<i>In Vitro</i> differentiation potential of protein iNPCs.

<p>(A) Human protein iNPC-derived neural cells exhibited typical neuronal morphology. (B) Quantification of MAP2⁺ neurons, GFAP⁺ astrocytes and O4⁺ oligodendrocytes among total cells. Data are represented as mean ± SD. (C) Differentiation potential of iNSCs into neurons by TUJ1, astrocytes as determined by GFAP, and oligodendrocytes by O4 antibody via immunocytochemistry. DAPI staining is shown in blue. (D) MAP2⁺ neurons derived from iNPCs. (E) Dotted Synapsin expression in proximity of the TUJ1⁺ nerve fibers suggested morphological synapse formation. (F) The major population of iNPC-derived neurons was GABA⁺. (G) A small population of neurons were TH⁺. TUJ: β-Tubulin class III; MAP2: Microtubule-associated protein 2; TH: Tyrosine hydroxylase.</p

Induction of Neural Progenitor-Like Cells from Human Fibroblasts via a Genetic Material-Free Approach

<div><p>Background</p><p>A number of studies generated induced neural progenitor cells (iNPCs) from human fibroblasts by viral delivering defined transcription factors. However, the potential risks associated with gene delivery systems have limited their clinical use. We propose it would be safer to induce neural progenitor-like cells from human adult fibroblasts via a direct non-genetic alternative approach.</p><p>Methodology/Principal Findings</p><p>Here, we have reported that seven rounds of TAT-SOX2 protein transduction in a defined chemical cocktail under a 3D sphere culture gradually morphed fibroblasts into neuroepithelial-like colonies. We were able to expand these cells for up to 20 passages. These cells could give rise to cells that expressed neurons and glia cell markers both <i>in vitro</i> and <i>in vivo</i>.</p><p>Conclusions/Significance</p><p>These results show that our approach is beneficial for the genetic material-free generation of iNPCs from human fibroblasts where small chemical molecules can provide a valuable, viable strategy to boost and improve induction in a 3D sphere culture.</p></div

<i>In Vivo</i> transplantation of iNPCs.

<p>(A) Transplanted GFP⁺ protein iNPCs were TUJ⁺ at 10 days post-transplantation into the brain of a rat pup. Nuclei were counterstained with DAPI. (B) GFAP⁺ astrocytes were derived from GFP⁺ iNPCs <i>in vivo</i>.</p

Assessment of TAT recombinant protein transduction.

<p>(A) Visualization of fusion proteins in in suspension cultured HFFs transduced with 10 μg/ml TAT-EGFP for 24 h. Scale bars represent 200 μM. (B) To determine protein stability, 10 μg/ml of TAT-EGFP proteins were added to HFFs for 24, 48 and 72 h, and the presence of transduced protein in the cells were analyzed by flow cytometery (M; monolayer and S; 3D sphere culture). (C) The uptake and stability of the TAT fusion protein were visualized by TAT immunostaining of transduced human fibroblasts after 24 and 48 h post-TAT-SOX2 transduction (Scale bars: 200 μm). (D) The middle panels represent double staining TAT and SOX2 with higher magnification in transduced cells at 24 h post-transduction (50 μm). The nuclei were counterstained with DAPI. (E) Determining the time required for protein transduction. Relative expression of neural markers (<i>SOX2</i> and <i>PAX6</i>) induced by 10 μg/ml TAT-SOX2 with a 48 h transduction interval (d0: Non-transduced cells).</p

Characterization of established protein iNPCs.

<p>(A) Phase contrast of established iNSCs morphology at higher passages (p20). (B) Real-time analysis of iNPC and ES-derived NPC (ES-NPC) for neural progenitor markers. (C) Expressions of Nestin, PAX6 and SOX2 were visualized by immunostaining. Nuclei were counterstained with DAPI. (D) Flow cytometric analysis of neural progenitor markers (PAX6 and SOX2) and proliferation marker Ki67 in iNPCs. (E) Single-cell-per-well experiments illustrated that the established protein iNPC grew clonally. (F) Real-time analysis of secondary spheres of iNPCs for neural progenitor markers. (G) Relative expression levels of region-specific marker genes over human fibroblasts, whose expression was considered to be 1 for all genes.</p