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.
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.
Opdagelsen af menneskelige inducerede pluripotente stamceller (hiPSCs) har revolutioneret inden for regenerativ medicin, hvilket giver en realistisk metode til generering af patientspecifikke stamceller 1-3. hiPSCs succes er blevet genereret fra forskellige somatiske celletyper, herunder fibroblaster 4,5, hæmatopoietiske celler 6,7, renale epitelceller fra urin 8 og keratinocytter 9,10. Til dato, hudfibroblaster og hæmatopoietiske celler repræsenterer de mest almindeligt anvendte cellekilder til generering patientspecifikke iPSCs. Velsagtens, dette skyldes det faktum, at hudbiopsier og blodprøvetagning er rutinemæssige medicinske procedurer og store biobanker af patientens blod eller hud prøver er blevet etableret i mange lande.
I modsætning til blodceller og hud fibroblast som kræver invasive ekstraktionsmetoder, repræsenterer keratinocytter et lettilgængeligt celletype for hiPSC generation. Keratinocytes er keratin-rige epitelceller, der danner det ydre epidermale barriere i huden og også findes i negle og hår 11. Især kan keratinocytter findes på den ydre rod kappe (ORS), i hårfollikler, et ydre cellulære lag, der dækkede håret sammen med den indre rod kappe (IRS) celler (12, figur 1). Da hår kollektion er en simpel procedure, der ikke kræver hjælp fra medicinsk personale, giver det en mulighed for patienter at indsamle og sende deres egne hårprøver til laboratorier, som i høj grad ville lette indsamlingen af patientprøver for hiPSC generation. Epidermiskeratinocytter har også en højere omprogrammering effektivitet og hurtigere omprogrammering kinetik i forhold til fibroblaster, hvilket bidrager til fordelene ved anvendelse keratinocytter som startende celler for hiPSC generation 9,13. Endvidere kan hiPSCs også genereres under anvendelse af andre cellepopulationer i hårsækken,herunder hudpapillen celler placeret i bunden af hårsækken 14,15.
Tidligere rapporter om iPSC generation bruger hår-afledte celler ofte udnytter retroviral eller lentiviral-baserede omprogrammering metoder 9,14,15. Men disse virale metoder indføre uønsket genomisk integration af udenlandske transgener under omprogrammering. Til sammenligning anvendelse af episomale vektorer udgør en realistisk, ikke-viral omprogrammering metode til at generere integration-fri iPSCs 4. Vi har tidligere udviklet en enkel, omkostningseffektiv og ikke-viral metode til effektivt at omprogrammere keratinocyt i hiPSCs hjælp episomale vektorer 13. Her giver vi en detaljeret protokol til generering af keratinocytafledte hiPSCs, herunder udledning af keratinocytter fra plukkede hår, udbygning og vedligeholdelse af keratinocytter og efterfølgende omprogrammering for at generere hiPSCs.
Generation af patient-specifikke hiPSCs tilbyder en unik tilgang til at studere patogenese i de syge celletyper in vitro, og giver også en platform for narkotika screening for at identificere nye molekyler, der kan redde sygdommen fænotyper. Denne sygdom modelmetode hjælp hiPSCs har givet lovende resultater for en række sygdomme, herunder Long QT-syndrom, Huntingtons sygdom, Parkinsons sygdom og amyotrofisk lateral sklerose 22. Flere initiativer er allerede i gang for at etablere store biblioteke…
The authors have nothing to disclose.
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.
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 |