HK1237369B - Chemical reprogramming of human glial cells into neurons for brain and spinal cord repair - Google Patents

Description

Chemical reprogramming of human glial cells into neural cells for brain and spinal cord repair

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/084,365, filed on November 25, 2014, and U.S. Provisional Application No. 62/215,828, filed on September 9, 2015, the disclosures of each of which are incorporated herein by reference.

Statement Regarding Federally Funded Research

This invention was made with government support under Contract Nos. MH083911 and AG045656 awarded by the National Institutes of Health. The government has certain rights in this invention.

Technical Field

The present disclosure relates generally to the prevention and treatment of disorders associated with glial scar tissue, and more particularly to compositions and methods comprising small molecules for converting intrinsic glial cells into functional neurons for brain and spinal cord repair.

Background Art

The regeneration of functional neurons in neurodegenerative diseases or after neural injury remains a major challenge in the field of neural repair. Current efforts are mainly focused on cell replacement therapy using exogenous cells derived from embryonic stem cells or induced pluripotent stem cells (Buhnemann et al., 2006; Emborg et al., 2013; Nagai et al., 2010; Nakamura and Okano, 2013; Oki et al., 2012; Sahni and Kessler, 2010). Despite its great potential, this cell transplantation approach faces significant obstacles in clinical application, such as potential immune injection, tumorigenesis, and uncertainty in differentiation (Lee et al., 2013; Liu et al., 2013b; Lukovic et al., 2014). Furthermore, although previous studies have shown that astrocytes can be directly converted into functional neurons in vitro (Guo et al., 2014; Heinrich et al., 2010) and in vivo (Grande et al., 2013; Torper et al., 2013; Guo et al., 2014), and that astrocytes can be converted into neuroblasts and then differentiated into neuronal cells in the injured mouse brain (Niu et al., 2013) or spinal cord (Su et al., 2014), these methods have the significant disadvantage of requiring viral infection in the brain. Therefore, these previous methods require the performance of complex brain surgery, intracranial injection of viral particles, and the considerable risks associated with these steps. Therefore, there is still an ongoing and unmet need for new compositions and methods for regenerating functional neurons in the central nervous system or peripheral nervous system without the need to introduce exogenous reprogrammed cells or viral constructs into human subjects.

Summary of the Invention

The present disclosure provides compositions and methods for chemically reprogramming glial cells into neurons. The present disclosure differs significantly from previous methods, at least in part, because it involves reprogramming glial cells using chemically synthesized compounds. Therefore, it does not include the risks associated with introducing exogenous genes, viral vectors, or engineered cells into patients, nor does it require manipulation of stem cells or other multipotent cells or somatic cells such as fibroblasts in culture to differentiate or reverse-differentiate them into neurons or otherwise prepare cells for administration to a subject. Instead, the present disclosure involves reprogramming glial cells already present in an individual's nervous system, thereby converting glial cells into neurons using a combination of small molecules described more fully below. It is expected that the compositions and methods provide a convenient and safe method for treating various neural injuries or neurodegenerative diseases involving, for example, reactive glial cells or glial scars. Those skilled in the art will recognize that glial scars can be caused by many causes known in the art, and they generally involve astrocyte proliferation following injury or disease processes in the central nervous system and peripheral nervous system, including the brain and spinal cord. Active astrocytes are the main cellular component of glial scars, followed by NG2 glial cells and microglia. Therefore, in embodiments, the present disclosure includes chemically inducible reprogramming of astrocytes to convert them into neurons. However, similar chemical reprogramming methods can also be used to convert NG2 glial cells or microglia, or other cell types surrounding cerebral blood vessels, into neurons.

It is apparent from the description, drawings and data presented in this disclosure that we have developed in vitro and in vivo data demonstrating that pre-existing, differentiated glial cells are reprogrammed into neurons. In particular, our data show that the sequential application of small molecules as described herein results in a majority of human astrocytes (~70%) being reprogrammed into neuronal cells in vitro. In addition, these small molecule-reprogrammed human neurons can survive in culture for more than five months and exhibit robust synaptic activity. Furthermore, injection of human astrocyte-converted neurons into mouse brains demonstrates that human neurons can be integrated into local brain circuits. Therefore, the data presented in this disclosure collectively demonstrate that chemical reprogramming of human astrocytes into functional neurons in damaged or diseased brains in vivo is now possible without the need to introduce individual cultured cells or viruses or other expression vectors or exogenous genes, a method never before achieved.

The present disclosure includes demonstrating that combining compounds that act together on signals including but not limited to transforming growth factor beta (TGF-β), bone morphogenetic protein (BMP), glycogen synthase kinase 3 (GSK-3) and gamma-secretase/Notch pathways can reprogram glial cells into neurons. In general, the present disclosure includes administering compounds capable of inhibiting these pathways to individuals in need. In one embodiment, the present disclosure includes administering a combination of compounds selected from the group consisting of thiazovivin, LDN193189, SB431542, TTNPB, CHIR99021, DAPT, VPA, SAG, 2,6,9-trisubstituted purines or pharmaceutically acceptable salts thereof or analogs of these compounds or compounds having the same or similar functional effects such that their administration reprograms glial cells into neurons and a variety of combinations of the aforementioned compounds. In one approach, the compound administered to the individual comprises at least three compounds selected from the four compounds of the core (which is not intended to be bound by any particular theory) and is considered to be required to achieve reprogramming. These compounds are SB431542, LDN193189, CHIR99021, and DAPT, which can also be substituted with functional analogs as described below. In one embodiment, the present disclosure includes the use of any of the following combinations: i) LDN193189/CHIR99021/DAP, ii) SB431542/CHIR99021/DAPT; iii) LDN193189/DAPT/SB431542, and iv) LDN193189/CHIR99021/SB431542. In one embodiment, three pharmaceutical compositions of SB431542/CHIR99021/DAPT are used.

The compositions can be administered to a subject in need thereof in any combination and can include simultaneous administration of a combination of at least two compounds, and can include sequential administration of any compounds and combinations, specific embodiments of which are described more fully below. In certain embodiments, a composition comprising LDN193189 and SB431542 is introduced to a subject as an initial administration, and a composition comprising CHIR99021 and DAPT is introduced to a subject as a subsequent administration.

The composition can be administered using any acceptable route and formulation, including but not necessarily limited to oral, intranasal, intravenous, and intracranial methods. In one aspect, the composition is administered orally.

In certain embodiments, the methods of the present disclosure are used for therapeutic purposes to induce the reprogramming of glial cells into neurons in an individual in need of neurons due to a disorder comprising neuronal loss and/or glial scarring. In certain embodiments, the individual is in need of generated neurons due to ischemic brain injury such as stroke, hypoxia or other brain trauma, or has been diagnosed with or is suspected of having Alzheimer's disease or other neurodegenerative disorders.

On the other hand, the present disclosure includes pharmaceutical compositions comprising a combination of at least two of thiazovivin, LDN193189, SB431542, TTNPB, CHIR99021, DAPT, VPA, SAG, and 2,6,9-trisubstituted purines, wherein the composition is used to reprogram glial cells into neurons. Pharmaceutical compositions comprising salts and analogs of these compounds and functionally related compounds (i.e., functional analogs) are also contemplated. In an embodiment, the pharmaceutical composition of the present disclosure includes at least two of SB431542, LDN193189, CHIR99021 and DAPT, and/or a pharmaceutically acceptable salt thereof. In an embodiment, the pharmaceutical composition includes all of SB431542, LDN193189, CHIR99021 and DAPT, and may also include additional compounds. In an embodiment, the present disclosure includes a composition containing as an active agent for reprogramming glial cells to neurons, one of the following groups: i) LDN193189/CHIR99021/DAPT, ii) SB431542/CHIR99021/DAPT, iii) LDN193189/DAPT/SB431542, and iv) LDN193189/CHIR99021/SB-431542. In one embodiment, any three members of the aforementioned groups are included. In one embodiment, the composition comprises or consists of a three-drug combination of SB431542/CHIR99021/DAPT.

In another aspect, the present disclosure includes an article of manufacture comprising packaging and at least one container, the container comprising a pharmaceutical composition comprising a combination of at least three compounds selected from the group consisting of thiazovivin, LDN193189, SB431542, TTNPB, CHIR99021, DAPT, VPA, SAG, 2,6,9-trisubstituted purines and pharmaceutically acceptable salts thereof, the packaging comprising printed information providing an indication that the pharmaceutical composition is used to treat a condition, wherein the condition is associated with a lack of functional neurons.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Sequential exposure to defined panels of small molecules converts human astrocytes into neurons. (A) Schematic illustration of our strategy for converting cultured human astrocytes into neurons using a cocktail of small molecules. Note that different subsets of small molecules were used at different stages of reprogramming. (B, C) Quantitative analysis of human astrocyte cultures (HA1800, ScienCell). The majority of cells in our human astrocyte cultures were immunopositive for the astrocyte marker GFAP (79.3 ± 4.9%), the astrocyte glutamate transporter GLT-1 (82.5 ± 4.3%), and to a lesser extent, S100β (39.3 ± 1.8%). No cells were immunopositive for the neuronal markers NeuN, MAP2, or doublecortin (DCX). HuNu, a human nuclear marker, was used for human cells. N = 3 batches. (D) Control human astrocyte cultures without small molecule treatment showed few cells immunopositive for the neuronal markers DCX (green), β-III tubulin (Tuj1, red), or MAP2 (cyan). (E) Sequential exposure of human astrocytes to small molecules yielded a large number of neurons immunopositive for DCX (green), Tuj1 (red), and MAP2 (cyan). MCM stands for master converting molecule and includes nine small molecules used collectively for reprogramming. Analysis was performed 14 days after the initial small molecule treatment. (F) Thirty days after the initial small molecule treatment, human astrocyte-converted neurons developed extensive dendrites (MAP2, green) and were immunopositive for the mature neuronal marker NeuN (red). (G) Small molecule-converted human neurons survived in culture for four months and displayed robust dendritic trees (MAP2, green) and extensive axons (SMI312, red). (H) Astroglial lineage labeled with GFAP::GFP retrovirally demonstrates that GFP+ cells are immunopositive for the neuronal marker NeuN (red) after small molecule treatment. N = 5 batches. (I and J) Small molecule treatment achieves high conversion efficiency after 8 days of exposure to MCM (67.1 ± 0.8%, Tuj1+ neurons/total DAPI-labeled cells, n = 4 batches). (K) Chemical reprogramming of human midbrain astrocytes into neurons. One month after initial small molecule treatment of human midbrain astrocytes (ScienCell), the majority of cells are immunopositive for the neuronal markers NeuN (red) and MAP2 (green). (L) After one month of culture in neuronal differentiation medium without small molecule treatment, control human midbrain astrocyte cultures show few cells immunopositive for NeuN (red) or MAP2 (green). (M) Quantitative analysis shows a large number of NeuN-positive neurons converted from human midbrain astrocytes 1 month after small molecule treatment (199.7 ± 9.2 per 40x field of view), while the control group had only a small number of NeuN+ cells (5.6 ± 1.4 per 40x field of view). N = 4 batches. Scale bar: Panel B, 50 μm; other images, 20 μm. ***P < 0.001, Student's t test. Data are expressed as mean ± SEM.

Figure 2. Functional analysis of neurons converted from human astrocytes induced by small molecule treatment. (A) Long-lived small molecule-induced human neurons (cultured for 5 months) and numerous synaptic points (SV2, red) along dendrites (MAP2, Green). Scale bar: 20 μm. (BD) Show representative traces of Na+ and K+ currents recorded from 1-month-old (B) and 2-month-old (C) human neurons induced by small molecules. Panel D shows the blockade of Na+ currents by TTX (2 μM). (E) Quantitative analysis of peak Na+ and K+ currents in neurons converted from human astrocytes from 2 weeks to 3 months of age by small molecules. (F) Representative traces of repetitive action potentials recorded in small molecule-induced human neurons 75 days after initial drug treatment. (G and H) Show representative traces of spontaneous synaptic activity in 2-month-old converted human neurons. Holding potential = -70 mV. (H) Expanded trace from (G). (I) Inhibitory GABAergic activity is shown in human astrocyte-converted neurons when the holding potential is held at 0 mV (2 months old). This activity is blocked by the GABAA receptor antagonist bicuculline (BIC, 10 μM). (JK) Show representative traces of spontaneous bursting activity in 3-month-old small molecule-induced human neurons. HP = -70 mV. (K) is an expanded view of the burst in (J). (L) Burst activity is blocked by TTX (2 μM). The glutamate receptor antagonist DNQX (10 μM) blocks most of the synaptic activity at -70 mV, suggesting that they are glutamatergic activities. (M) Dual whole-cell recordings show that small molecule-converted human neurons form a strong synaptic network and fire synchronously. (N) Ca ²⁺ ratio imaging further shows that small molecule-converted human neurons are highly connected and exhibit synchronous activity. Data are expressed as mean ± SEM.

Figure 3. Characterization of neurons converted from human astrocytes induced by small molecules. (A-C) Immunostaining for anterior-posterior neuronal markers showed that small-molecule-converted human neurons were positive for the forebrain marker FoxG1 (A) but negative for the hindbrain and spinal cord markers HOX B4 (B) and HOX C9 (C). (D-F) Immunostaining for cortical neuronal markers showed that small-molecule-converted human neurons were negative for the superficial layer marker Cux1 (D) and positive for the deep layer markers Ctip2 (E) and Otx1 (F). (G-H) Small-molecule-converted human neurons were also immunopositive for the general cortical neuronal marker Tbr1 (G) and the hippocampal neuronal marker Prox1 (H). (I) Quantitative analysis 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 bar: 20 μm. (J) MCM-transformed human neurons are immunopositive for VGluT1. (K) A small fraction of MCM-transformed human neurons are GAD67-positive. (L-N) MCM-transformed neurons are immunonegative for the cholinergic neuron marker vesicular acetylcholine transporter (VAChT) (L), the dopaminergic neuron marker tyrosine hydroxylase (TH) (M), or the spinal motor neuron marker Isl1 (N). (O) Quantitative analysis of small molecule-transduced human neurons (VGluT1, 88.3±4%, n=4 batches; GAD67, 8.2±1.5%, n=4 batches). Scale bar: 20 μm. Data are expressed as mean ± SEM.

Figure 4. Transcriptional and epigenetic regulation during chemical reprogramming of human astrocytes into neurons. (A-B) PCR arrays show substantial transcriptional activation of neural transcription factors (NGN1/2, NEUROD1, and ASCL1) and the immature neuronal gene DCX on day 4 (A) or day 8 (B) after small molecule treatment. Note that DCX increased by more than 2000-fold compared to the control at D8. Genes that showed significant changes in PCR array assays are provided (P<0.05, Mann-Whitney t-test). (C-F) Transcriptional changes over the time course are shown by real-time quantitative PCR analysis. Neural transcription factors NGN2 (C) and NEUROD1 (D) showed transcriptional peaks on D4 and D6, respectively; while astrocyte genes GFAP (E) and ALDH1L1 (F) were significantly downregulated. *P<0.05, **P<0.01, ***P<0.001; two-way ANOVA followed by Dunnett’s test. N=3 batches. (G-I) Epigenetic regulation of the GFAP promoter and transcription start site during chemical reprogramming. MeDIP-seq showed a significant increase in methylation of the GFAP promoter region (G, box region) after 8 days of small molecule treatment, which was confirmed by subsequent BS-seq (bisulfite sequencing) (H). It was noted that the hypermethylated sites were located in the flanking regions of two important transcription factor binding sites (STAT3 and AP1), which significantly inhibited the transcription of GFAP. BS-seq also showed a significant increase in the methylation levels of the GFAP transcription start site (TSS) and 5'UTR regulatory region (I), further indicating that GFAP transcription is inhibited by DNA methylation. (J-K) MeDIP-seq and BS-seq revealed a significant decrease in methylation in the promoter region of the neuronal gene NEFM (neurofilament M), indicating transcriptional activation of neuronal genes during chemical reprogramming of human astrocytes into neurons. (L-M) ChIP-qPCR revealed a significant increase in histone acetylation in the NGN2 promoter region following small molecule treatment, likely caused by the HDAC inhibitor VPA. (N-O) H3K4 methylation levels were significantly increased in the NGN2 promoter region (N), while H3K27 methylation at the NGN2 transcription start site was significantly decreased (O), indicating epigenetic activation of NGN2 through histone modification. Data are presented as mean ± SEM.

Figure 5. Increased protein expression levels of neural transcription factors during chemical reprogramming. (A-C) Representative images illustrate the gradual activation of endogenous neural transcription factors Ascl1 (A), Ngn2 (B), and NeuroD1 (C) following small molecule treatment for different days. (D-E) Representative images demonstrate the gradual increase in neuronal signals DCX (D) and NeuN (E) from D0 to D10. (F) Representative images demonstrate a decrease in the astrocyte marker GFAP from D0 to D10. Scale bar: 20 μm. (G-I) Quantitative analysis of protein expression levels of Ascl1 (G), Ngn2 (H), and NeuroD1 (I). Note the significant three-fold increase in Ascl1 on day 2, while Ngn2 peaks on day 4 and NeuroD1 peaks on day 6. N = 3 batches. (J) Quantitative data demonstrate a significant increase in NeuN from day 6 to day 10. N = 3 batches. (K) Quantitative data demonstrate a significant decrease in GFAP from D0 to D10. N = 3 batches. Data are expressed as mean ± SEM.

Figure 6. Evaluation of the essential role of each individual small molecule during astrocyte-neuron reprogramming. (A) Human astrocytes treated with 1% DMSO served as a control. NeuN, green; MAP2, red. (B) Defined combinations of nine small molecules induced the generation of numerous neurons (14 days after initial small molecule treatment, identical to the following depletion experiments). (C-F) Individual depletion of DAPT (C), CHIR99021 (D), SB431542 (E), or LDN193189 (F) from the nine-molecule pool significantly reduced the number of converted neurons. (G) Combined depletion of the sonic hedgehog agonists SAG and Purmo slightly reduced the number of converted neurons. (H) Depletion of VPA also slightly reduced the number of neurons. (I-J) Depletion of Tzv (I) or TTNPB (J) did not affect neuronal conversion. Scale bar: 20 μm. (K) Quantitative analysis showed that DAPT was the most effective reprogramming factor, followed by CHIR99021, SB431542, and LDN193189. *P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA followed by Sidak's multiple comparison test. N = 3 batches. Data are expressed as mean ± SEM.

Figure 7. In vivo survival and integration of small molecule-transformed human neurons in the mouse brain. (A) Schematic diagram showing the transplantation of small molecule-transformed human neurons into the mouse brain on day 1 after birth. (B) 7 days after cell injection (7DPI), GFP-positive cells were identified around the lateral ventricle. Many GFP-positive cells were also positive for DCX (red), and all GFP-positive cells were immunopositive for human nuclei (HuNu, Blue), indicating their human cell characteristics. N=6 mice. (C) At 11DPI, some GFP-positive cells were immunopositive for MAP2 (red), indicating the survival and growth of human neurons in the mouse brain in an in vivo manner. N=6 mice. (D) At 11DPI, some GFP-positive human neurons that were immunopositive for NeuN (red) and HuNu (cyan) migrated to the adjacent striatal region and extended long processes. (E) Human neurons labeled with NeuN (red) and HuNu (blue) survived for over a month in 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 shown by numerous synaptic puncta (SV2, red) along GFP-positive neurites (inset), indicating synaptic integration of transplanted human neurons into local neural circuits. N = 2 mice. Scale bar: 20 μm.

Figure 8, characterization of cultured human cortical astrocytes. (A) Human cortical astrocytes (HA1800, Sciencell) cultured in glial culture medium (GM, 10% FBS) or N2 culture medium (for reprogramming, without FBS) and immunostained with Musashi, Nestin and Sox2. Note that in both culture media, there are no neural progenitor cells. (B) Quantitative analysis of neural stem cell markers shows that the expression levels of Musashi, Nestin and Sox2 in cultured human astrocytes are lower than those in human neural progenitor cells (NPCs). ***P<0.0001, one-way analysis of variance, followed by Dunnett's test. N=3 batches. (C-D) Human astrocytes (HA1800, Sciencell) were cultured in neuronal differentiation medium (NDM) supplemented with BDNF, NT3 and NGF for 1 month to ensure neural differentiation, if there are any neural stem cells in the astrocyte cultures. Quantitative analysis showed that the majority of cells were immunopositive for the astrocyte markers S100β (74.7±1.5%), GFAP (83.6±1.2%), glutamate synthase (GS) (94.3±0.7%), and GLT-1 (91.4±1.5%). A small number of cells stained positively for DCX (5.18±0.67) and Tuj1 (8.98±0.75%), but no cells were positive for NeuN or NG2. N = 4 batches, scale bar = 20 μm. (E-H) Functional analysis of cultured human astrocytes (HA1800). Electrophysiological recordings revealed large K+ currents but no Na+ currents (E), gap junctional junctions between adjacent astrocytes (F-G), and glutamate (500 M) transporter currents (H). (F) shows dye binding between localized astrocyte domains after recording. Data are expressed as mean ± SEM.

Figure 9. Time-lapse imaging of astrocyte-neuron conversion during small molecule treatment. (A) Human astrocytes (HA) labeled with a CAG::GFP retrovirus expressing GFP (green) maintained astrocytic morphology at day 8 when treated with 1% DMSO as a control. Images were taken daily using a Nikon 2000 fluorescence microscope. In this time-lapse live cell imaging experiment, we used a CAG::GFP retrovirus instead of a GFAP::GFP retrovirus because the GFAP promoter is weak, and the GFP signal in GFAP::GFP-infected cells was too weak for live cell imaging. (B) Representative images show that GFP+ cells in the control group are immunopositive for the astrocyte marker GFAP (red) after 21 days of culture. (C) Two GFP-labeled human astrocytes were monitored from one day before to 10 days after small molecule treatment. A clear morphological transition from astrocytes at day 0 to neuron-like cells at day 9 was observed. (D) After time-lapse imaging, cells were fixed on day 21 and immunostained for the neuronal markers NeuN and Tuj1. GFP-labeled cells (green, arrows) after small molecule treatment were immunopositive for NeuN (red) and Tuj1 (cyan). Scale bar = 20 μm. (E-G) Human astrocytes were infected with the GFAP::GFP retrovirus for lineage tracing. All GFAP::GFP-infected cells were GFAP+ (E, red). Without small molecule treatment, GFP+ cells remained GFAP+ astrocytes after 18 days of culture (F, red), and no neurons were detected (G). N = 3 batches. Scale bar = 10 μm. For small molecule-treated groups, see Figure 8H. (H-J) Human astrocytes infected with the LCN2::GFP retrovirus were immunopositive for GFAP (H, red) and LCN2 (H, cyan). Without small molecule treatment, GFP+ cells retained astrocyte morphology and immunoreactivity for GFAP (I, red) after 18 days of culture. In contrast, after small molecule treatment, GFP+ cells became immunoreactive for NeuN (J, red). N = 3 batches. Scale bar = 10 μm.

Figure 10. Conversion of human astrocytes from different sources into neurons. (A) Characterization of human midbrain astrocytes (HA midbrain, Sciencell), which are immunopositive for the astrocyte markers GFAP, S100β, GS, and Glt1, but display low expression levels of the neural stem cell markers Sox2 and nestin. (B) Control human midbrain astrocyte cultures not treated with small molecules have few cells immunopositive for the neuronal markers DCX (green) or β-III tubulin (Tuj1, red). (C) Sequential exposure of human midbrain astrocytes to small molecules produces a large number of neuronal cells immunopositive for DCX (green) and Tuj1 (red). Analyzed 16 days after the initial small molecule treatment. (D-E) Immunostaining images of the axon marker SMI312 (green) in one-month-old cultures of human midbrain astrocytes that were not treated with small molecules (D) or treated with small molecules (E). (F) Long-term surviving human neurons converted from human midbrain astrocytes. A large number of synaptic points (SV2, red) are distributed along the dendrites (MAP2, green) in NeuN-positive (cyan) neurons. (G-H) Human astrocytes from different sources (Gibco) were also successfully reprogrammed into neurons using the same small molecule protocol with an efficiency of 41.1±3.6%. N=4 batches. (I) Immunostaining shows synaptophysin (red) and MAP2 (green) signals in 2-month-old neurons converted from Gibco human astrocytes. Therefore, human astrocytes from different sources can be successfully converted into neurons using the same small molecule strategy. The scale bars in panels A and I are 10 μm, and the rest are 20 μm.

Figure 11. Human astrocytes convert directly into neurons without undergoing a stem cell stage. (A-C) Representative images show low expression levels of the neural stem cell markers Sox2 (A, red), Nestin (B, green), and Pax6 (green) at different days of small molecule treatment compared to human NPC cultures. (D) Representative images show the lack of significant cell expansion during reprogramming, as indicated by the cell proliferation marker Ki67 (red). (E-F) BrdU was used to determine the birthdate of astrocyte-converted neurons before and after small molecule-mediated reprogramming. BrdU was added to the culture medium 1 day before (E) or 10 days after (F) small molecule treatment. Cells were fixed on day 30. Arrows in (E) point to cells where BrdU and NeuN colocalized. (G-I) Quantification of fluorescence intensities of Sox2 (G), Nestin (H), and Pax6 (I) during chemical reprogramming, normalized to the intensity at D0. Compared with D0, Sox2 expression levels increased slightly at D4-D10, but were much lower than those in NPC cells (G). On the other hand, nestin expression levels were very low compared with NPC cells (H). Similarly, there were very few Pax6+ cells compared with NPC cells (I). (J) Quantitative analysis of Ki67+ cells to assess cell proliferation rates during chemical reprogramming. Compared with D0, cell proliferation was significantly reduced on D2-D6 after small molecule treatment. The overall proliferation rate of human astrocytes was significantly lower than that of NPC cells. The reduced proliferation rate in the presence of small molecules indicates that cell expansion does not occur during chemical reprogramming. (K) Quantitative analysis of BrdU-labeled neurons in E-F. When BrdU was added before small molecule treatment, a large number of cells showed colocalization of BrdU and NeuN (77.3±3.8%). If BrdU was added after small molecule treatment, there were virtually no NeuN+ cells colocalizing with BrdU (D10 to D30, 1.75 ± 0.73%), indicating that most cells were converted into neurons in the presence of the small molecule. N = 3 batches. Scale bar = 20 μm. **P < 0.001; ***P < 0.0001; one-way ANOVA followed by Dunnett's multiple comparison test. Data are expressed as mean ± SEM.

Figure 12. Signaling pathways in small-molecule-mediated reprogramming. (A-B) PCR arrays demonstrate no significant gene expression changes on day 4 (A) or day 8 (B) in control human astrocyte cultures not treated with small molecules but treated with 1% DMSO (as a solvent control). (C-F) In control human astrocyte cultures (1% DMSO), real-time quantitative PCR also reveals minimal transcriptional changes in the neural transcription factors NGN2 (C) and NEUROD1 (D), or the glial genes GFAP (E) and ALDH1L1 (F). N = 3 batches. (G) Representative images show decreased levels of phosphorylated SMAD1/5/9 (green) in the nucleus after 2 days of small-molecule treatment. Quantitative analysis of p-SMAD1/5/9 fluorescence intensity indicates inhibition of the BMP signaling pathway. (H) Representative images and quantitative analysis demonstrate decreased levels of the D6 Notch intracellular domain (NICD) (green) after initial small-molecule treatment, indicating inhibition of the Notch signaling pathway. (I) Representative images and quantitative analysis demonstrate increased levels of phosphorylated GSK3β (green) after 6 days of small molecule treatment, indicating GSK3β inactivation. **P < 0.001; ***P < 0.0001; one-way ANOVA followed by Dunnett's multiple comparison test. N = 3 batches. Scale bar = 10 μm. Data are expressed as mean ± SEM.

Figure 13. In control human astrocytes without small molecule treatment, endogenous neural transcription factors showed no significant changes. (A-C) Immunostaining shows very low protein expression levels of the endogenous neural transcription factors Ascl1 (A), Ngn2 (B), and NeuroD1 (C) in control human astrocytes (1% DMSO). (D) NeuN staining shows almost no neurons under control conditions. (E) Representative images show constant expression of GFAP (red) under control conditions. (F-H) Quantitative analysis of fluorescence intensity of Ascl1 (F), Ngn2 (G), and NeuroD1 (H) from D2 to D10 of culture, normalized to the intensity at D0. (I) Quantitative analysis shows almost no NeuN-positive cells in control human astrocyte cultures from D0 to D10. (J) Quantitative analysis shows that GFAP expression in control human astrocyte cultures remains high from D0 to D10. N = 3 batches. Scale bar = 20 μm. Data are expressed as mean ± SEM.

Figure 14. Small molecules injected into the mouse cortex promote the differentiation of mouse cortical astrocytes into neural stem cells in vivo. (A) Six days after a single injection (dpi) of small molecules containing 0.1 nmol of SB431542, 0.01 nmol of LDN193189, 0.03 nmol of CHIR99021, 0.1 nmol of DAPT, 0.01 nmol of SAG, and 0.01 nmol of TTNPB (mixed in a total volume of 2 μl), mouse cortical astrocytes (GFAP, red) exhibit significant morphological changes and express high levels of nestin (green). Some cells expressing DCX (cyan) were observed around the injection site. N = 4 animals. (B) Quantitative analysis shows increased nestin expression in astrocytes of mice treated with small molecules in vivo (Student's t-test, ***P < 0.0001). (C) Cortical tissue treated with small molecules was isolated and cultured in vitro. Compared to control cortical tissue treated with PBS containing 6% DMSO, there are many more and larger primary neurospheres. (D-E) Quantitative analysis shows that cortical tissue treated with small molecules produces more neurospheres (D) and has larger size (E). Student's t-test, ***P<0.0001. (F) Primary neurospheres were subcultured and plated as single cells. Highly proliferative single cells continue to divide to form secondary neurospheres in suspension culture 3 days after plating. (G-H) Quantification shows that more secondary neurospheres with larger size (H) are formed in the small molecule-treated group (G). Student's t-test, **P<0.001, ***P<0.0001. (I) Cells from secondary neurospheres were cultured as a monolayer and were immunopositive for the neural stem cell markers Sox2 (green) and nestin (red). (J) Cells from secondary neurospheres were differentiated into neurons (Tuj1, green) in neuronal differentiation medium and into oligodendrocytes (CNPase, red) or astrocytes (GFAP, green) in glial medium. N = 3 batches. Scale bars: A, I, J = 20 μm. C, F = 200 μm. Data are expressed as mean ± SEM.

Figure 15. Data show that a total of four small molecules can successfully reprogram human glial cells into neurons. (A) The core drugs, SB431542 5μM, LDN193189 0.25μM, CHIR99021 1.5μM, and DAPT 5μM, were added to the human astrocyte cell line HA1800 for 6 days. The drug-containing medium was replaced every two days. 14 days after drug addition, cells were immunostained for the neuronal marker NeuN, demonstrating the conversion of many human glial cells into neurons. (B-C) SB431542 was replaced by its functional analog Repsox 1μM (B) or A-8301 0.25μM (C). (D) Quantification of NeuN-immunopositive cells in the four groups. Different batches were normalized to the core group. The conversion rate of the SB replaced by Repsox group was 88.5±5.0% of that of the core drug group, while the conversion rate of the SB replaced by A-8301 group was 86.8±5.0% of that of the core drug group.

Figure 16. Data showing the efficacy of the core drugs. (A) The core drugs (SB431542 5μM, LDN193189 0.25μM, CHIR99021 1.5μM, and DAPT 5μM) were added to the human astrocyte cell line HA1800 for 6 days. The drug-containing medium was replaced every two days. 14 days after drug addition, cells were immunostained for the neuronal marker NeuN. (B-C) LDN193189 was replaced by its functional analogs Dorsomorphin 1μM (B) and DMH1 1.5μM (C). (D) Quantification of NeuN-immunopositive cells in the four groups. Different batches were normalized to the core group. The conversion efficiency of the group where LDN193189 was replaced by Dorsomorphin was similar to that of the core group, while the conversion rate of the group where LDN193189 was replaced by DMH1 was 86.8±4.9% of that of the core group.

Figure 17. Data showing the efficacy of core drugs. (A) Core drugs (SB431542 5μM, LDN193189 0.25μM, CHIR99021 1.5μM, and DAPT 5μM) were added to the human astrocyte cell line HA1800 for 6 days. Drug-containing culture medium was replaced every two days. 14 days after drug addition, cells were immunostained for the neuronal marker NeuN. (B-C) CHIR99021 was replaced by its functional analog ARA014418 6μM (B) or SB216763 1μM (C). (D) Quantification of NeuN-immunopositive cells in the four groups. Different batches were normalized to the core group. The conversion rate in the group where CHIR was replaced by ARA014418 was 56.9±4.3% of that in the core group, while the conversion rate in the group where CHIR was replaced by SB216763 was 76.07±4.2% of that in the core group.

Figure 18. Data showing the efficacy of the core drugs. A) The core drugs (SB431542 5μM, LDN193189 0.25μM, CHIR99021 1.5μM, and DAPT 5μM) were added to the human astrocyte cell line HA1800 for 6 days. The drug-containing medium was replaced every two days. 14 days after drug addition, cells were immunostained for the neuronal marker NeuN. (B-C) DAPT was replaced by its functional analog BMS906024 2μM (B) and RO4929097 0.5μM (C). (D) Quantification of NeuN-immunopositive cells in the four groups. Different batches were normalized to the core group. The group in which DAPT was replaced by RO4929097 achieved a conversion rate similar to that of the core group, while the group in which DAPT was replaced by BMS906024 achieved a conversion rate that was 85.0±6.1% of that of the core group.

Figure 19. Data show that any combination of SB431542, LDN193189, CHIR99021, and DAPT can reprogram human glial cells into neurons. Drugs were added for 6 days, and immunostaining for the neuronal marker NeuN was performed 14 days after drug treatment. The 3-drug combination of SB431542/CHIR99021/DAPT appears to be more effective than SB431542/LDN193189/CHIR99021.

DETAILED DESCRIPTION

The present disclosure includes compositions and methods designed to convert human glial cells into functional neurons. In embodiments, the present disclosure includes, but is not necessarily limited to, the reversal of glial scars to neural tissue, which is expected to be useful in various therapies, non-limiting embodiments of which include brain and spinal cord repair. The method generally includes administering to an individual in need an effective amount of a combination of compounds including thiazovivin, LDN193189, SB431542, TTNPB, CHIR99021, DAPT, VPA, SAG, and 2,6,9-trisubstituted purines and combinations thereof, or a combination of compounds selected from the group consisting of thiazovivin, LDN193189, SB431542, TTNPB, CHIR99021, DAPT, VPA, SAG, and 2,6,9-trisubstituted purines and combinations thereof, such that the glial cells in the individual are converted into neurons. In embodiments, optional compounds are used, wherein these compounds have the same or similar effects as the above-mentioned compounds, and wherein the administration of the combination results in the conversion of glial cells into neurons.

In embodiments, the present disclosure is expected to be widely applicable to the treatment of any human subject that needs neurons to produce.Neuron production needs to grow is to affect neuronal function and/or reduce the result of any of the various conditions, disorders or damages of the number of functional neurons in an individual.Therefore, the present disclosure is directed to preventing and/or treating, including but not necessarily limited to, ischemic brain injury, such as caused by stroke, hypoxia or other brain trauma or caused by glial scar formation or neurodegenerative disease.In embodiments, the present disclosure is directed to treating neurodegenerative disorders, including but not limited to Alzheimer's disease or other conditions showing dementia, or chronic traumatic encephalopathy (CTE) such as athletes with a history of acute or recurrent brain trauma (i.e., concussion), or Parkinson's disease, or Huntington's disease, or multiple sclerosis, or glioma, or spinal cord injury or spinal muscular atrophy or amyotrophic lateral sclerosis (ALS).

The present disclosure is considered to be novel relative to previous methods because it does not include modified cells or viral constructs introduced into subjects.For example, although U.S. Patent Publication No. 20130183674 discloses the use of a cell culture medium containing compound SB431542, LDN1933189, SU5402, CHIR99021 and DAPT, for inducing pluripotent (pluripotent) stem cells or specialized (multipotent) stem cells to be formed into nociceptor cells (nociceptor cells), it is limited to using these compounds for in vitro differentiation of these stem cells, and importantly, this prior art method is different from our reprogramming of glial cells to neuronal cells, because stem cells can naturally differentiate into neurons, but glial cells cannot become neurons, unless undergoing reprogramming process, such as the reprogramming process demonstrated in the present disclosure. In addition, it will be appreciated by those skilled in the art that the stem cells cultivated or the neurons differentiated thereof are injected into human subjects, particularly brain, causing risks (srisk) to subjects. Likewise, as mentioned above, it has been demonstrated that astrocytes can be converted into neurons in vivo, but these approaches involve the introduction of viral vectors or other exogenous genes into the subject, which also poses particular risks to the subject.

In contrast to previous approaches, the present disclosure provides, in various embodiments, the use of completely cell- and virus-free pharmaceutical formulations comprising chemical compounds that act synergistically with each other to induce the conversion of glial cells into neurons, and provides in vivo demonstration of this process.

In an embodiment, the present disclosure includes administering to a subject in need thereof an effective amount of one or more compositions comprising a combination of compounds selected from thiazovivin, LDN193189, SB431542, TTNPB, CHIR99021, DAPT, VPA, SAG, and 2,6,9-trisubstituted purines as active ingredients. In an embodiment, different combinations of these compounds are administered sequentially. Each of these compounds is known in the art and is commercially available. The present disclosure includes compositions and methods containing any three, four, five, six, seven, eight, or all nine of these compounds, and may include additional compounds described herein or obvious to those skilled in the art that may benefit the present disclosure. The present disclosure includes pharmaceutically acceptable salts of these compounds, analogs of the compounds and salts, and compounds that function the same or similar to the compounds, provided that their combination is administered to an individual to cause the conversion of glial cells into neurons.

In one embodiment, the present disclosure includes administering a combination of compounds to a subject (simultaneously or sequentially), wherein the combination comprises or consists of at least three of SB431542, LDN193189, CHIR99021, and DAPT. Without wishing to be bound by theory, these four compounds are sometimes referred to herein as core compounds.

Of these compounds, it will be apparent to one skilled in the art that SB431542 is: -[4-(1,3-benzodiazol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide and has the chemical structure:

LDN193189 is: 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride and has the chemical structure:

CHIR99021 is: 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-nitrile pyridine and has the chemical structure:

DAPT is: N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester and has the chemical structure:

Those skilled in the art will recognize that, to the extent not expressly indicated in the chemical formulas and nomenclature presented in this disclosure, each compound described herein includes its pharmaceutically acceptable salt. It will also be recognized that SB-431542 is an inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. LDN-193189 is an inhibitor of bone morphogenetic protein type I receptors ALK2 and ALK3. CHIR99021 is a selective inhibitor of glycogen synthase kinase 3 (GSK-3), and DAPT is an inhibitor of gamma-secretase. Therefore, other compounds having these functions (i.e., functional analogs) are included within the scope of this disclosure. In this regard, the present disclosure provides data demonstrating that the reprogramming of glial cells to neurons can be achieved using a combination of only three drugs selected from a group of four core compounds comprising SB431542, LDN193189, CHIR99021, and DAPT. In addition, the disclosure provides that these four core drugs can be replaced with functional analogs and still have the evidence of similar effects, i.e., promote human glial cells to be converted into neurons. " Functional analog " used herein refers to a compound having similar physical, chemical, biochemical or pharmacological properties compared to another compound. Functional analogs may or may not have structures similar to each other. In the disclosure, it is demonstrated that the combined compound acting together on signal transduction by transforming growth factor β (TGF-β), bone morphogenetic protein (BMP), glycogen synthase kinase 3 (GSK-3) and gamma-secretase/Notch pathways can reprogram glial cells into neurons. This is specifically described using drug combinations i) LDN193189/CHIR99021/DAPT, ii) SB431542/CHIR99021/DAPT, iii) LDN193189/DAPT/SB431542 and iv) LDN193189/CHIR99021/SB431542 (see Example 9 and Figures 15-19). In addition, it was demonstrated that replacing these compounds with functional analogs can achieve the same results. For example, the TGF-β receptor inhibitor SB431542 can be replaced by other TGF-β receptor inhibitors, such as Repsox and A8301. Similarly, the BMP receptor inhibitor LDN193189 can be replaced by its functional analogs, as demonstrated using Dorsomorphin and DMH1. The GSK-3 inhibitor CHIR9902 can be replaced by functional analogs such as AR-A014418 and SB216763. Similarly, the γ-secretase/Notch1 signaling inhibitor DAPT can be replaced by pan-Notch inhibitors BMS906024 or RO4929097. Therefore, in various embodiments, the present disclosure includes reprogramming human glial cells into neurons by regulating TGF-β, BMP, GSK-3, and γ-secretase/Notch signaling pathways. Other functional analogs are described in Table 1. Thus, in certain embodiments, alternatives to SB431542, LDN193189, CHIR99021, and DAPT encompassed by the present invention include, but are not necessarily limited to, those described in this table:

Table 1

In an embodiment, the administered combination includes the Shh agonist smoothened agonist (SAG), which is an agonist of sonic hedgehog.

Therefore, it will be apparent from the description, examples and drawings of the present disclosure that we have found that by combining the small molecules described herein, human astrocytes can be directly reprogrammed into functional neurons. In making this discovery, we tested a number of small molecules that target signaling pathways that are considered important for activating neurogenesis while inhibiting gliosis. We found that the above-mentioned small molecule group can reprogram human astrocytes into neurons. In more detail, when human astrocytes are simultaneously exposed to a common library of nine small molecules, they experience severe cell death, and the neuronal reprogramming efficiency is low, less than 10%. In contrast, when a subset of nine small molecules is administered in a sequential manner, most human astrocytes (~70%) are reprogrammed into neuronal cells. We demonstrate that these small molecule reprogrammed human neurons can survive in culture for more than three months and show robust synaptic activity. Neurons converted from human astrocytes are injected into mouse brains, showing that these human neurons can be integrated into local brain circuits. Together, these data demonstrate the feasibility of purely chemically reprogramming human astrocytes into functional neurons, which is expected to lead to a convenient method of chemical delivery for treating various brain injuries and neurodegenerative disorders. Furthermore, our results are not limited to in vitro demonstration, as we demonstrate here that chemically reprogrammed human neurons administered to animals generate synaptic connections with endogenous neurons in the mouse brain.

Generally, the method of the present disclosure includes administering an effective amount of a compound as described herein to a subject so that the number of neurons in an individual increases. In embodiments, the neuroglia (e.g., astrocytes) in an individual are reprogrammed to convert them into neurons. In embodiments, the newly generated neurons mainly include glutamatergic neurons with a small amount of GABAergic neurons. In embodiments, by using the methods described herein (if necessary, those skilled in the art will make changes to the method in a manner that is obviously beneficial to the present disclosure), it is expected that the present disclosure will promote the development of neocortical forebrain neurons, or midbrain neurons, or hindbrain neurons, or spinal cord neurons, or a combination thereof. In embodiments, it is expected that the method of the present disclosure will result in an increase in the endogenous neural transcription factors in the cells converted into neurons. In embodiments, target cells show an increase in the expression of Ascl1, Ngn2, NeuroD1, and a combination. In embodiments, the reprogrammed neurons are characterized in that they express neuronal markers, which include but are not necessarily limited to Dcx and NeuN. In embodiments, cells in the brain, such as neuroglia, are converted into neurons. In embodiments, neurons are functional neurons. Functional neurons may exhibit properties including, but not necessarily limited to, firing repetitive action potentials, developing multiple dendritic branches, and releasing neurotransmitters including, but not necessarily limited to, glutamate (glutamate), dopamine, acetylcholine, serotonin, norepinephrine (noradrenaline), and gamma-aminobutyric acid (GABA).

Compositions comprising compounds of the present disclosure can be provided in pharmaceutical preparations. The form of the pharmaceutical preparation is not particularly limited, but generally comprises these active ingredients and at least one inactive ingredient. In certain embodiments, suitable pharmaceutical compositions can be prepared by mixing any one compound or a combination thereof with a pharmaceutically acceptable carrier, diluent or excipient and suitable components well known in the art. Some examples of these carriers, diluents and excipients can be found in the following literature: Remington: The Science and Practice of Pharmacy (2005) ^21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins. In embodiments, the pharmaceutical preparation is suitable for delivering the active ingredient across the blood-brain barrier and/or the spinal cord or other parts of the central nervous system. Such compositions can include, for example, lipid formulations or other nanoparticle-based delivery systems.

In one embodiment, the pharmaceutical preparation is suitable for oral administration and can therefore be provided in atomized, liquid or solid dosage forms. Solid dosage forms include but are not necessarily limited to tablets, capsules, caplets and strips for swallowing or oral dissolution, and can be provided for rapid release or delayed release, or release different compounds in a desired sequence over a period of time. Separate pharmaceutical compositions comprising two compounds or any combination of compounds can also be used. Therefore, the pharmaceutical preparation can include any two or any combination of SB431542, LDN193189, CHIR99021 and DAPT, as well as any other functional analogs. Therefore, in certain embodiments, for the purpose of stimulating the reprogramming of neurons in human subjects, a collection of three of LDN193189, SB431542, CHIR99021 and DAPT or these compounds or their functional analogs may be necessary. In an embodiment, the core compound may be necessary and sufficient for reprogramming glial cells into neurons.

Regarding the administration of the pharmaceutical preparation, the route of administration can be any suitable route. In an embodiment, the composition comprising the compound is delivered orally. In other non-limiting embodiments, the composition is administered intravenously, parenterally, subcutaneously, intraperitoneally, transdermally, intranasally, by implantation or intra-arterial administration. In an embodiment, an implantable medical device, such as a pump, including but not limited to an osmotic pump, can be used. In an embodiment, the composition comprising the compound is delivered via an intracranial route.

In view of the benefits of the present disclosure, the appropriate dosage of the compound can be determined in combination with the knowledge of those skilled in the art. In an embodiment, when determining the active ingredient effective amount and the dosage regimen, the individual's weight and age, personal history of neuronal damage or disease, and the risk of experiencing the same neuronal damage, or the presence of glial scarring or reactive gliomas can be considered. In an embodiment, the compound is administered in an amount of about 0.01 nmol to about 100 nmol or more per day, including this number and including all integers and ranges therebetween, depending on the delivery method used. In an embodiment, the compound is provided in a single, multiple or controlled release dosage regimen. In an embodiment, SB431542, LDN193189, CHIR99021 and DAPT and other small molecules according to the present disclosure are administered simultaneously or sequentially.

In certain embodiments, the present disclosure includes nutritional compositions designed to impart beneficial effects related to individual neuron health and/or function improvement. In certain embodiments, the compositions of the present invention can be used to improve the general health of an individual or the cognitive abilities of an individual, such as for improving memory or maintaining memory. In embodiments, the compositions can be used to improve any or all of short-term memory, long-term memory, or motor skills, including but not necessarily limited to rough and fine motor skills. Therefore, the present disclosure includes the use of nutritional supplements comprising small molecules as described herein.

In one embodiment, the present disclosure includes articles. In some aspects, articles include closed or sealed packages comprising two compounds described herein or a combination of compounds described herein, such as separate tablets, capsules, etc. The packaging can include one or more containers, such as closed or sealed vials, bottles, blister (blister) (bubble) packaging, or any other suitable packaging for sale, distribution, or pharmaceutical use. Therefore, the packaging can include a pharmaceutical composition comprising all SB431542, LDN193189, CHIR99021, and DAPT, or only three of these compounds, or functional analogs and/or other compounds described herein. Any two or all of these compounds can be included, and each can be provided separately in the same or different dosage formulations or in combination with one or more other substances so that they can be delivered simultaneously or sequentially. In one embodiment, LDN193189 or SB431542 or a combination thereof is provided separately from CHIR99021 or DAPT or a combination thereof.

In addition to the pharmaceutical composition, packaging can include printed information. The printed information can be provided on a label or on an inserted paper, or printed on the packaging material itself. The printed information can include the information of the instructions for identifying the active agent in the packaging, the amount and type of the inactive ingredient, the pharmaceutical composition for treating the disease and the instructions for taking the pharmaceutical composition, such as the dosage quantity taken in a given time period, the information of the order of taking the composition, etc. Therefore, in various embodiments, the present disclosure includes a pharmaceutical composition of the present invention that is packaged in the packaging material and identified by printing on or in the packaging material, and the composition is used to treat or prevent any disease, disease or disorder related to neuronal deterioration, neuronal deficiency or neuronal functional defect. In another embodiment, in addition to the pharmaceutical composition, the present disclosure includes nutritional preparations, and printed material provides information about using such preparations to improve cognitive function, memory, motor function, overall health, etc.

The following specific examples are provided to illustrate the invention but are not intended to be limiting in any way.Where reference is made to colors in the figures, the labels are provided as representative samples of the referenced colors.

Example 1

This embodiment demonstrates that human astrocytes are successfully reprogrammed into neurons by small molecules as described above. These experimental designs are used to develop a convenient method for reprogramming human astrocytes into neurons by small molecules using methods such as, but not limited to, oral administration drugs that can be easily taken by patients. Therefore, we studied whether small molecules can replace neural transcription factors and reprogram glial cells into neurons. We use human cortical astrocytes (HA1800, ScienCell, San Diego, CA, USA) for chemical reprogramming culture for clinical applications in human brain repair. Based on two main selection criteria: one ct inhibits the signal transduction pathway of glial cells, and the other is the signal transduction pathway that activates neurons, we select 20 kinds of small molecules as our starting candidate library. Including some molecules is because they can regulate DNA or histone structure to improve reprogramming efficiency. The 20 small molecules selected for our initial screening were: SB431542, RepSox, LDN193189, dorsomorphin, DAPT, BMS-299897, CHIR99021, TWS119, Thiazovivin, Y27632, SAG, 2,6,9-trisubstituted purine (purmorphamine), TTNPB, RA, VPA, forskolin, BIX01294, RG-108, ISX9, and Stattic.

We primarily used human cortical astrocytes (HA1800, ScienCell, San Diego, CA, USA) in primary culture for chemical reprogramming. Human astrocytes were isolated, passaged, and maintained in culture medium with 10% fetal bovine serum (FBS) to reduce possible progenitor cell contamination, as FBS stimulates the differentiation of progenitor cells. For initial testing, we co-applied a panel of small molecules to human astrocyte cultures, but observed substantial cell death after 2 days of drug treatment. To reduce cell death, we added smaller amounts of small molecules at different time points. Each molecule was tested with a range of different concentrations to find the optimal concentration for reprogramming. After testing hundreds of different combinations, we found that a combination of 9 small molecules was able to reprogram human astrocytes into neurons when added in a stepwise manner (Figure 1A). This group of 9 small molecules is hereafter referred to as the master conversion molecule (MCM). Specifically, human astrocytes were first treated with LDN193189 (0.25 μM), SB431542 (5 μM), TTNPB (0.5 μM) and thiazovivin (Tzv, 0.5 μM) for 2 days. SB431542 is an inhibitor of TGFβ/activated receptors, which participates in inhibiting the fate of neurons and promoting the fate of glial cells during early neural development. Similarly, LDN193189 is an inhibitor of BMP receptors, which are members of TGFβ receptors and are important for astrocyte differentiation. TTNPB is an agonist of retinoic acid receptors, which is reported to be crucial in central nervous system patterns. We use a combination of LDN193189, SB431542 and TTNPB to start the reprogramming process by inhibiting glial signaling pathways and simultaneously activating neuronal signaling pathways. Tzv, an inhibitor of Rho-associated kinase (ROCK), promotes cell survival and has been reported to enhance iPSC reprogramming efficiency. Tzv was administered throughout the 8-day reprogramming period. After the initial two-day priming period, we replaced the first set of three small molecules (LDN193189, SB431542, and TTNPB) with a second set of small molecules, including CHIR99021 (1.5 μM), DAPT (5 μM), and VPA (0.5 mM). CHIR99021 is an inhibitor of glycogen synthase kinase 3 (GSK3). GSK3 signaling promotes neural progenitor cell homeostasis and neocortical neural induction. DAPT (tert-butyl N-[N-(3,5-difluorophenylacetyl)-L-alanyl]-S-phenylglycine), a γ-secretase inhibitor that indirectly inhibits the Notch signaling pathway, effectively induced neural differentiation of progenitor cells. VPA (valproic acid), a histone deacetylase inhibitor, promotes histone acetylation. VPA was included in the reprogramming medium for only 2 days, as longer exposure increases cell death, whereas CHIR99021 and DAPT were present from days 3 to 6. On days 7 to 8, we used SAG (0.1 μM) and 2,6,9-trisubstituted purine (Purmo, 0.1 μM), two agonists for activating the sonic hedgehog (Shh) signaling pathway, to complete the reprogramming process. Shh signaling is a key determinant of CNS patterning. SAG and Purmo, or Shh itself, have been used to induce neuronal differentiation from pluripotent stem cells. On day 9, we removed SAG and Purmo from the culture medium and replaced them with neurotrophic factors (BDNF, NT3, and IGF-1) to promote neuronal maturation after astrocyte-to-neuron conversion. This successful reprogramming strategy is illustrated in Figure 1A.

Before reprogramming, we characterized the properties of human astrocytes in our cultures and found that the majority of cells were immunopositive for the astrocyte markers GFAP (79.3 ± 4.9%) and Glt1 (astrocyte-specific glutamate transporter, 82.5 ± 4.3%), with no neurons detected (Figures 1B-C). We found little contamination of neural stem cells in human astrocyte cultures, as shown by immunostaining for Sox2, Musashi, and nestin (Figures 8A-B), likely due to the presence of 10% FBS in our culture medium. After culturing human astrocytes for one month in neural differentiation medium supplemented with growth factors (BDNF, NT3, and NGF), this was further confirmed: the majority of cells were immunopositive for astrocyte markers (S100β, GFAP, glutamine synthetase, Glt1), but very few were positive for neurons or NG2 cells (Figures 8C-D). In addition, we performed patch clamp recordings and demonstrated that our cultured human astrocytes were functional, with large K+ currents and glutamate transporter currents, but no Na+ currents, and gap junctions formed between astrocytes (Figure 8E-H). In the control reprogramming medium (1% DMSO) without small molecules, almost no neurons were detected in our human cortical astrocyte cultures (Figure 1D). In contrast, after sequential exposure to small molecules, we found a large number of neuron-like cells that were immunopositive for neuronal markers such as doublecortin (DCX), β3-tubulin (Tuj1), MAP2, and NeuN (Figure 1E-F). Neurons converted from human astrocytes survived 4-5 months in our cultures and developed robust axons and dendrites (Figure 1G). In order to visualize the conversion process from astrocytes to neurons, we infected human astrocytes with 1 μl of EGFP-encoding retrovirus so that only a small amount of EGFP-positive astrocytes were observed in each coverslip (Figure 9). We performed imaging over time to monitor the morphological changes of human astrocytes. In the absence of small molecules, the morphology of human astrocytes did not change from day 0 to day 8 (Figure 9A), and was immunopositive for the glial cell marker GFAP (Figure 9B). In contrast, during the treatment with small molecules, there was a clear transition from astrocyte morphology to neuronal morphology as long axons extended from day 8 to day 10 (Figure 9C). After imaging over time, we fixed the cells and performed immunostaining on day 21. GFP-labeled neuron-like cells were indeed immunopositive for NeuN and Tuj1 (Figure 9D). We further used GFAP::GFP retrovirus to label astrocytes (91±6.7% of GFAP::GFP infected cells were GFAP ⁺ ) and traced the astrocyte-neuron conversion process (Figure 9E). 18 days after small molecule treatment, GFP-labeled astrocytes were effectively converted into NeuN ⁺ neurons (68.7±4.2%, Figure 1H, n=5 batches); while no neurons were detected in the control group that was not treated with small molecules (Figure 9F-G). Similar results were obtained using LCN2::GFP retrovirus (88.5±3% of LCN2::GFP labeled cells were GFAP ⁺ ) to trace astrocyte-neuron conversion (Figure 9H-J). 54.4±5.3% of LCN2::GFP labeled astrocytes became NeuN ⁺ neurons 18 days after small molecule treatment (n=3 batches). The transformation efficiency obtained by lineage tracing experiments was similar to the overall transformation efficiency induced by small molecule treatment ( Figure 1I-J ; control, 3.3±0.5% Tuj1 ⁺ , n=4 batches; MCM, 67.1±0.8% Tuj1 ⁺ , n=4 batches; p<0.0001, Student t test).

In order to study whether human astrocytes from different sources can be reprogrammed to neurons using the same small molecule scheme, we further tested human midbrain astrocytes and human spinal cord astrocytes from ScienCell. Interestingly, using our step-by-step 9 kinds of small molecule strategies human midbrain astrocytes are effectively reprogrammed to neurons (Figure 1K-M, Figure 10A-F), while using the same scheme human spinal cord astrocytes can not be reprogrammed to neurons (data not shown). This result shows that our chemical reprogramming scheme is more suitable for astrocytes from human brain origin. In order to further test whether our small molecule reprogramming strategy is generally applicable to human astrocytes from different sources, we purchased human astrocytes from Gibco, and found that they can also be reprogrammed to neurons (Figure 10G-I). To ensure that our chemical reprogramming method does not involve the redifferentiation of human astrocytes into neural progenitor cells, we monitored Sox2, nestin, Pax6, and Ki67 signals during chemical reprogramming from day 0 to day 10 and compared them with neural progenitor cells (Figure 11). Although Sox2 showed some increase during reprogramming, it never reached the level of neural progenitor cells (Figure 11A, G). Nestin and Pax6 did not increase significantly during small molecule treatment (Figure 11B-C, H-I). Ki67-labeled proliferating cells were significantly reduced after small molecule treatment (Figure 11D, J), indicating that there were no progenitor cells that could expand and generate neurons. Furthermore, when we labeled human astrocytes with BrdU before chemical treatment, many converted neurons were BrdU-positive (Figure 11E, K); however, when we labeled our cell cultures with BrdU on day 10 after small molecule treatment, essentially all converted neurons were BrdU-negative (Figure 11F, K), indicating that all glial-to-neuron conversion occurred in the presence of small molecules. In summary, we have developed a successful strategy for chemically reprogramming human astrocytes into neurons using a defined combination of small molecules.

Example 2

The present embodiment proves that the human neurons converted by the small molecules produced according to the present disclosure are fully functional in terms of exciting action potentials and releasing neurotransmitters. In particular, we found that the neurons converted by small molecules survived for a long time (>5 months) and showed strong synaptic points along dendrites (Fig. 2A). Similarly, neurons reprogrammed from midbrain human astrocytes and Gibco's human astrocytes also survived more than 2 months in culture, with many synaptic points along dendrites (Fig. 10F, I). Patch clamp recordings showed obvious sodium currents and potassium currents in neurons converted by astrocytes, and gradually increased during the maturation of neurons (Fig. 2B-E; 2 months: I _Na =1889 ± 197Pa, n=10; I _K =2722 ± 263Pa, n=10). These neurons can excite repetitive action potentials (Fig. 2F). More importantly, small molecule-converted neurons showed robust spontaneous synaptic activity, including excitatory postsynaptic currents (EPSC; frequency = 0.66 ± 0.14 Hz; amplitude = 24.8 ± 8.2 Pa, n = 15) (Figure 2G-H) and inhibitory postsynaptic currents (IPSC; frequency = 0.48 ± 0.21 Hz; amplitude = 23.3 ± 6.3 Pa, n = 2) (Figure 2I). Notably, 3 months after the initial small molecule treatment, human astrocyte-converted neurons showed large periodic bursting activity that was eliminated by TTX or DNQX (Figure 2J-L), indicating that these neurons formed functional networks and began to fire synchronously together. To support this view, we performed dual whole-cell recordings and demonstrated that two adjacent neurons showed synchronous bursting activity (Figure 2M). In addition, we used Fura-2Ca ²⁺ ratio imaging and recorded synchronous Ca ²⁺ spikes in chemically reprogrammed neurons (Figure 2N), indicating that these neurons have been functionally networked together. Thus, human astrocytes can be chemically reprogrammed into fully functional neurons using defined small molecules.

Example 3

This example demonstrates that the small molecules described herein reprogram human astrocytes into forebrain glutamatergic neurons. In order to characterize the neuronal properties after small molecule-induced reprogramming, we examined the neuronal markers expressed from the anterior to posterior nervous system. We found that most of the neurons converted by human astrocytes were immunopositive for the forebrain marker FoxG1 (97.1 ± 1.1%, Figure 3A, n = 3 batches), but negative for the hindbrain and spinal cord markers HoxB4 and HoxC9 (Figure 3B-C, n = 3 batches). We then performed a series of immunostaining with various cortical neuron markers. We found that most of the neurons converted by human astrocytes were immunonegative for the cortical surface marker Cux1 (Figure 3D), but immunopositive for the deep layer marker Ctip2 (Figure 3E, 71.4 ± 3%, n = 5 batches) and Otx1 (Figure 3F). Human astrocyte-converted neurons were also immunopositive for the forebrain neuron marker Tbr1 (Figure 3G, 86.4 ± 3.4%, n = 3 batches) and the hippocampal neuron marker Prox1 (Figure 3H). Figure 3I shows the quantitative results. Therefore, our chemically reprogrammed neurons are mainly deep forebrain neurons or hippocampal neurons.

We further investigated neuronal subtypes based on the neurotransmitters they contain. We found that the majority of small molecule-reprogrammed neurons were immunopositive for the glutamatergic neuron marker VgluT1 (Figure 3J). A small portion of the converted neurons were immunopositive for the GABAergic neuron marker GAD67 (Figure 3K). On the other hand, astrocyte-converted neurons were primarily immunonegative for the cholinergic marker VAChT (Figure 3L), the dopaminergic marker TH (Figure 3M), or the spinal motor neuron marker Isl1 (Figure 3N). Quantitative analysis of neuronal subtypes is shown in Figure 3O. (Vglut1, 88.3±4%, n=4 batches; GAD67, 8.2±1.5%, n=4 batches). These results indicate that glutamatergic neurons are the predominant subtype using our small molecule reprogramming protocol. Different small molecules may be required to reprogram human astrocytes into other neuronal subtypes.

Example 4

The present embodiment demonstrates the activation of endogenous neural transcription factors during chemical reprogramming. In order to understand the molecular mechanism of chemical reprogramming, we first used PCR arrays (Qiagen) to study gene spectrum changes. On the 4th day after small molecule treatment, we found that the transcription levels of several neural transcription factors including NGN1/2, NEUROD1 and ASCL1 and immature neuron marker DCX increased significantly by up to 300 times (Fig. 4A). On the 8th day, the most significant change in transcription level was the immature neuron gene DCX, which showed an increase of 2000 times (Fig. 4B), indicating that most of the newly converted cells at the end of small molecule treatment were immature neurons. By contrast, glial cell-related genes are typically downregulated (Fig. 4A-B). Then we carried out real-time quantitative PCR experiments to examine the transcriptional changes (Fig. 4C-F) of NGN2, NEUROD1 and astrocyte genes GFAP and ALDH1L1 in the time course during chemical reprogramming. Interestingly, we found that during small molecule treatment, NGN2 transcription peaked on day 4 (Figure 4C), while NEUROD1 peaked on day 6 (Figure 4D), which is consistent with their sequential expression during early brain development. For glial genes, GFAP transcript levels were significantly reduced by more than 200-fold on D4 (Figure 4E), consistent with the activation of neural transcription factors (Figures 4C-D). Similarly, transcript levels of another astrocyte gene, ALDH1L1, were also downregulated (Figure 4F). In contrast, control experiments without small molecule treatment showed minimal transcriptional changes (Figures 12A-F). Therefore, our small molecule treatment activates neural transcription factors while inhibiting astrocyte genes.

Example 5

This embodiment provides a study on whether epigenetic regulation is involved in our chemical reprogramming. DNA methylation in gene promoters affects the accessibility of transcription factor binding and is therefore the rate-limiting factor for pluripotent stem cell reprogramming. We performed methylated DNA immunoprecipitation and then sequencing (MeDIP-seq, methylated DNA immunoprecipitation sequencing) to examine the methylation levels of the gene of interest before and after small molecule treatment. As expected, the promoter region of the GFAP gene in human astrocytes was initially unmethylated before small molecule treatment (D0), but a significant increase in methylation was detected 8 days after small molecule treatment (Figure 4G). This increased methylation was further confirmed by targeted bisulfite sequencing (BS-seq) (Figure 4H). It is worth noting that the GFAP promoter region contains transcription factor binding sites for STAT3 and AP1, which have been shown to play a key role in the activation of the GFAP gene. BS-seq data show that the flanking sites of the STAT3 and AP1 binding regions are highly methylated (Figure 4H), which may explain why GFAP transcription is significantly downregulated after small molecule treatment (Figure 4E). Our MeDIP-seq analysis also revealed increased DNA methylation at the GFAP transcription start site (TSS) after small molecule treatment, which was also confirmed by BS-seq (Figure 4I). In contrast to the glial gene GFAP, the neuronal gene NEFM (a medium-sized neurofilament gene specific for neurons) showed decreased methylation signals in its promoter region after small molecule treatment (Figures 4J-K), indicating neuronal gene activation. We also investigated the epigenetic regulation of the transcription factor NGN2, a key gene involved in neuronal differentiation. MeDIP-seq analysis showed that, consistent with previous reports (Covic et al., 2010), methylation levels in the NGN2 promoter region were significantly reduced before and after small molecule treatment (data not shown). In addition to DNA methylation, histone modifications can also regulate gene expression. Therefore, we further investigated histone modifications in the NGN2 promoter region and transcription start site (Figures 4L-O). Consistent with the application of the HDAC inhibitor VPA during chemical reprogramming, a significant increase in histone acetylation was observed at day 8 (Figure 4M). Interestingly, H3K4me3 levels were significantly increased in the promoter region (Figure 4N), while H3K27me3 levels were significantly decreased at the transcription start site at D8 (Figure 4O), consistent with the transcriptional activation of NGN2 induced by small molecule treatment. Taken together, our results suggest that our chemical reprogramming process involves both transcriptional and epigenetic regulation.

To confirm our transcriptional and epigenetic analyses, we further performed immunostaining to examine changes in protein expression during chemical reprogramming (Figure 5). We found that Ascl1 expression levels first showed a significant increase after 2 days of treatment with LDN193189, SB431542, and TTNPB (Figures 5A and G). After small molecule treatment, Ngn2 expression levels showed a peak at D4 (Figures 5B and H; in the presence of CHIR99021, DAPT, and VPA). Compared to Ascl1 and Ngn2, NeuroD1 expression appeared delayed, reaching a peak at D6 after small molecule treatment (Figures 5C and I), which is consistent with our transcriptional studies (Figures 4C-D). In addition, immunostaining experiments also showed that after the peak expression of NeuroD1, some cells began to show neuronal markers such as DCX at D4-D6 (Figure 5D), and NeuN+ neurons appeared at D8-D10 (Figures 5E and J). In contrast to the increase in neuronal markers, the astrocyte protein GFAP showed a significant decrease after small molecule treatment (Figure 5F and K), which is consistent with epigenetic silencing and transcriptional downregulation of the GFAP gene. Control astrocytes cultured for 10 days without small molecule treatment did not show much change in the expression levels of neural transcription factors, neuronal protein NeuN, or astrocyte protein GFAP (Figure 13). These experiments indicate that our small molecule strategy has successfully activated endogenous neural transcription factors, which may play an important role in the reprogramming of astrocytes into neurons.

Example 6

This embodiment describes the analysis of the functional effects of each individual compound during chemical reprogramming. In order to analyze the contribution of each single molecule to reprogramming, we carried out a series of experiments (Fig. 6) by canceling each individual compound from our cocktail library (cocktail pool). Compared with sequential exposure to a total of 9 molecules, removing DAPT causes the most significant reduction in the number of converted neurons (Fig. 6A-C). Similarly, removing CHIR99021 or SB431542 or LDN193189 also significantly reduces reprogramming efficiency (Fig. 6D-F). Removing VPA or SAG+Purmo can slightly reduce reprogramming efficiency (Fig. 6G-H). Interestingly, removing Tzv or TTNPB has no significant effect on astrocyte-neuron reprogramming (Fig. 6I-J). Fig. 6K illustrates the summary data of drug cancellation experiment. Although Tzv is not surprising because it has no effect mainly as a cell survival factor, surprisingly, removing TTNPB has no effect. We include TTNPB because it is an agonist of retinoic acid receptors that are found to play an important role in neural differentiation. The lack of TTNPB contribution shows that retinoic acid may not be a necessary factor for astrocyte reprogramming to neurons. Therefore, the present disclosure includes the condition that compositions does not include TTNPB. On the other hand, it seems that inhibition of Notch signaling, GSK-3 β and BMP/TGF β signaling pathways is important for astrocyte reprogramming to neurons. In order to ensure that these signaling pathways are indeed inhibited during our small molecule treatment, we have carried out a series of immunostaining to phosphorylated SMAD1/5/9, Notch intracellular domain (NICD) and phosphorylated GSK3 β (Figure 12G-1). Our results show that after small molecule treatment, BMP/TGF β, Notch and GSK3 β signaling pathways are significantly inhibited (Figure 12G-I), which shows that the inhibition of these signaling pathways and the close connection between astrocyte conversion to neurons.

Example 7

The present embodiment provides the demonstration of the in vivo integration of human neurons in the mouse brain after reprogramming. We further study whether the neurons converted by human astrocytes can survive in vivo in the mouse brain. In order to distinguish the neurons converted by human astrocytes from the mouse neurons pre-existing in the brain, we use EGFP-lentivirus to infect human astrocytes before small molecule treatment, so that the neurons converted by human astrocytes are mostly marked by EGFP (Fig. 7A). 14 days after the initial small molecule treatment, we harvested the cells containing both converted neurons and unconverted astrocytes and injected them into the lateral ventricle of newborn mice (Fig. 7A). 7 days after cell injection (DPI), we found a cluster of EGFP-labeled cells in the lateral ventricle, which were all immunopositive to human nuclei (HuNu, Fig. 7B), which showed that these cells were derived from the human cells injected. Importantly, we found that many EGFP-labeled human cells were immunopositive for the neuronal markers DCX (Figure 7B), MAP2 (Figure 7C), and NeuN (Figure 7D), indicating that human astrocyte-converted neurons can survive in vivo in the mouse brain. Even one month after cell injection, we were still able to identify clusters of EGFP-labeled neurons in brain regions adjacent to the lateral ventricle, such as the thalamus and striatum (Figure 7E), suggesting that human astrocyte-converted neurons may have migrated out of the lateral ventricle and integrated into local neural circuits. Supporting this notion, we found numerous synaptic puncta along the dendrites of EGFP+ human neurons (Figure 7F), indicating that these transplanted human neurons have established synaptic connections with host neurons. In summary, these in vivo experiments demonstrate that our small molecule-reprogrammed human neurons can not only survive in the mouse brain but also integrate into local neural circuits.

We also attempted to use our small molecule strategy to reprogram mouse astrocytes into neurons in vitro and in vivo. We found that mouse astrocytes treated with small molecules expressed more nestin signaling in vivo than vehicle controls (Figure 14A-B). Therefore, we isolated cortical tissue surrounding the small molecule-injected area and cultured it in vitro. Interestingly, small molecule-treated cortical tissue generated more neurospheres than vehicle controls (Figure 14C-H). These neurospheres could be dissociated into neural stem cells and gave rise to neurons, astrocytes, and oligodendrocytes (Figure 14I-J).

Example 8

This example provides a description of the materials and methods used to obtain the data disclosed herein.

Human astrocyte cultures. Human astrocytes were purchased from ScienCell (HA1800, California) or Gibco (N7805-100). Human astrocytes are primary cultures obtained from human fetal brain tissue. They are isolated and cultured in the presence of 10% fetal bovine serum (FBS), which essentially induces differentiation of any progenitor cell. Human astrocytes were subcultured when they exceeded 90% confluency. For subculture, cells were trypsinized by TrypLE ^™ Select (Invitrogen), centrifuged at 900 rpm for 5 minutes, resuspended, and plated in a medium consisting of DMEM/F12 (Gibco), 10% fetal bovine serum (Gibco), penicillin/streptomycin (Gibco), 3.5 mM glucose (Sigma), and supplemented with B27 (Gibco), 10 ng/ml epidermal growth factor (EGF, Invitrogen), and 10 ng/ml fibroblast growth factor 2 (FGF2, Invitrogen). Cells were cultured at 37°C in a humidified atmosphere containing 5% _CO2 .

Human astrocytes are reprogrammed into neurons. In 24-well plates (BD Biosciences), astrocytes are cultured on poly-D-lysine (Sigma) coated coverslips (12 mm) at a density of 50,000 cells per coverslip. The cells are cultured to 90% confluence in human astrocyte culture medium. On day 0 before reprogramming, half of the culture medium is replaced with N2 culture medium consisting of DMEM/F12 (Gibco), penicillin/streptomycin (Gibco) and N2 supplement (Gibco). The following day (day 1), the culture medium is completely replaced by N2 culture medium supplemented with small molecules or an N2 culture medium control group containing 1% DMSO. For most experiments using 9 molecules for reprogramming (MCM treatment), astrocytes were treated for 2 days with TTNPB (0.5 μM, Tocris#0761), SB431542 (5 μM, Tocris#1614), LDN193189 (0.25 μM, Sigma#SML0559) and Thiazovivin (0.5 μM, Cayman#14245). On the third day, the culture medium was replaced with a different set of small molecules including CHIR99021 (1.5 μM, Tocris#4423), DAPT (5 μM, Sigma#D5942), VPA (0.5 mM, Cayman#13033) and Thiazovivin (0.5 μM). On day 5, VPA was withdrawn by replacing the medium with only CHIR99021 (1.5 μM), DAPT (5 μM), and Thiazovivin (0.5 μM). On day 7, the medium was replaced with SAG (0.1 μM, Cayman #11914), 2,6,9-trisubstituted purine (Purmo, 0.1 μM, Cayman #10009634), and Thiazovivin (0.5 μM). On day 9, the medium was completely replaced with Neuronal Differentiation Medium (NDM) containing DMEM/F12 (Gibco), 0.5% FBS (Gibco), 3.5 mM glucose (Sigma), penicillin/streptomycin (Gibco), and N2 supplement (Gibco). 200 μl of Neuronal Differentiation Medium was added to each well weekly to maintain constant osmotic pressure. To promote synaptic maturation of converted neurons, brain-derived neurotrophic factor (BDNF, 20 ng/ml, Invitrogen), insulin-like growth factor 1 (IGF-1, 10 ng/ml, Invitrogen), and neurotrophin 3 (NT-3, 10 ng/ml, Invitrogen) were added to the neuronal differentiation medium on day 9 and refreshed every four days until day 30 (Song et al., 2002).

To examine whether our human astrocytes contained any neural stem cells, we cultured human astrocytes for 1 month in neuronal differentiation medium supplemented with 20 ng/ml BDNF, 10 ng/ml NT3, and 10 ng/ml NGF. Growth factors were refreshed every 3–4 days.

Human neural progenitor cells (NPCs) derived from human pluripotent stem cells were a gift from Dr. Fred Gage. NPCs were cultured on poly-L-ornithine and laminin-coated coverslips in neuronal proliferation medium containing DMEM/F12, penicillin/streptomycin, B27 supplement, N2 supplement, and FGF2 (20 ng/ml) (Gibco).

Data and Statistical Analysis. Cell counts were performed by capturing images of several randomly selected fields on each coverslip and analyzing them using Image J software. Fluorescence intensity was analyzed using Image J software. Data are presented as mean ± SEM. Student's t-test was used to compare data between two groups. One-way analysis of variance with post hoc tests was used for statistical analysis of multiple data sets.

In vivo transplantation of small molecule-transduced human neurons. In vivo experiments were performed using wild-type C57/BL6 mice. Mice were housed on a 12-h light/dark cycle and provided with adequate food and water. The experimental protocol was approved by the Pennsylvania State University IACUC and is consistent with National Institutes of Health guidelines.

Human astrocytes cultured in T25 flasks were transduced with 10 μl FUGW-GFP lentiviral suspension for efficient infection. One day after viral transduction, cells were dissociated with TrypLE and plated on poly-D-lysine-coated coverslips in 24-well plates at a density of 50,000 cells per coverslip. When the cells reached 90% confluence, approximately 70% of the cells were GFP positive. After GFP infection, human astrocytes were treated with small molecules according to the above protocol. On the 14th day after the initial small molecule treatment, a mixture of human astrocytes and converted neurons was dissociated with Accutase (Gibco) and resuspended with 20 μl neuronal differentiation medium supplemented with 10 ng/ml BDNF, 10 ng/ml NT3, and 10 ng/ml IGF-1. A cell suspension containing 2 × 10 ⁵ cells was injected into the lateral ventricle of newborn mouse pups (day 1 after birth, P1), with 2 μl injected into each hemisphere. Using a stereotaxic apparatus (Hamilton), cells were injected 1.5 mm anterior and 1.5 mm lateral to the lambda suture and at a depth of 1 mm. Brains were collected 7, 11, 14 days and 1 month after injection for analysis.

Immunocytochemistry. For brain section staining, mice were anesthetized with 2.5% Avertin and then perfused with ice-cold _artificial cerebrospinal fluid (ACSF) containing 124 mM NaCl, ₂₆ mM NaHCO₃, 10 mM glucose, 1.3 mM _MgSO₄ , 1.25 mM _NaH₂PO₄ , _2.5 mM KCl, and 2.5 mM CaCl₂. The brains were removed and fixed overnight at 4°C in 4% paraformaldehyde (PFA). Brains from young mice (<1 month old) were dehydrated with 30% sucrose for 2 days and sliced at 50 μm using a cryostat (Leica). Brains from adult mice (>1 month old) were sliced at 45 μm using a vibratome (Leica). Coronal brain sections were incubated in 2.5% normal goat serum, 2.5% normal donkey serum, and 0.3% Triton X-100 in phosphate-buffered saline (PBS, pH 7.4) for 2 hours and then incubated in primary antibodies overnight.

For cell culture staining, cultures were fixed in 4% PFA in PBS for 15 minutes at room temperature. Cells were first washed three times with PBS and then incubated in 2.5% normal goat serum, 2.5% normal donkey serum, and 0.1% Triton X-100 in PBS for 30 minutes. Primary antibodies were incubated with brain sections or cultures overnight at 4°C in 3% normal goat serum, 2% normal donkey serum, and 0.1% Triton X-100 in PBS. After another wash in PBS, samples were incubated with appropriate secondary antibodies conjugated to Alexa Fluor 488, Alexa 546, Alexa 647 (1:800, Molecular Probes), FITC, TRITC, or Dylight (1:500, Jackson ImmunoResearch) for 1 hour at room temperature, followed by extensive washing in PBS. Finally, coverslips were mounted on slides using antifade mounting solution containing DAPI (Invitrogen). Slides were analyzed using a fluorescence microscope (Keyence BZ-9000) or a confocal microscope (Olympus FV1000). Z-stack digital images were acquired and analyzed using FV10-ASW3.0 Viewer software (Olympus).

Electrophysiology. For neurons converted from human astrocytes, whole-cell recordings were performed using a Multiclamp 700A patch clamp amplifier (Molecular Devices, Palo Alto, CA) using known techniques. The recording chamber was continuously perfused with a bath consisting of 128mM NaCl, 30mM glucose, 25mM HEPES, 5mM KCl, 2mM _CaCl2 , and 1mM _MgCl2 . The bath pH was adjusted to 7.3 with NaOH, and the osmotic pressure was 315-325mOsm/L. The patch pipette was pulled out of borosilicate glass (4-6MΩ) and filled with a pipette solution consisting of 10mM KCl, 125mM potassium gluconate, 5mM sodium phosphocreatine, 10mM HEPES, 2mM EGTA, 4mM MgATP, and _0.5mM Na2GTP, adjusted to pH 7.3 with KOH. The series resistance was typically 10-25MΩ. For voltage-clamp experiments, the membrane potential was typically maintained at -70 mV, except when recording IPSCs, when the holding potential was set to 0 mV. Drugs were administered via a gravity-driven drug delivery system (VC-6, Harvard Apparatus, Hamden, CT). To monitor gap junctions between human astrocytes, 2 mM sulforhodamine B (SRB) dye (MW = 559 Da) was added to the pipette solution.

Data were acquired using pCamp 9 software (Molecular Devices, Palo Alto, CA), sampled at 10 kHz, and filtered at 1 kHz. Na+ and K+ currents and action potentials were analyzed using pClamp 9 Clampfit software. Spontaneous synaptic activity was analyzed using MiniAnalysis software (Synaptosoft, Decatur, GA). All experiments were performed at room temperature (22-24°C).

RNA extraction

RNA was extracted from human cortical astrocytes using an RNA kit on days 0, 2, 4, 6, and 10 of chemical treatment. 350 μl of lysis buffer was added to each well of a 24-well plate, and the cell lysate was collected. RNA was purified using RNA columns, and the purified RNA was eluted with 40 μl of RNase-free _H₂O , yielding an RNA concentration of 100 to 300 ng/μl per well. RNA concentration was measured and RNA quality was checked using a NanoDrop. All isolated RNA had an _A₂₀ / _A₂₀ ratio between 2 and 2.1, indicating RNA purity. The isolated RNA was stored at -80°C.

cDNA synthesis and quantitative real-time PCR

For real-time quantitative PCR (qRT-PCR), cDNA synthesis was performed using Quanta Biosciences qScript™ cDNA SuperMix. For each sample, 1 μg of RNA was used per 20 μl total reaction volume. The reaction mixture was incubated at 25°C for 5 minutes, 42°C for 30 minutes, and 85°C for 5 minutes, maintained at 4°C. The cDNA product was diluted 5-fold with RNase/DNase-free _H2O . Primer sets were designed using Applied Biosystems Primer Express software and are listed in Table 2. RT-qPCR was performed using Quanta Biosciences Green SuperMix, ROX ^™ . A real-time cycler, Applied StepOnePlus ^™ , was used. 5 μl of cDNA corresponding to 1 μg of total RNA was used in a final reaction volume of 25 μl. 40 PCR cycles of 95°C for 15 seconds and 65°C for 45 seconds were performed for amplification. Melting curve analysis was performed after PCR cycling. The comparative Ct method was used to quantify and calculate gene expression fold changes. GAPDH was used as an internal control gene, and relative gene expression was analyzed relative to that of a control human astrocyte group at day 0. RT-qPCR data were obtained from three replicate PCR reactions for each sample.

PCR array

Before (D0) and after (D4 and D8) small molecule treatment, human astrocytes were subjected to RT ² analyzer PCR arrays (Qiagen, PAHS-404ZC-12). cDNA was synthesized from isolated RNA (using an RNAi kit) using the QIAGEN RT ² First Strand Kit (Qiagen #330401). For each 96-well PCR array plate, 0.5 μg of total RNA was mixed with 19.5 μl of reverse transcription mix and incubated at 42°C for 15 minutes, followed by incubation at 95°C for 5 minutes. 20 μl of cDNA product was diluted with 81 μl of RNase-free H ₂ O. For each 96-well PCR array plate, 101 μl of diluted cDNA was mixed with RT ² SYBR Green QPCR mastermix (Qiagen #330522) to a total volume of 2700 μl. 25 μl of the QPCR mix was transferred to each well of the PCR array plate. An Applied StepOnePlus ^™ real-time cycler was used for PCR reactions and data acquisition. 40 cycles of 95°C for 15 seconds and 60°C for 1 minute were performed, followed by melting curve analysis. Gene thresholds were set to the same value for all RT2 ^Profiler PCR Array runs within the same analysis. Quantification was performed using QIAGEN ^RT2 Profiler PCR Array Data Analysis Software, version 3.5. Gene expression at D0 was set as a control.

Virus production

The Pcag::GFP-IRES-GFP retroviral vector was a gift from Dr. Fred Gage (Salk Institute, CA). The human GFAP promoter gene was subcloned from the Hgfap promoter-Cre-MP-1 (Addgene) and the CAG promoter was replaced to generate the Pgfap::GFP-IRES-GFP retroviral vector (Guo et al., 2014). The mouse LCN2 promoter sequence was subcloned from the mouse genome and the CAG promoter was replaced to generate the Plcn2::GFP-IRES-GFP retroviral vector. The FUGW-EGFP lentiviral vector was generously provided by Dr. Roger Nicoll (University of California, San Francisco, San Francisco, USA). Retroviral particles were packaged in GPG helper-free HEK (human embryonic kidney) cells to produce VSV-G (vesicular stomatitis virus glycoprotein)-pseudotyped retrovirus as previously described (Guo et al., 2014; Tashiro et al., 2006). Lentiviral particles were packaged in HEK293T cells as previously described (Naldini et al., 1996). The titer of viral particles was determined after transduction of HEK cells and was approximately ¹⁰⁸ particles/ml.

Imaging over time

Human astrocytes cultured in T25 flasks were transduced with 1 μl of Pcag::GFP-IRES-GFP retroviral suspension. Two hours after viral transduction, cells were dissociated with TrypLE and plated on poly-D-lysine-coated coverslips at a density of 50,000 cells per coverslip in a 24-well plate. On day 0, only one or two GFP-positive cell clusters were found in each well. One GFP-positive cluster was imaged under a fluorescence microscope (Nikon TE-2000-S) on days 0, 2, 4, 6, 8, and 10 without or with small molecule treatment, as described above. To visualize the reprogramming process induced by the sequential application of nine molecules, images were taken at each time point before changing the culture medium containing the next set of small molecules.

Lineage tracing experiments

Human astrocytes were cultured on poly-D-lysine-coated coverslips and infected overnight with 2 μl of a Pgfap::GFP-IRES-GFP retroviral suspension. For Plcn2::GFP-IRES-GFP retroviral infection, cultured human astrocytes were pretreated with 100 ng/ml lipopolysaccharide (LPS) to render them reactive and express LCN2. Retrovirus-infected cells were then treated with small molecules or 1% DMSO. Cells were cultured for 18 days and then fixed for immunostaining.

BrdU date of birth determination

One day before small molecule treatment, human cortical astrocytes were incubated with 5-bromo-2-deoxyuridine (BrdU) at a final concentration of 10 μM for 12 hours. The next day, the BrdU-containing medium was completely removed and fresh human astrocyte culture medium was carefully added to the culture. Approximately 70-80% of human astrocytes were labeled with BrdU at D0. Human astrocytes labeled with BrdU were treated with small molecules and fixed on day 30 after the initial small molecule treatment. In another group, 10 μM BrdU was added to the neural differentiation medium on day 10 after small molecule treatment and renewed every 3-4 days until day 30. On day 30, the cells were fixed with 4% PFA for 15 minutes at room temperature and then treated with 2M HCl for 20 minutes at 37°C to denature the DNA. After washing five times with PBS, cells were blocked in blocking buffer (2.5% normal donkey serum, 2.5% normal goat serum, 0.1% Triton in PBS) for 1 h at room temperature and then incubated in anti-BrdU primary antibody (Dako, 1:500) at 4°C overnight.

Calcium imaging

The calcium indicator Fura-2AM (Life Technology) was loaded into the cells by incubating the neurons converted from human astrocytes in a culture medium containing Fura-2AM (2 μg/ml) in an incubator (37°C) for 30 minutes. The plasma calcium concentration was monitored using a Nikon 20× Super Fluor objective lens (NA 0.75), a Hamamatsu ORCA-ER digital camera (Hamamatsu, Shiota, Japan) and a Sutter DG5 optical switcher (Sutter Instrument, Novato, CA) for rapidly changing the excitation wavelength. Data acquisition and analysis were performed using Hamamatsu's Simple PCI software.

Methylated DNA immunoprecipitation (MeDIP) and high-throughput sequencing

MeDIP experiments were performed according to the manufacturer's protocol (Active Motif). The enriched methylated DNA was purified using the Qiagen DNA purification kit and used for library preparation using the NEBNext ChIP-Seq Library Prep Reagent Set for Illumina according to the manufacturer's protocol. In brief, 25 ng of genomic DNA or experimental enriched DNA was used for each library construction. After connecting the adapter, 150-300 bp DNA fragments were selected using AMPure XP beads (Beckman Coulter). An Agilent 2100 BioAnalyzer was used to quantify the amplified DNA and QPCR was used to accurately quantify the library concentration. 20 μM diluted libraries were used for sequencing. 50 cycles of single-end sequencing were performed using an Illumina HISeq 2000. Image processing and sequence extraction were performed using standard Illumina internal processes (pipeline).

Targeted BS-seq

DNA samples were applied to the EpiTect Bisulfite Kit (Qiagen) according to the manufacturer's instructions. PCR amplicons were then purified using Ampure XP beads and eluted in 50 μl of _H₂O . Concentrations were quantified using the Qubit High Sensitivity Kit, and each sample was pooled at equal molar concentrations. The pooled amplicons were then subjected to library preparation and Miseq deep sequencing (100× or greater) according to standard Illumina protocols. Image analysis and base calling were performed using standard Illumina protocols.

To determine the DNA methylation status of the GFAP transcription start site, genomic DNA was treated with sodium bisulfite using the EZ DNA Methylation-Gold kit (Zymo Research) according to the manufacturer's instructions. Bisulfite-converted DNA was amplified using nested PCR. The purified PCR amplicon was then ligated into a TOPO-TA vector (Invitrogen). The reconstructed plasmid was purified and each clone was sequenced. Ten clones were randomly selected from each time point. Data are from two independent experiments.

Bioinformatics analysis

Bioinformatics analysis of MeDIP-seq was performed using known techniques. Briefly, FASTQ sequence files were aligned to the HG19 reference genome using Bowtie. Peaks were identified by model-based analysis using ChIP-Seq (MACS) software.

For BS-seq, paired-end reads were first preprocessed using Trimmomatic 0.20 to remove adapter sequences and low-quality sequences at the 3' and 5' ends. The preprocessed reads were then aligned to the C-to-T and G-to-A sequences at the sites of interest using Bowtie 0.12.9 (-m1-130-n0-e90-X550). Only uniquely aligned reads were retained, and PCR duplicates were removed using MarkDuplicates (Picard Tools 1.82). To avoid counting reference positions covered by overlapping paired-end reads, the overlapping regions were cut off and the overlapping regions with higher quality were retained. The original computationally converted C and G were restored, and the C reads and T reads were counted for each reference cytosine position using SAMTools mpileup.

Chromatin immunoprecipitation (ChIP)-quantitative PCR

Chromatin immunoprecipitation (ChIP) experiments were performed using conventional methods with minor modifications. Briefly, cultured human astrocytes were fixed with 1% formaldehyde for 10 minutes before and after small molecule treatment and quenched with 0.125 M glycine for 5 minutes. Chromatin was sonicated using a Bioruptor (Diagenode Inc.) to a fragment size range of 300-500 base pairs. Following the ChIP procedure, eluted DNA samples were purified using a DNA cleanup and concentration kit (Zymo Research). The degree of enrichment was determined by qPCR and normalized to the total input.

Stereotaxic injection of small molecules into the mouse brain

Brain surgery was performed on 2-month-old wild-type C57BL6 mice. Mice were anesthetized by injecting 20 mL/kg 0.25% Avertin (a mixture of 25 mg/ml tribromoethanol and 25 μl/ml tert-amyl alcohol) into the peritoneum and then placed in a stereotactic apparatus. Artificial eye ointment was used to cover and protect the eyes. The animals underwent a midline scalp incision and a drill hole in the skull above the somatosensory cortex. Each mouse received a single injection of a small molecule mixture or PBS containing 6% DMSO using a 2 μl syringe and a 34-gauge needle (coordinates: AP 1.25 mm, ML 1.4 mm, DV-1.5 mm). The injection volume and flow rate were controlled to 2 μl at 0.2 μl/min. After injection, the needle was kept in place for at least 5 minutes and then slowly removed.

In vitro cell suspension culture

Six days after the small molecule injection (dpi), the animals were sacrificed by exposure to CO _2. The brain was dissected, and the cortical brain tissue of about 1.5 mm around the injection site was separated and cut into 0.1 × 0.1 mm blocks, treated with 0.5% trypsin (Gibco) at 37 ° C for 30 minutes, and then centrifuged at 900G for 8 minutes. The cell pellet was resuspended in neuronal proliferation culture medium supplemented with 20 ng/ml FGF2 and 20 ng/ml EGF, and about 100 cells in 10 ml culture medium were seeded in a 6-well plate (Corning#3471) with an ultra-low attachment surface. Growth factors were renewed every 2-3 days. One week after the initial inoculation, neurospheres were observed and counted under a 10x microscope (Nikon). In order to subculture, one-week-old primary neurospheres were collected by centrifugation at 900G for 3 minutes and incubated at 37 ° C for 5 minutes using cell digestion solution (Gibco). The cell pellet was centrifuged at 900G for 5 minutes and ground into single cells, then suspended in neuronal proliferation medium. Three days after subculture, the second-generation neurospheres were observed and counted under a 10× microscope. For monolayer culture, 4-day-old second-generation neurospheres were trypsinized and resuspended according to the above protocol. Single cells were plated on poly-L-ornithine/laminin-coated coverslips and cultured in neuronal proliferation medium with 20ng/ml FGF2 and 20ng/ml EGF. When the cells reached 60-70% confluence, they were fixed with 4% PFA or induced to differentiate with neuronal differentiation medium or glial medium containing DMEM/F12, 5% FBS, 50mg/ml NaHCO ₃ and penicillin/streptomycin.

The following primary antibodies were used in this study:

Polyclonal anti-green fluorescent protein (GFP, chicken, 1:1000, Abcam, AB13970), polyclonal anti-glial fibrillary acidic protein (GFAP, rabbit, 1:1000, Abcam, Z0334), polyclonal anti-glial fibrillary acidic protein (GFAP, chicken, 1:1000, Millipore, AB5541), monoclonal anti-S100β (mouse, 1:800, Abcam, ab66028), polyclonal anti-vesicular glutamate transporter 1 (vGluT1, rabbit, 1:1000, Synaptic Systems), polyclonal anti-vesicular glutamate transporter 2 (SV2, mouse, 1:2000, Developmental Research Hybridoma Bank, Iowa City, IA), polyclonal anti-microtubule-associated protein 2 (MAP2, chicken, 1:2000, Abcam, AB5392), polyclonal anti-T-box 1 (brain, polyclonal anti-T-box 1) (mouse, 1:800, Abcam, ab66028), polyclonal anti-mouse glutamate transporter 2 (vGluT1, rabbit, 1:1000, Synaptic Systems), polyclonal anti-vesicular glutamate transporter 2 (SV2, mouse, 1:2000, Developmental Research Hybridoma Bank, Iowa City, IA), polyclonal anti-microtubule-associated protein 2 (MAP2, chicken, 1:2000, Abcam, AB5392), polyclonal anti-brain T-box 1 (T-box 1) (mouse, 1:800, Abcam, ab66028), polyclonal anti-mouse glutamate transporter 2 (VGluT1, rabbit, 1:1000, Synaptic Systems), polyclo anti-T-box, brain, 1, Tbr1, 1:300, rabbit, Abcam, AB31940), polyclonal anti-Prox1 (rabbit, 1:1000, ReliaTech GmbH, 102-PA32), polyclonal anti-musashi-1 (rabbit, 1:500, Neuromics, RA14128), monoclonal anti-SRY (sex determining region Y)-box2 (Sox-2, mouse, 1:500, Abcam, AB79351), polyclonal anti-SRY (sex determining region Y)-box2 (Sox-2, rabbit, 1:500, Millipore, AB5603), monoclonal anti-Biii tubulin (Tuj1, mouse, 1:1000, COVANCE, MMS-435P), polyclonal anti-doublecortin (DCX, rabbit, 1:500, Abcam, AB18723), polyclonal anti-NeuN (rabbit, 1:1000, Millipore, ABN78), monoclonal anti-NG2 (mouse, 1:200, Abcam, AB50009), monoclonal anti-ubiquitin - axonal neurofilament marker (SMI 312, 1:1000, mouse, Covance, SMI-312R), polyclonal anti-glial glutamate transporter GLT-1 (EAAT2) (Glt1, guinea pig, 1:2000, Millipore, AB1783), monoclonal anti-NeuroD1 (mouse, 1:1000, Abcam, ab60704), monoclonal anti-human nuclear (HuNu, mouse, 1:1000, Millipore, MAB1281), monoclonal anti-synaptobrevin (mouse, 1:800, Millipore, MAB368), polyclonal anti-CDP (Cux1, rabbit, 1:500, Santa Cruz, CA), monoclonal anti-human leukocyte antigen (HUNE), monoclonal anti-synaptobrevin (HLE), monoclonal anti-mouse leukocyte antigen (HLE), monoclonal anti-mouse leukocyte antigen (HLE), monoclonal anti-human leukocyte antigen (HLE), monoclonal anti-human leukocyte antigen (HLE), monoclonal anti-human leukocyte antigen (HLE), monoclonal anti-human leukocyte antigen (HLE), Cruz, sc-13024), monoclonal anti-Ctip2 (rat, 1:600, Abcam, ab18465), anti-Otx1 (mouse, 1:200, Developmental Studies Hybridoma Bank, Iowa City, otx-5F5), anti-HoxC9 (mouse, 1:200, Developmental Studies Hybridoma Bank, Iowa City, 5B5-2), anti-HoxB4 (mouse, 1:200, Developmental Studies Hybridoma Bank, Iowa City, I12 anti-Hoxb4), polyclonal anti-FoxG1 (goat, 1:1000, Abcam ab3394), polyclonal anti-vesicular acetylcholine transporter (VAChT, guinea pig, 1:800, Millipore, AB1588), monoclonal anti-GAD67 (mouse, 1:1000, Millipore, MAB5406), anti-Isl1 (mouse, 1:200, Developmental Research Hybridoma Bank, Iowa City, IA 39.4D5), monoclonal anti-tyrosine hydroxylase (TH, mouse, 1:600, Millipore, MAB318), polyclonal anti-neurogenin 2 (Ngn2, rabbit, 1:600, Abcam, ab26190), monoclonal anti-NeuroD1 (mouse, 1:800, Abcam, ab60704), polyclonal anti-MASH1/Acheatescute homolog 1 (Ascl1, rabbit, Abcam, ab74065), monoclonal anti-nestin (mouse, 1:800, , Neuromics, MO15056), polyclonal anti-Ki67 (rabbit, 1:800, Abcam, ab15580), monoclonal anti-N200 (mouse, 1:1000, Sigma, N0142), monoclonal anti-BrdU (mouse, 1:500, Dako, 074401-8), monoclonal anti-glutamine synthetase (GS, mouse, 1:800, Millipore, MAB302), monoclonal anti-phospho- Acidified GSK-3β (Ser9) (Ser9) (5B3) (rabbit, 1:100, Cellsignaling, 9323), monoclonal anti-phospho-signaling molecule 1 (Smad1) (Ser463/465)/signaling molecule 5 (Smad5) (Ser463/465)/signaling molecule 9 (Smad9) (Ser465/467) (D5B10) (rabbit, 1:600, Cell signaling, 13820), monoclonal anti-cleaved Notch1 (Val1744) (D3B8) (rabbit, 1:200, Cellsignaling, 4147), monoclonal anti-CNPase (mouse, 1:800, Abcam, ab6319), polyclonal anti-lipocalin-2/NGAL (LCN2, goat, 1:1000, R&D, AF1857).

The following antibodies were used to pull down DNA in the CHIP assay: polyclonal anti-acetyl histone H3 (rabbit, Millipore, 06-599); polyclonal anti-trimethyl histone H3 (Lys27) (H3K27Me3, rabbit, Millipore, 07-449); and polyclonal anti-H3K4me3 (rabbit, Active Motif 39159).

Example 9

This example extends the foregoing disclosure and demonstrates that the use of four drugs and even three drugs is sufficient to achieve the reprogramming of glial cells into neurons. Specifically, this example demonstrates that reprogramming using a combination of SB431542 (TGF-β inhibitor), LDN193189 (BMP inhibitor), CHIR99021 (GSK-3 inhibitor) and DAPT (γ-secretase and Notch inhibitor) can successfully reprogram human glial cells into functional neurons. In addition, we also tested each of these four drugs in combination with other drugs with similar effects and demonstrated that they can all convert human astrocytes into neurons. Therefore, the present disclosure includes the reprogramming of glial cells into neurons using a combination of drugs that act on one or a combination of the following signaling pathways: TGF-β, BMP, GSK-3 and γ-secretase/Notch signaling pathways.

The data presented in Figures 15-19 demonstrate that various drugs with similar activity can be substituted while still achieving reprogramming. Specific combinations demonstrated to reprogram include: i) LDN193189/CHIR99021/DAPT, ii) SB431542/CHIR99021/DAPT, iii) LDN193189/DAPT/SB431542, and iv) LDN193189/CHIR99021/SB431542. Furthermore, we demonstrated that LDN193189 can be replaced by its functional analogs Dorsomorphin and DMH1; SB431542 can be replaced by Repsox or A8301; CHIR99021 can be replaced by its functional analogs ARA014418 and SB216763; and DAPT can be replaced by its functional analogs BMS906024 and RO4929097. Therefore, the present disclosure demonstrates that any three-drug combination from the group of SB431542, LDN193189, CHIR99021 and DAPT can reprogram human glial cells into neurons, while any one or more drugs in these combinations can be replaced by their functional analogs and still achieve reprogramming.

While the invention has been described with reference to specific embodiments, routine modifications will be apparent to those skilled in the art, and such modifications are intended to be within the scope of the invention.

Claims (4)

1. Use of a combination of compounds in the preparation of a medicament for reprogramming human glial cells in an individual brain, wherein said use comprises contacting said human glial cells with compounds that co-inhibit the transforming growth factor β (TGF-β), bone morphogenetic protein (BMP), glycogen synthase kinase 3 (GSK-3), and γ-secretase/Notch pathways in said glial cells, wherein said combination of compounds is at least one of the following combinations: i) LDN193189/CHIR99021/N-[(3,5- The compound applied is either 1) difluorophenylacetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT), ii) SB431542/CHIR99021/DAPT, iii) LDN193189/DAPT/SB431542, or iv) LDN193189/CHIR99021/SB431542, wherein the applied compound is cell-free and virus-free, and wherein the application is sufficient to reprogram at least some of the glial cells into neurons. 2. Use of a combination of compounds in the preparation of a medicament for generating neurons in the brain of an individual, wherein the use comprises administering the combination of compounds to the individual, wherein the combination of compounds comprises at least three compounds selected from the group consisting of LDN193189, SB431542, CHIR99021 and N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT). 3. The use according to claim 2, wherein the combination of said compounds comprises each of LDN193189, SB431542, CHIR99021 and DAPT. 4. The use according to claim 1, wherein the combination of said compounds includes one of LDN193189, SB431542, and CHIR99021, and wherein said use further includes contacting said human glial cells with DAPT.