Dose-dependent accumulation and subpassage of CJD prions in iPSC-derived astrocytes. (A) Immunoblot analysis of astrocytes exposed to vCJD (MM) brain homogenate at five concentrations for 24 h and assayed immediately (0 dpe) or after recovery in fresh media for 3 d (3 dpe). MM (iPSC1) astrocytes replicate PrP^Sc in a concentration-dependent manner. MV (iPSC2) and VV (iPSC3) astrocytes failed to replicate PrP^Sc at 3 dpe. The experiment was conducted twice with similar results. (B) MM (iPSC1) astrocytes exposed to 1% spin-filtered vCJD brain homogenate. Both first and second passage were analyzed at 8 dpe. In the second passage experiment, the naive MM (iPSC1) astrocytes were exposed to vCJD-infected cell homogenate diluted to match PrP^Sc level of original vCJD brain homogenate used for exposure of astrocytes in the first passage. n = 2, in duplicate. (C) VV (iPSC3) astrocytes exposed to 1% spin-filtered sCJD (VV2) brain homogenate (first passage) and sCJD VV2–infected astrocyte homogenate (second passage). The naive VV (iPSC3) astrocytes were exposed to a whole sCJD-infected cell homogenate (1:1), i.e., cell lysate of a single well served as an inoculum for a new well because of a lower efficiency of sCJD prion propagation. Both passages were analyzed at 8 dpe. n = 2, in triplicate. Blots were developed using anti-PrP (A and B) 3F4 and (C) HuM-P antibodies. Molecular mass is indicated in kilodaltons. (B and C) Data are plotted with mean. PK-resistant PrP^Sc signal values in cell lysates were normalized by the PrP^Sc signal value of the inoculum used in each individual experiment. (D) Representative PrP and GFAP immunolabeling in VV (iPSC3) astrocytes exposed to 1% spin-filtered sCJD (VV2) brain homogenate (first passage, middle). VV astrocytes exposed to spin-filtered cell homogenate of astrocytes propagating sCJD (VV2; second passage, right). Both were analyzed at 8 dpe. PrP immunolabeling intensity (bright green) showed an increase in cell-associated PrP^Sc when astrocytes of first sCJD passage were compared with PrP^C of unexposed control cells (control, left), and PrP^Sc signal appeared more abundant in astrocytes in the second passage. n = 2, in triplicate. (E) Maximum intensity projection of Z stacks of VV (iPSC3) astrocytes immunolabeled for PrP at 8 dpe in cells exposed to sCJD (VV2) infected cell homogenate (right) and unexposed control (left). (D and E) Cells were immunolabeled with anti-PrP antibody 3F4. Nuclei were stained with DAPI (blue). Bars: (D) 20 µm; (E) 5 µm.