Skip to main content
Fig. 1 | Stem Cell Research & Therapy

Fig. 1

From: Apoptosis and failure of checkpoint kinase 1 activation in human induced pluripotent stem cells under replication stress

Fig. 1

hiPS cells generated with an mRNA-based integration-free method display typical characteristics of hES cells. a Timeline and essential steps for the reprogramming of human fibroblasts into mRNA-induced iPS cells. Human fibroblasts were plated 1 day before the first transfection in FBSm media, on a human feeder-coated dish. Cells were transfected daily with synthetic mRNA encoding the factors OCT4, SOX2, KLF4, C-MYC, and LIN28 in Pluriton™ reprogramming medium plus B18R (all provided by Stemgent) and kept in norm-oxygen (21 %) conditions. iPS colonies appeared between days 15 and 21, when they were mechanically picked and moved onto feeders in KSR medium. b Morphological changes of human fibroblasts throughout the reprogramming period in norm-oxygen. Typical fibroblast morphology at the start (day 0), transitioning cells with epithelial morphology half-way (day 10), until embryonic stem cell-like colonies have formed (day 19). Magnification: 40× (background images), digital zoom (smaller windows). c hiPS cell lines express typical intracellular and extracellular pluripotency markers. Immunofluorescence staining with monoclonal antibodies of stem cell markers OCT4, TRA-1-81, and SSEA4 (red) and Hoechst DNA counterstain (blue) shown for iPS cell lines MIFF1 and MIFF3. Magnification: 40× (background images), digital zoom (smaller windows). d Established hiPS cells are able to differentiate and induce markers of three germ layers in a 7-day EB differentiation assay. Early ectoderm marker PAX6, mesoderm marker brachyury, and endoderm marker AFP are upregulated in day 7 EBs, as analyzed by RT-PCR. Nanog, a marker of undifferentiated hES cells, decreases its expression upon differentiation. Beta-actin is used as a housekeeping gene. e Representative images of H & E-stained microsections of a teratoma generated after injection of hiPS cells into immunocompromised mice. Teratomas were extracted 9–12 weeks after injection and fixed in formaldehyde before embedding and sectioning. Sections show the presence of cartilage (mesoderm), intestinal glandular-like structure (endoderm), and neural tissue (ectoderm), representing derivative of the three germ layers. Magnification: 160×. AFP alpha-fetoprotein, UD undifferentiated cells, EB embryoid body, FBS fetal bovine serum, hES human embryonic stem, MIFF mRNA-induced foreskin fibroblast, PAX6 paired box 6, SSEA stage-specific embryonic antigen

Back to article page