Method for inducing dorsal root node satellite glial cells to be reprogrammed into neurons by using small molecule compound and application of method

Abstract

The invention discloses a method for inducing dorsal root node satellite glial cells to be reprogrammed into neurons by using a small molecule compound and application of the method. The small molecule compound composition is prepared from ISX-9, RO4929097, CHIR99021, VPA, RepSox, Forskolin, SC79 and VC, and growth factors BDNF, GDNF, NT-3 and NGF beta, neuron culture additives B27 and N2 additives are added at the same time, so that survival and maturation of neurons are promoted and induced. Wherein each component is a safe and low-cost small molecule compound and a neurotrophic factor, and the pharmaceutical composition and the growth factor can be used for reprogramming rat dorsal root ganglion satellite glial cells into neurons with specific phenotypes and functional characteristics in vitro. And a new seed cell is provided for basic research of clinical nerve injury diseases and clinical cell treatment development.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a method for inducing the reprogramming of dorsal root ganglion satellite glial cells into neurons using small molecule compounds and its application. Background Technology

[0002] Neuronal degeneration, apoptosis, and necrosis caused by nerve injury or neurodegenerative diseases pose a significant challenge to nerve repair due to the inability of damaged neurons to regenerate. Current clinical treatments for nerve injury primarily include surgery, drug delivery, post-treatment rehabilitation, electrical stimulation, and stem cell-based tissue engineering. However, due to the non-regenerative nature of endogenous neurons and the complexity of the nervous system, these treatments have minimal impact on nerve regeneration and poor prognoses. While progress has been made in stem cell replacement therapy using induced pluripotent stem cells (iPSCs), exogenous cell therapy faces ethical concerns, immune rejection, high carcinogenicity, and limited cell sources, restricting its clinical application. Therefore, somatic cell reprogramming into neurons to promote nerve injury repair has significant basic research and clinical translational application value.

[0003] Numerous studies have shown that adding small molecules during reprogramming to pluripotency or direct cell fate conversion can improve efficiency and / or survival, and in some cases allows for the chemical substitution of single or complete replacement of viral transduced transcription factors. Lineage specifiers play a crucial role in coordinating the restoration of pluripotency by substituting pluripotency-associated transcription factors. Furthermore, combinations of small molecules can acquire pluripotency from somatic cells without any transgenes, providing an easily tractable platform for precise analysis of the induction and maintenance of cell identity. Chemical stimulation through exposure to small molecules offers an alternative approach to manipulate cell fate in a simple and highly controllable manner. Small molecule compounds refer to active compounds with a molecular weight less than 900 Da. Due to their low molecular weight, these compounds readily penetrate cells. Small molecule compounds can exert their effects by binding to receptors on the cell surface, in the cytoplasm, or in the nucleus, influencing signaling pathways and thus disease progression by modulating the activity of their protein targets.

[0004] The development of the peripheral nervous system plays a crucial role in the normal functioning of the nervous system. Peripheral nerve injury can lead to a reduction in dorsal root ganglion (DRG) neurons, disruption of neuronal connections, and abnormal sensory conduction, causing neurological dysfunction and affecting the development, differentiation, and plasticity of the nervous system. Satellite glial cells (SGCs) are a group of cells surrounding sensory neurons in the DRG. They not only provide nutrition, support, and protection to sensory neurons, but also maintain neuronal homeostasis through gap junctions, ion channels, chemokines, and neurotransmitters, ensuring that neurons and SGCs are in a normal state. They also communicate and transmit information with the surrounding neuronal cell bodies and other adjacent satellite glial cells through these mechanisms. Studies have found that sensory neurons and their surrounding SGCs form an independent functional unit, and the number of SGCs around the neuronal cell body is directly proportional to the size of the neuronal cell body. SGCs are closely related to sensory neurons, interconnected, and participate in the occurrence and development of neuropathic pain. Sensory dysfunction following peripheral nerve injury is related to apoptosis of DRG sensory neurons. Neurogenesis or an increase in the number of neurons is observed in the dorsal root ganglion (DRG) of rats. Following peripheral nerve injury, SGCs proliferate and express NTS and p75 neurotrophin receptor (p75NTR) at high levels, with increased expression of TGF-α and BDNF. Two weeks after sciatic nerve injury in mice, new gap connections can be established between SGCs in the DRG. Soares et al. found that SGCs express the c-fos gene after nerve injury, indicating that the signal of nerve injury has been transmitted to SGCs. Furthermore, fatty acid synthesis in SGCs can promote the repair and regeneration of injured sensory neurons. During embryonic development, SGCs develop later than neurons, mediated by Notch signal-dependent collateral inhibition. In this collateral inhibition, neuronal proliferation activates the Notch cascade signaling pathway, inhibiting precursor cell differentiation into neurons and promoting cell proliferation and glial cell differentiation. In conclusion, SGCs possess good potential and application value for reprogramming. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for inducing the reprogramming of dorsal root ganglion satellite glial cells into neurons using small molecule compounds, and its application. This invention induces rat dorsal root ganglion satellite glial cells into neurons in vitro. Morphological observation and immunofluorescence staining are used to identify changes in cell identity. Calcium imaging experiments monitor intracellular calcium ion changes to reflect the activity of functional neuronal networks, further demonstrating that the induced differentiated cells are functional neurons.

[0006] To solve the above technical problems, the technical solution of the present invention is as follows: a method for inducing dorsal root ganglion satellite glial cells to reprogram into neurons using small molecule compounds, the method is operated as follows: purified DRG-SGCs are passaged in passage medium, then induced in neuron-inducing medium containing small molecule compounds is used for induction culture, and then neurons are obtained by culture in mature neuron medium.

[0007] The small molecule compounds are ISX-9, RO4929097, CHIR99021, VPA, RepSox, Forskolin, SC79, and Vc.

[0008] Preferably, the induced neuron culture medium also contains growth factors BDNF, GDNF, NT-3, NGFβ, neuron culture additive B27, and N2 additive.

[0009] Preferably, the formulation of the neuron-inducing culture medium comprises: DMEM / F12 medium, Neurobasal medium, 2-5 μmol ISX9, 1-4 μmol RO4929097, 1-5 μmol CHIR99021, 100-10000 μmol VPA, 1-5 μmol RepSox, 2-10 μmol Forskolin, 0.01-2 μmol SC79, 100-400 μmol VC, 3-20 ng / mL BDNF, 3-20 ng / mL GDNF, 3-20 ng / mL NT-3, 3-20 ng / mL LNGFβ, 1-5% B27, 0.1-3% N2, L-alanyl-glutamine solution, and 0.1-1% penicillin-streptomycin solution.

[0010] Preferably, the mature neuron culture medium comprises: Neurobasal medium, 100-500 μmol VC, 10-50 ng / mL BDNF, 10-50 ng / mL GDNF, 10-50 ng / mL NT-3, 10-50 ng / mL LNGFβ, 1-5% B27, 0.1-3% N2, 0.1-3% L-alanyl-glutamine solution, and 0.1-1% penicillin-streptomycin solution.

[0011] As a preferred method, when the cell density reaches 60-70% through passage culture, the induced neuron culture medium should be replaced for further induction culture.

[0012] As a preferred method, the culture medium for induced neurons is changed every 1-2 days. After culturing for 6-15 days, the culture medium is changed to the culture medium for mature neurons and cultured for another 3-8 days, changing the culture medium for mature neurons every 1-2 days.

[0013] The present invention also provides neurons cultured by the method described above. The neurons obtained by the present invention have sensory neuron-specific molecular markers and physiological response characteristics to specific stimuli, such as changes in intracellular calcium activity of neurons.

[0014] This invention also provides the application of the aforementioned neurons in the preparation of drugs for nerve injury or neurodegenerative diseases. The method of reprogramming endogenous satellite glial cells into neurons according to this invention can be used for basic research in the field of somatic cell reprogramming and for translational applications in clinical nerve injury repair cell replacement therapy.

[0015] In this invention, Isx9 can increase the survival rate of induced neurons, RO4929097 inhibits the Notch signaling pathway, CHIR99021 is a Wnt signaling pathway activator that inhibits GSK-3 signal transduction, and VPA is a histone deacetylasase inhibitor that can activate the expression of many neuronal development-related genes. Histone acetylation modification is controlled by histone deacetylases (HDACs) and histone deacetylases. Histone deacetylases add acetyl groups to lysine residues, resulting in chromatin being in a more open state, making transcription easier. HDAC removes the acetyl group from lysine residues, resulting in a more condensed chromatin state, which is usually associated with gene silencing; RepSox inhibits the TGFβ signaling pathway, replacing the role of transcription factor Sox2; Forskolin is a cAMP activator that increases intracellular cAMP levels; SC79 is an AKT activator, which is beneficial for SGCs to transdifferentiate into neuron-like cells; VC is an antioxidant that alleviates cell senescence; BDNF is brain-derived neurotrophic factor that promotes neuronal survival and synapse formation; Glial cell line-derived neurotrophic factor (GDNF) regulates the differentiation of neurons into specific subtypes; Neurotrophin-3 (NT-3) promotes the survival and maturation of sensory neurons; Beta-nerve growth factor β (NGFβ) regulates neuronal proliferation, differentiation, and survival; L-alanyl-glutamine is used to replace glutamine in cell culture because of its high stability in aqueous solution, which can maintain a stable nutrient supply for a long time and avoid the cytotoxicity caused by decomposition products.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses a combination of small molecule compounds and neurotrophic factors to induce satellite glial cells to be reprogrammed into neurons; wherein each component is a safe and low-cost small molecule compound and neurotrophic factor, and the pharmaceutical composition combined with growth factors can reprogram rat dorsal root ganglion satellite glial cells into neurons with specific phenotypes and functional characteristics in vitro, providing new seed cells for basic research on clinical neurological injury diseases and the development of clinical cell therapy. Attached Figure Description

[0017] Figure 1 A schematic diagram of a method for inducing DRG-SGCs to reprogram into neurons by combining small molecule compounds with neurotrophic factors;

[0018] Figure 2 Representative images at various time points during the DRG-SGCs culture process;

[0019] Figure 3 Immunofluorescence assay was used to visualize the expression of SGC markers GS, S100β, GFAP, and FABP7 in cells, with a scale bar of 50 μm.

[0020] Figure 4 Representative microscopic images of morphological changes in SGCs induced by a combination of small molecule compounds and neurotrophic factors. Scale Bar = 50 μm. a. Uninduced SGCs after passage. bf. Cells induced for 24 h, 48 h, 72 h, 7 d, 14 d, and 21 d, respectively.

[0021] Figure 5 Immunofluorescence results of SGCs induced by a combination of small molecule compounds and neurotrophic factors on days 7 and 14 after differentiation, Scale Bar = 50 μm. A. Almost all Tuj1-positive cells expressed GS 3 days after differentiation. B. Immunofluorescence identification of early neuronal markers Tuj1 (β-tubulin III) and mature neuronal marker Map2 (microtubule-associated protein 2) in cells differentiated on days 7 and 14. C. Quantitative cell counting analysis of immunofluorescence of early neuronal markers Tuj1 and mature neuronal markers Map2 in cells differentiated on days 7 and 14 (n = 3, mean ± SD);

[0022] Figure 6 Immunofluorescence results of SGCs induced by a combination of small molecule compounds and neurotrophic factors for 45 days showed that the cell phenotype was positive for neuron-specific markers PRPH (peripheral protein), SYN (synapsin), and NeuN (neuronal core antigen), with a scale bar of 20 μm.

[0023] Figure 7 After SGCs were induced to differentiate for 21 days by a combination of small molecule compounds and neurotrophic factors, they expressed multiple sensory neuron markers. Scale Bar = 50 μm.

[0024] Figure 8 Calcium flux analysis for iNs-21d. A. Fluorescence changes, Scale Bar = 20 μm. B. Quantitative fluorescence curve. Detailed Implementation

[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions. The small molecule compounds used in the following embodiments and their mechanisms of action are shown in Table 1.

[0026] Table 1. Combinations of small molecule compounds and neurotrophic factors used in neuronal induction.

[0027]

[0028]

[0029]

[0030] Example 1: Combination of small molecule compounds induces DRG-SGCs to be reprogrammed into neurons

[0031] The process by which small molecule compound combinations induce DRG-SGCs to reprogram into neurons is as follows: Figure 1 As shown, the specific experimental procedure is as follows.

[0032] Experimental animals: Newborn SD rats aged 0-24 hours were purchased from the Department of Experimental Animals, Kunming Medical University. This invention was approved by the Medical Ethics Committee of Kunming Medical University.

[0033] Instruments required for DRG collection: stereomicroscope, small surgical scissors, spring scissors, fine straight forceps, and fine curved forceps.

[0034] In the absence of Ca 2+ and Mg 2+ Poly-L-lysine hydrobromide powder was dissolved in sterile DPBS to prepare a 50 μg / mL working solution.

[0035] Coat the surface of the culture dish and culture plate with poly-L-lysine working solution (e.g., 1 mL per well in a 6-well plate, 5 mL in a 10 cm culture dish), and incubate the culture dish and culture plate at room temperature for 1 hour.

[0036] Discard the poly-L-lysine solution and rinse the culture dishes and plates three times with sterile PBS or distilled water. Since excess poly-L-lysine is toxic to cells, ensure thorough rinsing of the culture dishes and plates.

[0037] Remove PBS or distilled water, and allow the surfaces of the petri dishes and plates to dry completely for 2 hours before use.

[0038] Purified rat dorsal root ganglion satellite glial cells (DRGs) were obtained in vitro: Newborn SD rats within 24 hours of birth were disinfected with alcohol, decapitated, and their spines were dissected. The spine was cut open to expose the intervertebral foramina as much as possible. Under a stereomicroscope, blood vessels and spinal cord were cleaned, and the DRGs were removed. Nerve fibers and membranes on the DRGs were thoroughly removed to minimize the risk of fibroblast and Schwann cell contamination. The processed DRGs were cultured in DRG-SGCs medium and seeded into six-well plates. 500 μL of DRG-SGCs medium was added to each well, with approximately 2 mm spacing between DRGs. The plates were then incubated at 37°C in a 5% CO2 incubator. After the DRGs adhered to the plates, the DRG-SGCs medium was changed every other day. Then, satellite glial cells were induced to migrate out of the DRG. When the cell density reached 70%-80% (approximately day 10 of DRG culture), the cells were passaged. 0.05% trypsin was added for digestion. The digestion time depended on the DRG culture time and the potency of the trypsin. FBS was then added to stop the digestion. After gently pipetting with a Pasteur pipette, the cells were transferred to centrifuge tubes and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in DRG-SGCs culture medium and seeded in 10 cm culture dishes for further culture.

[0039] The specific formulation of DRG-SGCs medium includes DMEM / F12, B27, penicillin-streptomycin bispecific antibody, L-propionamide-glutamine, BSA (bovine serum albumin), NRG1-β1 (neuroregulatory protein), dexamethasone, insulin, T3 (triiodothyronine sodium), and T4 (tetraiodothyronine).

[0040] Each 50 mL of DRG-SGCs culture medium contains 46–48 mL of DMEM / F12, 0.8–1.2 mL of B27, 0.4–0.6 mg of penicillin-streptomycin, 0.4–0.6 mg of L-glutamine, 15–20 mg of BSA, 1–1.2 μg of NRG1-β1, 1.8–2 μg of dexamethasone, 280–300 μg of insulin, 0.5–0.6 μg of T3, and 20–24 μg of T4.

[0041] Figure 2 Representative images at various time points during the culture of DRG-SGCs: On the first day of culture, a small number of oval-shaped cells can be seen migrating out of the DRG tissue block. Figure 2 (a and d). After about 3 days of culture, a large number of cells migrated out from around the DRG. Figure 2 (b and e), after approximately 10 days of culture, the number of cells can be used for subculture. Figure 2 c and f). Figure 3 Images of DRG-SGCs after passage: The results showed that all cultured cells were positive for GS, S100β, FABP7 and GFAP, and the cultured cells were identified as SGCs. These are four biomarkers of SGCs proven in in vitro and in vivo studies.

[0042] Purified DRG-SGCs were passaged into 10cm culture dishes at a cell density of 70%, with cells exhibiting excellent growth and high refractive index for induction. 6ml of induced neuron medium was added, and the medium was changed every two days until day 10. The medium was then replaced with mature neuron medium, and the medium was changed every two days, continuing culture for 3-10 days. Induced neurons are prone to detachment. When removing used medium, the pipette tip should not touch the cells; when adding fresh medium, it should be done gently towards the culture plate.

[0043] The formulation of the neuron-inducing medium includes: DMEM / F12 medium, Neurobasal medium, 3 μmol ISX9, 2 μmol RO4929097, 3 μmol CHIR99021, 500 μmol VPA, 2 μmol RepSox, 5 μmol Forskolin, 1 μmol SC79, 200 μmol VC, 10 ng / ml BDNF, 10 ng / ml GDNF, 10 ng / ml NT-3, 10 ng / ml NGFβ, 2% B27, 1% N2, 1% L-alanyl-glutamine solution, and penicillin-streptomycin.

[0044] The formulation of mature neuron culture medium includes: Neurobasal medium, 200 μmol VC, 20 ng / ml BDNF, 20 ng / ml GDNF, 20 ng / ml NT-3, 20 ng / ml NGFβ, 2% B27, 1% N2, 1% L-alanyl-glutamine solution and penicillin-streptomycin.

[0045] The above three culture media (DRG-SGCs Medium, Induced-neuron Medium, and Matured-neuron Medium) are stable for 2 weeks when stored at 2-8°C in the dark. The penicillin-streptomycin solution is for preventing cell contamination and does not need to be added.

[0046] The formulation of 50 mL of neuron-inducing medium is shown in Table 2.

[0047] Table 2

[0048]

[0049] The formulation of 50 mL of mature neuron culture medium is shown in Table 3.

[0050] Table 3

[0051]

[0052]

[0053] Observe the morphological changes of cells at different time points during induction under bright field, such as Figure 4 As shown: with increasing induction time, the cell bodies become rounder and fuller, cell processes lengthen, and gradually connect to form a well-developed neural network. The results are as follows... Figure 5 As shown in Figure A, SGCs expressed almost no early neuronal marker Tuj1. At iNs-24h, some cells co-expressed both GS and Tuj1, and the number of co-expressing cells increased over time. After 72h of induced differentiation, almost all Tuj1-positive cells expressed GS, indicating that these neurons originated from SGCs. Figure 5 B and Figure 5 As shown in Figure C, the mature neuronal marker Map2 begins to appear on day 7 of induction and is highly expressed on day 14 of induction. Figure 6 As shown, the differentiated neuron-like cells can survive for 45 days in the mature culture medium and can continuously express mature neuron markers such as presynaptic protein (SYN), neuronal nuclear protein NeuN, and peripheral protein (PRPH), indicating that the present invention has successfully obtained neurons.

[0054] Peripheral sensory neurons are characterized by their size, molecular features, and physiological responses to specific stimuli. Nociceptors are specialized cell types in the peripheral nervous system (PNS) and are crucial for transmitting information about the presence of noxious stimuli from the body to the higher brain centers of the spinal cord and somatosensory system. To further characterize DRG-SGCs-derived sensory neurons, pansenistous neuron markers were detected in neuron-like cells induced for 21 days using immunofluorescence, such as... Figure 7As shown, iNs-21d expresses the pansensory neuron transcription factor Brn3a, the sensory axon marker peripheral protein PRPH, the neurotrophic factor receptor TrkA, the neuropeptide secreted by sensory nerve endings—calcitonin-related peptide (CGRP), and the neuronal-specific structural protein—ankyrin G.

[0055] When neurons are stimulated, changes occur in ion channels on the cell membrane, leading to alterations in membrane potential. These changes are then transmitted to the next neuron via synapses. The cell membrane contains numerous channels and receptors involved in calcium influx. The most notable are voltage-gated calcium channels (VDCCs). VDCCs are present on the surface of many excitatory cell membranes, such as neurons, cardiac muscle, skeletal muscle, and smooth muscle cells. They have many subtypes. Based on their biophysical and pharmacological properties, they can be classified into L, T, N, P / Q, and R types. L-type channels are mainly distributed in skeletal muscle and cardiac muscle cells and are closely related to excitation-contraction coupling; T-type channels are distributed in cardiac muscle and neurons; N, P / Q, and R-type channels are mainly distributed in neural tissue and are involved in neurotransmitter release. Figure 8 As shown, iNs-21d cells were used as experimental subjects. Cells were exposed to 25 mM KCl, 10 μM L-type calcium channel activator Bay K, 5 μM L-type calcium channel inhibitor Nisoldipine, and 1 μM capsaicin. The concentrations of agonists and inhibitors were selected to ensure that only the corresponding homologous receptors were activated. Changes in intracellular calcium activity induced by different agonists and inhibitors were detected using high-resolution confocal microscopy.

[0056] This invention also provides the application of the aforementioned neurons in the preparation of drugs for nerve injury or neurodegenerative diseases. The method of reprogramming endogenous satellite glial cells into neurons according to this invention can be used for basic research in the field of somatic cell reprogramming and for translational applications in clinical nerve injury repair cell replacement therapy.

[0057] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention extends to all other methods and applications having the same function.

Claims

1. A method for inducing the reprogramming of dorsal root ganglion satellite glial cells into neurons using small molecule compounds, characterized in that, The method is as follows: purified DRG-SGCs are passaged in a subculture medium, then induced in a neuron-inducing medium containing small molecule compounds, and finally cultured in a mature neuron-inducing medium to obtain neurons. The small molecule compounds are multiples of ISX-9, RO4929097, CHIR99021, VPA, RepSox, Forskolin, SC79, and VC.

2. The method for inducing dorsal root ganglion satellite glial cells to reprogram into neurons using small molecule compounds according to claim 1, characterized in that, The induced neuron culture medium also contains growth factors BDNF, GDNF, NT-3, NGFβ, neuron culture additive B27, and N2 additive.

3. The method for inducing dorsal root ganglion satellite glial cells to reprogram into neurons using small molecule compounds according to claim 2, characterized in that, The formulation of the neuron-inducing medium comprises: DMEM / F12 medium, Neurobasal medium, 2-5 μmol ISX9, 1-4 μmol RO4929097, 1-5 μmol CHIR99021, 100-10000 μmol VPA, 1-5 μmol RepSox, 2-10 μmol Forskolin, 0.01-2 μmol SC79, 100-400 μmol VC, 3-20 ng / mL BDNF, 3-20 ng / mL GDNF, 3-20 ng / mL NT-3, 3-20 ng / mL LNGFβ, 1-5% B27, 0.1-3% N2, 0.1-3% L-alanyl-glutamine solution, and 0.1-1% penicillin / streptomycin solution.

4. The method for inducing the reprogramming of dorsal root ganglion satellite glial cells into neurons using small molecule compounds according to claim 1, characterized in that, The formulation of the mature neuron culture medium includes: Neurobasal medium, 100-500 μmol VC, 10-50 ng / mL BDNF, 10-50 ng / mL GDNF, 10-50 ng / mL NT-3, 10-50 ng / mL LNGFβ, 1-5% B27, 0.1-3% N2, 0.1-3% L-alanyl-glutamine solution, and 0.1-1% penicillin / streptomycin solution.

5. The method for inducing the reprogramming of dorsal root ganglion satellite glial cells into neurons using small molecule compounds according to claim 1, characterized in that, When the cell density reaches 60-80% during passage culture, replace the inducing neuron culture medium for further induction.

6. The method for inducing the reprogramming of dorsal root ganglion satellite glial cells into neurons using small molecule compounds according to claim 1, characterized in that, Change the induced neuron culture medium every 1-2 days. After culturing for 6-15 days, change the culture medium to mature neuron culture medium and continue culturing for 3-8 days, changing the mature neuron culture medium every 1-2 days.

7. A neuron cultured according to any one of claims 1-6.

8. The use of the neuron according to claim 7 in the preparation of drugs for nerve injury or neurodegenerative diseases.

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