We have previously developed a cocktail of nine small molecules to convert human fetal astrocytes into neurons, but a nine-molecule recipe is difficult for clinical applications. Here, we identify a chemical formula with only three to four small molecules for astrocyte-to-neuron conversion. We demonstrate that modulation of three to four signaling pathways among Notch, glycogen synthase kinase 3, transforming growth factor β, and bone morphogenetic protein pathways is sufficient to change an astrocyte into a neuron. The chemically converted human neurons can survive >7 months in culture, fire repetitive action potentials, and display robust synaptic burst activities. Interestingly, cortical astrocyte-converted neurons are mostly glutamatergic, while midbrain astrocyte-converted neurons can yield some GABAergic neurons in addition to glutamatergic neurons. When administered in vivo through intracranial or intraperitoneal injection, the four-drug combination can significantly increase adult hippocampal neurogenesis. Together, human fetal astrocytes can be chemically converted into functional neurons using three to four small molecules, bringing us one step forward for developing future drug therapy.

In this study, we identify a chemical protocol composed of only three to four small molecules (DAPT, CHIR99021, SB431542, and LDN193189) that can more efficiently reprogram HAs into neurons. By substituting each of these four drugs (core drugs) with functional analogs, we demonstrate that simultaneous modulation of four signaling pathways including Notch, glycogen synthase kinase 3β (GSK-3β), transforming growth factor β (TGF-β), and bone morphogenetic protein (BMP) pathways, is sufficient to reprogram HAs into neurons. Even modulating three out of the four signaling pathways can convert HAs into neurons. Our chemically converted human neurons are highly functional and can survive >7 mo in cell culture. Moreover, when applied in vivo, core drugs significantly increase the adult neurogenesis in the mouse hippocampus. Therefore, we have identified a simple chemical formula for astrocyte-to-neuron conversion, bringing us one step closer toward developing future drug therapy for brain repair.

We have recently demonstrated that reactive astrocytes and NG2 cells can be directly reprogrammed into functional neurons inside mouse brains with the expression of a single neural transcription factor, NEUROD1 (). Other transcription factors, such as neurogenin 2 (NGN2), ASCL1, and SOX2, have also been reported to reprogram glial cells into neurons both in vitro and in vivo (). Direct conversion from glial cells into neurons inside the brain or spinal cord without cell transplantation can avoid the problems of tumor formation, aberrant differentiation, and immunorejection that are often associated with stem cell transplantation (). The majority of glia-to-neuron conversion research has been carried out using virus-mediated ectopic expression of transcription factors, which requires production of viruses and sophisticated intracranial or intra-spinal injection procedures. However, small-molecule-mediated chemical reprogramming has been developed to allow cell trans-differentiation without viruses (). Our lab recently developed a chemical protocol to reprogram human astrocytes (HAs) into functional neurons using a cocktail of nine small molecules (). These nine molecules need to be sequentially administered to reprogram HAs into neurons, making its clinical translation quite difficult due to the large number of small molecules used and the complicated timing of drug application.

Neuronal loss is the leading cause of symptoms in patients with neural injury or neurodegenerative disorders. Following nerve injury, glial cells including astrocytes, NG2 cells, and microglia will proliferate and become reactive glial cells to form glial scarring in order to protect neighboring tissues from further damage (). However, the continuous presence of glial scars also inhibits neuronal growth and synaptic transmission in the injured area (). Thus, reactive glial cells are a double-edged sword that can have both neuroprotective and neuroinhibitory functions during the progression of injury or diseases. Glial scarring has been widely reported after traumatic brain injury, stroke, and spinal cord injury, but efforts to remove the neuroinhibitory effect of glial scarring has only resulted in limited success ().

We next further tested whether our core drugs can pass through blood-brain barrier (BBB) to regulate adult neurogenesis through intraperitoneal (i.p.) administration (daily i.p. injection of core drugs for 3 weeks, and bromodeoxyuridine (BrdU) was i.p. injected every 3–4 days from day 5 to day 26 to identify newborn cells) ( Figure 7 F). It is important to note that the intracranial injection of core drugs was one-time injection, whereas the i.p. injection was repeat injections over 3 weeks. Interestingly, i.p. injection of core drugs also resulted in a significant increase of DCX-labeled newborn neurons and Ki67-positive cells in the dentate granule layer ( Figures 7 G–7J). Moreover, BrdU-labeled cells that were dually immunopositive for DCX or NEUN increased significantly after i.p. injection of core drugs ( Figures 7 K–7N), with more newborn neurons migrating toward the outer layer of the dentate granule layer than in control group ( Figure 7 M, arrowhead), suggesting an accelerated neuronal maturation after core drug treatment. These results suggest that core drugs can regulate adult neurogenesis in vivo.

With successful conversion of cultured HAs into neurons in vitro, we then injected the core drugs into mouse brains to test whether they can convert astrocytes into neurons in vivo. After spending several years using various methods to deliver small molecules in the mouse brain in vivo, we have not achieved definitive success of chemical conversion inside mouse brains despite the observation of a few neurons after chemical treatment. This is rather disappointing, but we are still continuously trying direct in vivo chemical conversion in the mouse brain. The biggest challenge for in vivo chemical conversion is how to maintain a constant concentration of small molecules inside the brain without causing a severe invasive damage to the brain. We have tried using biomaterial to encapsulate small molecules, but, perhaps because our small molecules are too small or we have not found the right biomaterial for such small molecules, the small molecules we applied might not stay for a long time inside the brain. We also tried an osmotic minipump (Alzet) but the tip of the insertion caused significant tissue damage inside the brain, and the injury induced many DCX+ cells that were mainly reactive astrocytes 2 weeks after drug treatment ( Figures S7 I–S7K). On the other hand, during our vigorous testing of in vivo chemical reprogramming, we accidentally found that core drugs significantly increased adult neurogenesis in the mouse hippocampus ( Figure 7 ). We initially injected core drugs through intracranial injection into the hippocampus and sacrificed the mice 7 days later ( Figure 7 A). We observed remarkable increase of DCX-labeled newborn neurons together with Ki67-labeled proliferative cells in the dentate granule layer ( Figures 7 B–7E). When separating the four core drugs into two-drug combinations (CHIR99021 + DAPT; and SB431542 + LDN193189), we found that the two-drug combinations could also increase adult hippocampal neurogenesis ( Figures S7 A–S7H). However, the four drugs together were more potent in promoting neuronal maturation, as shown by more complex dendritic morphology and more neurons migrated into the outer granule layer ( Figures S7 A–S7H).

(G–J) Representative images (G and H) and quantitative analyses (I and J) showing that core drugs significantly increased the cell number of dividing cells (Ki67, red) and newborn neurons (DCX, green) compared with vehicle control with 20% captisol (CPTS) injection (Student's t test, ∗ p < 0.01, ∗∗∗ p < 0.0001, N = 4 mice). Scale bar, 100 μm. The total cell number of Ki67+ or DCX+ cells in one side of the dentate gyrus was quantified based on 10 stacked images with a total thickness of 18 μm and a volume of ∼0.014 mm 3 .

(F) Schematic drawing illustrating intraperitoneal injection of core drugs (SB 431542, 165 μM; LDN193189, 8.25 μM; CHIR99021, 49.5 μM; and DAPT, 165 μM; all in 20% captisol, 10 mL/kg) daily for 23 days. BrdU was administrated every 3–4 days from day 5 to day 26. The mice were sacrificed at day 32 for immunostaining.

To investigate whether the four-drug cocktail (SLCD) is the minimal combination capable of reprogramming HAs into neurons, we further tested all possible combinations of three drugs, two drugs, and each individual drug, among the four-drug cocktail (SLCD). Interestingly, all three-drug combinations were capable of reprogramming HAs into neurons but with lower efficiency compared with the four-drug group ( Figures 6 A–6E). Notably, the combination of three drugs SB431542/CHIR99021/DAPT reached >80% of the four-drug efficiency ( Figure 6 Q, One-way ANOVA followed with post hoc Dunnett's multiple comparison test, n ≥ 3 batches). On the other hand, all combinations of two drugs showed significantly diminished reprogramming efficiency, with the highest one (LDN193189/CHIR99021) still <40% of that induced by the four-drug cocktail ( Figures 6 F–6K, quantified in Figure 6 Q). None of the individual drugs among the four core drugs showed any significant difference from the DMSO control ( Figure 6 L–6P, quantified in Figure 6 Q). real-time PCR analysis also showed that the core drugs and combinations of three or two drugs all downregulated astrocytic genes such as S100β and Glast (SLC1A3) ( Figures S6 H–S6I). These studies demonstrate that HAs may be chemically reprogrammed into neurons by simultaneously modulating at least three or two signaling pathways, but not by changing a single signaling pathway.

(B–E) Three-drug combinations, including S/C/D (B), S/L/D (C), L/C/D (D), and S/L/C (E), among the four core drugs could also reprogram a significant number of HAs into neurons, although with lower efficiency compared with the four core drugs.

(A) Core drug (S for SB431542, L for LDN193189, C for CHIR99021, or D for DAPT)-induced chemical reprogramming of HA into neurons (NEUN, green) as a control. For all experiments in this figure, immunostaining was performed at 14 days after initial drug treatment.

After identifying the four core drugs (SLCD), we further tested whether these particular four drugs were unique for chemical reprogramming, or rather they could be replaced by other functional analogs modulating similar signaling pathways. For example, SB431542 is a TGF-β inhibitor and inhibits a subfamily of activin receptor-like kinase (ALK) receptors including ALK4, ALK5, and ALK7 (). We therefore substituted SB431542 with its functional analogs Repsox (1 μM) or A-8301 (0.25 μM), which also inhibit TGF-β pathway. Interestingly, we found that HAs were also efficiently reprogramed into neurons by substituting SB431542 with Repsox or A-8301 among the four-drug combination ( Figures 5 A–5C). Thus, inhibiting the TGF-β signaling pathway in general, rather than any particular inhibitor, is critical for chemical reprogramming. Similarly, replacing LDN193189, an inhibitor of BMP through inhibiting another ALK subfamily (ALK2 and ALK3), with other two BMP inhibitors Dorsomorphin (1 μM) or DMH 1 (1.5 μM), also resulted in a significant neuronal reprogramming ( Figures 5 D–5F). CHIR99021 is a selective inhibitor of GSK-3 and indirectly activates the WNT signaling pathway. CHIR99021 has been used in several studies to generate neurons from ESCs or iPSCs (), and WNT has been shown to regulate postnatal and adult neurogenesis (). Replacement of CHIR99021 by two other GSK-3 inhibitors, SB216763 (1 μM; different from TGF-β inhibitor SB431542) or AR-A014418 (6 μM), resulted in a large number of neurons but less than the effect of CHIR99021 itself ( Figures 5 G–5I). Lastly, DAPT inhibits Notch signaling pathway by targeting γ-secretase and is involved in the maintenance of the proliferative and undifferentiated state of embryonic stem cells during development (). Replacing DAPT with other two Notch inhibitors, BMS906024 (2 μM) or RO4929097 (0.5 μM), resulted in comparable reprogramming efficiency ( Figures 5 J–5L), suggesting an important role of Notch signaling in neuronal conversion. After testing each individual signaling pathway with at least three different inhibitors, we replaced all of the original four core drugs (SLCD) with their functional analogs, Repsox, Dorsomorphin, SB216763, and RO4929097 (RDSRo). The four functional analogs (RDSRo) together also generated a large number of converted iNs, with the conversion efficiency reaching ∼70% of the original core drug group ( Figures 5 M–5O). Similar to the core drug group (SLCD), the RDSRo group also induced mainly glutamatergic neurons but not GABAergic or dopaminergic neurons ( Figures S6 A–S6C) and formed local synaptic connections ( Figure S6 D). Consistently, real-time PCR analysis revealed that both core drug group or their various substitution groups all downregulated glial genes such as Glt1 (SLC1A2) ( Figures S6 E and S6F). Furthermore, real-time PCR experiments identified significant changes of gene targets that are correlated with the four signaling pathways, including NEUROGENIN 1 for the GSK-3β pathway (), UNC13A for the Notch pathway (), ID1 for the BMP pathway (), and COL1A1 for the TGF-β pathway () ( Figure S6 G). Altogether, these results suggest that modulation of four signaling pathways including TGF-β, BMP, GSK-3, and Notch in HAs is sufficient for reprogramming into functional neurons.

(M–O) Replacing the four core drugs (M) with their corresponding functional analogs RDSRo (N, SB431542 replaced by Repsox, LDN193189 by Dorsomorphin, CHIR99021 by SB216763, and DAPT by RO4929097) resulted in lower reprogramming efficiency (66% ± 16%) (O).

(A–C) Among core drugs, replacing SB431542 with its functional analog A-8301 (A) or Repsox (B) yielded similar numbers of reprogrammed neurons (87% ± 4% for A-8301 and 89% ± 6% for Repsox replacement group) (C). Immunostaining of NEUN was performed 14 days after the start of drug treatment.

We further investigated whether the core drug-converted iNs were functionally connected to each other. Different from mouse neuron cultures, we found that the core drug-converted human neurons could survive for 3–7 months in our cell culture condition and form robust synaptic connections ( Figures 4 A and 4B). Some core drug-converted human neurons could survive as long as 7.5 months ( Figure 4 B), providing an extended time window for future studies on drug screening or disease modeling. Functionally, patch-clamp recordings revealed that our chemically converted iNs showed large sodium and potassium currents ( Figure 4 C) as well as repetitive action potentials ( Figure 4 F). As expected, converted iNs showed a clear developmental time course of functional maturation when analyzed from 1- to 3-month cultures after chemical conversion. Specifically, sodium currents increased from 1 nA at 1-month culture to 3 nA at 3-month culture, while potassium currents increased from ∼2 nA at 1-month culture to ∼3.5 nA at 3-month culture ( Figures 4 D and 4E). The number of cells firing repetitive action potentials increased from 12/35 at 1-month culture to 27/29 at 3-month culture ( Figure 4 G). The membrane capacitance also increased ( Figure S5 G, indicating cell growth), together with a decrease of membrane resistance ( Figure S5 H, an indication of channel and receptor expression) from 1- to 3-month cultures. We also detected glutamate- and GABA-induced receptor currents in 2-month-old converted iNs ( Figure 4 H), which was consistent with the immunostaining of glutamate receptors (GluR1 and GluR2 subunits) ( Figures S5 J and S5K) and GABAreceptors (γ2 and α5 subunits) ( Figures S5 L and S5M). Moreover, we recorded robust spontaneous synaptic activities, some in burst activity pattern, in 3-month-old cultures after chemical conversion ( Figure 4 I), suggesting the formation of a highly integrated neural network among the converted iNs. Notably, most of the synaptic events were blocked by glutamate receptor antagonist DNQX ( Figure 4 I), consistent with their glutamatergic identity shown in Figure 3 . Therefore, the four core drugs (SLCD) can chemically reprogram HAs into highly functional neurons.

(F and G) Whole-cell recordings revealed an increase of action potential firing from 1- to 3-month-old cultures after core drug-mediated chemical conversion (F), with quantitative result showing the ratio of converted iNs having repetitive action potential firing at different time points (G).

We next characterized the neuronal identity after chemical reprogramming by performing immunostaining on a series of neuronal markers. The core drug-converted human neurons (3 months after drug treatment) were mainly immunopositive for PROX1, CTIP2 (BCL11B), and TBR1 ( Figures 3 A–3C), but negative for other neuronal markers such as CUX1, NURR1 (NR4A2), and HOXB4 ( Figures 3 D–3G; quantified in Figure 3 H). These results were further confirmed with real-time PCR analysis ( Figure 3 I). When examining neurotransmitter subtypes, we found that the majority of neurons converted from human cortical astrocytes were immunopositive for VGlut1 (SLC17A7) (78%), a glutamatergic neuron marker, but very few were GABAergic (GAD67 [GAD1], 2%) or dopaminergic neurons (TH, 1%) ( Figures 3 J–3M). However, when testing our core drug combination in midbrain HAs (ScienCell, 1850), we found a large proportion of converted iNs co-expressing GABA and GAD67 ( Figure S5 A–S5F), suggesting that different lineages of astrocytes originated from different brain regions may be chemically converted into different subtypes of iNs. Therefore, human cortical astrocytes can be converted into cortical glutamatergic neurons, and different lineages of astrocytes may have their own intrinsic factors to influence the final cell fate after conversion.

(H) Real-time PCR experiments were performed at 1 month after core drug treatment to assess different neuronal gene expression level. All data were normalized to human astrocyte control. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, unpaired t test after log 2 transformation. Data are represented in means ± SEM. N = 3 batches.

Besides transcriptional regulation of NGN2 and NEUROD1, we further discovered a significant change of REST (RE1-silencing transcription factor) and MECP2 (methyl CpG binding protein 2) signals during core drug-mediated chemical reprogramming ( Figures 2 C–2F). REST is a transcriptional repressor that can silence neuronal genes in stem cells and non-neuronal cells (). During neuronal differentiation, REST is downregulated in neurons. We found a significant decrease of REST expression during chemical reprogramming process, particularly in Tuj1neurons ( Figure 2 C, quantified in Figure 2 E), indicating the suppression of REST by core drugs during chemical conversion. In contrast, the expression level of MECP2, a nuclear protein that can regulate the expression of many genes including suppressing REST (), increased significantly after core drug treatment ( Figure 2 D, quantified in Figure 2 F). MECP2 has been reported to be highly expressed in neurons, much more than that in astrocytes (). Consistently, we found that the increase of MECP2 signal was coinciding with an increase of Tuj1 and a decrease of GFAP signal after core drug treatment. Together, the changes of REST and MECP2 expression level indicate a cell fate change from astrocytes to neurons induced by core drug treatment.

What is the molecular mechanism underlying the core drug reprogramming? Our real-time PCR experiments showed that the basic-helix-loop-helix neural transcription factors, including both NEUROD1 and NGN2, were significantly upregulated at day 1 and day 4 by core drug treatment ( Figures 2 A and 2B), whereas the astrocytic GFAP expression was downregulated ( Figure S4 A). Interestingly, each individual drug among the four core drugs upregulated NGN2 level ( Figure 2 A), and the NEUROD1 level was upregulated by LDN193189, SB431542, and DAPT ( Figure 2 B). VPA, an HDAC inhibitor that alters histone acetylation and gene transcription, was found to induce a significant increase of both NEUROD1 and NGN2 expression ( Figures 2 A and 2B). However, when VPA was added together with the four core drugs, it unexpectedly decreased the reprogramming efficiency ( Figures S4 B and S4C). We then further tested core drugs in combination with other individual drugs including ROCK inhibitor Tzv, retinoic acid receptor agonist TTNPB, sonic hedgehog activator SAG, and Purmo. Addition of Tzv to the core drugs showed no effect ( Figure S4 D), while addition of TTNPB decreased the reprogramming efficiency ( Figure S4 E and quantified in S4G). Addition of SAG and Purmo significantly increased astrocytic proliferation, resulting in overgrown astrocytes and decrease of neurons (data not shown). These results suggest that alteration of extra signaling pathways in addition to the four pathways modulated by core drugs might result in reduced conversion efficiency.

(A and B) Real-time qPCR analyses revealed transcriptional activation of NGN2 (A) and NEUROD1 (B) by core drug treatment. Note than NGN2 was activated earlier than NEUROD1, and that the core drug group showed higher levels of NGN2 and NEUROD1 than the nine-drug group. Among individual drugs, SB431542, CHIR99021, LDN193189, DAPT, and VPA increased NGN2 to a significant level, whereas SB431542, LDN193189, DAPT, TTNPB, and VPA significantly increased the expression of NEUROD1. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, one-way ANOVA after log 2 transformation, Dunnett's multiple comparison test. Data are represented in means ± SEM. N = 3 batches.

We further tested whether our core drugs could reprogram different sources of HAs into neurons. After purchasing a different line of HAs from Lonza (cc-2565), we found that our core drugs (4-day treatment of 2.5 μM SB431542, 0.125 μM LDN193189, 0.75 μM CHIR99021, and 2.5 μM DAPT) also induced a large number of iNs ( Figures S3 K–S3N, immunostained at day 10). Therefore, we conclude that HAs from different sources can be chemically converted into neurons.

Can HAs spontaneously turn into neurons during our experimental process? To test this idea, we performed DCX and TUJ1 (TUBB3) staining but did not observe a significant number of newborn neurons in the DMSO control group from day 2 to day 7 ( Figures S3 A and S3B, quantified in Figures S3 C–S3F). In the core drug-treated group, DCX and TUJ1 signals did not change much at day 2 and day 4, but dramatically increased at day 7 ( Figures S3 A–S3F), suggesting that it takes ∼1 week for HAs to be chemically converted into neurons. At day 14 of the DMSO control group, we did see a few neurons, which were eliminated if the N2 supplement was removed from the culture medium ( Figures S3 G–S3J), suggesting that the N2 supplement may have a very weak neural induction role in cell cultures.

We then performed a lineage-tracing experiment using GFAP:GFP retroviruses to label HAs with GFP before drug treatment ( Figure S2 A). The GFP-labeled astrocytes became NeuN+ (RBFOX3) neurons after core drug treatment ( Figure S2 A, top row), but remained GFAPastrocytes in the DMSO control group ( Figure S2 A, bottom row). To observe the astrocyte-to-neuron conversion process, we performed long-term time-lapse imaging for eight consecutive days on a group of GFP-labeled HAs during and after core drug treatment ( Video S1 ). As shown in the video, the initial flat HAs infected with GFP lentiviruses gradually changed their morphology into neuron-like cells with extended long neurites after core drug treatment (see Video S1 in Supplemental Information ). Following time-lapse imaging, we further performed post-fix immunostaining and confirmed that the GFP-labeled neuron-like cells after core drug treatment were indeed immunopositive for NeuN ( Figure S2 B). Importantly, we did not observe significant cell proliferation during the 8 days of time-lapse imaging ( Video S1 ), consistent with the Ki67 immunostaining results showing an inhibition of cell proliferation by core drug treatment ( Figures S2 C and S2D, ∼130 Ki67+ cells/field before drug treatment and ∼10 Ki67+ cells/field after drug treatment). Moreover, NESTIN staining rarely showed neuroprogenitor cells induced by core drugs ( Figures S2 E and S2F). Together, through lineage tracing, time-lapse imaging, and Ki67 and NESTIN staining, we show that HAs can be directly converted into neurons by four molecules.

After characterizing HA properties, we repeated and confirmed successful chemical conversion of HAs into neurons using the nine-molecule cocktail () ( Figures 1 C and 1D). We then investigated how to reduce the number of drugs for chemical reprogramming by studying the effect of each individual drug among the nine-molecule cocktail. Interestingly, four molecules including SB431542 (S), LDN193189 (L), CHIR99021 (C), and DAPT (D) appeared to be most critical in maintaining high reprogramming efficiency (). Therefore, we tested these four drugs (SLCD) together to determine their reprogramming capability. Unexpectedly, when we applied SB431542 (5 μM) and LDN193189 (0.25 μM) for 2 days, and then replaced with CHIR99021 (1.5 μM) and DAPT (5 μM) for 4 days, it yielded more neurons than the nine-drug formula ( Figures 1 E and 1G). When we applied the four drugs (SLCD) altogether for 6 days, it yielded even more neurons ( Figures 1 F–1H). Quantitatively, the neuronal yield (converted neurons/astrocyte number before drug treatment) reached 71% when the four drugs (SLCD) were applied together ( Figures S1 H and S1I). Therefore, the four drugs together (SLCD) are referred as “core drugs” hereafter, and the core drug-converted neurons are referred to as induced neurons or “iNs” to be consistent with other studies (). It is worth mentioning that the chemically converted iNs and the remaining non-converted astrocytes formed a neuron-astrocyte co-culture condition, with the iNs migrating onto the surface of astrocytes, and the astrocytes served as a feeder layer to promote neuronal survival and maturation ( Figure S1 J) ().

We have recently identified a combination of nine small molecules, including SB431542, LDN193189, TTNPB, Thiazovivin, CHIR99021, DAPT, valproic acid (VPA), smoothened agonist (SAG), and purmorphamine (Purmo), to reprogram human fetal astrocytes into functional neurons (). However, for therapeutic purpose, a nine-molecule cocktail added in a sequential manner is rather difficult to translate into clinical applications. Here, to search for a more practical formula for chemical reprogramming, we purchased human fetal astrocytes (HA1800) from ScienCell (San Diego, CA) as described previously (). Because human fetal astrocytes might contain neuroprogenitors, we have subcultured the astrocytes for at least ten generations in the presence of 10% fetal bovine serum (FBS) to minimize progenitors (). After more than ten passages, the majority of our astrocytes were immunopositive for astrocytic markers such as glial fibrillary acidic protein(GFAP), S100β, and glutamine synthetase ( Figures 1 A, 1B, and S1 A), but rarely for neuroprogenitor markers SOX2 ( Figures S1 B and S1C) and NESTIN ( Figures S2 E and S2F). Long-term culture of our HAs in neural differentiation medium for 1 month also rarely yielded any neurons ( Figures S1 D and S1E). At the transcriptome level ( Figure S1 F), in comparison with published datasets on HAs () or human neural stem cells (hNSCs) (), our HA samples (red box, GSE123397 ) were closely related to the fetal astrocytes from human brain, but clearly different from the hNSCs (green box). Figure S1 G shows the heatmap of gene expression of typical astrocyte and NSC markers. Our HA samples showed low expression of STMN1, DLX1, and NES but high expression of FN1, COL1A1, VIM, and MYC, which was opposite to that of hNSCs. Together, the significant difference between our HA samples and NSCs suggests that after ten passages in 10% FBS, neuroprogenitors are minimized in our HA cultures.

(G) Quantified data (NEUN) showing higher reprogramming efficiency when four drugs (DCSL) were added in sequence or together as a mixture. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, one-way ANOVA followed with Dunnett's multiple comparison test. Data are represented in means ± SEM. The immunostaining was performed at 14 days after the beginning of drug treatment. N = 3 batches.

(E and F) The four core drugs (DAPT, CHIR99021, SB431542, and LDN193189) were administered either in sequence (E) or together (F) for 6 days, and both yielded a significant number of neurons.

Discussion

In this work, we identified a chemical formula using only three to four small molecules to reprogram HAs into neurons. Through replacement of different functional analogs, we demonstrate that modulating multiple signaling pathways, but not a single signaling pathway, is important for chemical reprogramming of astrocytes into neurons. The chemically converted iNs can survive more than 7 months and are highly functional with bursts of synaptic activities. Besides upregulation of neural transcription factors such as NEUROD1 and NGN2, we found that the neural suppressor gene REST was significantly downregulated, while MECP2 was upregulated during chemical conversion. During our attempt to chemically convert astrocytes into neurons in the mouse brain in vivo, we accidentally discovered that our core drugs can potently regulate adult neurogenesis in the mouse hippocampus. Together, our chemical formula with only three to four small molecules to reprogram HAs into neurons brings us one step closer toward a potential drug therapy for brain repair.

Hu et al., 2015 Hu W.X.

Qiu B.L.

Guan W.Q.

Wang Q.Y.

Wang M.

Li W.

Gao L.F.

Shen L.

Huang Y.

Xie G.C.

et al. Direct conversion of normal and Alzheimer's disease human fibroblasts into neuronal cells by small molecules. Li et al., 2015 Li X.

Zuo X.H.

Jing J.Z.

Ma Y.T.

Wang J.M.

Liu D.F.

Zhu J.L.

Du X.M.

Xiong L.

Du Y.Y.

et al. Small-molecule-driven direct reprogramming of mouse fibroblasts into functional neurons. Zhang et al., 2015 Zhang L.

Yin J.C.

Yeh H.

Ma N.X.

Lee G.

Chen X.A.

Wang Y.M.

Lin L.

Chen L.

Jin P.

et al. Small molecules efficiently reprogram human astroglial cells into functional neurons. Chambers et al., 2009 Chambers S.M.

Fasano C.A.

Papapetrou E.P.

Tomishima M.

Sadelain M.

Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Gao et al., 2017 Gao L.

Guan W.

Wang M.

Wang H.

Yu J.

Liu Q.

Qiu B.

Yu Y.

Ping Y.

Bian X.

et al. Direct generation of human neuronal cells from adult astrocytes by small molecules. One discovery made in this study is the synergistic modulation of multiple signaling pathways that are critical for chemical reprogramming. Modulating a single signaling pathway alone is not sufficient to change the cell fate. For example, while CHIR99021 alone increased NGN2 by 120-fold, it did not convert astrocytes into neurons, suggesting that modulating a single signaling pathway is not sufficient for chemical conversion. Nevertheless, CHIR99021 is one of the most widely used small molecules in chemical reprogramming studies (). Application of SB431542 and LDN193189 together has been reported to induce neurogenesis from stem cells (), but they are not sufficient to reprogram HAs into neurons, suggesting that HAs, as terminally differentiated cells, are very different from neural stem cells in terms of reprogrammability. By substituting each core drug with its functional analogs, we further demonstrate that it is not the specific drugs (SLCD) per se but rather the four signaling pathways (Notch, GSK-3β, TGF-β, and BMP) that are regulated by the four core drugs are critical for chemical reprogramming. Interestingly, modulating any three out of the four signaling pathways can successfully reprogram HAs into neurons, suggesting that these four pathways may have some overlapping functions. It is worth mentioning that, during the progress of this study, Dr. Pei's group reported that human adult astrocytes isolated from patients with brain tumor can be reprogrammed into neurons by a six-small-molecule cocktail VCRFBI (VPA, CHIR99021, Repsox, forskolin, i-Bet151, and ISX-9) (). This is a very interesting work because it represents another major step toward a future clinical application to directly convert adult HAs into neurons using a drug approach. On the other hand, it should be cautioned that any astrocytes isolated from the brain and put into Petri dishes may lose their in vivo identity. Therefore, it is still pivotal to test whether small molecules can convert HAs directly in a brain circuit in vivo. Interestingly, we have unexpectedly found that in vivo injection of the core drugs can significantly increase adult neurogenesis in the mouse hippocampus. This finding of small molecules passing through the BBB and regulating adult neurogenesis may have important implications for future development of drug therapies.

Cheng et al., 2015 Cheng L.

Gao L.

Guan W.

Mao J.

Hu W.

Qiu B.

Zhao J.

Yu Y.

Pei G. Direct conversion of astrocytes into neuronal cells by drug cocktail. Gao et al., 2017 Gao L.

Guan W.

Wang M.

Wang H.

Yu J.

Liu Q.

Qiu B.

Yu Y.

Ping Y.

Bian X.

et al. Direct generation of human neuronal cells from adult astrocytes by small molecules. Li et al., 2015 Li X.

Zuo X.H.

Jing J.Z.

Ma Y.T.

Wang J.M.

Liu D.F.

Zhu J.L.

Du X.M.

Xiong L.

Du Y.Y.

et al. Small-molecule-driven direct reprogramming of mouse fibroblasts into functional neurons. Guo et al., 2014 Guo Z.Y.

Zhang L.

Wu Z.

Chen Y.C.

Wang F.

Chen G. In vivo direct reprogramming of reactive glial cells into functional neurons after brain injury and in an Alzheimer's disease model. Heinrich et al., 2010 Heinrich C.

Blum R.

Gascon S.

Masserdotti G.

Tripathi P.

Sanchez R.

Tiedt S.

Schroeder T.

Gotz M.

Berninger B. Directing astroglia from the cerebral cortex into subtype specific functional neurons. Zhang et al., 2015 Zhang L.

Yin J.C.

Yeh H.

Ma N.X.

Lee G.

Chen X.A.

Wang Y.M.

Lin L.

Chen L.

Jin P.

et al. Small molecules efficiently reprogram human astroglial cells into functional neurons. Gao et al., 2017 Gao L.

Guan W.

Wang M.

Wang H.

Yu J.

Liu Q.

Qiu B.

Yu Y.

Ping Y.

Bian X.

et al. Direct generation of human neuronal cells from adult astrocytes by small molecules. Another discovery of this study is that, regardless of nine- or four-molecule cocktails, a common outcome of our chemical reprogramming protocols is the upregulation of neural transcription factors NEUROD1 and NGN2. Consistent with our discovery, recent work on chemical reprogramming of cultured fibroblasts or astrocytes into neurons also reported an upregulation of NEUROD1 and NGN2 together with other transcription factors (). Therefore, it seems that our chemical reprogramming approach is an upstream process that may trigger the upregulation of neural transcription factors, which then carry on the reprogramming process. Furthermore, we and other groups have reported that overexpression of neural transcription factors NEUROD1 () and NGN2 () can both convert astrocytes into glutamatergic neurons. Interestingly, both our nine- and four-molecule cocktails convert HAs mainly into glutamatergic neurons as well (), possibly through the upregulation of NEUROD1 and NGN2. Dr. Pei's group recently also reported that a six-drug cocktail (VCRFBI) converted adult HAs into glutamatergic neurons associated with upregulation of neural transcription factors including NEUROD1 and NGN2 (). Notably, our nine-drug cocktail generated a small percentage of GABAergic neurons but a four-drug cocktail did not, suggesting that the small molecules removed in this current study (TTNPB, SAG/Purmo, VPA, TZV) might be responsible for the small number of GABAergic neurons. Further work is needed to find various chemical cocktails that can reprogram HAs into other neuronal subtypes such as GABAergic and dopaminergic neurons. In addition, besides the four signaling pathways identified in this study, other signaling pathways that may also be important for chemical reprogramming of astrocytes into neurons, or to reprogram other glial cells such as NG2 glia into neurons, should be investigated.

In summary, this study reveals a drug formula that uses only three or four small molecules to efficiently reprogram HAs into functional neurons. Such a simple chemical reprogramming protocol makes it possible to develop a potentially useful drug therapy for neuroregeneration and brain repair, although great challenges such as chemical toxicity and CNS drug delivery are yet to be solved in future studies.