wong, S.S.C. Hung) and operational infrastructure support from the Victorian Government.
Materials
| Antibiotic Mix: | |||
| 250 ng/ml Antimycotic amphotericin B | Sigma | A2942-20ml | Antibiotic mix is made up in PBS. |
| 1X Penicillin/Streptomycin | Invitrogen | 15140-122 | |
| PBS (-) | Invitrogen | 14190-144 | |
| Knockout Serum Replacement (KSR) medium: | KSR medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. bFGF is added fresh to the media before use. | ||
| 20% knockout serum replacement (KSR) | Invitrogen | 10828-028 | |
| DMEM/F12 with glutamax | Invitrogen | 10565-042 | |
| 1× MEM non-essential amino acid | Invitrogen | 11140-050 | |
| 0.5× Penicillin/Streptomycin | Invitrogen | 15140-122 | |
| 0.1 mM β-mercaptoethanol | Invitrogen | 21985 | |
| bFGF (10 ng/ml, added fresh) | Millipore | GF003 | |
| Keratinocyte medium: | |||
| EpiLife with 60 µM Calcium | Invitrogen | M-EPI-500-CA | |
| 1× Human keratinocyte growth supplement (HKGS) | Invitrogen | S-001-5 | |
| Fetal Bovine Serum (FBS) medium: | FBS medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. | ||
| 10% fetal bovine serum (FBS) | Invitrogen | 26140079 | |
| DMEM | Invitrogen | 11995-073 | |
| 0.5× Penicillin/Streptomycin | Invitrogen | 15140-122 | |
| 2 mM L-glutamine | Invitrogen | 25030 | |
| 0.25% trypsin-EDTA | Invitrogen | 25200-056 | |
| Extracellular Matrix (ECM): | |||
| Matrigel | Corning | 354234 | Aliquot Matrigel stock and store in -80°C following manufacturer’s instructions. Stock concentration of Matrigel varies slightly from batch to batch (~9mg/ml). We recommend to use 200µl matrigel for coating a 12-well plate (~150µg/well). |
| Coating Matrix Kit | Invitrogen | R-011-K | |
| Plasmids: | Note that pCXLE-eGFP is only used for monitoring transfection efficiency and is not required for reprogramming. | ||
| - pCXLE-eGFP | Addgene | 27082 | |
| - pCXLE-hOct3/4-shP53F | Addgene | 27077 | |
| - pCXLE-hSK | Addgene | 27078 | |
| - pCXLE-hUL | Addgene | 27080 | |
| Transfection reagent Fugene HD | Promega | E231B | |
| Gelatin (from porcine skin) | Sigma | G1890 | Make up 0.1% gelatin in distilled water. Autoclave before use. |
| Reduced Serum medium: OPTI-MEM | Invitrogen | 31985062 | |
| Accutase | Sigma | A6964-100ml | |
| Mouse embryonic fibroblast (MEF) feeder | MEF can be inactivated by mitomycin C treatment or irradiation as described previously 16. | ||
| 26G needle | Terumo | NN2613R | |
| 6-well plate (tissue culture treated) | BD Biosciences | 353046 | |
| 12-well plate (tissue culture treated) | BD Biosciences | 353043 | |
| 10 cm dish (tissue culture treated) | BD Biosciences | 353003 | |
| Dispase | Invitrogen | 17105-041 | Use at 10mg/ml |
| Collagenase IV | Invitrogen | 17104-019 | Use at 1mg/ml |
| TRA-160 antibody | Millipore | MAB4360 | Use at 5µg/ml |
| OCT4 antibody | Santa Cruz | SC-5279 | Use at 5µg/ml |
| NANOG antibody | R&D Systems | AF1997 | Use at 10µg/ml |
| MycoAlert Detection kit | Lonza | LT07-418 |
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