A Preliminary Study On The Differentiation Of Teratoma In Vitro Which Derived From Human Induced Pluripotent Stem Cells

Objective Induced pluripotent stem cells which were reprogramed from normalhuman keratinocytes were performanced teratomas assay in vivo to prove that they havethe potential to form several types of germ layer cells.Methods The Suspended iPSCs were injected into nude mice, tumor growth wasobserved in planting site. Eight weeks later, the tumor was excised under sterile conditions,and was divided into two equal parts. One was used for HE staining andimmunohistochemistry respectively, another was digested into single cell suspension, thencultured in KC, M254, DMEM media in vitro, respectively. The cultured cells wereobserved by inverted microscope and immunofluorescence microscopy and were stainedwith some markesResults After8weeks, the noculation tumors growth to0.36cm3, tumor cells werepatchy distribution groups, large stained nucleus, prominent nucleoli, abundantcytoplasm, more uniform size and shape, mitotic easy to see in HE staining. Tumor tissuewas subjected to immunohistochemical staining: Vimentin (+), Ki-67(+), CK (pan)(+),Oct3/4(+), GFAP (-), AFP (-).Cultured cells were observed under inverted microscopeafter a week.The cells were polygonal, closely arranged, adherent growth with part of athree-dimensional growth in KC culture medium. The cells were long fusiform adherentgrowth with sparse dendritic structure in M254culture medium. The cells were adherentcell growth with fusiform or ovaladherent in DMEM culture medium. Three medium ofcells expressed strong fluorescence in immunofluorescence microscopy. Cells from KCculture medium were subjected to immunohistochemical staining: Vimentin (+), CK (pan)(+), Oct3/4(+), Ki-67(+), GFAP (-), AFP (-).Conclusion Teratomas assay in vivo can generate tumors contain two germ layers of cells,proved KC-derived iPSCs have the potential to differentiate into multiplegerm layers of cells.