A reliable and economical method for gaining mouse embryonic fibroblasts capable of preparing feeder layers
Tóm tắt
Mouse embryonic fibroblasts (MEFs) are widely used to prepare feeder layers for culturing embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in vitro. Transportation lesions and exorbitant prices make the commercially obtained MEFs unsuitable for long term research. The aim of present study is to establish a method, which enables researchers to gain MEFs from mice and establish feeder layers by themselves in ordinary laboratories. MEFs were isolated from ICR mouse embryos at 12.5–17.5 day post-coitum (DPC) and cultured in vitro. At P2–P7, the cells were inactivated with mitomycin C or by X-ray irradiation. Then they were used to prepare feeder layers. The key factors of the whole protocol were analyzed to determine the optimal conditions for the method. The results revealed MEFs isolated at 12.5–13.5 DPC, and cultured to P3 were the best choice for feeder preparation, those P2 and P4–P5 MEFs were also suitable for the purpose. The P3–P5 MEFs treated with 10 μg/ml of mitomycin C for 3 h, or irradiated with X-ray at 1.5 Gy/min for 25 Gy were the most suitable feeder cells. Treating MEFs with 10 μg/ml of mitomycin C for 2.5 h, 15 μg/ml for 2.0 h, or irradiating the cells with 20 Gy of X-ray at 2.0 Gy/min could all serve as alternative methods for P3–P4 cells. Our study provides a reliable and economical way to obtain large amount of qualified MEFs for long term research of ESCs or iPSCs.
Tài liệu tham khảo
Abraham S, Riggs MJ, Nelson K, Lee V, Rao RR (2010) Characterization of human fibroblast-derived extracellular matrix components for human pluripotent stem cell propagation. Acta Biomater 6:4622–4633
Aoshima K, Baba A, Makino Y, Okada Y (2013) Establishment of alternative culture method for spermatogonial stem cells using knockout serum replacement. PLoS One 8:e77715
Burt RK, Chen YH, Verda L, Lucena C, Navale S, Johnson J, Han X, Lomasney J, Baker JM, Ngai KL, Kino A, Carr J, Kajstura J, Anversa P (2012) Mitotically inactivated embryonic stem cells can be used as an in vivo feeder layer to nurse damaged myocardium after acute myocardial infarction: a preclinical study. Circ Res 111:1286–1296
Chen HF, Chuang CY, Shieh YK, Chang HW, Ho HN, Kuo HC (2009) Novel autogenic feeders derived from human embryonic stem cells (hESCs) support an undifferentiated status of hESCs in xeno-free culture conditions. Hum Reprod 24:1114–1125
Eiselleova L, Peterkova I, Neradil J, Slaninova I, Hampl A, Dvorak P (2008) Comparative study of mouse and human feeder cells for human embryonic stem cells. Int J Dev Biol 52:353–363
Genbacev O, Krtolica A, Zdravkovic T, Brunette E, Powell S, Nath A, Caceres E, McMaster M, McDonagh S, Li Y, Mandalam R, Lebkowsk J, Fisher SJ (2005) Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil Steril 83:1517–1529
Hongisto H, Vuoristo S, Mikhailova A, Suuronen R, Virtanen I, Otonkoski T, Skottman H (2012) Laminin-511 expression is associated with the functionality of feeder cells in human embryonic stem cell culture. Stem Cell Res 8:97–108
Hu K, Yu J, Suknuntha K, Tian S, Montgomery K, Choi KD, Stewart R, Thomson JA, Slukvin II (2011) Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells. Blood 117:e109–e119
Jozefczuk J, Drews K, Adjaye J (2012) Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells. J Vis Exp 64:e3854
Kibschull M, Mileikovsky M, Michael IP, Lye SJ, Nagy A (2011) Human embryonic fibroblasts support single cell enzymatic expansion of human embryonic stem cells in xeno-free cultures. Stem Cell Res 6:70–82
Klimanskaya I, Chung Y, Meisner L, Johnson J, West MD, Lanza R (2005) Human embryonic stem cells derived without feeder cells. Lancet 365:1636–1641
Ludwig TE, Bergendahl V, Levenstein ME, Yu J, Probasco MD, Thomson JA (2006) Feeder-independent culture of human embryonic stem cells. Nat Methods 3:637–646
Nieto A, Cabrera CM, Catalina P, Cobo F, Barnie A, Cortes JL, Barroso del Jesus A, Montes R, Concha A (2007) Effect of mitomycin-C on human foreskin fibroblasts used as feeders in human embryonic stem cells: immunocytochemistry MIB1 score and DNA ploidy and apoptosis evaluated by flow cytometry. Cell Biol Int 31:269–278
Park SP, Lee YJ, Lee KS, AhShin H, Cho HY, Chung KS, Kim EY, Lim JH (2004) Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers. Hum Reprod 19:676–684
Prowse AB, McQuade LR, Bryant KJ, Marcal H, Gray PP (2007) Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast conditioned media. J Proteome Res 6:3796–3807
Richards M, Fong CY, Chan WK, Wong PC, Bongso A (2002) Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 20:933–936
Richards M, Tan S, Fong CY, Biswas A, Chan WK, Bongso A (2003) Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. Stem Cells 21:546–556
Roy A, Krzykwa E, Lemieux R, Neron S (2001) Increased efficiency of gamma-irradiated versus mitomycin C-treated feeder cells for the expansion of normal human cells in long-term cultures. J Hematother Stem Cell Res 10:873–880
Skottman H (2010) Derivation and characterization of three new human embryonic stem cell lines in Finland. In Vitro Cell Dev Biol Anim 46:206–209
Stojkovic P, Lako M, Stewart R, Przyborski S, Armstrong L, Evans J, Murdoch A, Strachan T, Stojkovic M (2005) An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 23:306–314
Strom S, Holm F, Bergstrom R, Stromberg AM, Hovatta O (2010) Derivation of 30 human embryonic stem cell lines—improving the quality. In Vitro Cell Dev Biol Anim 46:337–344
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676
Tecirlioglu RT, Nguyen L, Koh K, Trounson AO, Michalska AE (2010) Derivation and maintenance of human embryonic stem cell line on human adult skin fibroblast feeder cells in serum replacement medium. In Vitro Cell Dev Biol Anim 46:231–235
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147
Unger C, Gao S, Cohen M, Jaconi M, Bergstrom R, Holm F, Galan A, Sanchez E, Irion O, Dubuisson JB, Giry-Laterriere M, Salmon P, Simon C, Hovatta O, Feki A (2009) Immortalized human skin fibroblast feeder cells support growth and maintenance of both human embryonic and induced pluripotent stem cells. Hum Reprod 24:2567–2581
Vallier L, Alexander M, Pedersen RA (2005) Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J Cell Sci 118:4495–4509
Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19:971–974
Xu C, Jiang J, Sottile V, McWhir J, Lebkowski J, Carpenter MK (2004) Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth. Stem Cells 22:972–980
Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA (2009) Human induced pluripotent stem cells free of vector and transgene sequences. Science 324:797–801
Zhang K, Cai Z, Li Y, Shu J, Pan L, Wan F, Li H, Huang X, He C, Liu Y, Cui X, Xu Y, Gao Y, Wu L, Cao S, Li L (2011) Utilization of human amniotic mesenchymal cells as feeder layers to sustain propagation of human embryonic stem cells in the undifferentiated state. Cell Reprogram 13:281–288