Generation af 3D hud Organoid fra ledningen blod-afledte induceret pluripotente stamceller
Summary
Vi foreslår en protokol, der viser hvordan du differentiere inducerede pluripotente stamceller-afledt keratinocytter og fibroblaster og generere en 3D hud organoid, ved hjælp af disse keratinocytter og fibroblaster. Denne protokol indeholder en yderligere skridt til at generere en humaniseret mus model. Teknikken præsenteret her vil forbedre dermatologiske forskning.
Abstract
Huden er kroppens største organ og har mange funktioner. Huden fungerer som en fysisk barriere og protektor af kroppen og regulerer kropsfunktioner. Biomimetik er efterligning af modeller, systemer og elementer i naturen med henblik på at løse komplekse menneskelige problemer1. Huden Biomimetik er et nyttigt værktøj til in vitro-sygdom forskning og in vivo regenerativ medicin. Menneskelige inducerede pluripotente stamceller (iPSCs) har karakteristisk for ubegrænset spredning og differentiering evne til tre Kim lag. Menneskets iPSCs er genereret ud fra forskellige primære celler, såsom blodlegemer, keratinocytter, fibroblaster og meget mere. Blandt dem, ledningen blod mononukleære celler (CBMCs) er dukket op som en alternativ celle kilde fra perspektivet af allogene regenerativ medicin. CBMCs er nyttige i regenerativ medicin, fordi human leukocyt antigen (HLA) at skrive er afgørende for cellen banksystem. Vi leverer en metode for differentiering af CBMC-iPSCs til keratinocytter og fibroblaster og generation af en 3D hud organoid. CBMC-iPSC-afledte keratinocytter og fibroblaster har kendetegn svarende til en primær cellelinie. 3D hud organoids genereres ved at lægge en epidermale lag på en dermal lag. Ved hjælp af transplantation denne 3D hud organoid, genereres en humaniseret mus model. Denne undersøgelse viser, at en 3D menneskelige iPSC-afledte hud organoid kan være en roman, alternative værktøj til dermatologiske research in vitro- og in vivo.
Introduction
Huden dækker den yderste overfladen af kroppen og beskytter indre organer. Huden har forskellige funktioner, herunder beskyttelse mod patogener, absorberende og opbevaring af vand, regulering af kropstemperatur, og udskiller kroppen affald2. Hudtransplantation kan klassificeres afhængig af huden kilde; grafts ved hjælp af huden fra en anden donor kaldes allografts, og grafts ved hjælp af patientens egen hud er autografts. Selv om en autograft er den foretrukne behandling på grund af sin lave afvisning risiko, er hud biopsier svære at udføre på patienter med alvorlige læsioner eller et utilstrækkeligt antal hudceller. Hos patienter med alvorlige forbrændinger er tre gange antallet af hudens celler nødvendige for at dække store områder. De begrænsede mængder af hudceller fra en patient krop resulterer i situationer, hvor allogenous transplantation er nødvendige. En allograft bruges midlertidigt, indtil autolog transplantation kan udføres, da det normalt er afvist af værtens immunsystem efter ca 1 uge3. For at overvinde afvisning af patientens immunsystem, skal grafts komme fra en kilde med de samme immune identitet som den patient4.
Menneskets iPSCs er en spirende kilde af celler for stamcelle terapi5. Menneskets iPSCs er genereret fra somatiske celler, ved hjælp af omprogrammering faktorer såsom OCT4, SOX2, Klf4 og c-Myc6. Ved hjælp af menneskelige iPSCs overvinder de etiske og immunologiske aspekter af embryonale stamceller (økonomiske)7,8. Menneskelige iPSCs har pluripotency og kan differentiere sig til tre Kim lag9. Forekomsten af HLA, en kritisk faktor for regenerativ medicin, bestemmer immunresponset og muligheden for afvisning10. Brugen af patient-afledte iPSCs løser problemerne med celle-kilde begrænsning og immunsystemet afvisning. CBMCs er også dukket op som en alternativ celle kilde for regenerativ medicin11. Obligatorisk HLA maskinskrivning, som opstår under CBMC bank, kan nemt bruges til forskning og transplantation. Yderligere, homozygot HLA-type iPSCs kan bredt anvende til forskellige patienter12. En CBMC-iPSC bank er en roman og effektiv strategi for Celleterapi og allogen regenerativ medicin12,13,14. I denne undersøgelse, vi bruger CBMC-iPSCs, differentierede keratinocytter og fibroblaster, og generere stratificeret 3D hudlag. Resultaterne fra denne undersøgelse tyder på, at en CBMC-iPSC-afledte 3D hud organoid er en roman værktøj til in vitro- og in vivo dermatologiske forskning.
Protocol
Representative Results
Discussion
Menneskets iPSCs er blevet foreslået som et nyt alternativ til personlig regenerativ medicin17. Patient-afledte personlig iPSCs afspejle patient karakteristika, der kan anvendes til sygdom modellering, stof screening og autolog transplantation18,19. Brugen af patient-afledte iPSCs kan også overvinde problemer vedrørende primærelementer, mangel på passende celle numre og immun reaktioner5,<sup class="…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af en bevilling fra Korea Healthcare Technology R & D-projektet, Ministeriet for sundhed, velfærd og familiespørgsmål, Republikken Korea (H16C2177, H18C1178).
Materials
| Adenine | Sigma | A2786 | Component of differentiation medium for fibroblast |
| AggreWell Medium (EB formation medium) | STEMCELL | 05893 | EB formation |
| Anti-Fibronectin antibody | abcam | ab23750 | Fibroblast marker |
| Anti-KRT14 antibody | abcam | ab7800 | Keratinocyte marker |
| Anti-Loricrin antibody | abcam | ab85679 | Stratum corneum marker |
| Anti-p63 antibody | abcam | ab124762 | Keratinocyte marker |
| Anti-Vimentin antibody | Santa cruz | sc-7558 | Fibroblast marker |
| BAND AID FLEXIBLE FABRIC | Johnson & Johnson | – | Bandage |
| Basement membrane matrix (Matrigel) | BD | 354277 | Component of differentiation medium for fibroblast |
| BLACK SILK suture | AILEEE | SK617 | Skin graft |
| CaCl2 | Sigma | C5670 | Component of epithelial medium for 3D skin organoid |
| Collagen type I | BD | 354236 | 3D skin organoid |
| Collagen type IV | Santa-cruz | sc-29010 | Component of differentiation medium for keratinocyte |
| Defined keratinocyte-Serum Free Medium | Gibco | 10744-019 | Component of differentiation medium for keratinocyte |
| DMEM, high glucose | Gibco | 11995065 | Component of differentiation medium |
| DMEM/F12 Medium | Gibco | 11330-032 | Component of differentiation medium |
| Essential 8 medium | Gibco | A1517001 | iPSC medium |
| FBS, Qualified | Corning | 35-015-CV | Component of differentiation medium for fibroblast and keratinocyte |
| Glutamax Supplement | Gibco | 35050061 | Component of differentiation medium for fibroblast |
| Insulin | Invtrogen | 12585-014 | Component of differentiation medium for fibroblast and keratinocyte |
| Iris standard curved scissor | Professional | PC-02.10 | Surgical instrument |
| Keratinocyte Serum Free Medium | Gibco | 17005-042 | Component of differentiation medium for keratinocyte |
| L-ascorbic acid 2-phosphata sesquimagnesium salt hydrate | Sigma | A8960 | Component of differentiation medium for keratinocyte |
| MEM Non-Essential Amino Acid | Gibco | 1140050 | Component of differentiation medium for fibroblast |
| Meriam Forceps Thumb 16 cm | HIROSE | HC 2265-1 | Surgical instrument |
| NOD.CB17-Prkdc SCID/J | The Jackson Laboratory | 001303 | Mice strain for skin graft |
| Petri dish 90 mm | Hyundai Micro | H10090 | Plastic ware |
| Recombinant Human BMP-4 | R&D | 314-BP | Component of differentiation medium for keratinocyte |
| Recombinant human EGF protein | R&D | 236-EG | Component of differentiation medium for keratinocyte |
| Retinoic acid | Sigma | R2625 | Component of differentiation medium for keratinocyte |
| T/C Petridish 100 mm, 240/bx | TPP | 93100 | Plastic ware |
| Transferrin | Sigma | T3705 | Component of epithelial medium for 3D skin organoid |
| Transwell-COL collagen-coated membrane inserts | Corning | CLS3492 | Plastic ware for 3D skin organoid |
| Vitronectin | Life technologies | A14700 | iPSC culture |
| Y-27632 Dihydrochloride | peprotech | 1293823 | iPSC culture |
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