Small Molecules Efficiently Reprogram Human Astroglial Cells into Functional Neurons

Abstract

We have recently demonstrated that reactive glial cells can be directly reprogrammed into functional neurons by a single neural transcription factor, NeuroD1. Here we report that a combination of small molecules can also reprogram human astrocytes in culture into fully functional neurons. We demonstrate that sequential exposure of human astrocytes to a cocktail of nine small molecules that inhibit glial but activate neuronal signaling pathways can successfully reprogram astrocytes into neurons in 8-10 days. This chemical reprogramming is mediated through epigenetic regulation and involves transcriptional activation of NEUROD1 and NEUROGENIN2. The human astrocyte-converted neurons can survive for >5 months in culture and form functional synaptic networks with synchronous burst activities. The chemically reprogrammed human neurons can also survive for >1 month in the mouse brain in vivo and integrate into local circuits. Our study opens a new avenue using chemical compounds to reprogram reactive glial cells into functional neurons.

Figure 1. 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, 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), β-III tubulin (Tuj1, red) or MAP2 (cyan). (E) Sequential exposure of human astrocytes to small molecules resulted into 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 9 small molecules for reprogramming together. 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 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 8 days exposure to 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 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 40x field), whereas control group only had a few NeuN+ cells (5.6 ± 1.4 per 40x field). N = 4 batches. Scale Bars: 50 μm for panel B; 20 μm for other images. *** P < 0.001, Student's t test. Data are represented as mean ± SEM.

Figure 2. Functional analyses of human astrocyte-converted neurons induced by small molecule treatment

(A) Long-term survival of small molecule-induced human neurons (5 months in culture) and massive number of synaptic puncta (SV2, red) along the dendrites (MAP2, Green). Scale bar: 20 μm. (B-D) Representative traces showing Na⁺ and K⁺ currents recorded from 1-month (B) and 2-month (C) old human neurons induced by small molecules. Panel D shows the blockade of Na⁺ currents by TTX (2 μM). (E) Quantitative analyses of peak Na⁺ and K⁺ currents in 2-week to 3-month old neurons converted from human astrocytes by small molecules. (F) Representative trace of repetitive action potentials recorded in small molecule-induced human neurons at 75 days post initial drug treatment. (G and H) Representative traces showing spontaneous synaptic events in 2-month old converted human neurons. Holding potential = −70 mV. (H) Expanded trace from (G). (I) Inhibitory GABAergic events revealed in human astrocyte-converted neurons when holding potential was held at 0 mV (2-month old). The events were blocked by GABA_A receptor antagonist bicuculline (BIC, 10 μM). (J-K) Representative traces showing spontaneous burst activities in 3-month old small molecule-induced human neurons. HP = −70mV. (K) Expanded view of a burst in (J). (L) The burst activities were blocked by TTX (2 μM). The majority of synaptic events at −70 mV were blocked by glutamate receptor antagonist DNQX (10 μM), suggesting that they were glutamatergic events. (M) Dual whole-cell recordings illustrating that small molecule-converted human neurons formed robust synaptic networks and fired synchronously. (N) The Ca²⁺ ratio imaging further illustrating that the small molecule-converted human neurons were highly connected and showed synchronous activities. Data are represented as mean ± SEM.

Figure 3. Characterization of the human astrocyte-converted neurons induced by small molecules

(A-C) Immunostaining with anterior-posterior neuronal markers revealed that the small molecule-converted human neurons were positive for forebrain marker FoxG1 (A), but negative for hindbrain and spinal cord marker HOX B4 (B) and HOX C9 (C). (D-F) Immunostaining with cortical neuron markers revealed that small molecule-induced human neurons were negative for superficial layer marker Cux1 (D), but positive for deep layer marker Ctip2 (E) and Otx1 (F). (G-H) The small molecule-converted human neurons were also immunopositive for general cortical neuron marker Tbr1 (G) and hippocampal neuron marker Prox1 (H). (I) Quantitative analyses of small molecule-induced human neurons (FoxG1, 97.1 ± 1.1%, n = 3 batches; Cux1, 3.1 ± 1.9%, n = 4 batches; Ctip2, 71.4 ± 3%, n = 4 batches; Otx1, 87.4 ± 3.2%, n = 3 batches; Tbr1, 86.4 ± 3.4%, n = 3 batches; Prox1, 73.4 ± 4.4%, n = 4 batches). Scale bars: 20 μm. (J) MCM-converted human neurons were immunopositive for VGluT1. (K) A small portion of MCM-converted human neurons were GAD67-positive. (L-N) MCM-converted neurons were immunonegative for cholinergic neuronal marker vesicular acetylcholine transporter (VAChT) (L), dopaminergic neuronal marker tyrosine hydroxylase (TH) (M), or spinal motor neuron marker Isl1 (N). (O) Quantitative analyses of small molecule converted human neurons (VGluT1, 88.3 ± 4%, n = 4 batches; GAD67, 8.2 ± 1.5%, n = 4 batches). Scale bars: 20 μm. Data are represented as mean ± SEM.

Figure 4. Transcriptional and epigenetic regulation during chemical reprogramming of human astrocytes into neurons

(A-B) PCR array revealed substantial transcriptional activation of neural transcription factors (NGN1/2, NEUROD1, and ASCL1) and immature neuronal gene DCX at day 4 (A) or day 8 (B) after small molecule treatment. Note that DCX increased >2000-fold at D8 compared to the control. The genes showing significant change in PCR array assay were presented (P < 0.05, Mann-Whitney t test). (C-F) The time course of transcriptional changes revealed by quantitative real-time PCR analyses. Neural transcriptional factors NGN2 (C) and NEUROD1 (D) showed a peak transcription at D4 and D6, respectively; whereas astroglial genes GFAP (E) and ALDH1L1 (F) were significantly downregulated. * P < 0.05, ** P < 0.01, *** P < 0.001; Two-way ANOVA followed with Dunnett's test. N = 3 batches. (G-I) Epigenetic regulation of GFAP promoter and transcription start site during chemical reprogramming. MeDIP-seq revealed a significant increase of methylation in the GFAP promoter region (G, box region) after 8 days of small molecule treatment, which was confirmed by subsequent BS-seq (H). Note that the hypermethylated sites were located in the flanking region of two important transcription factor-binding sites, STAT3 and AP1, which will significantly inhibit the transcription of GFAP. BS-seq also showed a significant increase of the methylation level at GFAP transcription start site (TSS) and 5’ UTR regulatory region (I), further suggesting an inhibition of GFAP transcription through DNA methylation. (J-K) MeDIP-seq and BS-seq revealed a significant decrease of methylation at the promoter region of a neuronal gene NEFM (neurofilament-M), suggesting transcriptional activation of neuronal genes during chemical reprogramming of human astrocytes into neurons. (L-M) CHIP-qPCR revealed a significant increase of histone acetylation in the NGN2 promoter region after small molecule treatment, likely caused by HDAC inhibitor VPA. (N-O) The methylation level of H3K4 increased significantly in the NGN2 promoter region (N), whereas H3K27 methylation at the NGN2 transcription start site showed a significant decrease (O), indicating epigenetic activation of NGN2 through histone modification. Data are represented as mean ± SEM.

Figure 5. Increase of the protein expression level of neural transcription factors during chemical reprogramming

(A-C) Representative images illustrating the gradual activation of endogenous neural transcription factors Ascl1 (A), Ngn2 (B), and NeuroD1 (C) at different days of small molecule treatment. (D-E) Representative images showing the gradual increase of neuronal signal DCX (D) and NeuN (E) during the conversion process from D0 to D10. (F) Representative images showing the decrease of astrocytic marker GFAP from D0 to D10. Scale bars: 20 μm (G-I) Quantitative analyses of the protein expression level of Ascl1 (G), Ngn2 (H), and NeuroD1 (I). Note that Ascl1 significantly increased at day 2 by 3-fold, while Ngn2 peaked at day 4 and NeuroD1 peaked at day 6. N = 3 batches. (J) Quantified data showing a significant increase of NeuN from day 6 to day 10. N = 3 batches. (K) Quantified data showing a significant decrease of GFAP from D0 to D10. N = 3 batches. Data are represented as mean ± SEM.

Figure 6. Evaluating the essential role of each individual small molecule during astrocyte-neuron reprogramming

(A) Human astrocytes treated with 1% DMSO as a control. NeuN, green; MAP2, red. (B) A defined combination of 9 small molecules induced a massive number of neurons (14 days post initial small molecule treatment, the same for the following removal experiments). (C-F) Individual removal of DAPT (C), CHIR99021 (D), SB431542 (E) or LDN193189 (F) from the 9 small molecule pool significantly reduced the number of converted neurons. (G) Removal of sonic hedgehog agonists SAG and Purmo together slightly reduced the number of converted neurons. (H) Removal of VPA also slightly reduced the neuronal number. (I-J) Removal of Tzv (I) or TTNPB (J) did not affect the neuronal conversion. Scale bars: 20 μm. (K) Quantitative analyses showing that DAPT is the most potent reprogramming factor, followed by CHIR99021, SB431542, and LDN193189. * P < 0.05; ** P < 0.01; *** P < 0.001; one-way ANOVA followed with Sidak's multiple comparison test. N = 3 batches. Data are represented as mean ± SEM.

Figure 7. In vivo survival and integration of small molecule-converted human neurons in the mouse brain

(A) Schematic drawing showing the transplantation of small molecule-converted human neurons into the mouse brains at postnatal day 1. (B) GFP-positive cells were identified around lateral ventricles at 7 days post cell injection (7 DPI). Many GFP-positive cells were also positive for DCX (red), and all of the GFP-positive cells were immunopositive for human nuclei (HuNu, Blue), indicating their human cell identity. N = 6 mice. (C) At 11 DPI, some GFP-positive cells were immunopositive for MAP2 (red), indicating the survival and growth of human neurons in the mouse brain in vivo. N = 6 mice. (D) Some GFP-positive human neurons, which were immunopositive for NeuN (red) and HuNu (cyan), migrated into the adjacent striatum areas and extended long neurites at 11 DPI. (E) Human neurons, labeled by NeuN (red) and HuNu (blue), survived for more than 1 month inside the mouse brain and were surrounded by mouse neurons (NeuN positive but HuNu negative). N = 2 mice. (F) GFP-positive human neurons were innervated by surrounding neurons as indicated by many synaptic puncta (SV2, red) along the GFP-positive neurites (inset), suggesting the synaptic integration of the transplanted human neurons into the local neural circuit. N = 2 mice. Scale bars: 20 μm.