Journal: Aging Cell

Article Title: Protracted Fate Acquisition and Epigenetic De‐Aging During Induced Neural Stem Cell Conversion of Human Blood Cells

doi: 10.1111/acel.70751

Figure Lengend Snippet: CD71‐positive EPCs can be programmed into DACH1‐expressing NSCs within less than 2 weeks. (A) Peripheral blood‐derived EPCs were programmed into isogenic sets of directly converted iNSCs and iPS‐NSCs. Samples were collected at different stages of iNSC conversion, for example around the time point of clone picking (D14), at low passage (LP) right before high temperature treatment and at a stage when established iNSC lines were transgene‐free (TF), in order to investigate the dynamics of cellular age reprogramming in the process of iNSC conversion, using embryonic stage‐like iPS‐NSCs as controls. In the center of the figure, the components of the utilized iNSC media are listed. Components printed in bold are also constituents of the iPS‐NSC medium. Components marked with asterisks are only used until Day 10 of iNSC conversion. (B) Exemplary immunofluorescence images of cells stained against the EPC marker CD71 and the NSC marker DACH1 at different time points of iNSC conversion. Days indicate the time after SeV infection. Scale bars = 50 μm. Differences in cell size are most likely due to the cyto‐spin procedure used to adhere suspension cells prior to the staining procedure. (C) At Day 12 of conversion, arising iNSCs were stained against the NSC marker PAX6. Scale bar = 50 μm. (D) Representative immunofluorescence image of an established iNSC line stained against the NSC markers SOX2 and NES. This picture represents an excerpt of Figure . Scale bar = 50 μm.

Article Snippet: Afterwards, cells were stained with rabbit polyclonal IgG antibody to DACH1 (1:100; Proteintech, Planegg‐Martinsried, Germany) in 1× DPBS containing 1% BSA and 0.3% Triton‐X100 for 2 h at room temperature.

Techniques: Expressing, Derivative Assay, Immunofluorescence, Staining, Marker, Infection, Suspension