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. 2023 Jul 3;133(13):e164637. doi: 10.1172/JCI164637

Figure 7. Blocking JAK/STAT with tofacitinib in MS patient astrocyte/NGN2-neuron cocultures.

Figure 7

(A) iPSC-derived astrocytes cocultured with NGN2-neurons were treated with tofacitinib (100/1,000 nM) or IL-13 plus IL-21 (50 ng/mL each) for 24 hours, followed by a treatment with TNF-α/IL-17A (50 ng/mL) for 24 hours. Inhibition of JAK/STAT by tofacitinib led to failed neurite protection, as shown by an increase in SMI32/SMI31 ratio. Immunofluorescently stained SMI32/SMI31 neurons were analyzed with Imaris (Bitplane) and presented as surface ratio of SMI32/SMI31 ± SD, normalized to the control. Each data point represents a microscopic field of view (641 × 479 μm) of 3 independent experiments (different symbols); pooled data represent the mean from 3 individual patients (different colors) and 3 independent experiments (different symbols). Statistical significance was tested with a Kruskal-Wallis test; ***P < 0.001, ****P < 0.0001. (B) Supernatants of tofacitinib-treated samples were collected after the cytokine stimulation period of 24 hours. Relative changes of cytokine concentration in tofacitinib-treated versus control cocultures were analyzed. LIF and TGF-β1 levels were decreased, whereas M-CSF, VEGF, and BDNF were increased. Changes are displayed in percentage compared with (control) tCBMS (100%); each data point represents a single supernatant measurement. Statistical significance was tested with a Mann-Whitney U test; ***P < 0.001, ****P < 0.0001.