Figure 1.
iPSC colonies on different feeder layers.
Three types of irradiated feeder layers have been used: murine embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and human bone marrow derived mesenchymal stromal cell (MSCs) (A). The morphology of iPSC colonies is exemplarily presented on each of these feeder layers (B). iPSC colonies were further characterized by immunofluorescent staining for the pluripotency markers OCT4 (green), TRA-1-60 (red) and SSEA3 (red) with nuclear counterstaining (DAPI, blue) (C,D). The initial colony-frequency and increase of colony-size was compared on different feeder layers: colony-frequency was significantly higher on irradiated MEFs than on HDFs (P = 0.03), or MSCs (P = 0.01; total colony counts (in four experiments): 333 with MEF, 225 with HDF, and 153 with MSC) (E) and the colony-size increased faster on MEFs than on HDFs or MSCs (P = 0.01 and P = 0.13, respectively) (F). Alternatively, induced fibroblasts were re-seeded in wells without preformed irradiated feeder cells. Non-induced fibroblasts from the parental cell preparation then grew out to provide a stromal support for iPSC colonies but their frequency was lower than using preformed irradiated feeders (total colony counts (in three experiments): 215 with HDF and 132 without feeder) (G). (** = P<0.01; * = P<0.05; n.s. = not significant).
Figure 2.
Generation of iPSCs in bulk culture.
Time axis for reprogramming of human dermal fibroblasts with episomal plasmids (A). Exemplary phase contrast images of bulk-cultured colonies are depicted in the course of culture-expansion. Already at day 6 the first changes in cell morphology could be noticed (arrows). Bulk cultured colonies revealed a typical ESC-like morphology after 10 passages (B).
Figure 3.
Comparison of cloned and bulk-cultured iPSC colonies.
Three weeks after transfection, individual ESC-like colonies were either manually picked from culture wells corresponding to independent transfections (to ensure clonal derivation from different parental cells), or all colonies per well were dissociated using collagenase IV and re-seeded on new feeder layer (bulk-culture) (A). Comparison of clonal and bulk-cultured iPSCs at passage 10 did not reveal any differences in immunofluorescence staining for OCT4 (green), TRA-1-60 and SSEA4 (red) with DAPI counterstaining (blue) (B). Loss of episomal plasmids over time is exemplified in fibroblasts which were transfected with an episomal vector for green fluorescent protein (GFP; representative results of three independent experiments are demonstrated) (C). Quantitative PCR analysis of the reprogramming factors (OCT3/4, LIN28, and SOX4) encoded by the three episomal vectors and their endogenous counterparts did not reveal stable integration in clonal and bulk-cultured colonies: episomal plasmids were only detected at day 7 and day 22 after transfection but not in any of the established iPSCs at passage 10 (Representative results of three independent experiments are demonstrated) (D).
Figure 4.
Gene expression profiles and in vitro differentiation of iPSCs.
Hierarchical clustering of global gene expression profiles revealed clear separation of non-transfected fibroblasts (F1, F2; green) and iPSCs (bulk: black; clonal: red). iF2_B1 and iF2_C1 grew nicely until passage 3, but were then lost due to bacterial contamination. iF4 was only cultured clonally without bulk counterpart. (A). Scatterplot analysis of mean signal intensity demonstrated very similar gene expression profiles of clonal and bulk-cultured iPSCs and statistical analysis did not reveal significant differences (B). PluriTest analysis supported the notion that bulk-cultured as well as clonally-derived iPSCs are pluripotent as they grouped with established pluripotent cells (red area) and not with somatic samples (blue area) (C). In vitro differentiation potential of iPSC colonies was evaluated by an embryoid body (EB) assay. After 10 days, each cell preparation revealed cystic structures (black arrows), areas with cells of epithelial morphology (white arrows) and spontaneous beating areas indicating cardiac differentiation (D). Up-regulation of differentiation markers AFP (endodermal marker), cTnT (mesodermal marker) and nestin (ectodermal marker) could be induced in all bulk-cultured and clonally derived iPSCs (E). Expression of ectodermal (GFAP, NKX6-1, PAX6, SOX1), mesodermal (PECAM1, CD34, MYH6), and endodermal markers (AFP, ALB, SOX17) was assessed by RT-qPCR: all of these genes were up-regulated upon differentiation, whereas OCT4 expression was down-regulated (F).