4.13. Differentiation to Neural Progenitor Cells (NPCs) and Induced Astrocytes (iAs)

Both Ctrl2-SV4F-1 and GLDC27-FiPS4F-1 iPSC lines underwent differentiation into Neural Progenitor Cells (NPCs) following the supplier’s instructions. We implemented rigorous standardization of iPSC reprogramming and differentiation. Both cell lines were matched for time in culture and passages. Cells plating densities were optimized to prevent potential alterations due to local growth factors. A panel of cell markers was used to assess cell types along the differentiation pathway, and the quality of cultures was monitored by evaluating cellular functionality.

Briefly, iPSCs were seeded at a density of 250,000 cells per well in Matrigel-coated 6-well plates (Corning, Corning, NY, USA) using mTesR plus (STEMCell Technologies, Vancouver, BC, Canada) containing 10 μM ROCK inhibitor Y-27632 (STEMCell Technologies). On day 3, the medium was switched to a STEMdiff^TM SMADi Neuronal Induction Kit (STEMCell Technologies) and refreshed daily. By day 8, the cells were replated at a density of 250,000 cells/cm² on Matrigel-coated 6-well plates. This process was repeated twice more. After the third passage, the culture medium was changed to STEMdiff^TM Neural Progenitor Medium, with daily replacements. At this differentiation stage, cells were analyzed using RT-qPCR and immunofluorescence for expression of the NPC markers SOX1, PAX6, and NES. Cell banks in this passage were cryopreserved in STEMdiff^TM Neural Progenitor Freezing Medium, employing a slow freezing protocol (approximately 1 °C/min reduction) according to the supplier’s guidelines.

For astrocyte differentiation, cryopreserved NPCs were thawed in Matrigel-coated 6-well plates. Once 80–90% confluence was reached, NPCs were harvested using Acutase^TM (Millipore, Burlington, MA, USA) and plated at a density of 2 × 10⁵ cells/cm² in STEMdiff^TM Astrocyte Differentiation Medium (STEMCell Technologies) in Matrigel^® (Corning)-precoated 6-well plates. After seven days in culture, with daily medium changes, the first cell colony passage was performed. The cells were dissociated using Acutase^TM (Millipore) and seeded at 1.5 × 10⁵ cells/cm² in new Matrigel^®-treated 6-well plates (Corning). This process was repeated twice. From the second passage (day 14 of the differentiation process), the culture medium was changed every 48 h. Following the third passage, the culture medium was switched to STEMdiff^TM Astrocyte Maturation. After three further passages in STEMdiff^TM Astrocyte Maturation, the developed cell line was characterized through confocal microscopy, RT-qPCR analysis of specific markers, and functional evaluation of glutamate transport. Additionally, expression analysis of proteins related to NKH pathology, serine–glycine–one-carbon metabolism, and metabolite measurement in the culture medium was conducted at this stage.