Saç türetilmiş keratinositlerden Kullanarak Entegrasyon serbest İnsan uyarılmış pluripotent kök hücrelerin üretilmesi
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
İnsan uyarılmış pluripotent kök hücreleri (hiPSCs) keşif hastaya özgü kök hücrelerin 1-3 üretimi için uygun bir yöntem sağlayan, rejeneratif tıp alanında devrim yarattı. hiPSCs başarılı fibroblastlar 4,5, hematopoietik hücrelerin 6,7, idrar 8 böbrek epitel hücreleri ve 9,10 keratinositler dahil olmak üzere çeşitli somatik hücre tiplerinin, elde edilmiştir. Bugüne kadar, deri fibroblastları ve hematopoietik hücreler hastaya özgü iPSCs üretilmesi için en yaygın olarak kullanılan hücre kaynağıdır. Muhtemelen, bu deri biyopsileri ve kan toplama rutin tıbbi prosedürler ve birçok ülkede kurulmuştur hastanın kan veya cilt örneklerinin büyük biyobankalar olmasından kaynaklanmaktadır.
Kan hücreleri ve invazif çıkarma yöntemleri gerektiren deri fibroblast aksine, keratinositler hiPSC üretimi için kolay erişilebilir bir hücre tipini temsil etmektedir. Keratinocytes cildin dış epidermal bariyer oluşturur ve ayrıca çivi ve saç 11'de bulunan keratin bakımından zengin epitel hücreleri vardır. Özellikle, keratinositler saç köklerinin, iç kök kılıfı (IRS) hücreleri (12, Şekil 1) ile birlikte saç gövdesine kaplı bir dış hücresel tabakanın dış kök kılıfı (ORS) bulunabilir. Saç toplama sağlık personelinin yardım gerektirmeyen, basit bir prosedür olduğu gibi hastaların toplamak ve büyük ölçüde hiPSC nesil için hasta örneklerinin koleksiyonu kolaylaştıracak laboratuvarlar, kendi saç örneklerini göndermek için, bir fırsat sağlar. Epidermal keratinositler, aynı zamanda hiPSC üretimi 9,13 başlangıç hücreleri gibi keratinositleri kullanmanın avantajları ekleyerek, fibroblastlar ile karşılaştırıldığında daha yüksek bir yeniden programlama verimliliği ve daha hızlı bir yeniden programlama kinetiği sahiptir. Ayrıca, hiPSCs da saç folikülü içinde diğer hücre popülasyonları kullanılarak üretilebilir,saç folikülü 14,15 tabanında bulunan dermal papilla hücrelerini dahildir.
Saç türetilmiş hücreleri kullanarak iPSC nesil Önceki raporlar genellikle retroviral veya lentiviral tabanlı yeniden programlama yöntemlerini 9,14,15 kullanmaktadır. Ancak, bu viral yöntemler yeniden programlama sırasında yabancı transgenlerin istenmeyen genomik entegrasyon tanıtmak. Karşılaştırma olarak, epizomal vektörlerin kullanımı entegrasyonu içermeyen iPSCs 4 oluşturmak için uygun bir non-viral yeniden programlama yöntemi temsil eder. Biz daha önce verimli epizomal vektörler 13 kullanan hiPSCs içine keratinosit yeniden programlamak için basit, düşük maliyetli ve viral olmayan bir yöntem geliştirdik. Burada koparıp saç büyüme ve keratinositler bakım ve hiPSCs oluşturmak için daha sonra yeniden programlama ile ilgili keratinosit türetilmesi dahil olmak üzere, keratinosit türetilmiş hiPSCs, üretimi için ayrıntılı bir protokol sağlar.
Protocol
Representative Results
Discussion
Hastaya özgü hiPSCs üretilmesi, in vitro olarak hastalıklı hücre tiplerinde patojenezinin araştırılması için eşsiz bir yaklaşım sunmaktadır ve ayrıca hastalığın fenotipleri kurtarabilir yeni moleküllerin belirlenmesi için ilaç tarama için bir platform sağlar. HiPSCs kullanılarak bu hastalığa modelleme yaklaşımı uzun QT sendromu, Huntington hastalığı, Parkinson hastalığı ve amiyotrofik lateral skleroz 22 de dahil olmak üzere çeşitli hastalıkların, umut vaat ede…
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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