Human iPSC-derived Neural Progenitor Cells

Cytotoxicity of 35 DNT chemicals on Cos-7, HepG2, iPSC, and NPC

The OECD test guideline 426 for assessing developmental neurotoxicity (DNT) of chemicals relies heavily on animal testing, which is costly, time-consuming, and raises ethical concerns. Kamata et al. presents an alternative protocol using human-induced pluripotent stem cells (iPSC) and their differentiation into neural progenitor cells (NPC) to evaluate DNT in a simpler, quicker, and less expensive manner.

Thirty-five DNT chemicals and acetaminophen (negative control) were tested on four cell types (Cos-7, HepG2, iPSC, and NPC) at various concentrations. After two days, cell survival was assessed using mitochondrial MTS reduction and cellular ATP level assays, and IC₅₀ values were calculated (Fig. 1). Most DNT chemicals showed concentration-dependent inhibition, though some, like aldicarb, n-hexane, thalidomide, and toluene, were not cytotoxic at their highest concentrations. The IC₅₀ values for most DNT chemicals were significantly lower in iPSC and NPC compared to Cos-7 and HepG2 cells (Fig. 2). Differences in IC₅₀ values were more pronounced between Cos-7 and HepG2 cells than between iPSC and NPC (Fig. 3). NPC passage numbers did not affect the results.

NPC Surface Modification with Esbp-PEG-lipid

Cell-based therapy offers a potential solution for stroke-related disorders, which have limited treatment options after 4.5 hours. Goel et al. explored enhancing stroke treatment using NPCs modified with Esbp-PEG-lipid. NPCs were surface-modified with Esbp-PEG-lipid of different PEG molecular weights (5 and 40 kDa) and optimized for binding to stroke area surfaces, such as recombinant E-selectin and TNF-α activated endothelium.

To confirm surface modification, FITC-Esbp-PEG-lipid-modified cells were analyzed by flow cytometry and confocal microscopy, and the number of Esbp molecules on the cell surface was quantified. Confocal images (Fig. 4(a) and (b)) showed green fluorescence from FITC on modified NPCs, but not on unmodified cells. Quantitative measurements (Fig. 4(c) and (d)) revealed a 50-fold increase in fluorescence intensity in treated NPCs compared to untreated cells, indicating successful incorporation of Esbp onto the NPC surface via PEG-lipid. The number of incorporated FITC-Esbp-PEG-lipids (Fig. 4(e)) depended on the feed concentration, with a maximum of approximately 2.0 × 10⁸ molecules for 40k and 2.0 × 10¹⁰ for 5k at concentrations above 120 µM. At 25 µM, the number of incorporated molecules was about 4.0 × 10⁷ for 40k and 1.0 × 10¹⁰ for 5k. These numbers were deemed sufficient for surface modification, so these cells were used for further experiments. The significant difference in the number of incorporated 5k and 40k PEG lipid molecules may be due to the larger size of 40k PEG causing more steric hindrance on cell surfaces.