Extended Data Fig. 2: Cortical neurons are generated directly from human adult fibroblasts using a combinatorial transcription-factor and small-molecule protocol.
From: Proteostasis and lysosomal repair deficits in transdifferentiated neurons of Alzheimer’s disease
(a) Transdifferentiation scheme using transcription factors Brn2, Ascl1, Myt1l, and Ngn2 (herein BAMN factors) that induces a global transcriptional change in fibroblasts to constitute a new neuronal cell state. Fibroblasts are plated one day before transdifferentiation. On Day 0, cells are infected with lentiviruses expressing BAMN factors and harvested four days after doxycycline-induction. Transduced cells expressing PSA-NCAM are magnetically isolated and replated to vitronectin/laminin-coated plates on Day 4 and then switched to reprogramming medium (DMEM/F12/Neurobasal) supplemented with small molecules 5 µM Forskolin, 10 µM SB 431542, 2 µM Dorsomorphin and 2 µM XAV939 on Day 5. 10 ng/mL BDNF and NT-3 are added into the reprogramming medium after one week. Cells are switched to maturation medium (Neuronal) on Day 20 and used for experiments after Day 35. (b) Fibroblasts are infected with lentiviruses expressing BAMN factors and GFP and monitored for morphological changes after lentiviral induction. Transduced cells undergo sorting based on PSA-NCAM+ gate in flow cytometry on Day 4. Scale bar: 200 μm. (c,d) IF images of neuronal markers in tNeurons at PID 35. Quantification of the percentage of cells positive for both NeuN and Tuj1 (c) and the numbers of Syn-1 puncta in the soma (d). Tuj1/DAPI, MAP2/DAPI & MAP2/Tuj1: n = 1208 (young), 678 (aged) and 789 (aged/sAD) cells; NeuN/DAPI and NeuN/Tuj1: n = 822 (young), 579 (aged) and 759 (aged/sAD) cells. Syn-1: n = 40 (young), 42 (aged) and 41 (aged/sAD) cells. Scale bars: 100 μm (c) and 20 μm (d). In panels c and d, the boxes show median and 1st and 3rd quartile and the whiskers extending 1.5 times the interquartile range from the boxes. Data show box-and-whisker plots of two to four independent experiments and cells from independent HC and AD patients. Statistical analysis is performed using One-Way ANOVA followed by Bonferroni post-hoc analysis. *P < 0.05 and ***P < 0.001. Source numerical data are provided.
