Fig 2: Characterization of TSC2 +/+ and TSC2 -/- cell lines. (A) A hES line harboring a TSC2 knockout (TSC2 -/-) and an isogenic control line (TSC2 +/+) were generated at La Roche-Hoffmann Ltd. and NSCs derived from these hES lines were characterized immunologically. No differences could be observed in the expression of the pluripotency markers SOX2, Nanog, and Oct4 in the TSC2 +/+ and TSC2 -/- hES. Also, TSC2 +/+ and TSC2 -/- NSCs expressed the NSC markers Nestin and Pax6 in a similar manner. (B) Neuronal differentiation of TSC2 +/+ and TSC2 -/- hES was performed by dual SMAD inhibition for 80 days. After 10 days, the neuroepithelial sheet consisting of Pax6-positive NSCs had formed in both cultures followed by Pax6-positive neural rosettes around day 18. Rosettes in TSC2 -/- cultures tended to be bigger and cell-denser as in wildtype cultures. After 80 days of maturation, both lines generated TUJ1-positive neurons and few GFAP-positive astrocytes. However, TSC2 -/- neuronal cultures were found to be denser despite a similar initial plating density.

Fig 3: Impact of a low glucose concentration on cellular output in neuronal differentiation. Cortical neuronal differentiation was performed on TSC2 +/+ and TSC2 -/- hES in either 25 mM or 5 mM glucose media. Cellular compositions were examined at day 80 using immunofluorescence (A) and flow cytometry (B) for the postmitotic marker HuC/HuD; neuronal marker TUJ-1 and glial marker GFAP (n = 1 for each differentiation). For HuC/HuD, no differences were observed in TSC2 +/+ cultures between the differentiations in high or low glucose as both media led to the generation of approximately 25% postmitotic neurons at day 80 and TSC2 -/- cells produced just under 20% HuC/HuD-positive neurons in 25 mM glucose media. However, lowering the glucose concentration to 5 mM nearly abolished the generation of postmitotic neurons in TSC2 -/- cultures. The opposite could be observed with regard to GFAP-positivity, which did not differ for TSC2 +/+ cultures in both media, whereas GFAP-positivity increased to nearly 95% in TSC2 -/- cultures kept at 5 mM glucose. No differences between cell lines and media were found for TUJ1, which was detected in approx. 95% of all cells in all cultures.