Small Molecules Efficiently Reprogram Human Astroglial Cells into Functional Neurons
Sequential Exposure to a Defined Group of Small Molecules Converts Human Astroglial Cells into Neuronal Cells
(A) Schematic illustration of our strategy to convert cultured human astrocytes into neurons using a cocktail of small molecules. Note that different subsets of small molecules were used at different reprogramming stages.
(B and C) Quantitative analysis of the human astrocyte cultures (HA1800, ScienCell). The majority of cells in our human astrocyte cultures were immunopositive for astrocytic marker GFAP (79.3% ± 4.9%), astrocytic glutamate transporter GLT-1 (82.5% ± 4.3%), and to a lesser degree S100β (39.3% ± 1.8%). No cells were immunopositive for neuronal markers NeuN, MAP2, or Doublecortin (DCX). HuNu, human nuclei, marker for human cells. n = 3 batches.
(D) Control human astrocyte cultures without small-molecule treatment had very few cells immunopositive for neuronal markers DCX (green), β3-tubulin (Tuj1, red), or MAP2 (cyan).
(E) Sequential exposure of human astrocytes to small molecules resulted in a massive number of neuronal cells, which were immunopositive for DCX (green), Tuj1 (red), and MAP2 (cyan). MCM stands for master conversion molecules, including the nine small molecules used together for reprogramming. Samples were analyzed at 14 days after initial small-molecule treatment.
(F) At 30 days post initial small-molecule treatment, human astrocyte-converted neurons developed extensive dendrites (MAP2, green) and were immunopositive for mature neuronal marker NeuN (red).
(G) Small-molecule-converted human neurons survived for 4 months in culture and showed robust dendritic trees (MAP2, green) as well as extensive axons (SMI312, red).
(H) Astroglial lineage tracing with GFAP::GFP retrovirus showing that GFP+ cells were immunopositive for neuronal marker NeuN (red) after small-molecule treatment. n = 5 batches.
(I and J) Small-molecule treatment achieved high conversion efficiency after cells were exposed to 8 days of MCM (67.1% ± 0.8%, Tuj1+ neurons/total cells labeled by DAPI, n = 4 batches).
(K) Chemical reprogramming of human midbrain astrocytes into neurons. At 1 month post initial small-molecule treatment of human midbrain astrocytes (ScienCell), most cells were immunopositive for neuronal marker NeuN (red) and MAP2 (green).
(L) Control human midbrain astrocyte cultures without small-molecule treatment had very few cells immunopositive for NeuN (red) or MAP2 (green) at 1 month of culture in neuronal differentiation medium.
(M) Quantitative analysis revealed a large number of NeuN-positive neurons converted from human midbrain astrocytes at 1 month post small-molecule treatment (199.7 ± 9.2 per 40× field), whereas the control group only had a few NeuN+ cells (5.6 ± 1.4 per 40× field). n = 4 batches.
Scale bars represent 50 μm for (B) and 20 μm for other images. ∗∗∗p < 0.001, Student’s t test. Data are represented as mean ± SEM.