Generatie van Integratie-vrije mens geïnduceerde pluripotente stamcellen Met behulp van Hair-afgeleide keratinocyten
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
De ontdekking van de mens geïnduceerde pluripotente stamcellen (hiPSCs) heeft een revolutie op het gebied van regeneratieve geneeskunde, die een haalbare methode voor het genereren van patiënt-specifieke stamcellen 1-3. hiPSCs succes gegenereerd uit verschillende somatische celtypen, waaronder fibroblasten 4,5, 6,7 hematopoëtische cellen, epitheelcellen van nier urine 8 en keratinocyten 9,10. Tot op heden huidfibroblasten en hematopoietische cellen zijn de meest gebruikte cel voor het genereren van patiëntspecifieke iPSCs. Dit is wellicht te wijten aan het feit dat de huid biopten en bloedafname zijn routinematige medische procedures en grote biobanken van de patiënt bloed of huidmonsters hebben in veel landen vastgesteld.
In tegenstelling tot bloedcellen en de huid fibroblast die invasief extractie vereisen, keratinocyten vormen een gemakkelijk toegankelijke celtype voor iPSC generatie. Keratinocytes zijn keratine-rijke epitheliale cellen die de buitenkant epidermale barrière van de huid te vormen en zijn ook gevonden in nagels en haren 11. In het bijzonder kunnen keratinocyten te vinden op de buitenste wortelschacht (BHS) van haarfollikels, een uitwendige cellaag die de haarschacht bedekt met de binnenste schede (IRS) cellen (12, figuur 1). Zoals haar collectie is een eenvoudige procedure die de hulp van medisch personeel niet vereist, het biedt een mogelijkheid voor patiënten te verzamelen en sturen hun eigen haar monsters naar laboratoria, die sterk het verzamelen van monsters van patiënten voor iPSC generatie zou vergemakkelijken. Epidermale keratinocyten hebben ook een hogere efficiëntie en snellere herprogrammering herprogrammering kinetiek vergeleken met fibroblasten, waardoor de voordelen van het gebruik keratinocyten als uitgangspunt cellen voor iPSC productie 9,13. Bovendien kan hiPSCs ook opgeroepen met andere celpopulaties in de haarfollikel,waaronder de dermale papilla cellen aan de onderkant van de haarfollikel 14,15.
Vorige meldingen van iPSC generatie met behulp van haar afgeleide cellen gebruiken vaak retrovirale of-lentivirale gebaseerde herprogrammering methoden 9,14,15. Echter, deze virale methoden introduceren ongewenste genomische integratie van buitenlandse transgenen tijdens het herprogrammeren. Ter vergelijking, het gebruik van episomale vectoren vertegenwoordigt een mogelijke, niet-virale herprogrammering methode-integratie vrije iPSCs 4 te genereren. We hebben eerder ontwikkelde een eenvoudige, kosteneffectieve en non-virale methode om efficiënt herprogrammeren keratinocyten in hiPSCs middels episomale vectoren 13. Hier geven we een gedetailleerd protocol voor het genereren van uit keratinocyt verkregen hiPSCs, waaronder de afleiding van keratinocyten uit geplukt haren, uitbreiding en onderhoud van de keratinocyten en daaropvolgende herprogrammering hiPSCs genereren.
Protocol
Representative Results
Discussion
Generatie van patiënt-specifieke hiPSCs biedt een unieke benadering voor het bestuderen van de pathogenese van de zieke celtypen in vitro, en biedt ook een platform voor drug discovery om nieuwe moleculen die de ziekte fenotypes kan redden identificeren. Deze ziektemodel benaderingswijze via hiPSCs heeft veelbelovende resultaten opgeleverd voor diverse ziekten, waaronder lange QT-syndroom, ziekte van Huntington, ziekte van Parkinson en amyotrofe laterale sclerose 22. Diverse initiatieven zijn al aan…
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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