Method for inducing human pluripotent stem cells into otic-like neurons

Abstract

Provided is a method for inducing human pluripotent stem cells into otic-like neurons, comprising the following steps: S1: inducing human pluripotent stem cells into otic neural precursor cells; S2: purifying the otic neural precursor cells; and S3: expanding and differentiating the otic neural precursor cells into otic-like neurons. Provided is a new and efficient method for differentiating hPSCs into ONs. The method not only enables the induction of pluripotent stem cells into ONs using growth factors, but also optimizes the induction and expansion of ONs by means of the regulation of a signaling pathway and the modification of a culture medium. The technique can not only be applied to the treatment of diseases such as auditory neuropathy, but also provides new treatment strategies for various diseases such as sensory nervous system injuries.

Description

A method for inducing human pluripotent stem cells into otic neuron-like cells TECHNICAL FIELD

[0001] The present application belongs to the technical field of induced cell, and particularly relates to a method for inducing human pluripotent stem cells (hPSCs) into otic neuron-like cells (ONs). BACKGROUND

[0002] Auditory neuropathy is a common deafness disease, which seriously affects the learning, communication and working ability of patients. The common cochlear implant therapy is only effective for patients with outer hair cell damage in the cochlea, but there is no effective treatment for patients with cochlear auditory nerve damage. With the continuous development of biotechnology, stem cell treatment for deafness has become one of the research hotspots in otology in recent years.

[0003] Human pluripotent stem cells (hPSCs) are a kind of stem cells with potential application prospect, and the human pluripotent stem cells include human embryonic stem cells (hESCs) and induced pluripotent stem cells (hIPSs).

[0004] The hPSCs cells including hESCs and hIPSs can be differentiated into otic neurons (ONs) in a specific environment, and the transplantation of the otic neurons into the damaged auditory system can promote the self-repair and regeneration of the auditory loop to restore the auditory function. The current research mainly focuses on the method of using growth factors to induce hESCs to differentiate into target cell types, however, this method has the problems of low efficiency and insufficient accuracy, and therefore, a new method is needed to efficiently convert the pluripotent stem cells into otic neurons.

[0005] The spiral ganglion neurons (SGNs) of the inner ear work together with the hair cells to send sound pulses to the brainstem. The outer ear detects sound, and the hair cells are responsible for translating mechanical signals into chemical and electrical signals so that they can propagate downstream through oscillation. The SGN is a typical bipolar neuron, which is an adult cell lacking regenerative ability. The SGN is responsible for transmitting peripheral sound information to the cochlear nucleus of the brainstem and then to the auditory cortex. Hearing impairment associated with hair cell damage can be treated by cochlear implantation to restore hearing, however, the degeneration or loss of SGNs can lead to irreversible hearing loss because they lack regenerative ability.

[0006] The inventors of the present application induced hESCs into SGN-like cells using the action of growth factors FGF3 and FGF10 in 2012 (Nature, PMID: 22972191).

[0007] A study published by the same group in 2017 showed that the combined administration of WNT agonists and TGFβ / SMAD inhibitors significantly enhanced the development of neurospheres during the in vitro induction process, as well as the potential of growing auditory neural precursors and inducing inner ear neural precursor cells. By simulating the development and differentiation of SGNs in vivo, this study gradually induced and differentiated SGN-like cells without using FGF3 and FGF10 (Stem Cells Translational Medicine, PMID: 28186679), in which the separation and purification of very important inner ear neural precursor cells (ONPs) were purified using a flow cytometry sorting technique. SUMMARY

[0008] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a method for inducing human pluripotent stem cells (hPSCs) into auditory neuron-like cells (ONs) to solve the problems of low cell purity and insufficient induction accuracy in traditional induction methods.

[0009] To achieve the above-mentioned purpose, the first aspect of the present application provides a method for inducing human pluripotent stem cells into auditory neuron-like cells, comprising the following steps:

[0010] S1: inducing human pluripotent stem cells into auditory neural precursor cells;

[0011] S2: purification of auditory neural precursor cells;

[0012] S3: proliferation and differentiation of auditory neural precursor cells into auditory neuron-like cells;

[0013] In the induction process of S1, the following steps are taken:

[0014] The human pluripotent stem cells are plated, and when the cell density reaches about 60%, the induction is started:

[0015] Days 0-3: 50nM LDN193189 2HCl and 2μM IWP-2 are added to the BGM medium containing 20μM SB431542 for culture;

[0016] Days 4-5: LDN193189 2HCl is stopped, and 2μM IWP-2 is continued to be added to the BGM medium for culture;

[0017] Days 5-7: continue to use BGM medium for culture;

[0018] Days 8-13: continue culturing with BGM medium and add 50 ng / ml of human recombinant FGF3 and FGF10 into the medium;

[0019] Days 14-19: replace BGM medium with DFBN medium and add 50 ng / ml of human recombinant FGF3 and FGF10 into the medium.

[0020] According to the present application, the purification of S2 is to selectively digest the mixed cell population with collagenase IV at 37°C, and separate the auditory neural precursor cells from non-auditory neural precursor cells according to the different sensitivities of different cells to collagenase IV.

[0021] According to the present application, the proliferation and differentiation of S3 is to induce the differentiation of auditory neural precursor cells into auditory neuron-like cells before or after the proliferation stage, wherein:

[0022] In the proliferation stage, the auditory neural precursor cells are placed in DFNB medium supplemented with FGF3 and FGF10 for expansion for 2-3 months;

[0023] In the differentiation stage, the auditory neural precursor cells are continuously cultured with differentiation medium for 1-2 weeks, and the differentiation medium is replaced every other day;

[0024] The differentiation medium is DFNB medium added with 10 ng / mL BDNF, 10 ng / mL NT3, 10 ng / mL β-NGF, 10 ng / mL GDNF, 0.5 mM dibutyryl-cAMP, 200 μM L-ascorbic acid, and 1% penicillin / streptomycin.

[0025] According to the present application, the human pluripotent stem cells include human embryonic stem cells and induced pluripotent stem cells.

[0026] According to a preferred embodiment of the present application, the human embryonic stem cells include hESCs-H1, hESCs-H9, and IPS cells.

[0027] According to the present application, the formula of the BGM medium is as follows:

[0028] 2% (m / v) bovine serum albumin, 10 ng / ml Heregulin β-1, 10 μg / ml bovine transferrin, 200 ng / ml recombinant human IGF-I LR3, 50 μg / ml (+)-L-sodium ascorbate, 1% trace elements A, B, and C, 1% non-essential amino acids, 8 ng / ml bFGF, 1% penicillin / streptomycin, 0.18% β-mercaptoethanol, and 20 μM SB431542 in DMEM / F12.

[0029] In a second aspect, the application provides a method for evaluating the ability of a nerve cell to establish synaptic connection with a rat cochlear nucleus, comprising the following steps:

[0030] S1: co-culturing the nerve cell with the rat cochlear nucleus cell;

[0031] S2: performing immunofluorescence detection on the co-cultured cells;

[0032] In S2, the immunofluorescence detection is for detecting the expression of Synapsin 1, VGLUT, TrkB and TrkC in the co-cultured cells; if the detection result shows that the cochlear nucleus cell and the nerve cell both express the four proteins and the expression can be superimposed, it is indicated that synaptic connection is established between the two cells.

[0033] According to the application, the culture medium for the co-culture in S1 is DFNB culture medium, with the addition of 10 ng / ml IGF-1, 10 ng / ml bFGF and 10 ng / ml EGF.

[0034] According to the application, the nerve cell is previously genetically modified with a light channel.

[0035] According to a preferred embodiment of the application, the genetic modification of the light channel is the introduction of a green fluorescent protein gene.

[0036] The application has the following advantages:

[0037] 1. The application provides a novel and efficient method for differentiating hPSCs into ONs, which can not only induce pluripotent stem cells into ONs through growth factors, but also optimize the induction and propagation of ONs through the regulation of signal pathways and the adjustment of culture medium. This technology can be applied not only to the treatment of auditory neuropathy and other diseases, but also to the provision of new treatment strategies for sensory nervous system damage and other diseases.

[0038] 2、In order to effectively and as much as possible non-destructively purify ONP, the present application uses Collagenase IV to purify and separate ONP according to the different sensitivities of ONP and its surrounding cells. Compared with our own research in 2012, the development signal is more and more complex. Compared with the research of the same period in 2017, the present application proposes a more convenient and easy-to-implement solution based on the principle of early auditory development, and does not require more potential harmful steps to precursor cells such as FACS. The method of the present application has higher purity, and after purification, the purity can be as high as 80-90%. The method is more convenient and easy to implement, and the technical details are different (F3 and F10), and does not need to be sorted by flow cytometry, so that the activity of the cells can be better maintained. The results of cell qPCR, cell immunofluorescence staining and electrophysiological characteristic detection show that the differentiated cells have typical SGN-like molecular marker characteristics at the RNA level and protein level and electrophysiological characteristics.

[0039] 3、In order to prove the potential integration ability of the differentiated cells in the future in the process of transplantation with cochlear nucleus cells, or the ability to establish a neural loop, the present application co-cultures auditory neuron-like cells (ONs) integrated with light channels with cochlear nuclei at different levels of cells and brain slices, and the results show that the ONs differentiated by the present application have the ability to establish an auditory neural loop with cochlear nucleus cells at the cell level and the organ level. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is a cell induction flowchart of the present application.

[0041] Figure 2 is the immunofluorescence staining results of pluripotent markers (NANOG, OTC4, SOX2 and TRA-1-60) in pluripotent stem cell lines (H9-ChR2-eYFP cell lines), and the results of DAPI staining of cell nuclei with immunostaining images (scale: 100 μm).

[0042] Figure 3 shows cell morphology at different culture periods, wherein a is the cell before induction, b is the cell at the stage before the otic placode (PP stage), c is the auditory nerve precursor cell, d is the lower resolution auditory nerve cell, and e is the higher resolution auditory nerve cell.

[0043] Figure 4 shows the expression results of different genes such as Nanog and OCT4 at different stages by real-time fluorescence quantitative PCR analysis, wherein a and b are the expression comparison of Nanog and OCT4 at ES and PP stages, respectively, and c, d, e, f and g are the expression comparison of PAX2, EYA1, SIX1, GATA3 and FOXG1 at ES, PP and ONP stages, respectively (****, p<0.0001; ***, p<0.001; **, p<0.01; t test).

[0044] Figure 5 shows the results of identification of ONP marker from hESCs induction, wherein:

[0045] A is the results of chemical staining of ONP stage PAX6+ cells (left column), SIX1+ cells before purification (middle column), and SIX1+ cells after purification (right column) and the corresponding DAPI staining results;

[0046] B is the quantification of Pax6 and Six1 expression levels at ONP stage before purification using immunocytochemical staining;

[0047] C is the quantification of Six1 expression levels after ONP purification with collagenase IV;

[0048] D shows the immunostaining expression of neural progenitor cell markers Nestin (a), Pax8 (b), TuJ1 (c), FoxG1 (d), Pax2 (e) and Brn3A (f);

[0049] E shows the proportion of ONP markers FoxG1, Pax2, Brn3A, TuJ1, Nestin and Pax8 expression detected by immunostaining cells after 19 days of induction and subsequent purification.

[0050] Figure 6 is the results of immunocytochemical staining of ONs differentiated from ONPs, showing the expression results of auditory neuron markers TuJ1, tyrosine kinase receptors 2 / 3 (TrkB and TrkC), synaptophysin 1 (SYN) and VGLUT.

[0051] Figure 7 is a qPCR analysis quantifying the relative expression levels of N-cam, S-100, TrkB, TUBB3, VGLUT and TrkC at ONP and ON stages (****, p<0.0001, t-test).

[0052] Figure 8 is the evaluation of cochlear nucleus neurons (CNNs) and induced ONs (green) after 10 days of co-culture, showing the immunofluorescence staining of CNNs and induced ONs (green) after 10 days of co-culture and examination (scale bar: 25 pm); wherein a is synaptophysin SYN, b is vesicular glutamate transporter VGLUT, c is tyrosine kinase receptor TrkB, d is tyrosine kinase receptor TrkC; in addition, the nerve fibers of EGFP positive ONs are connected to the nerve fibers of CNNs that do not express fluorescent proteins.

[0053] Figure 9 shows the results of immunohistochemistry of co-cultures of ONs and brain sections, wherein A shows co-cultures between CN sections and induced ON spheres; B shows phase and fluorescence images of CN sections and induced ONs after specific co-culture duration (a-d, scale bar: 500 μm; e, scale bar: 100 μm); C shows immunofluorescent staining of brain sections, induced ONs after 7-10 days of co-culture, suggesting the presence of SYN and VGLUT in CN sections and ON spheres (green) in co-culture, showing structural connections at different levels of detail (scale bar: top line, 200 μm; bottom line, 25 μm).

[0054] Figure 10 shows various electrical properties generated on ONs by patch clamp electrophysiology examination, wherein a and b are action potentials, c is resting membrane potential, d is threshold potential, e is peak potential, f is half duration, g is time constant, and h is input resistance (n = 5).

[0055] Figure 11A shows the results of immunostaining of connections formed by ONs with cochlear nuclei, showing expression of synaptic proteins and VGLUT in co-cultured CN brain sections and ONs. In addition, nuclei are labeled with DAPI. Figure 11B shows that blue light stimulation activates cochlear nucleus neurons to cause INa + / IK + increase (n = 5, p < 0.05) (a-d); electrical signals of action potentials measured from CNN before and after exposure to light stimulation (e and f) (n = 4). DETAILED DESCRIPTION

[0056] The technical solutions of the present application will be described below in conjunction with specific embodiments, but those skilled in the art should understand that the embodiments described below are only used to illustrate the present application and should not be regarded as limiting the scope of the present application.

[0057] Compared with existing methods for converting pluripotent stem cells into auditory neurons, the present application is based on induction using combined administration of WNT agonists and TGFβ / SMAD inhibitors, and continues to use FGF3 and FGF10, a pair of growth factors that are very important for the development of the inner ear. In addition, in order to effectively and as much as possible non-destructively purify ONPs, the present application uses collagenase IV to purify and separate ONPs based on their different sensitivities to ONPs and surrounding cells. The results of cell qPCR, cell immunofluorescent staining, and electrical physiological property detection show that the differentiated cells have typical SGN-like molecular marker characteristics at the RNA level and protein level and electrical physiological properties.

[0058] In the present application, the differentiation stages are similar to the SGN-like cells induced from hESCs, which are divided into: non-neural ectoderm, ear placode and otic capsule, spiral ganglion neural precursor cells and SGN-like neurons. The pre-ear placode stage (PP stage), hESCs are induced to form pre-placodal cells, which show specific cell morphology, and the expression of pluripotency markers of hESCs is reduced, and the expression of pre-matrix markers is increased. Then, under the action of FGF3 and FGF10, the cells are induced into ONPs with auditory fate, and then the ONPs can be maintained and proliferated for more than 10 generations (60-90 days, the specific time depends on the batch). Whether there is a proliferation stage of ONPs or not, the ONPs can be differentiated into SGN-like neurons after 1-2 weeks, and show the expression of SGN markers and certain electrophysiological activity. In summary, the method of the present application is based on the principle of early auditory to differentiate into SGN-like cells, and the phenotype and electrophysiological activity are similar to those of SGN in vivo.

[0059] In addition, the isolation of high-purity cell populations is a key to stem cell biology research. Similarly, purifying neural stem cells (NSCs) is a primary task for cell therapy based on NSCs. The differentiation of NSCs can be affected by contamination or mixing with other cells, which can cause side effects and hinder the process of transformation. According to the research of Matsuoka et al., spiral ganglion precursor cells were induced from hPSCs and purified by fluorescence-activated cell sorting (FACS). However, during the process of cell sorting by FACS, cells are subjected to various stresses, including high and reduced pressure, acceleration, high speed, force, laser illumination, electric charge, and rapid temperature change. These stresses can change the structure and function of cells, trigger stress pathways, and affect cell metabolism. Any of these forces can cause changes in cell health, viability, and gene expression.

[0060] The present application proposes a more convenient and easy-to-implement solution based on the principle of early auditory development, which does not require more potentially harmful steps to the precursor cells like FACS. The induced cells in the ONP stage are a mixed cell population, and Six1+ cells account for a part of the cell population on day 19. Different cell populations are dissociated at different times and sequences using collagenase IV to identify and isolate Six1+ cells and other cell types in the ONP stage. Unlike other cell types, Six1+ cells can be kept in culture dishes for a long time without changing, and then separated by collagenase IV. Therefore, high-purity Six1+ cells or ONPs are produced. A large part of the differentiated ear neurons (ON) in cell culture also comes from Six1+ precursor cells. In summary, the direct induction method of ONP is based on the principle of early auditory development. It eliminates the need for flow cytometry sorting and other potentially harmful steps, and the ONPs and ONs produced thereby have high purity.

[0061] In addition, to demonstrate the future potential of these differentiated cells to integrate with cochlear nucleus cells during transplantation, or to establish neural circuitry, the present application co-cultured the auditory neuron-like cells (ONs) with the cochlear nucleus at different levels of cells and brain slices, and the results showed that the ONs we differentiated have the ability to establish auditory neural circuitry with cochlear nucleus cells. Specifically, to determine whether synapses and electrophysiological activity can be observed between hPSCs-derived SGN-like cells and neurons from the cochlear nucleus (CN), we used a cell co-culture method to establish an organotypic co-culture system of CN brain slice cells or tissues and organs and hPSCs-derived neurons to verify synapse formation and neuronal innervation. However, how to integrate stem cell-derived neurons into the natural nervous system remains to be further revealed in auditory research. To confirm the functional connection between the parts of the neural circuit, optogenetic technology can provide a solution to selectively stimulate a specific group of cells by pre-labeling cells with light-sensitive ion channels. However, the potential neural circuit between the exogenous neurons and the CN or CN neurons has not been evaluated using this method. The present application provides an alternative and more direct induction protocol that mimics the development of SNPs in vitro and genetically modifies the light channel in the auditory neuron-like cells produced by differentiation from the hESCs stage, so that the SGNs have the light channel ChR2. The present application uses differential selection of collagenase to purify ONP precursor cells. Immunocytochemistry, qPCR and electrophysiological data confirm the SGN-like characteristics of the differentiated neural cells. Immunostaining and optogenetic results confirm the establishment of functional synapses between neural networks and neural networks at the intracellular and organotypic levels after co-culture. The results of the present application show that SGN-like cells from hESCs have great clinical potential and can help to create a new method for treating diseases related to sensorineural hearing loss (SNHL).

[0062] The experimental materials used in the following examples, unless otherwise specified, can be obtained by conventional commercial channels.

[0063] The % and ‰ involved in the following examples, unless otherwise specified, are mass ratios.

[0064] Example 1, Construction of EGFP-hESCs cell line and optogenetic ESCs cell line

[0065] This embodiment constructs EGFP-hESCs cell line and optogenetic ESCs cell line with human embryonic stem cells hESCs-H1, hESCs-H9 and IPS cells respectively. The human embryonic stem cells hESCs-H1 and hESCs-H9 used in this embodiment are from the Stem Cell Bank of Chinese Academy of Sciences, and the IPS cells are commercial products. The construction methods of the three are the same, so only hESCs-H9 is described in detail below.

[0066] The formula of the expansion culture medium used in this embodiment is as follows:

[0067] 48.5mL Knockout TM DMEM medium, add 10mL KSR, 500μL PS, 500μL NEAA, 500μL GlutaMAX, 90μL β-Mercaptoethanol, 20μL 20ng / mL bFGF.

[0068] 1.1, Construction of hESCs-H9-EGFP cell line

[0069] 1. Select hESCs-H9 cells cultured without feeder layer and stably passaged for more than 10 generations, remove the culture medium, and digest the cells with Accutase at 37°C for 3-5min. After the cells are digested into single cells, centrifuge to collect the cells.

[0070] 2. Add 20μL Opti-MEM TM I to resuspend the cells, then add EGFP plasmid (Addgene company) and corresponding packaging plasmids pSPAX2 and pMD2G (3μg in total) to the cells, mix them evenly, and then transfer them to the electroporation cup.

[0071] 3. Set the relevant parameters according to the instructions of the electroporator and perform electroporation. The relevant parameters are set as follows:

[0072] In Decay mode, set the breakdown voltage to 150V, the breakdown duration to 10ms, the breakdown interval to 10ms, the driving voltage to 20V, the driving duration to 50ms, the driving interval to 50ms, and the total cycle number to 10.

[0073] 4. After the completion of the electroporation program, add 150μL Opti-MEM TM I to the cells, mix the cells gently by blowing, and transfer the cell suspension after electroporation to a T12.5 cell culture bottle coated with Matrigel (Corning company), and add 3mL of mTeSR1 culture medium (STEMCELL Technologies company).

[0074] 5. Remove supernatant and resuspend cells with 1 mL hESCs expansion medium; after 3-5 days, hESCs-H9-EGFP clones can be observed under green fluorescence channel of fluorescence microscope, and appropriate clones can be selected and passaged.

[0075] According to the above steps, H9 cells are replaced by H1 and IPS, and hESCs-H1-EGFP and hESCs-IPS-EGFP cell lines are constructed, respectively.

[0076] 1.2. Construction of hESCs-H9-ChR2-eYFP cell line

[0077] 1. Plasmid transformation:

[0078] Thaw competent DH5a on ice, and add 1 μg of hChR2(H134R)-eYFP (Addgene company, hereinafter referred to as ChR2-eYFP), pCMVR8.74 and psPAX2 plasmids (Addgene company) into 50 μL competent DH5a, respectively, and follow the conventional transformation procedure.

[0079] 2. Plasmid extraction: According to the instructions, use Omega endotoxin-free plasmid extraction kit to extract ChR2-eYFP lentivirus vector and psPAX2 and pCMVR8.74 packaging vector from the bacterial solution obtained in the previous step.

[0080] 3. Preparation of 293T cells before plasmid transfection: use T25 cell culture flask and 293T culture medium, and subculture 293T cells at a density of 5 × 105 cells / cm 2 the day before virus packaging.

[0081] 4. Preparation of transfection reagent: add 5 μL Lipofectamine 2000 to 250 μL Opti-MEM TM I, mix gently and reserve. Take 120 μL Opti-MEM TM I into a new EP tube, and add ChR2-eYFP, pCMVR8.74 and psPAX2 plasmids to different EP tubes in a mass ratio of 5:1.5:3, respectively. Gently blow with a pipette for about 10 times, mix the plasmids with Opti-MEM TM I, and then mix with 120 μL Opti-MEM TM I containing Lipofectamine 2000, and stand at room temperature for 20 min.

[0082] 5. Transfect 293T cells with ChR2-eYFP, pCMVR8.74 and psPAX2 plasmids.

[0083] 6. Collect virus, prepare infection: Collect virus twice, mix ChR2-eYFP, pCMVR8.74 and psPAX2 three kinds of collected lentivirus containing medium, filter with 0.45 μm filter membrane, and reserve for use.

[0084] 7. Infection of virus: infect hESCs-H9 cells with collected lentivirus twice, then discard the infection medium, add new mTeSRl medium to the culture bottle to restore the state of the cells, and start the second infection 16 h later to increase the infection efficiency.

[0085] 8. ChR2-eYFP expression: 3-5 days after infection of virus, ChR2-eYFP expressing hESCs-H9 can be observed under fluorescence microscope, and the cells are digested and passaged with Accutase at 37°C. The hESCs expansion medium is used for culture.

[0086] 9. Selection of clones: 3-5 days later, ChR2-eYFP expressing hESCs form clones under the green fluorescence channel of fluorescence microscope, and the hESCs-ChR2-eYFP are expanded and passaged using mTeSRl medium.

[0087] 10. Selection of hESCs-H9-ChR2-eYFP stably passaged for more than 10 generations, and immunofluorescence identification of stem cell markers is performed, and the identification results are shown in Figure 2. The pluripotency markers NANOG, OCT4, SOX2 and TRA-1-60 can be expressed in all cells, indicating that the cells transfected with plasmid still maintain the potential for multi-directional differentiation.

[0088] According to the above steps, H9 cells are replaced by H1 and IPS, and hESCs-H1-ChR2-eYFP cell line and hESCs-IPS-ChR2-eYFP cell line are respectively constructed.

[0089] Example 2, Induction of human embryonic stem cells (hESC) into auditory neuron-like cells (ON)

[0090] In this example, hESCs-H9-EGFP, hESCs-H1-EGFP, hESCs-IPS-EGFP, hESCs-H9-ChR2-eYFP, hESCs-H1-ChR2-eYFP and hESCs-IPS-ChR2-eYFP cell lines obtained in Example 1 are taken as examples, and are induced to differentiate into auditory neuron-like cells. Since the induction and differentiation methods of the six kinds of cells are the same, only hESCs-H9-EGFP is taken as an example for detailed description. The flow chart of induction is shown in Figure 1.

[0091] The formula of BGM medium used in this example is as follows:

[0092] DMEM / F12 (GIBCO) supplemented with 2% (m / v) bovine serum albumin (Fraction v, without fatty acids, without endotoxins (BSA), Millipore), 10 ng / ml of Heregulin β-1 (Peprotech), 10 μg / ml of bovine transferrin (Holo form, Invitrogen), 200 ng / ml of animal component-free recombinant human IGF-I LR3 (Peprotech), 50 μg / ml of (+)-L-sodium ascorbate (Invitrogen), 1% of trace elements A / B / C (Cellgro), 1% of non-essential amino acids (GIBCO), 8 ng / ml of bFGF (Peprotech), 1% of penicillin / streptomycin (fdbioscience), β-mercaptoethanol (Invitrogen) and 20 μM of SB431542 (Selleck).

[0093] 2.1. Induction of hESCs into auditory neural progenitor cells (ONP)

[0094] The induction of hESCs into the pre-placodal stage (PP stage) was initiated 72 hours after the single cell plating of hESCs. The induction medium of hESCs was switched from mTeSR1 to the basal growth medium (BGM medium). The cell morphology at the start of induction is shown in Figure 3a.

[0095] hPSCs were plated and induction was initiated when the cell density reached about 60%, as follows:

[0096] Days 0-3: The cells were cultured in BGM medium (basal medium) containing 20 μM SB431542, with the addition of small molecule inhibitors LDN193189 2HC1 (50 nM) and IWP-2 (2 μM).

[0097] Days 4-5: The use of LDN193189 2HC1 was discontinued, and the cells were cultured in basal medium with the addition of IWP-2 (2 μM).

[0098] Days 5-7: The cells were cultured in basal medium. After 7 days of induction, the cells were induced into pre-placodal cells (PPs), and the cell status at this time is shown in Figure 3b. QPCR detection of the cells induced for 7 days showed a decrease in the expression of pluripotency genes NANOG and OCT4, as shown in Figures 4a and 4b, which demonstrated that the cells had lost pluripotency, marking the successful initiation of differentiation.

[0099] Days 8-13: Culture with basal medium with 50 ng / ml of human recombinant FGF3 and FGF10. The hESCs are induced into ONPs with auditory fate under the influence of FGF3 and FGF10, and the ONPs can be maintained and expanded for more than 10 passages, usually 60-90 days. The cells are passaged when the density is too high, usually at about 50% confluence. Early cell aggregates of small and dark ONPs can be seen at about 13 days, which appear crowded and clumped.

[0100] Days 14-19: The basal medium is replaced with DFBN medium with 50 ng / ml of human recombinant FGF3 and FGF10. The cells enter the stage of otic neural progenitors (ONPs) at this time, and the cell morphology is shown in Figure 3c. QPCR analysis of cells induced for 7 and 19 days is shown in Figures 4c-g. PAX2 and EYA1 are markers of pre-placodal cells, which are highly expressed at 7 days and decreased at 19 days. The expression of inner ear related genes SIX1, GATA3 and FOXG1 gradually increases. These results indicate that the cells are differentiating towards an inner ear progenitor fate.

[0101] 2.2, Purification of ONPs from hESCs

[0102] After 19 days of induction of hESCs, the mixed cell population is selectively digested with collagenase IV at 1 mg / ml at 37°C. The putative ONP population is separated from non-ONPs based on the differential sensitivity of different cells to collagenase IV.

[0103] Add 1x volume of 0.1% collagenase IV (Sigma) to the flask and incubate at 37°C for 30 min, observing every 10 min. At this time, non-target cells (e.g. interstitial-like cells) are digested first, and more non-target cells are loosened and fall off after gently tapping or shaking the flask. The non-target cell population is removed from the flask, and the ONPs are retained in the flask for subsequent passaging and differentiation. The ONPs at the bottom of the flask are removed, and the supernatant is removed. Add trypsin at 37°C, and just cover the cells. Place the flask in a 37°C CO2 incubator for 3-5 min. When the cells are digested into single cells, add 3x volume of mTesRl medium to stop the digestion. Collect the cells into a centrifuge tube and centrifuge at 180g for 5 min. Remove the supernatant and resuspend in basal medium at 60-80,000 cells / cm 2The density of the cells was passaged into Matrigel-coated culture flasks. The flasks were placed in a 37°C, 5% CO2 incubator.

[0104] After purification, the proportion of the auditory nerve precursor cells was greatly increased. SIX1 is a marker of ONPs, and the immunofluorescence identification results are shown in Figures 5a, 5b, and 5c. It can be seen that the proportion of cells expressing SIX1 has increased compared to before purification, proving that the proportion of ONPs has increased after purification. The immunofluorescence identification results shown in Figures 5d and 5e show that the purified ONPs can express the markers Nestin, PAX8, TUJ1, FOXG1, PAX2, and BRN3A of auditory nerve precursor cells, confirming the differentiation state of the cells at this time.

[0105] 2.3, Proliferation and differentiation of ONPs into ONs

[0106] After the ONPs are purified, they are induced to differentiate into ONs before or after the proliferation stage. In the proliferation stage, the ONP cells are expanded in DFNB medium supplemented with FGF3 and FGF10 for 2-3 months. In the proliferation stage, the differentiation medium (DM medium) used includes DFNB medium and is supplemented with 10 ng / mL BDNF, 10 ng / mL NT3, 10 ng / mL β-NGF, 10 ng / mL GDNF, 0.5 mM dibutyryl-cAMP, 200 μM L-ascorbic acid, and 1% penicillin / streptomycin.

[0107] The differentiation of ONs requires a continuous culture period of 1-2 weeks, with the differentiation medium being replaced every two days. Figure 3d shows the morphology of the auditory nerve cells (ONs) obtained after 45 days of induction, and Figure 3e is a high-resolution photograph of the ONs in Figure 3d. Cell immunofluorescence identification was performed on the induced and differentiated ONs, and the results are shown in Figure 6. The induced ONs can express the neural cell marker TUJ1, the auditory nerve markers TrKB and TrKC, the synaptic protein SYN, and the glutamatergic nerve marker VGLUT, which indicate from the perspective of marker molecules that the cells have differentiated into mature auditory neurons.

[0108] Real-time quantitative PCR was performed on the ONPs and ONs to detect the expression of nerve-related genes, and the results are shown in Figure 7. The expression of the auditory nerve-related genes N-cam, TUBB3, TRKB, TRKC, and VGLUT in the ONs stage was significantly higher than in the ONPs stage, indicating that the cell differentiation fate has changed from a neural precursor to a mature neuron.

[0109] Through the above steps, six hESC cells were induced to differentiate into ON cells.

[0110] Example 3, Co-culture of auditory nerve cells and cochlear nucleus cells

[0111] 3.1, Harvesting of cochlear nucleus cells

[0112] Harvest cochlear nucleus from 12-day-old Sprague-Dawley rats and divide into small tissue pieces. Transfer the cochlear nucleus tissue pieces to a clean 15 mL centrifuge tube and centrifuge at 200 g for 2 min at room temperature. Discard the supernatant and add 2 mL of Accutase digestion solution pre-warmed at 37 °C. Digest at 37 °C. After a 20 min digestion period, repeatedly pipette the tissue pieces to dislodge as many single cells as possible from the cochlear nucleus tissue pieces. Centrifuge the cell suspension at 100 g for 3 min at room temperature and collect the cochlear nucleus cells. Resuspend the cochlear nucleus cells in co-culture medium and drop onto Matrigel-coated Cover slips cell slides and place in a 37 °C incubator. Allow the primary cochlear nucleus cells to adhere for the first 2 days and then replace or add fresh medium as needed.

[0113] The co-culture medium is DFNB medium supplemented with 10 ng / ml IGF-1, 10 ng / ml bFGF (Peprotech), and 10 ng / ml EGF (Peprotech).

[0114] 3.2, Co-culturing

[0115] After observing sufficient cochlear nucleus cells under the microscope, drop the suspension of Example 2 expressing EGFP auditory nerve cells (ON) onto the cell slides and allow the cells to adhere for the first 2 days. Observe daily and replace the medium every other day.

[0116] After 2-4 weeks of co-culturing, observe the connection of neurites between the foreign nerve cells and the rat cochlear nucleus cells under the microscope. This co-culture system is used for subsequent immunofluorescence detection or other physiological detection evaluation.

[0117] The co-culture medium is DFNB medium supplemented with 10 ng / ml IGF-1, 10 ng / ml bFGF (Peprotech), and 10 ng / ml EGF (Peprotech).

[0118] 3.3, Immunofluorescence identification

[0119] The cell slides were taken out of the culture medium and washed with PBS for three times to remove the unattached cells or dead cells. The slides were fixed in 4% paraformaldehyde solution at room temperature for 15 minutes, and then used for cell immunofluorescence detection. The first antibody label was selected as Synapsin 1, that is, a vesicle protein, to observe whether the cochlear nucleus cells and the exogenous auditory nerve cells in the co-culture can express Synapsin 1, and whether the Synapsin 1 produced by both parties can be superimposed.

[0120] The identification results are shown in FIG. 8. It is found in the experiment that the co-cultured ONs and cochlear nucleus cells can express the synaptic protein marker Synapsin 1, which indicates that synaptic connections are indeed formed between the two cells. Synapsin 1 is a presynaptic protein, and its expression is usually closely related to the formation and function of synapses. Therefore, this finding further confirms that there is not only structural contact between ONs and cochlear nucleus cells, but also functional connection. The neurons expressing EGFP form structural connections with cochlear nucleus cells that do not exhibit green fluorescence. The nerve fibers of the neurons overlap with the neurites of the cochlear nucleus cells, which is a typical morphological feature of synapse formation. This overlap increases the possibility of establishing synaptic connections between the two cells, thereby facilitating the transmission of auditory information. VGLUT immunostaining positive indicates that the transport mode of this synapse is based on glutamate transport vesicles. VGLUT is a kind of glutamate transporter, which is responsible for releasing glutamate from presynaptic neurons to the synaptic cleft, and is a key molecule for excitatory synaptic transmission. Therefore, the expression of VGLUT further proves that functional synaptic connections are formed between ONs and cochlear nucleus cells. TrkB and TrkC expression is also detected in ONs with EGFP fluorescent labels. TrkB and TrkC are receptors for nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), which play an important role in the growth, differentiation and synaptic plasticity of neurons. Therefore, their expression may promote synaptic connections and neural pathway regeneration between ONs and cochlear nucleus cells. In summary, the induced ONs-EGFP and cochlear nucleus cells form synaptic connections after co-culturing for a period of time.

[0121] Example 4, auditory nerve cells co-cultured with rat brain slices

[0122] This example establishes a co-culture system composed of ON spheres and rat brainstem slices to study their interaction.

[0123] The SD rat brain was taken and immersed in artificial cerebrospinal fluid (ACSF) cutting solution. The ACSF was prepared from ice water mixed with continuously supplied 95% oxygen and 5% carbon dioxide gas. After the brain tissue was quickly dissected, the extra brain regions were cut off and the brainstem containing the cochlear nucleus was pasted on the cutting stage with adhesive. The 200 μm thick brain slices were cut using a Leica VT 1200s slicer and placed on the insert coated with Matrigel coating using a brush. The next day, the ON spheres were transplanted into the co-culture medium near the AVCN region of the brain slice.

[0124] The co-culture medium was composed of DMEM high glucose medium (GIBCO) supplemented with 5% horse serum (Beyotime Biotechnology), 10% fetal bovine serum (GIBCO), 1% penicillin / streptomycin / amphotericin B (Beyotime Biotechnology), 25 mM HEPES (GIBCO), 1% N2 supplement, 2% B27 supplement and 10 ng / ml β-NGF (Peprotech). The co-culture medium was changed daily.

[0125] The co-cultured brain slice and ONs-EGFP were tracked as shown in FIGS. 9A, B. The ONs-EGFP neural spheres were still round in shape when they were just transplanted, indicating that the cells maintained a good aggregation state in the initial stage. Over time, the neural spheres expressing green fluorescent label began to migrate on the semi-permeable membrane and extend neural fibers to the cochlear nucleus region of the brain slice (without green fluorescent label). Through continuous observation at different time points, the dynamic process of neural sphere migration and neural fiber projection can be seen.

[0126] The immunofluorescence evaluation was performed after 10 days of co-culture. The results of the immunofluorescence detection are shown in FIG. 9C. Different colored arrows indicate the location of the connection between hESCs-ONs-EGFP and the cochlear nucleus cells of the brain slice, showing that the neural fibers of the cells expressing EGFP (ONs) and the cells not expressing EGFP (CN) are structurally connected, and both cells express the neuronal marker TUJ1 and the glutamatergic neuronal marker VGLUT1. The expression of TUJ1 proves that these cells indeed have the characteristics of neurons. The expression of VGLUT1 further confirms that the synaptic connection between the two cells is functional and can participate in glutamatergic excitatory synaptic transmission.

[0127] Figure 11A further demonstrates the results of immunofluorescence detection of co-cultured ONs-EGFP and cochlear nucleus cells of brain slices. Neurites expressing Synapsin 1 and VGLUT1 extend from the exogenous ONs positive for EGFP to multisynaptic cells in the cochlear nucleus tissue. The expression of these two proteins further confirms that structural and functional connections are formed between the ONs and the cochlear nucleus cells. This indicates that the neural network of ONs is able to establish extensive connections with the cochlear nucleus on organotypic slices, providing a structural basis for the transmission of auditory signals.

[0128] Example 5, Electrophysiological evaluation

[0129] During recording, the glass slice containing the induced ONs was transferred to the electrophysiological recording chamber, and the temperature of the recording solution was maintained at 28±2°C. Neurons with smooth surfaces and clear outlines were selected for whole-cell recording under a microscope. The recording solution used was ACSF, continuously perfused with mixed gas.

[0130] First, spontaneous excitatory postsynaptic currents (sEPSCs) were recorded using cesium methanesulfonate electrode internal solution, with an electrode impedance of 4-7 MΩ. After membrane rupture, cells with a pathway resistance (Ra) of less than 20 MΩ were statistically recorded. During sEPSC recording, the holding potential was limited to -60 mV.

[0131] Second, when recording neuron action potentials (APs), potassium gluconate electrode internal solution was used, with an electrode impedance of 4-7 MΩ. The cell was voltage clamped, and when the resistance exceeded 1 GΩ, the membrane was ruptured, and then the current clamp mode was switched to record APs. The protocol was as follows: the starting injection current was -50 pA, the recording time was 500 ms, and the sweep was 10 times with an increment of 10 pA, and the action potential discharge was observed. The resting membrane potential was represented by the voltage at the current baseline during AP recording; the action potential threshold was the minimum voltage at the onset of the action potential; the input resistance and time constant (τ) were fitted using Clampfit software; the action potential amplitude was measured from the threshold to the peak; and the action potential duration was the time course of the half-peak amplitude.

[0132] Third, potassium (K + ) currents were recorded using potassium gluconate electrode internal solution, with an electrode impedance of about 4-7 MΩ. In voltage clamping mode, the cell was patched, and the potassium current after membrane rupture was recorded. The protocol was set as follows: the starting injection voltage was 0 mV, the recording time was 500 ms, and the sweep was 10 times with an increment of 10 mV, sweeping to +50 mV to observe potassium ion current emission. + When recording sodium (Na

[0133] All data were recorded using a MultiClamp 700B amplifier with a sampling frequency of 5 KHz. The filter frequency was set to 1 KHz to filter the interfering current. P / N leakage subtraction, leak current removal and current analysis were performed using Clampfit 11.

[0134] The solutions were as follows:

[0135] Cesium methanesulfonate internal solution preparation: Cesium methanesulfonate 125 mM, CsCl 5 mM, Hepes 10 mM, EGTA 0.2 mM, MgCl2 1 mM, Mg-ATP 4 mM, Na-GTP 0.3 mM, Creatine phosphate disodium hydrate 10 mM, QX314 5 mM; pH 7.30, osmolarity 280 mOsm / kg.

[0136] Potassium gluconate internal solution preparation: k-gluconate 120 mM, KCl 6 mM, Hepes 10 mM, EGTA 0.1 mM, Mg-ATP 4 mM, Na-GTP 0.3 mM; pH 7.30, osmolarity 280 mOsm / kg.

[0137] Cesium methanesulfonate internal solution preparation: Cesium methanesulfonate 125 mM, CsCl 5 mM, Hepes 10 mM, EGTA 0.2 mM, MgCl2 1 mM, Mg-ATP 4 mM, Na-GTP 0.3 mM, Creatine phosphate disodium hydrate 10 mM, QX314 5 mM; pH 7.30, osmolarity 280 mOsm / kg.

[0138] 1. In vitro detection of the electrophysiological characteristics of ONs

[0139] The results of the electrophysiological detection of ONs are shown in Figure 10. ONs generated action potentials similar to those of spiral ganglion neurons (SGNs) in vivo, as shown in Figures 10a-b, which indicates that these cells have acquired the basic electrophysiological characteristics of neurons. The parameters of the resting membrane potential (c), threshold potential (d), peak potential (e), half-duration (f), time constant (g) and input resistance (h) of ONs further confirm that the embryonic stem cell-derived ONs have similar electrophysiological characteristics to SGNs.

[0140] 2. Optogenetic detection of the electrophysiological characteristics of rat cochlear nucleus cells co-cultured with ONs-ChR2-eYFP

[0141] The co-culture method is as described in Example 4. The tissue co-cultured for 10 days was taken for detection, and the tissue was irradiated and activated with 473 nm blue light. The potential characteristics of the rat cochlear nucleus cells on the semi-permeable membrane before and after blue light irradiation were recorded. The experimental results are shown in Figure 11B. After 473 nm blue light stimulation, the I Na+ / IK+ All of them were significantly increased (p<0.05), as shown in Figure 11B a-d. No action potentials were generated without light stimulation; action potentials released by downstream cochlear nucleus neurons after light stimulation on upstream ONs were significantly increased, as shown in Figure 11B e-f. These results indicate that the synaptic connections between ONs-Chr2-eYFP and brain slice cochlear nucleus cells are physiologically functional and can effectively transmit neural signals.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that the technical solutions described in the foregoing embodiments can be modified, or some or all of the technical features can be replaced by equivalents, without departing from the concept, spirit and scope of the present application; and these modifications or replacements all fall within the scope of the present application.

Claims

1. A method of inducing a human pluripotent stem cell into an auditory neuron-like cell, characterized by The method comprises the following steps: S1: inducing human pluripotent stem cells into auditory neural precursor cells; S2: purification of the auditory neural precursor cells; S3: proliferation and differentiation of the auditory neural precursor cells into auditory neuron-like cells; The induction process of S1 is as follows: The human pluripotent stem cells are plated, and when the cell density reaches about 60%, the induction is started: Days 0-3: the cells are cultured in BGM medium containing 20 μM SB431542, 50 nM LDN193189 2HCl and 2 μM IWP-2; Days 4-5: the use of LDN193189 2HCl is stopped, and the cells are continuously cultured in BGM medium containing 2 μM IWP-2; Days 5-7: the cells are continuously cultured in BGM medium; Days 8-13: the cells are continuously cultured in BGM medium, and 50 ng / ml human recombinant FGF3 and FGF10 are added into the medium; Days 14-19: the BGM medium is replaced by DFBN medium, and 50 ng / ml human recombinant FGF3 and FGF10 are added into the medium.

2. The method of claim 1, wherein, The purification of S2 is that the mixed cell population is selectively digested by collagenase IV at 37°C, and the auditory neural precursor cells are separated from non-auditory neural precursor cells according to the different sensitivities of different cells to collagenase IV.

3. The method of claim 1, wherein, The proliferation and differentiation of S3 are that the auditory neural precursor cells are induced to differentiate into auditory neuron-like cells before or after the proliferation stage, wherein: In the proliferation stage, the auditory neural precursor cells are expanded in DFNB medium supplemented with FGF3 and FGF10 for 2-3 months; In the differentiation stage, the auditory neural precursor cells are continuously cultured in a differentiation medium for 1-2 weeks, and the differentiation medium is replaced every other day; The differentiation medium is that 10 ng / mL BDNF, 10 ng / mL NT3, 10 ng / mL β-NGF, 10 ng / mL GDNF, 0.5 mM dibutyryl-cAMP, 200 μM L-ascorbic acid and 1% penicillin / streptomycin are added into DFNB medium.

4. The method of claim 1, wherein, The human pluripotent stem cells include human embryonic stem cells and induced pluripotent stem cells.

5. The method of claim 4, wherein, The human embryonic stem cells include hESCs-H1, hESCs-H9 and IPS cells.

6. The method of claim 1, wherein, The formula of the BGM medium is as follows: 2% (m / v) bovine serum albumin, 10 ng / ml Heregulin β-1, 10 μg / ml bovine transferrin, 200 ng / ml recombinant human IGF-I LR3, 50 μg / ml (+)-L-sodium ascorbate, 1% trace elements A, B and C, 1% non-essential amino acids, 8 ng / ml bFGF, 1% penicillin / streptomycin, 0.18% β-mercaptoethanol and 20 μM SB431542 are added into DMEM / F12.

7. A method for evaluating the ability of a neural cell to establish synaptic connections with a rat cochlear nucleus, characterized by The method comprises the following steps: S1: co-culturing neural cells with rat cochlear nucleus cells; S2: immunofluorescence detection of the co-cultured cells; The immunofluorescence detection in S2 is to detect the expression of Synapsin 1, VGLUT, TrkB and TrkC in the co-cultured cells; if the detection result is that cochlear nucleus cells and nerve cells both express the four proteins and the expression can be superimposed, it indicates that the synapse connection between the two kinds of cells is established.

8. The evaluation method according to claim 7, characterized in that The co-cultured medium in S1 is DFNB medium, and 10 ng / ml of IGF-1, 10 ng / ml of bFGF and 10 ng / ml of EGF are added.

9. The evaluation method according to claim 7, characterized in that The nerve cells are previously genetically modified with a light channel.

10. The evaluation method according to claim 7, characterized in that The genetic modification of the light channel is to introduce a green fluorescent protein gene.

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