Method for constructing inner ear precursor cells and inner ear organoids and application of inner ear precursor cells and inner ear organoids

Abstract

The invention relates to a method for constructing inner ear precursor cells and inner ear organoids and application, in particular to a preparation method of the inner ear precursor cells, which comprises the following steps: S1, in the presence of a first culture medium, culturing human pluripotent stem cells by contacting with an extracellular matrix, the first culture medium is a basic culture medium containing BMP, FGF and TGF beta signal transduction inhibitors; and S2, culturing the cells obtained in the S1 in a first culture medium containing a BMP inhibitor and a Wnt signal transduction inhibitor, and differentiating to obtain the inner ear precursor cells. The inner ear precursor cells obtained by the method provided by the invention can still be differentiated into mature inner ear neuron organoid after being preserved at low temperature, and the difference between batches is small.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to a method and application of constructing inner ear precursor cells and inner ear organoids. Background Art

[0002] Deafness is the most common peripheral sensory impairment. Mammalian spiral ganglion neurons (SGNs), located within the modiolus of the cochlea, are bipolar neurons and the sole afferent neurons of the auditory system. These neurons establish synaptic connections with cochlear hair cells and play a crucial role in sound perception by transmitting auditory signals from the hair cells to the cochlear nucleus in the brain. During development, SGNs originate from the otic placode, an ectodermal structure of the preplaque ectoderm (PPE) that ultimately forms the inner ear. Induced by transcription factors such as SOX2, EYA1, PAX2, NEUROD1, and NEUROG1, the otic placode invaginates to form the otic vesicle, which then undergoes stratification and differentiation to form SGNs. Factors such as ototoxic drugs, aging, and noise exposure can lead to SGN damage and degeneration, which not only interferes with sound transmission and causes difficulties with hearing clarity, speech comprehension, and sound localization, but also leads to permanent septal hyperplasia (SNHL) due to the inability of SGNs to regenerate. In addition, cochlear implantation is a common treatment for severe SNHL. It works by bypassing damaged hair cells and directly stimulating the remaining SGNs. Its efficacy depends on the presence of functional SGNs. However, the biology and regeneration of SGNs currently face some major challenges. First, because SGNs are located deep in the cochlea and are small in size and complex in structure, it is particularly difficult to obtain SGNs. In addition, human cochlear tissue for research is scarce and can usually only be obtained after death or during surgical procedures such as cochlear implantation. This limitation affects the quantity and quality of samples, thereby limiting direct research on human SGNs. Currently, SGN research models are mainly experimental animals, but animal models cannot fully replicate human auditory physiology or pathology. Therefore, it is very necessary to develop an in vitro model with human SGNs.

[0003] Introducing exogenous stem cells or inducing neighboring supporting cells to differentiate into glial cells in animal models can partially compensate for damaged SGNs. Human iPSC-derived inner ear lineages are particularly noteworthy because they recapitulate the development of inner ear cells, including inner ear neurons, in vitro and are widely used to simulate hearing loss in vitro. However, current methods for obtaining ear-derived neurons using hPSCs differentiation are not yet suitable for translational applications because they are heavily dependent on the initial cell population and cannot be reliably replicated between different research groups. The problem of inconsistent performance between batches may have led to the clinical failure of neural transplantation in the past. In addition, obtaining mature ear-derived neurons from hPSCs is a lengthy process, which may result in significant heterogeneity in the obtained inner ear neurons. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the first purpose of the present invention is to develop a method for differentiating inner ear precursor cells from human pluripotent stem cells (hPSCs) that can be stored at low temperatures.

[0005] A second objective of the present invention is to provide a method for differentiating inner ear neuronal organoids from human pluripotent stem cells (hPSCs). This preparation method is simple and can be performed in a single, continuous, self-assembly-based differentiation process from pluripotent stem cells into inner ear organoids. Alternatively, it can be performed in two steps: the first step involves stable induction of differentiation to produce inner ear precursor cells (inner ear progenitor cells), which can be cryopreserved for later use. The second step involves further differentiation of these precursor cells into inner ear organoids, thus splitting the continuous induction of differentiation to produce three-dimensional neurons into two discrete steps.

[0006] To achieve the above object, the present invention adopts the following technical solutions: In one aspect, the present invention provides a method for preparing inner ear precursor cells from pluripotent stem cells, the method comprising the following steps: S1: culturing human pluripotent stem cells in contact with an extracellular matrix in the presence of a first culture medium, wherein the first culture medium is a basal culture medium containing bone morphogenetic factor (BMP), fibroblast growth factor (FGF), and a transforming growth factor β (TGFβ) signaling inhibitor; S2: Culturing the cells obtained in S1 in a first culture medium containing a BMP inhibitor and a Wnt signaling inhibitor to differentiate and obtain the inner ear precursor cells.

[0007] Preferably, the BMP is selected from one or more of BMP2, BMP4 and BMP7.

[0008] Preferably, the FGF is bFGF.

[0009] Preferably, the TGFβ signaling inhibitor is selected from one or more of SB-431542, A-83-01, LY2157299 and RepSox.

[0010] Preferably, the BMP inhibitor is selected from one or more of DMH1, LDN-212854, doxorubicin, Nogin, LDN-193189, activin binding protein, tenascin and gramlin.

[0011] Preferably, the Wnt signaling inhibitor is selected from one or more of XAV939, Endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, triptolide, KYA1797K, JW55, JW67, JW74, cardiomyogenin 1, NLS-StAx-h, TAK715, PNU74654, iCRT3, iCRT14, WIF-1, DKK1, isoquercitrin, lanatoside C, dendrobium phenol, RCM-1, WIKI4, IQ-1, PRI-724 and tigatrabectan.

[0012] Preferably, the working concentration of the BMP is 5-15 ng / mL.

[0013] Preferably, the working concentration of FGF is 5-15 ng / mL.

[0014] Preferably, the working concentration of the TGFβ signaling inhibitor is 0.5-2 μM.

[0015] Preferably, the working concentration of the BMP inhibitor is 0.01-0.5 μM.

[0016] Preferably, the working concentration of the Wnt signaling inhibitor is 1-5 μM.

[0017] Preferably, the method comprises performing step S0 before step S1, and step S0 comprises: culturing a single-cell suspension of human pluripotent stem cells in contact with an extracellular matrix in the presence of a second culture medium, and then subjecting the cultured cells to step S1.

[0018] Preferably, the second culture medium is a basal culture medium containing a ROCK inhibitor, and the working concentration of the ROCK inhibitor is 5-15 μM.

[0019] Preferably, the culturing time in step S1 is 1 to 3 days.

[0020] Preferably, the culturing time in step S2 is 4 to 8 days.

[0021] Preferably, the culturing time in step S0 is 3 to 5 days.

[0022] In one aspect, the present invention provides inner ear precursor cells prepared according to any of the methods described above.

[0023] In one aspect, the present invention provides a method for preparing inner ear organoids from inner ear precursor cells, comprising culturing inner ear precursor cells obtained by any of the methods described above or inducing the differentiation of the inner ear precursor cells described above into inner ear organoids.

[0024] In one aspect, the present invention provides a method for discontinuously differentiating pluripotent stem cells into inner ear organoids, the method comprising the following steps: S3: thawing the frozen inner ear precursor cells obtained by any of the above methods or the inner ear precursor cells described above; S4: suspending the inner ear progenitor cells revived in step S3 and culturing them in a third culture medium for 4 to 8 days, wherein the third culture medium is a basal culture medium containing a Wnt agonist, FGF, IGF-1, and a ROCK inhibitor; S5: culturing the cells after suspension culture in step S4 for 5 to 9 days in a fourth culture medium, wherein the fourth culture medium is a basal culture medium containing ATRA, SHH agonist, FGF, IGF-1, and EGF; S6: Culturing the cells cultured in step S5 in a fifth culture medium to obtain the inner ear organoid, wherein the fifth culture medium is a basal culture medium containing db-cAMP, BDNF, NT3, IGF-1, and a ROCK inhibitor.

[0025] Preferably, the Wnt agonist is a GSK3 inhibitor, and the GSK3 inhibitor includes one or more of SB216763, GSK3β inhibitor VII, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-azacamparolone, indirubin, and CHIR99021.

[0026] Preferably, the ROCK inhibitor includes one or more of AR-12286, Y-27632 and SNJ-1656.

[0027] Preferably, the SHH agonist comprises one or more of SHH, SHH C25II, SAG, SAG 21K, Hh-Ag1.5, 20a-hydroxycholesterol, and purmorphamine.

[0028] Preferably, the working concentration of the Wnt agonist is 1-5 μM.

[0029] Preferably, the working concentration of FGF is 5-15 ng / mL.

[0030] Preferably, the working concentration of IGF-1 is 30-60 ng / mL.

[0031] Preferably, the working concentration of the ROCK inhibitor is 5-15 μM.

[0032] Preferably, the working concentration of ATRA is 0.5-3 μM.

[0033] Preferably, the working concentration of the SHH agonist is 400-600 ng / mL.

[0034] Preferably, the working concentration of EGF is 10-30 ng / mL.

[0035] Preferably, the working concentration of db-cAMP is 80-120 μM.

[0036] Preferably, the working concentration of BDNF is 10-30 ng / mL.

[0037] Preferably, the working concentration of NT3 is 10-30 ng / mL.

[0038] Preferably, the step S6 specifically comprises: culturing the cells cultured in the step S5 in the fifth culture medium for 1 to 3 days, then adjusting the working concentration of the ROCK inhibitor in the fifth culture medium to 0.5 to 3 μM, and continuing the culture.

[0039] In one aspect, the present invention provides an inner ear organoid obtained according to any one of the methods described above.

[0040] On the one hand, the present invention provides inner ear precursor cells obtained according to any one of the methods described above, the inner ear precursor cells described above, inner ear organoids obtained by any one of the methods described above, or the use of the inner ear organoids described above in the preparation of products related to the treatment of deafness.

[0041] The present invention has the following beneficial effects: The present invention provides a novel and efficient method for differentiating inner ear precursor cells from pluripotent stem cells. The obtained precursor cells can be used to directly differentiate into inner ear organoids, or the precursor cells can be frozen and preserved, then revived and differentiated into otodermal neuron spheres to promote the regeneration of otodermal neurons without relying on the original human embryonic stem cells. Therefore, this method is conducive to conducting experiments in stages.

[0042] Furthermore, the precursor cells (pool) obtained by the method provided by this invention can self-assemble into spheres under the same differentiation culture conditions, and the differentiation process can be paused, resulting in highly uniform inner ear neuron organoids. This invention provides valuable insights into SGN regeneration and the development of diagnostic and treatment options for deafness. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the inner ear neuron organoid differentiation and culture process of the present invention.

[0044] Figure 2 To identify the human inner ear precursor cells (SOX2p75 NTR ) Schematic diagram of the results.

[0045] Figure 3 Organoid morphology at day 4 and day 15 of the 3D differentiation process of inner ear neuronal organoids derived from uncryopreserved and cryopreserved inner ear progenitor cells.

[0046] Figure 4 Comparison of inner ear neuron expression levels in inner ear neuron organoids from different differentiation batches.

[0047] Figure 5 Immunofluorescence results of neuronal differentiation and maturation of inner ear neuron organoids obtained by self-assembly of inner ear precursor cells.

[0048] Figure 6 Figure 3. Calcium activity assay results of inner ear neuronal organoids obtained by self-assembly of inner ear progenitor cells. A is a schematic diagram of calcium imaging results of 3D differentiated organoids incubated with Fluo 4-AM calcium dye on day 25 of differentiation. B is a schematic diagram of GDP-like events in organoids on day 25 of differentiation. C is a schematic diagram of the response of organoids on day 25 of differentiation to glutamatergic neuron-specific activators and inhibitors. DETAILED DESCRIPTION

[0049] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0050] Cryopreservation technology uses ultra-low temperatures to preserve cell structural and functional integrity, ensuring a stable supply of quality-controlled, freshly thawed cells for screening, while eliminating the need for repeated validation of the parental cell line. Cryopreserved precursor or progenitor cells derived from hPSCs have demonstrated feasibility in translational applications. For example, cryopreserved hPSC-derived midbrain dopamine precursors have been shown to efficiently generate A9 midbrain dopamine neurons in vivo and rescue the disease phenotype in a rat model of Parkinson's disease. Furthermore, cryopreserved hPSC-derived midgut monolayer endoderm cells enables the reproducible and scalable production of small intestinal organoids without the need for redifferentiation of hPSCs. Based on this, the inventors of this application, after extensive and long-term experimental research, have developed a cryopreservation strategy to preserve human embryonic stem cell-derived ear-derived precursor (progenitor) cells, thereby promoting the regeneration of ear-derived neuron spheres and ear-derived neurons, independent of the original human embryonic stem cells. In the present invention, we cryopreserved inner ear progenitor cells differentiated from human pluripotent stem cells and systematically evaluated their ability to generate self-assembled human inner ear neuronal organoids after cryopreservation. We found that SGN-like neurons can efficiently and stably differentiate in three-dimensional (3D) spheroids.

[0051] The term "a" or "an" refers to one or more, for example, "a molecule" should be understood to mean one or more molecules. Therefore, the terms "a" or "an", "one or more" and "at least one" can be used interchangeably herein.

[0052] In the claims and description of the present invention, unless the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, that is, specifying the presence of the stated features but not excluding the presence or addition of other features in various embodiments of the present invention.

[0053] As used herein, the term "about" encompasses a range of ±25% of a given value. In other embodiments, the term "about" encompasses a range of ±20%, ±15%, ±10%, or ±5% of a given value. For example, in one embodiment, "about 3 grams" refers to a value of 2.7-3.3 grams (i.e., 3 grams ±10%), etc.

[0054] In one aspect, the present invention provides a method for preparing inner ear precursor cells from pluripotent stem cells, the method comprising the following steps: S1: culturing human pluripotent stem cells in contact with an extracellular matrix in the presence of a first culture medium, wherein the first culture medium is a basal culture medium containing bone morphogenetic factor (BMP), fibroblast growth factor (FGF), and a transforming growth factor β (TGFβ) signaling inhibitor; S2: Culturing the cells obtained in S1 in a first culture medium containing a BMP inhibitor and a Wnt signaling inhibitor to differentiate and obtain the inner ear precursor cells.

[0055] As used herein, "pluripotent stem cells" suitable for use according to the methods of the present invention are cells that have the ability to differentiate into cells of all three germ layers. The pluripotent stem cells used in the present invention can be human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPS). These cells can be derived from any organism. Preferably, the pluripotent stem cells are human pluripotent stem cells. More preferably, the pluripotent stem cells are human induced pluripotent stem cells.

[0056] The pluripotent stem cells may be derived from any cell line. In a preferred embodiment, the pluripotent stem cells are induced pluripotent stem cells.

[0057] In one embodiment, the BMP is selected from one or more of BMP2, BMP4 and BMP7.

[0058] In a specific and preferred embodiment, the BMP is selected from BMP4.

[0059] In a certain embodiment, the FGF is bFGF.

[0060] In one embodiment, the TGFβ signaling inhibitor is selected from one or more of SB-431542, A-83-01, LY2157299 (Galunisertib), and RepSox.

[0061] In a specific and preferred embodiment, the TGFβ signaling inhibitor is selected from SB-431542.

[0062] In one embodiment, the BMP inhibitor is selected from one or more of DMH1, LDN-212854, doxorubicin, Nogin, LDN-193189, activin binding protein, tenascin, and gremlin.

[0063] In a specific and preferred embodiment, the BMP inhibitor is selected from LDN-193189.

[0064] In one embodiment, the Wnt signaling inhibitor is selected from one or more of XAV939, Endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, triptolide, KYA1797K, JW55, JW67, JW74, cardiomyogen 1, NLS-StAx-h, TAK715, PNU74654, iCRT3, iCRT14, WIF-1, DKK1, isoquercitrin, lanatoside C, dendrobium phenol, RCM-1, WIKI4, IQ-1, PRI-724 and tigatrabectan.

[0065] In a specific and preferred embodiment, the Wnt signaling inhibitor is selected from IWP-2.

[0066] In one embodiment, the working concentration of the BMP is 5-15 ng / mL, for example, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, and 15 ng / mL.

[0067] In one embodiment, the working concentration of the BMP is 8-12 ng / mL.

[0068] In one embodiment, the working concentration of FGF is 5-15 ng / mL, for example, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, and 15 ng / mL.

[0069] In one embodiment, the working concentration of FGF is 8-12 ng / mL.

[0070] In one embodiment, the working concentration of the TGFβ signaling inhibitor is 0.5-2 μM, such as 0.5 μM, 1 μM, 1.5 μM, or 2 μM.

[0071] In one embodiment, the working concentration of the TGFβ signaling inhibitor is 0.8-1.5 μM.

[0072] In one embodiment, the working concentration of the BMP inhibitor is 0.01 to 0.5 μM, for example, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.12 μM, 0.15 μM, 0.16 μM, 0.18 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, or 0.5 μM.

[0073] In one embodiment, the working concentration of the Wnt signaling inhibitor is 1-5 μM, for example, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM.

[0074] In one embodiment, the method includes performing step S0 before step S1, and step S0 includes: culturing a single-cell suspension of human pluripotent stem cells in contact with an extracellular matrix in the presence of a second culture medium, and then subjecting the cultured cells to step S1.

[0075] In one embodiment, the second culture medium is a basal culture medium containing a ROCK inhibitor, and the working concentration of the ROCK inhibitor is 5-15 μM, for example, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, or 15 μM.

[0076] In one embodiment, the ROCK inhibitor comprises one or more of AR-12286, Y-27632, and SNJ-1656.

[0077] In one embodiment, the ROCK inhibitor is Y-27632.

[0078] A digestion step is also included before the S0 step. In one embodiment, the digestion is performed using a cell dissociation reagent. In the present invention, the cell dissociation reagent is a reagent well known to those skilled in the art. In the present invention, the type of cell dissociation reagent is not limited, as long as it can dissociate hPCS into single cells. In the present invention, examples of suitable cell dissociation reagents include StemPro TM Accutase TM Cell dissociation reagent (Thermo Fisher Scientific).

[0079] In one embodiment, the culturing time in step S1 is 1 to 3 days, for example, 1 day, 2 days, or 3 days.

[0080] In one embodiment, the culture period in step S2 is 4 to 8 days, for example, 4 days, 5 days, 6 days, 7 days, or 8 days. In steps S1 and S2, the culture medium is replaced every 16 to 36 hours.

[0081] In one embodiment, the seeding density of the pluripotent stem cells in step S1 is 1×10 3 ~1×10 5 pieces / cm 2 .

[0082] In one embodiment, the culture period in step S0 is 3 to 5 days, for example, 3 days, 4 days, or 5 days. In step S0, the culture medium is replaced every other day.

[0083] The cell culture medium used in the present invention includes any suitable cell culture medium subject to the limitations provided herein. Cell culture medium generally contains a number of components that are necessary to support the maintenance of the cultured cells. Taking into account the content disclosed below, those skilled in the art can easily prepare suitable combinations of components. The culture medium of the present invention is generally a nutrient solution containing standard cell culture ingredients, such as amino acids, vitamins, inorganic salts, carbon sources and buffers, as described in more detail below.

[0084] The culture medium of the present invention is typically prepared in deionized distilled water. It is typically sterilized prior to use, for example, by ultraviolet light, heating, radiation, or filtration to prevent contamination. The culture medium may be frozen (e.g., at -20°C or -80°C) for storage or transportation. The culture medium may contain one or more antibiotics to prevent contamination.

[0085] A preferred cell culture medium is a defined synthetic medium buffered to pH 7.4 (preferably having a pH of 7.2-7.6 or at least pH 7.2 and not higher than pH 7.6) with a carbonate-based buffer, while culturing the cells in air containing 5% to 10% CO2, or at least 5% and not more than 10% CO2, preferably 5% CO2.

[0086] Those skilled in the art will appreciate the types of culture media that can be used as the base medium in the cell culture medium of the present invention based on common knowledge. Potentially suitable cell culture media are commercially available and include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), KO-DMEM, Glasgow Minimal Essential Medium (G-MEM), Basal Medium of Eagle's (BME), DMEM / Ham's F12, Modified DMEM / Ham's F12, Iscove's Modified Dulbecco's Medium, Minimal Essential Medium (MEM), and RPMI1640 medium.

[0087] In the present invention, the basal medium of the first culture medium, the third culture medium, and the fourth culture medium is a medium containing 1% (v / v) N2, 2% (v / v) B27, 0.1 mM β-mercaptoethanol, and 50 μg / ml Normocin. TM The base of the second culture medium is 50µg / ml Normocin TM The base medium of the fifth culture medium includes 48% (v / v) DMEM-F12 medium, 48% (v / v) Neurobasal medium, 1% (v / v) N2, 2% (v / v) B27 and 50 μg / ml Normocin. TM .

[0088] In the present invention, the extracellular matrix is ​​matrigel, which contains very low levels of growth factors.

[0089] The present invention provides inner ear precursor cells prepared according to any one of the above methods.

[0090] The present invention provides a method for differentiating human pluripotent stem cells (hPSCs) into inner ear neuron organoids. This method can be performed in one step to continuously differentiate pluripotent stem cells into inner ear organoids, or it can be performed in two steps. In the first step, inner ear precursor cells (inner ear progenitor cells) are stably induced to differentiate, and these precursor cells can be cryopreserved for later use. In the second step, the precursor cells are further differentiated into inner ear organoids (see the inner ear neuron organoid differentiation and culture process of the present invention). Figure 1 ).

[0091] In one aspect, the present invention provides a method for preparing inner ear organoids from inner ear precursor cells, comprising culturing inner ear precursor cells obtained by any of the methods described above or inducing the differentiation of the inner ear precursor cells described above into inner ear organoids.

[0092] In one aspect, the present invention provides a method for discontinuously differentiating pluripotent stem cells into inner ear organoids, the method comprising the following steps: S1: culturing human pluripotent stem cells in contact with an extracellular matrix in the presence of a first culture medium, wherein the first culture medium is a basal culture medium containing bone morphogenetic factor (BMP), fibroblast growth factor (FGF), and a transforming growth factor β (TGFβ) signaling inhibitor; S2: culturing the cells obtained in S1 in a first culture medium containing a BMP inhibitor and a Wnt signaling inhibitor to differentiate and obtain the inner ear precursor cells; optionally, cryopreserving the inner ear precursor cells.

[0093] S3: thawing the frozen inner ear precursor cells obtained by any of the above methods or the inner ear precursor cells described above; S4: suspending the inner ear progenitor cells revived in step S3 and culturing them in a third culture medium for 4 to 8 days, wherein the third culture medium is a basal culture medium containing a Wnt agonist, FGF, IGF-1, and a ROCK inhibitor; S5: culturing the cells after suspension culture in step S4 for 5 to 9 days in a fourth culture medium, wherein the fourth culture medium is a basal culture medium containing ATRA, SHH agonist, FGF, IGF-1, and EGF; S6: The cells cultured in step S5 are cultured in a fifth culture medium to obtain the inner ear organoid, wherein the fifth culture medium is a basal culture medium containing db-cAMP, BDNF, NT3, IGF-1, and a ROCK inhibitor.

[0094] In one embodiment, the Wnt agonist is a GSK3 inhibitor, and the GSK3 inhibitor includes one or more of SB216763, GSK3β inhibitor VII, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-azacamparolone, indirubin, and CHIR99021.

[0095] In one embodiment, the GSK3 inhibitor is CHIR99021.

[0096] In one embodiment, the ROCK inhibitor comprises one or more of AR-12286, Y-27632, and SNJ-1656.

[0097] In one embodiment, the ROCK inhibitor is Y-27632.

[0098] In one embodiment, the SHH agonist comprises one or more of SHH, SHH C25II, SAG, SAG 21K, Hh-Ag1.5, 20a-hydroxycholesterol, and purmorphamine.

[0099] In one embodiment, the SHH agonist is SHH.

[0100] In one embodiment, the FGF in step S4 is FGF2.

[0101] In one embodiment, the FGF in step S5 is bFGF.

[0102] In one embodiment, the working concentration of the Wnt agonist is 1 to 5 μM, for example, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM.

[0103] In one embodiment, the working concentration of the Wnt agonist is 2-4 μM.

[0104] In one embodiment, the working concentration of FGF is 5-15 ng / mL, for example, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, and 15 ng / mL.

[0105] In one embodiment, the working concentration of FGF is 8-12 ng / ml.

[0106] In one embodiment, the working concentration of IGF-1 is 30-60 ng / mL, for example, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, etc.

[0107] In one embodiment, the working concentration of IGF-1 is 45-55 ng / mL.

[0108] In one embodiment, the working concentration of the ROCK inhibitor is 5-15 μM, for example, 5 μM, 6 μM, 7 ng / mL, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, or 15 μM.

[0109] In one embodiment, the working concentration of the ROCK inhibitor is 8-12 μM.

[0110] In one embodiment, the working concentration of ATRA is 0.5-3 μM, for example, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, or 3 μM.

[0111] In one embodiment, the working concentration of ATRA is 0.5-1.5 μM.

[0112] In one embodiment, the working concentration of the SHH agonist is 400-600 ng / mL, for example, 420 ng / mL, 440 ng / mL, 450 ng / mL, 460 ng / mL, 480 ng / mL, 500 ng / mL, 520 ng / mL, 550 ng / mL, 560 ng / mL, 580 ng / mL, 600 ng / mL.

[0113] In one embodiment, the working concentration of the SHH agonist is 480-520 ng / mL.

[0114] In one embodiment, the working concentration of EGF is 10-30 ng / mL, for example, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL.

[0115] In one embodiment, the working concentration of EGF is 15-25 ng / mL.

[0116] In one embodiment, the working concentration of db-cAMP is 80-120 μM, for example, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 105 μM, 110 μM, 115 μM, or 120 μM.

[0117] In one embodiment, the working concentration of db-cAMP is 90-110 μM.

[0118] In one embodiment, the working concentration of BDNF is 10-30 ng / mL, for example, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL.

[0119] In one embodiment, the working concentration of BDNF is 15-25 ng / mL.

[0120] In one embodiment, the working concentration of NT3 is 10-30 ng / mL, for example, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL.

[0121] In one embodiment, the working concentration of NT3 is 15-25 ng / mL.

[0122] In one embodiment, the seeding density of the inner ear precursor cells in step S4 is 1×10 3 ~1×10 5 pieces / cm 2 .

[0123] In one embodiment, the step S5 specifically comprises: culturing the cells after suspension culture in step S4 in a fourth culture medium for 1-2 days, and then transferring the cells to a low-viscosity culture vessel. Preferably, the culture vessel is a culture dish, a culture plate, or a culture flask.

[0124] In the present invention, in step S5, the frequency of changing the culture medium is 24 to 60 hours.

[0125] In one embodiment, the step S6 specifically comprises: culturing the cells cultured in the step S5 in the fifth culture medium for 1 to 3 days, then adjusting the working concentration of the ROCK inhibitor in the fifth culture medium to 0.5 to 3 μM (e.g., 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM), and continuing the culture.

[0126] In the present invention, the continued culture time is at least 10 days, and inner ear organoids that meet the requirements can be obtained; in a preferred example, the continued culture time is at least 50 days, and in a specific and preferred embodiment, the culture is continued for about 150 days, and inner ear organoids that meet the requirements can still be obtained.

[0127] In one embodiment, the continued culture time is 10 to 30 days, for example, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, etc.

[0128] The method provided by the present invention can be used to self-assemble and differentiate inner ear organoids from frozen inner ear precursor cells. It can self-assemble and induce differentiation of long-term frozen inner ear precursor cells (for example, frozen for 1 year, 2 years, 3 years, 4 years, 5 years or more) into inner ear organoids.

[0129] In one aspect, the present invention provides an inner ear organoid obtained according to any one of the methods described above.

[0130] In one aspect, the present invention provides an inner ear organoid obtained according to any one of the methods described above, or the use of the inner ear organoid described above in the preparation of products for treating deafness.

[0131] The present invention is further illustrated below by means of specific examples. Unless otherwise specified, "%" represents volume percentage. The materials and reagents used in the following examples, unless otherwise noted, are commonly used in the art and are commercially available or synthesized by known methods. Experimental procedures in the following examples, where conditions are not specified, were generally performed according to conventional experimental conditions or those recommended by the manufacturers of the relevant reagents (kits).

[0132] Example 1 Preparation of inner ear precursor cells Generate large numbers of inner ear progenitor cells by differentiating human induced pluripotent stem cells (hPSCs). TM Accutase TM hPSCs were dissociated into single cells and suspended in E8 medium supplemented with 10 μM Y27632 and 100 μg / ml Normocin. TM . The cell suspension was then seeded onto a 6-well plate pre-coated with 1% low-growth factor (GFR) matrix gel at a density of 10,000 cells / cm2. The day of seeding was marked as monolayer differentiation day 0, and the medium was fully replaced on the second day of differentiation. Small molecules and growth factors were then added at the corresponding time points to induce hPSC to differentiate towards the inner ear. Specifically, on the 4th day of differentiation, in NB-CDM medium (DMEM-F12 medium, 1% N2, 2% B27, 0.1 mM β-mercaptoethanol, 50µg / ml Normocin TM ) were supplemented with 10 ng / ml BMP4, 10 ng / ml bFGF, and 1 μM SB431542, and the medium was changed every day. On the 6th day of monolayer differentiation, 10 ng / ml BMP4, 10 ng / ml bFGF, 0.1 μM LDN193189, 2 μM IWP2, and 1 μM SB431542 were added to the NB-CDM medium, and the medium was changed every day until the 11th day of monolayer differentiation, and inner ear precursor cells (such as Figure 2 shown).

[0133] Example 2 Cryopreservation of Inner Ear Precursor Cells The inner ear precursor cells prepared according to Example 1 were cryopreserved as follows: Accutase TM The cells were discretized into single cells, centrifuged and suspended in freezing solution. After counting, the cells were divided into 0.5-1.0×10 7Aliquot each cell into cryovials. The cryopreservation solution consists of 90% NB-CDM:10% DMSO. Place the cells to be frozen into a gradient freezing box and store at -80°C for 48 hours. Then, transfer to a liquid nitrogen tank for long-term storage.

[0134] Example 3 Preparation of cochlear neuron organoids According to experimental requirements, the corresponding number of inner ear progenitor cells that had been frozen for 1096 days were taken out of the liquid nitrogen tank and quickly thawed in a 37°C water bath. After centrifugation at 1200 rpm for 3 minutes, the freezing solution was aspirated and the cell pellet was suspended in NB-CDM medium supplemented with 3 μM GSK3β inhibitor CHIR99021 (supplier: Selleck), 10 ng / ml FGF2, 50 ng / ml IGF-1 (supplier: Pepro Tech) and 10 μM Y27632. These cells were then seeded into low-adhesion 96-well V-bottom plates at 10,000 cells per well. This day was marked as day 0 of 3D differentiation. On the 6th day of 3D differentiation, the medium was completely changed, and the culture medium in the well plate was replaced with NB-CDM medium supplemented with 1 μM ATRA, 500 ng / ml sonic hedgehog (SHH), 10 ng / ml bFGF, 50 ng / ml IGF-1, and 20 ng / ml EGF. After 24 hours of culture, the spheres were transferred from the well plate to a low-viscosity 10 cm culture dish, and the culture dish was placed in a 3D shaker and cultured for another 6 days. The medium was changed every other day, and the culture medium composition was the same as on the 6th day. On the 13th day of 3D differentiation, the culture medium composition was NIM medium (48% DMEM-F12 medium, 48% Neurobasal medium, 1% N2, 2% B27, and 50 µg / ml Normocin). TM ) were supplemented with 100 μM db-cAMP, 20 ng / ml BDNF, 20 ng / ml NT3, 20 ng / ml IGF-1, and 10 μM Y27632 and cultured for 48 hours. On day 15 of 3D differentiation, the Y27632 concentration in the culture medium was changed to 1 μM. Inner ear neuronal organoids were obtained after 25 days of 3D differentiation.

[0135] At the same time, the inner ear precursor cells obtained according to the preparation method of Example 1 were not frozen and directly subjected to the above differentiation culture to obtain inner ear neuron organoids. The cell morphology and morphological comparison statistical analysis results on the 4th and 15th days of 3D differentiation are shown in Figure 3 ,Depend on Figure 3It can be seen that freezing of inner ear progenitor cells does not affect the 3D differentiation morphology of inner ear neuron organoids. In addition, there is no significant difference in the expression levels of early marker genes of the inner ear neuron lineage in spheroids at day 0 and day 15 of 3D differentiation of inner ear progenitor cells from different thawed batches (hPSCs1 and hPSCs2 were differentiated into inner ear progenitor cells, respectively) (e.g. Figure 4 shown).

[0136] On the 25th day after self-assembly of cryopreserved inner ear progenitor cells, organoids expressed inner ear neuron maturation markers MAP2, vGLUT1, and CALB2. Morphological analysis was performed after the organoids were isolated and plated for 7 days, revealing bipolar characteristics of surviving neurons. These findings indicate that the organoids at this stage are morphologically mature inner ear nerve organoids. See the immunofluorescence images for details. Figure 5 .Depend on Figure 6 As far as we know, calcium imaging was performed on 3D differentiated organoids on day 25 and later by incubating them with Fluo 4-AM calcium dye to analyze calcium activity. More calcium release was found in the entire sphere ( Figure 6 A), and more GDP-like events ( Figure 6 B in the middle is one of the electrophysiological signs of mature neuronal function) and showed specific responses to stimulation by specific activators and inhibitors of glutamatergic neurons ( Figure 6 Middle C), indicating that the inner ear organoids are functionally mature at this stage.

[0137] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. A method for preparing inner ear precursor cells from pluripotent stem cells, characterized in that: The method comprises the following steps: S1: culturing human pluripotent stem cells in contact with an extracellular matrix in the presence of a first culture medium, wherein the first culture medium is a basal culture medium containing BMP, FGF, and a TGFβ signaling inhibitor; S2: Culturing the cells obtained in S1 in a first culture medium containing a BMP inhibitor and a Wnt signaling inhibitor to differentiate and obtain the inner ear precursor cells.

2. The method according to claim 1, characterized in that BMP is selected from one or more of BMP2, BMP4 and BMP7; and / or, FGF is bFGF; and / or, the TGFβ signaling inhibitor is selected from one or more of SB-431542, A-83-01, LY2157299, and RepSox; and / or, the BMP inhibitor is selected from one or more of DMH1, LDN-212854, doxorubicin, Nogin, LDN-193189, activin binding protein, tenascin, and gramlin; And / or, the Wnt signaling inhibitor is selected from one or more of XAV939, Endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, triptolide, KYA1797K, JW55, JW67, JW74, cardiomyogen 1, NLS-StAx-h, TAK715, PNU74654, iCRT3, iCRT14, WIF-1, DKK1, isoquercitrin, lanatoside C, dendrobium phenol, RCM-1, WIKI4, IQ-1, PRI-724 and tigatrabectan.

3. The method according to claim 1, characterized in that The method comprises performing step S0 before step S1, wherein step S0 comprises: culturing a single-cell suspension of human pluripotent stem cells in contact with an extracellular matrix in the presence of a second culture medium, and then subjecting the cultured cells to step S1.

4. The method according to claim 3, characterized in that The second culture medium is a basal culture medium containing a ROCK inhibitor, and the working concentration of the ROCK inhibitor is 5 to 15 μM; And / or, the culturing time in step S0 is 3 to 5 days.

5. The method according to any one of claims 1 to 4, characterized in that The culturing time in step S1 is 1 to 3 days; And / or, the culturing time in step S2 is 4 to 8 days.

6. Inner ear precursor cells prepared according to the method according to any one of claims 1 to 5.

7. A method for preparing inner ear organoids from inner ear precursor cells, characterized in that: Cultivate the inner ear precursor cells obtained by the method according to any one of claims 1 to 5 or the inner ear precursor cells according to claim 6 and induce them to differentiate into inner ear organoids.

8. A method for discontinuous differentiation of pluripotent stem cells into inner ear organoids, characterized in that: The method comprises the following steps: S3: thawing the frozen inner ear precursor cells obtained by the method according to any one of claims 1 to 5 or the inner ear precursor cells according to claim 6; S4: suspending the inner ear progenitor cells revived in step S3 and culturing them in a third culture medium for 4 to 8 days, wherein the third culture medium is a basal culture medium containing a Wnt agonist, FGF, IGF-1, and a ROCK inhibitor; S5: culturing the cells after suspension culture in step S4 for 5 to 9 days in a fourth culture medium, wherein the fourth culture medium is a basal culture medium containing ATRA, SHH agonist, FGF, IGF-1, and EGF; S6: Culturing the cells cultured in step S5 in a fifth culture medium to obtain the inner ear organoid, wherein the fifth culture medium is a basal culture medium containing db-cAMP, BDNF, NT3, IGF-1, and a ROCK inhibitor.

9. The method according to claim 8, characterized in that The Wnt agonist is a GSK3 inhibitor, and the GSK3 inhibitor includes one or more of SB216763, GSK3β inhibitor VII, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-azacamparolone, indirubin, and CHIR99021; and / or, the ROCK inhibitor comprises one or more of AR-12286, Y-27632, and SNJ-1656; And / or, the SHH agonist includes one or more of SHH, SHH C25II, SAG, SAG 21K, Hh-Ag1.5, 20a-hydroxycholesterol, and purmorphamine.

10. The method according to claim 8, characterized in that The step S6 specifically comprises: culturing the cells cultured in the step S5 in the fifth culture medium for 1 to 3 days, then adjusting the working concentration of the ROCK inhibitor in the fifth culture medium to 0.5 to 3 μM, and continuing the culture.

11. An inner ear organoid obtained according to the method of any one of claims 7 to 10.

12. Use of the inner ear precursor cells obtained according to any one of claims 1 to 5, the inner ear precursor cells according to claim 6, the inner ear organoids obtained according to any one of claims 7 to 10, or the inner ear organoids according to claim 11 in the preparation of products for treating deafness.

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