This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.
For nylig har tiltrukket sig opmærksomhed iPSCs som en ny kilde af celler til regenerative terapier. Selv om den oprindelige fremgangsmåde til generering iPSCs påberåbte dermale fibroblaster opnået ved invasiv biopsi og retroviral genomisk indsættelse af transgener, har der været mange forsøg på at undgå disse ulemper. Humane perifere T-celler er en unik cellekilde til generering iPSCs. iPSCs afledt af T-celler indeholder omlejringer af T-cellereceptoren (TCR) gener og er en kilde til antigen-specifikke T-celler. Derudover T-cellereceptor omlejring i genomet har potentiale til at mærke individuelle cellelinier og skelne mellem transplanterede og donorceller. For sikker klinisk anvendelse af iPSCs, er det vigtigt at minimere risikoen for at udsætte nyligt genererede iPSCs for skadelige stoffer. Selv kalvefosterserum og fødeceller har været af afgørende betydning for pluripotente stamcelle kultur, foretrækkes det at fjerne dem fra dyrkningssystemet at mindske risikoenaf uforudsigelige patogenicitet. For at løse dette, har vi etableret en protokol til generering iPSCs fra humane perifere T-celler under anvendelse af Sendai-virus for at reducere risikoen for at udsætte iPSCs udefinerede patogener. Selvom håndtering Sendai-virus kræver udstyr med passende level-Sendai virus inficerer aktiverede T-celler uden indføring genom, men med høj effektivitet. I denne protokol, viser vi dannelsen af iPSCs fra humane perifere T-celler i fødefri betingelser under anvendelse af en kombination af aktiveret T-celle-kultur og Sendai-virus.
iPSCs har tiltrukket stor opmærksomhed som en banebrydende kilde til celler til regenerativ medicin 1-3. Til dato er der rapporteret diverse metoder til at generere iPSCs 4,5. Blandt disse har iPSCs genereret fra humane T-celler været af særlig interesse på grund af de mindre invasiv metode til celleprøveudtagningsproceduren 6-8. Derudover iPSCs afledt af T-celler indeholder omlejringer af T-cellereceptoren (TCR) genet og er således en kilde til antigen-specifikke T-celler 9,10. Derfor generere T-celle afledt iPSCs sikkert er nyttig til forløber regenerativ medicin.
Denne metode er baseret på princippet om at reducere risikoen for uforudsigelige patogenicitet. For sikker klinisk anvendelse af iPSCs er det vigtigt at reducere risikoen for eksponering for patogener 11. Tidligere i mange dyrkningssystemer af pluripotente stamceller, har kalvefosterserum og fødeceller blevet anvendt som væsentlige reagenss 12. Dog er udtagelse af begge disse reagenser fra dyrkningssystemet er at foretrække for iPSC generation til at reducere risikoen for uforudsigelige patogenicitet.
Desuden er denne fremgangsmåde har den fordel, at man undgår invasive celleprøveudtagningsproceduren fra patienter og besværlig fremstilling af fødeceller. Fordi T-celle-afledte iPSCs allerede er blevet anvendt med succes i sygdom forskning 13,14, er denne fremgangsmåde også anvendelig og nyttig til frembringelse af sygdomsspecifikke iPSCs fra patienter.
Blandt T-celle-omprogrammering metoder, ved hjælp af Sendai-virus (SeV) vektor som et gen køretøj er en metode, der kan generere iPSCs med høj effektivitet 7,16. Desuden, fordi SeV er en enkeltstrenget RNA-virus og behøver ikke en DNA fase for replikation, dets anvendelse i iPSC generation undgår at bryde værtsgenomet 17-19. Derfor har vi etableret protokoller til generering iPSCs fra humane perifere T-celler i serum-frEE og fødefri betingelser ved anvendelse af en kombination af matrigel, mTeSR medium og SeV-vektorer.
We describe a protocol for generating iPSCs from human peripheral T cells in serum-free and feeder-free conditions using a combination of matrigel, mTeSR medium, and SeV vectors. For clinical applications of iPSCs, it is important to have a protocol for stably generating iPSCs and a less-invasive method for cell sampling. Although generating iPSCs with the combination of matrigel and mTeSR medium showed lower reprogramming efficiency than that with knockout serum replacement (KSR) medium and feeder-cells, this combination achieves stable generation of iPSCs from donors16. Stable iPSC generation and less-invasive cell sampling has the advantage of being able to increase the number of donors for iPSC generation.
SeV vectors, a minus-strand RNA virus, is not integrated into the host genome. Therefore the risks of tumorigenesis can be avoided at the step of reprogramming factor induction20-24. Additionally, the feeder-free conditions, which use a combination of defined culture medium and matrigel instead of a feeder layer, make it possible to minimize the potential risks of exposure to unknown exogenous factors. The fusion of these techniques provides a less invasive and safer iPSC technology for regenerative medicine16.
Important steps in this protocol are the step of activating human T cells (Step 1) and infecting them with SeV vectors (Step 2). When no ESC-like colony is obtained in the culture dish at an optimal time after SeV infection, the following should be considered. First, the confluency of the mononuclear cells before T cell activation may not be appropriate because optimal activation of T cells is critical for SeV infection25,26. Too high a density of mononuclear cells leads to cell death, interfering with the proper activation of T cells. Too low a density of mononuclear cells also disturbs the proper activation and proliferation of T cells. Therefore, the confluency of mononuclear cells should be checked and adjusted accordingly. Second, the dosage of SeV vectors may not be sufficient. The induction efficiency of iPSC colonies depends on the dosage of the virus6. If no ESC-like colony is observed after SeV infection, the option of increasing the virus dosage up to an MOI of 15-20 should be considered. If signs of iPSC colony differentiation are observed, the frequency at which medium is changed may be increased up to every other day, or to every day when colonies are larger.
The limitation in this protocol consists of this method not being virus free. Although SeV for cell reprogramming is commercially available with ease, users need to prepare equipment according to the appropriate biosafety level. As another method for generating integration-free iPSCs, episomal vectors have been used until now27-29. Although episomal vectors can be used with equipment with a lower level of biosafety, episomal vectors might insert into the host genome at extremely low rates. Therefore, additional checks are required to confirm the disappearance of transgenes, as when using SeV.
There is another limitation in that this technique is not absolutely free from animal-derived products. Some substrates, such as matrigel, anti-CD3 mAb, dissociation solution and SeV solution are derived from animal products and are therefore associated with a risk of transferring xenogeneic pathogens. However, the reduction of animal-derived substrates in the culture system is meaningful for the clinical application of iPSC technology due to the lower risk.
The authors have nothing to disclose.
Vi takker Yoshiko Miyake, Sayaka Kanaami, Chihana Fujita, Miho Yamaguchi, Natsuko Henmi, og Rei Ohno fra Keio University School of Medicine til teknisk bistand. Dette arbejde blev delvist finansieret af en R & D Systems støtteprogram til at fremskynde den praktiske anvendelse af sundheds- forskningsresultater, og Highway Program for realiseringen af regenerativ medicin.
Ficoll-Paque PREMIUM | GE Healthcare | 17-5442-02 | |
Purified NA/LE mouse anti-human CD3 | BD Pharmingen | 555336 | |
KBM502 medium | KOHJIN BIO | 16025020 | Warm in 37 ℃ water bath before use |
Bovine albumin fraction V solution | Gibco | 15260-037 | |
BD Matrigel Matrix Growth Factor Reduced | BD Biosciences | 354230 | Thaw completely at 4℃ overnight and dilute it 50 times with Dulbecco's Modified Eagle's Medium before coating culture dishes |
mTeSR1 medium kit | STEM CELL | 5850 | Warm at room temperature before use |
Dissociation Solution | ReproCELL | RCHETP002 | |
D-PBS(–) | Wako | 045-29795 | |
SeV Vector kit CytoTune-iPS ver.1.0 | DNAVEC | DV-0303c | Thaw on ice before use |
100-mm tissue culture dish | Falcon | 353003 | |
96-well tissue culture plate | Falcon | 353078 | |
6-well tissue culture plate | Falcon | 353046 | |
15ml Centrifuge Tube | Greiner Bio-One | 188271 | |
50ml Centrifuge Tube | Greiner Bio-One | 227261 |