Transplantation of Human Neural Progenitor Cells

Human NPCs were obtained according to NIH Ethical Guidelines and have been fully described in previous studies^{21 (link)–23 (link, link)}. Briefly, NPCs were grown for 13 doublings in ultra-low attachment culture flasks under feeder-free conditions in serum and xeno-free Eagle’s essential medium (Hyclone, Logan, UT, USA), supplemented with Gem21 (Gemini Bio-Products, Sacramento, CA, USA), epidermal growth factor (Peprotech, Rocky Hill, NJ, USA), basic fibroblast growth factor (Peprotech), transforming growth factor alpha (Peprotech), insulin-like growth factor I (Peprotech), leukemia inhibitory factor (Millipore, Temecula, CA, USA), calcium chloride (Fisher Scientific, Waltham MA, USA), Glutamax (Invitrogen, Carlsbad, CA, USA), non-essential amino acids (Hyclone), and an N2 supplement (Invitrogen), all of which were added at proprietary concentrations. The dead NPCs were implemented as a negative control for any paracrine effects from the live NPC inoculations. The dead NPCs were obtained from a live NPC population and then were placed without cryoprotectant into a −20°C freezer for 30 min to freeze kill the cells, and then stored in −80°C freezer until use. The suspended dead NPCs in culture medium were thawed for 1 h and then used immediately for transplantation in sHW mutants.