Figure 2: hiPSCs proliferation during long-term expansion
A. Cumulative cell population doublings for hiPSCs cultured on the FN1 motifs surface as well as on the Matrigel-coated surface for 20 successive passages. B. Cell population doubling time for hiPSCs cultured on the FN1 motifs surface as well as on the Matrigel-coated surface for 20 successive passages. C. Cell population doubling time for hiPSCs cultured on the FN1 motifs surface, the Matrigel-coated surface as well as on the Synthemax II-SC-coated surface for 10 successive passages. The results represent a mean of three independent wells.
hiPSCs expanded on Eppendorf CCCadvanced™ FN1 motifs surface maintain a functional pluripotency
To ensure that fundamental properties of undifferentiated hiPSCs expanded in this new well-defned culture system have not changed, extensive pluripotent cell characterizations were performed. During the entire expansion process, very low levels of spontaneous cell differentiation were observed for hiPSCs expanded on the FN1 motifs surface (< 2% across the entire surface). These observations, initially based on cell morphology, were confrmed by high level expression and activity of a specifc alkaline phosphatase (ALP) enzyme isoform present in pluripotent stem cells. hiPSCs expanded on the FN1 motifs surface exhibited a high and stable level of ALP expression/ activity during the entire expansion pro cess (Figure 3). Moreover, the ALP expression/activity high lighted was similar to that observed for hiPSCs expanded on the biological Corning Matrigel-coated surface.

Figure 3: Alkaline phosphase staining during long-term expansion of hiPSCs
hiPSCs cultured on the CCCadvanced FN1 motifs surface as well as on the Corning Matrigel-coated surface showed high and stable levels of ALP expression. The images show representative areas at passage numbers 3 and 20, respectively. Scale bar indicates 100 µm.
In order to complete the qualitative data set and to confrm the preservation of the cell pluripotency status after long term expansion on the FN1 motifs surface, the expression of several other key self-renewal markers was assessed using immunostaining and flow cytometry analysis. After 25 successive passages on the FN1 motifs surface, hiPSCs continued to exhibit a high expression level of two pluripotent specifc surface proteins, TRA-1-60 and SSEA4, and of two self- renewal-associated nuclear transcription factor proteins, SOX2 and OCT4 (Figure 4).

Figure 4: Immunofluorescence staining of pluripotency markers after long-term expansion of hiPSCs

After 25 successive passages on the CCCadvanced FN1 motifs surface, hiPSCs expressed the pluripotency markers SOX2, TRA-1-60, SSEA4 and OCT4, as evaluated through immunofluorescent staining. Cells were counterstained with a classic nuclear marker, DAPI. The images show representative areas of hiPSCs stained after 25 passages in culture. Scale bar indicates 400 µm.