Figure 4

Blocking proteasome activity in hESC. Proteasome inhibition causes differentiation of hESCs (A). HS181 colonies (p56) were grown 4 days on hFF and then treated 40 hours with the proteasome inhibitor MG132. Control HS181 colonies express POU5F1 (scale bar 25 μ) and display a normal karyotype. Upon treatment with MG132, HS181 colonies differentiated. No effect of proteasome inhibition on hFF (B). HFF express the fibroblastic marker P4H and display a normal karyotype. HFF were cultured with MG132 during 40 hours. No morphological alteration was observed. No effect of proteasome inhibition on hES-dF-HD90 (C). The HD90 hESC line was differentiated into fibroblasts like cells (hES-dF) that display morphologic feature of dermal fibroblasts and express P4H: bottom left. An undifferentiated colony of HD90 P4H^-, starts to differentiate at the edges into P4H⁺ hES-dF (upper left). hES-dF-HD90 cells were cultured with MG132 during 40 hours. No morphological alteration was observed. Down regulation of pluripotency transcription factor in hESC by proteasome blockage (D). The expression of POU5F1/OCT4, NANOG and SOX2 was measured by semiquantitative RT-PCR in HS181 hESC after 40 h culture with MG132. GAPDH was used as a control. Flow cytometry analysis after proteasome inhibition (E). HFF, hES-dF-HS181 and HS181 cells were treated 40 hours with MG132, and then the cell surface fibroblastic markers CD13 and CD44, and the pluripotent cell surface marker TRA-1-60 were measured by FACS. Markers for fibroblastic cells were not altered with MG132 treatment, whereas the marker for pluripotency dropped to barely detectable level. Y-axis: percentage of the control (untreated) sample.