Human Induced Pluripotent Stem Cell Technology Research

Aim1) Obtain fibroblasts from human skin and hair follicle cells from plugged hair cells.Besides, reprogramming fibroblasts with retrovirus pMX-hOct4, -hSox2, -hKlf4 and–hc-Myc to generate iPS cells.2) Cryopreservate iPS Cells with ministraws.Methods1. Methods of generating iPS cells1) With the informed consent, fibroblasts were generated by digestion from patients'skin, while whole plugged human hair follicle were cultured in neural stem cells medium to get hair follicle cells.2) Retrovirus were proved correctly by enzyme digestion: pMX-OCT4 weredigested by Xho I while pMX-hSOX2,pMX-hKLF4, and pMX-hc-Myc were digested by Not I.3) Retrovirus pMX-OSKM were proved efficiency by successful package and infection of pMX-GFP retrovirus. Flow cytometric analysis was recruited to Detect infection efficiency.4) Reprogramming fibroblasts to iPS cellsMixture of OSKM retrovirus at a ratio of 1:1:1:1 and 10ng/ml polybrene were added to 1x105 fibroblasts which were plated 24 hours before infection. Infection were repeated 24 hours later, and could be repeated as more as 3 times. 24 hours after last infection, fibroblasts were transferred to feeder layer cells, then cultured as ES cells until first ES like colony appeared.5) Characterization of iPS cells from fibroblasts. ES like colonies were stained withalkaline phosphatase and TRA-1-81 live cell staining antibody. Karyotype,ES specific markers,methylation of oct4 primer,differentiation in vivo and in vitro were executed on iPS cells post P10.2. Methods of Cryopreservation iPS Cells with ministraws.Colonies of characterized iPS cells were dissected into pieces containing approximately 100-200 cells using mechanical methods, then sequentially treated with 10%,20% vitrification solution, and sealed in ministraws together with the second solution. Ministraws were then dropped into liquid nitrogen immediately for vitrification and cryopreservation. To thaw iPS cells after vitrification preservation, after ministraws were thawed at room tempreture, iPS cells were immediately balanced in 0.2M sucrose solution and then in 0.1M sucrose solution, and at last plated on feeder layer cells.Results1. Establishment of human iPS cells. Fibroblasts from human dermal skin exhibited typical fibroblast morphology and normal karyotype; Besides, cells with cobblestone appearance grow around the plugged hair follicle root in neural stem cells medium. Post infection of retrovirus OSKM, fibroblasts were reprogrammed to iPS cells which exhibited all the characters of ES cells.2. Post vitrification and thawing, iPS cells maintain properties of pluripotent stem cells, including normal morphology, alkaline phosphatase staining,OCT4 and SOX2 expression, and differentiation to three germs or neural cells. Calculation of colony recovery rates after vitrification and thawing procedures indicates that approximate 77.4%±13.12% of iPS cell colonies survived the freezing and thaw procedures.Conclusion1. We generated fibroblasts from human dermal skin and hair follicle cells from plugged hair follicle cells; Meanwhile, fibroblasts can be reprogrammed to iPS cells via retrovirus OSKM.2. iPS cells from fibroblasts exhibit ES properties and maintain these properties in long term culture in vitro.3. Vitrification with ministraws is a very useful and effective cryopreservation method for iPS cells without negative effect.4. This experiment introduced and optimized iPS cells genenration related technologies, which laid a foundation for further iPS cells research and application in our lab.