Methods and applications for constructing inner ear precursor cells and inner ear organoids

Abstract

This invention relates to a method and application for constructing inner ear progenitor cells and inner ear organoids. Specifically, the method for preparing inner ear progenitor cells includes 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 TGFβ signaling inhibitors; S2: culturing the cells obtained in S1 in a first culture medium containing BMP inhibitors and Wnt signaling inhibitors to differentiate into the inner ear progenitor cells. The method provided by this invention allows the inner ear progenitor cells to differentiate into mature inner ear neuronal organoids after cryopreservation, with minimal batch-to-batch variability.

Description

Technical Field

[0001] This invention relates to the field of cell biology technology, specifically to a method and application for constructing inner ear progenitor cells and inner ear organoids. Background Technology

[0002] Hearing loss is the most common peripheral sensory disorder. Spiral ganglion neurons (SGNs) in mammals, located within the cochlear axis, are bipolar neurons and the only afferent neurons in the auditory system. These neurons establish synaptic connections with cochlear hair cells, playing 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 basal plate of the ear, an ectodermal structure of the anterior basal plate ectoderm (PPE), which eventually forms the inner ear. Under the induction of transcription factors such as SOX2, EYA1, PAX2, NEUROD1, and NEUROG1, the basal plate invaginates to form auricular vesicles, which then undergo stratification and differentiation to form SGNs. Ototoxic drugs, aging, and noise exposure can lead to SGN damage and degeneration, not only interfering with sound transmission and causing difficulties in hearing clarity, speech comprehension, and sound localization, but also, because SGNs cannot regenerate, resulting in permanent non-hearing auditory hallucination (SNHL). Furthermore, cochlear implantation is a common treatment for severe non-hearing neutrophils (SNHL). It works by directly stimulating the remaining auditory nerve root (SGN) to bypass damaged hair cells, and its efficacy depends on the presence of a functional SGN. However, research on the biology and regeneration of the SGN currently faces several significant challenges. First, obtaining the SGN is particularly difficult due to its small size and complex structure, located deep within the cochlea. Additionally, human cochlear tissue for research is scarce, typically only obtainable post-mortem or during surgical procedures such as cochlear implantation. This limitation affects the quantity and quality of samples, thus restricting direct research on the human SGN. Currently, SGN research models are primarily experimental animals, but animal models cannot fully replicate human auditory physiology or pathology. Therefore, it is essential to develop in vitro models with human-derived SGNs.

[0003] In animal models, the introduction of exogenous stem cells or the induction of neighboring supporting cells to differentiate into glial cells can partially compensate for damaged SGNs. Human iPSC-derived inner ear lineages are particularly noteworthy because they reproduce inner ear cell development, including inner ear neuron development, in vitro and are widely used to simulate hearing loss. However, current methods for obtaining otogenic neurons through hPSC differentiation are not yet suitable for translational applications because they heavily rely on initial cell populations and cannot be reliably replicated between different research groups. Batch-to-batch performance inconsistencies may have contributed to previous clinical failures in neural transplantation. Furthermore, obtaining mature otogenic neurons from hPSCs is a lengthy process, which can lead to significant heterogeneity in the obtained inner ear neurons. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the first objective of this invention is to develop a method for obtaining inner ear progenitor cells differentiated from human pluripotent stem cells (hPSCs) that can be cryopreserved.

[0005] The second objective of this invention is to provide a method for differentiating human pluripotent stem cells (hPSCs) into inner ear neuronal organoids. This preparation method is simple, and it can be implemented either in one step, continuously differentiating pluripotent stem cells into inner ear organoids based on self-assembly, or in two steps. The first step involves stable induction and differentiation to obtain inner ear progenitor cells, which can be cryopreserved for later use. The second step involves differentiating these progenitor cells into inner ear organoids, thus splitting the continuous induction and differentiation process into two discontinuous processes to obtain three-dimensional neurons.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] On one hand, the present invention provides a method for preparing inner ear progenitor cells from pluripotent stem cells, the method comprising the following steps:

[0008] S1: Human pluripotent stem cells are cultured in contact with the extracellular matrix in the presence of a first culture medium, wherein the first culture medium is a basal culture medium containing inhibitors of bone morphogenetic factor (BMP), fibroblast growth factor (FGF), and transforming growth factor β (TGFβ) signaling.

[0009] S2: The cells obtained in S1 are cultured in a first culture medium containing BMP inhibitors and Wnt signal transduction inhibitors to differentiate into the inner ear progenitor cells.

[0010] Preferably, BMP is selected from one or more of BMP2, BMP4 and BMP7.

[0011] Preferably, the FGF is bFGF.

[0012] Preferably, the TGFβ signaling inhibitor is selected from one or more of SB-431542, A-83-01, LY2157299 and RepSox.

[0013] Preferably, the BMP inhibitor is selected from one or more of DMH1, LDN-212854, doxorphine, nifedipine, LDN-193189, activin-binding protein, tendinin, and graemlin.

[0014] 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, Tripterygium wilfordii lactone, KYA1797K, JW55, JW67, JW74, myocardial progenitor 1, NLS-StAx-h, TAK715, PNU74654, iCRT3, iCRT14, WIF-1, DKK1, isoquercitrin, lanolin C, dendrobine, RCM-1, WIKI4, IQ-1, PRI-724, and tigatrafetan.

[0015] Preferably, the working concentration of the BMP is 5~15 ng / mL.

[0016] Preferably, the working concentration of FGF is 5~15 ng / mL.

[0017] Preferably, the working concentration of the TGFβ signaling inhibitor is 0.5~2μM.

[0018] Preferably, the working concentration of the BMP inhibitor is 0.01~0.5μM.

[0019] Preferably, the working concentration of the Wnt signaling inhibitor is 1~5 μM.

[0020] Preferably, the method includes performing an S0 step before the S1 step, the S0 step comprising: culturing a human pluripotent stem cell single-cell suspension in contact with an extracellular matrix in the presence of a second culture medium, and then performing the cultured cells in the S1 step.

[0021] Preferably, the second culture medium is a basal culture medium containing a ROCK inhibitor, wherein the working concentration of the ROCK inhibitor is 5~15μM.

[0022] Preferably, the culture time in step S1 is 1 to 3 days.

[0023] Preferably, the culture time in step S2 is 4 to 8 days.

[0024] Preferably, the culture time in step S0 is 3 to 5 days.

[0025] On the one hand, the present invention provides an inner ear progenitor cell prepared according to any one of the above methods.

[0026] On one hand, the present invention provides a method for preparing inner ear organoids from inner ear progenitor cells, wherein the inner ear progenitor cells obtained by the method described above are cultured or the inner ear progenitor cells described above are induced to differentiate into inner ear organoids.

[0027] On one hand, the present invention provides a method for discontinuous differentiation of pluripotent stem cells into inner ear organoids, the method comprising the following steps:

[0028] S3: Revive the frozen inner ear progenitor cells obtained by any of the above methods or the inner ear progenitor cells described above.

[0029] S4: The inner ear progenitor cells revived in step S3 are suspended in the third culture medium and cultured for 4-8 days. The third culture medium is a basic culture medium containing Wnt agonist, FGF, IGF-1 and ROCK inhibitor.

[0030] S5: The cells after suspension culture in step S4 are cultured in the fourth culture medium for 5 to 9 days. The fourth culture medium is a basic culture medium containing ATRA, SHH agonist, FGF, IGF-1 and EGF.

[0031] S6: The cells cultured in step S5 are cultured in the fifth culture medium to obtain the inner ear organoids. The fifth culture medium is a basic culture medium containing db-cAMP, BDNF, NT3, IGF-1, and ROCK inhibitors.

[0032] Preferably, the Wnt agonist is a GSK3 inhibitor, which includes one or more of SB216763, GSK3β inhibitor VII, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-azacamparone, indirubin, and CHIR99021.

[0033] Preferably, the ROCK inhibitor includes one or more of AR-12286, Y-27632 and SNJ-1656.

[0034] Preferably, the SHH agonist includes one or more of SHH, SHH C25II, SAG, SAG 21K, Hh-Ag1.5, 20α-hydroxycholesterol, and puromorphamine.

[0035] Preferably, the working concentration of the Wnt agonist is 1~5 μM.

[0036] Preferably, the working concentration of FGF is 5~15 ng / mL.

[0037] Preferably, the working concentration of IGF-1 is 30~60 ng / mL.

[0038] Preferably, the working concentration of the ROCK inhibitor is 5~15μM.

[0039] Preferably, the working concentration of the ATRA is 0.5~3μM.

[0040] Preferably, the working concentration of the SHH agonist is 400~600 ng / mL.

[0041] Preferably, the working concentration of the EGF is 10~30 ng / mL.

[0042] Preferably, the working concentration of the db-cAMP is 80~120μM.

[0043] Preferably, the working concentration of BDNF is 10~30 ng / mL.

[0044] Preferably, the working concentration of NT3 is 10~30 ng / mL.

[0045] Preferably, step S6 specifically involves culturing the cells cultured in step S5 in a 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.

[0046] On the one hand, the present invention provides an inner ear organoid obtained by the method according to any one of the preceding claims.

[0047] On the one hand, the present invention provides the use of inner ear progenitor cells obtained by the method according to any one of the above-described methods, the inner ear progenitor cells described above, the inner ear organoids obtained by the method according to any one of the above-described methods, or the inner ear organoids described above in the preparation of products related to the treatment of deafness.

[0048] The present invention has the following beneficial effects:

[0049] This invention provides a novel and efficient method for differentiating pluripotent stem cells into inner ear progenitor cells. The obtained progenitor cells can be used to directly differentiate into inner ear organoids, or the progenitor cells can be cryopreserved and then revived to differentiate into otogenic neuronal spheres, promoting the regeneration of otogenic neurons, without relying on the original human embryonic stem cells. Therefore, this method is conducive to conducting experiments in stages.

[0050] Furthermore, the precursor cells (pools) obtained through the method provided by this invention can self-assemble into spheres under the same differentiation culture conditions, allowing for a paused differentiation process and the acquisition of highly homogeneous inner ear neuron organoids. This invention provides valuable insights for the development of SGN regeneration and diagnostic and therapeutic solutions for deafness. Attached Figure Description

[0051] Figure 1 This invention provides a process for differentiating and culturing organoids of inner ear neurons.

[0052] Figure 2To identify the human inner ear progenitor cells (SOX2p75) obtained in Example 1 using immunofluorescence methods NTR (Result diagram)

[0053] Figure 3 Organoid morphology images on day 4 and day 15 of the 3D differentiation process of inner ear progenitor cells (both unfrozen and frozen) into inner ear neuronal organoids.

[0054] Figure 4 The results show the comparison of expression levels of inner ear neurons in organoids from different differentiation batches.

[0055] Figure 5 Immunofluorescence results of the differentiation and maturation of inner ear neuronal organoids obtained from the self-assembly of inner ear progenitor cells.

[0056] Figure 6 The results show the calcium activity of inner ear neuron organoids obtained from the self-assembly of inner ear progenitor cells. In the figure, A is a schematic diagram of the calcium imaging results of the 3D differentiated organoids incubated with Fluo 4-AM calcium dye on day 25, B is the GDP-like event of the differentiated organoids on day 25, and C is a schematic diagram of the response of the differentiated organoids on day 25 to glutamatergic neuron-specific activators and inhibitors. Detailed Implementation

[0057] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show 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 pertains.

[0058] Cryopreservation technology maintains the structural and functional integrity of cells by using ultra-low temperatures, ensuring a stable supply of quality-controlled freshly thawed cells for screening, without requiring repeated validation of parental cell lines. Cryopreserved hPSC-derived precursor or progenitor cells have shown feasibility in transformational applications. For example, cryopreserved hPSC-derived midbrain dopamine precursors have been shown to effectively 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 enable reproducible and large-scale production of small intestinal organoids without the need for hPSC redifferentiation. Based on this, the inventors of this application, through extensive and long-term experimental research, have developed a cryopreservation strategy to preserve human embryonic stem cell-derived otogenic precursor cells (progenitor cells), thereby promoting the regeneration of otogenic neurobulbs and otogenic neurons without relying on the original human embryonic stem cells. In this invention, we cryopreserved inner ear progenitor cells obtained from human pluripotent stem cell differentiation, systematically evaluated the ability of cryopreserved cells to generate self-assembled human inner ear neuron organoids, and found that SGN-like neurons can differentiate efficiently and stably in a three-dimensional (3D) sphere.

[0059] The terms “a” or “an” refer to one or more molecules, for example, “a molecule” should be understood to represent one or more molecules. Therefore, the terms “a” or “an”, “one or more”, and “at least one” are used interchangeably in this document.

[0060] In the claims and description of this invention, unless the context requires otherwise due to the language of expression or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” are used in the sense of inclusion, that is, specifying the presence of the said feature, but not excluding the presence or addition of other features in various embodiments of the invention.

[0061] As used herein, the term "about" covers a range of values ​​within ±25% of a given value. In other embodiments, the term "about" covers a range of values ​​within ±20%, ±15%, ±10%, or ±5% of a given value. For example, in one embodiment, "about 3 grams" represents a value of 2.7–3.3 grams (i.e., 3 grams ± 10%).

[0062] On one hand, the present invention provides a method for preparing inner ear progenitor cells from pluripotent stem cells, the method comprising the following steps:

[0063] S1: Human pluripotent stem cells are cultured in contact with the extracellular matrix in the presence of a first culture medium, wherein the first culture medium is a basal culture medium containing inhibitors of bone morphogenetic factor (BMP), fibroblast growth factor (FGF), and transforming growth factor β (TGFβ) signaling.

[0064] S2: The cells obtained in S1 are cultured in a first culture medium containing BMP inhibitors and Wnt signal transduction inhibitors to differentiate into the inner ear progenitor cells.

[0065] As used in this invention, the "pluripotent stem cell" suitable for use in the method according to the invention is a cell capable of differentiating into cells of all three germ layers. The pluripotent stem cells used in this invention can be human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). These cells can be derived from any organism. Preferably, the pluripotent stem cell is a human pluripotent stem cell. More preferably, the pluripotent stem cell is a human induced pluripotent stem cell.

[0066] The pluripotent stem cells can be derived from any cell line. In a preferred embodiment, the pluripotent stem cells are induced pluripotent stem cells.

[0067] In one embodiment, BMP is selected from one or more of BMP2, BMP4 and BMP7.

[0068] In a specific and preferred embodiment, BMP is selected from BMP4.

[0069] In one implementation, FGF is bFGF.

[0070] In one embodiment, the TGFβ signaling inhibitor is selected from one or more of SB-431542, A-83-01, LY2157299 (Galunisertib), and RepSox.

[0071] In a specific and preferred embodiment, the TGFβ signaling inhibitor is selected from SB-431542.

[0072] In one embodiment, the BMP inhibitor is selected from one or more of DMH1, LDN-212854, doxorphine, nifedipine, LDN-193189, activin-binding protein, tendinin, and gremlin.

[0073] In a specific and preferred embodiment, the BMP inhibitor is selected from LDN-193189.

[0074] 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, Tripterygium wilfordii lactone, KYA1797K, JW55, JW67, JW74, myocardial progenitor 1, NLS-StAx-h, TAK715, PNU74654, iCRT3, iCRT14, WIF-1, DKK1, isoquercitrin, lanolin C, dendrobine, RCM-1, WIKI4, IQ-1, PRI-724, and tigatrafetan.

[0075] In a specific and preferred embodiment, the Wnt signaling inhibitor is selected from IWP-2.

[0076] 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, or 15 ng / mL.

[0077] In one embodiment, the working concentration of the BMP is 8~12 ng / mL.

[0078] In one embodiment, the working concentration of the 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, 15 ng / mL.

[0079] In one embodiment, the working concentration of the FGF is 8~12 ng / mL.

[0080] In one embodiment, the working concentration of the TGFβ signaling inhibitor is 0.5~2μM, for example 0.5μM, 1μM, 1.5μM, 2μM.

[0081] In one embodiment, the working concentration of the TGFβ signaling inhibitor is 0.8~1.5 μM.

[0082] In one embodiment, the working concentration of the BMP inhibitor is 0.01~0.5 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.

[0083] In one embodiment, the working concentration of the Wnt signaling inhibitor is 1~5μM, for example 1μM, 1.5μM, 2μM, 2.5μM, 3μM, 3.5μM, 4μM, 4.5μM, 5μM.

[0084] In one embodiment, the method includes performing an S0 step prior to the S1 step, the S0 step comprising: culturing a human pluripotent stem cell single-cell suspension in contact with an extracellular matrix in the presence of a second culture medium, and then performing the cultured cells in the S1 step.

[0085] In one embodiment, the second culture medium is a basal culture medium containing a ROCK inhibitor, wherein the working concentration of the ROCK inhibitor is 5~15μM, for example 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 11μM, 12μM, 13μM, 14μM, or 15μM.

[0086] In one embodiment, the ROCK inhibitor includes one or more of AR-12286, Y-27632, and SNJ-1656.

[0087] In one embodiment, the ROCK inhibitor is Y-27632.

[0088] A digestion step is included before step S0. In one embodiment, the digestion is performed using a cell dissociation reagent. In this invention, the cell dissociation reagent is a reagent well known to those skilled in the art. The type of cell dissociation reagent is not limited in this invention, as long as it can dissociate hPCS into single cells. Examples of suitable cell dissociation reagents in this invention include StemPro. TM Accutase TM Cell dissociation reagent (Thermo Fisher Scientific).

[0089] In one embodiment, the culture time in step S1 is 1 to 3 days, for example, 1 day, 2 days, or 3 days.

[0090] In one embodiment, the culture time 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 changed every 16 to 36 hours.

[0091] In one embodiment, the seeding density of the pluripotent stem cells in step S1 is 1×10⁻⁶. 3 ~1×10 5 pcs / cm 2 .

[0092] In one embodiment, the culture time in step S0 is 3 to 5 days, for example, 3 days, 4 days, or 5 days. In step S0, the culture medium is changed every other day.

[0093] The cell culture media used in this invention include any suitable cell culture media subject to the limitations set forth herein. Cell culture media generally contain a number of components essential for supporting the maintenance of cultured cells. Suitable combinations of components can be readily formulated by those skilled in the art in light of the following disclosure. The culture media of this invention are typically nutrient solutions containing standard cell culture components such as amino acids, vitamins, inorganic salts, carbon sources, and buffers, as described in more detail below.

[0094] The culture media of this invention are generally prepared in deionized distilled water. The culture media of this invention are generally sterilized before use by, for example, ultraviolet light, heating, radiation, or filtration to prevent contamination. The culture media may be frozen (e.g., at -20°C or -80°C) for storage or transport. The culture media may contain one or more antibiotics to prevent contamination.

[0095] The preferred cell culture medium is a defined synthetic medium buffered to pH 7.4 (preferably having pH 7.2-7.6 or at least pH 7.2 and not higher than pH 7.6) with 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.

[0096] Those skilled in the art will understand from common sense the types of culture media that can be used as basal media in the cell culture media of the present invention. Suitable cell culture media are commercially available and include, but are not limited to: Dalberg modified Eagle medium (DMEM), minimum essential medium (MEM), KO-DMEM, Glasgow minimum essential medium (G-MEM), Eagle basal medium (BME), DMEM / Ham's F12, modified DMEM / Ham's F12, Iskoff modified Dulbeco medium and minimum basal medium (MEM), and RPMI 1640 medium.

[0097] In this invention, the basal culture media for the first, third, and fourth culture media consist of 1% (v / v) N2, 2% (v / v) B27, 0.1 mM β-mercaptoethanol, and 50 µg / ml Normocin. TM The first medium was DMEM / F12. The second medium was a basal medium of 50 µg / ml Normocin. TM The fifth medium consisted of E8 medium. The basal medium included 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 .

[0098] In this invention, the extracellular matrix is ​​matrix gel. In this invention, the matrix gel contains extremely low levels of growth factors.

[0099] The present invention provides an inner ear progenitor cell prepared according to any one of the above methods.

[0100] This invention provides a method for differentiating human pluripotent stem cells (hPSCs) into inner ear neuron organoids. This method can be implemented either in a single, continuous differentiation process from pluripotent stem cells into inner ear organoids, or in two steps. The first step involves stable induction and differentiation to obtain inner ear progenitor cells, which can be cryopreserved for later use. The second step involves differentiating these progenitor cells into inner ear organoids (the inner ear neuron organoid differentiation and culture process of this invention is described in [link to invention]). Figure 1 ).

[0101] On one hand, the present invention provides a method for preparing inner ear organoids from inner ear progenitor cells, wherein the inner ear progenitor cells obtained by the method described above are cultured or the inner ear progenitor cells described above are induced to differentiate into inner ear organoids.

[0102] On one hand, the present invention provides a method for discontinuous differentiation of pluripotent stem cells into inner ear organoids, the method comprising the following steps:

[0103] S1: Human pluripotent stem cells are cultured in contact with the extracellular matrix in the presence of a first culture medium, wherein the first culture medium is a basal culture medium containing inhibitors of bone morphogenetic factor (BMP), fibroblast growth factor (FGF), and transforming growth factor β (TGFβ) signaling.

[0104] S2: The cells obtained in S1 are cultured in a first culture medium containing BMP inhibitors and Wnt signaling inhibitors to differentiate into the inner ear progenitor cells; optionally, the inner ear progenitor cells are cryopreserved.

[0105] S3: Revive the frozen inner ear progenitor cells obtained by any of the above methods or the inner ear progenitor cells described above.

[0106] S4: The inner ear progenitor cells revived in step S3 are suspended in the third culture medium and cultured for 4-8 days. The third culture medium is a basic culture medium containing Wnt agonist, FGF, IGF-1 and ROCK inhibitor.

[0107] S5: The cells after suspension culture in step S4 are cultured in the fourth culture medium for 5 to 9 days. The fourth culture medium is a basic culture medium containing ATRA, SHH agonist, FGF, IGF-1 and EGF.

[0108] S6: The cells cultured in step S5 are cultured in the fifth culture medium to obtain the inner ear organoids. The fifth culture medium is a basic culture medium containing db-cAMP, BDNF, NT3, IGF-1, and ROCK inhibitors.

[0109] In one embodiment, the Wnt agonist is a GSK3 inhibitor, which includes one or more of SB216763, GSK3β inhibitor VII, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-azacamparone, indirubin, and CHIR99021.

[0110] In one embodiment, the GSK3 inhibitor is CHIR99021.

[0111] In one embodiment, the ROCK inhibitor includes one or more of AR-12286, Y-27632, and SNJ-1656.

[0112] In one embodiment, the ROCK inhibitor is Y-27632.

[0113] In one embodiment, the SHH agonist includes one or more of SHH, SHH C25II, SAG, SAG 21K, Hh-Ag1.5, 20α-hydroxycholesterol, and puromorphamine.

[0114] In one embodiment, the SHH agonist is SHH.

[0115] In one embodiment, FGF in step S4 is FGF2.

[0116] In one embodiment, the FGF in step S5 is bFGF.

[0117] In one embodiment, the working concentration of the Wnt agonist 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, 5 μM.

[0118] In one embodiment, the working concentration of the Wnt agonist is 2-4 μM.

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

[0120] In one embodiment, the working concentration of the FGF is 8~12 ng / ml.

[0121] In one embodiment, the working concentration of IGF-1 is 30~60 ng / mL, such as 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, etc.

[0122] In one embodiment, the working concentration of IGF-1 is 45~55 ng / mL.

[0123] In one embodiment, the working concentration of the ROCK inhibitor is 5~15μM, for example 5μM, 6μM, 7 ng / mL, 8μM, 9μM, 10μM, 11μM, 12μM, 13μM, 14μM, 15μM.

[0124] In one embodiment, the working concentration of the ROCK inhibitor is 8-12 μM.

[0125] In one embodiment, the working concentration of the ATRA is 0.5~3μM, for example 0.5μM, 1μM, 1.5μM, 2μM, 2.5μM, 3μM.

[0126] In one embodiment, the working concentration of the ATRA is 0.5~1.5 μM.

[0127] 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, or 600 ng / mL.

[0128] In one embodiment, the working concentration of the SHH agonist is 480~520 ng / mL.

[0129] In one embodiment, the working concentration of the EGF is 10~30 ng / mL, for example 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, or 30 ng / mL.

[0130] In one embodiment, the working concentration of the EGF is 15~25 ng / mL.

[0131] In one embodiment, the working concentration of db-cAMP is 80~120μM, for example 80μM, 85μM, 90μM, 95μM, 100μM, 105μM, 110μM, 115μM, 120μM.

[0132] In one embodiment, the working concentration of db-cAMP is 90~110μM.

[0133] 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, or 30 ng / mL.

[0134] In one embodiment, the working concentration of BDNF is 15~25 ng / mL.

[0135] 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, or 30 ng / mL.

[0136] In one embodiment, the working concentration of NT3 is 15~25 ng / mL.

[0137] In one embodiment, the seeding density of the inner ear progenitor cells in step S4 is 1×10⁻⁶. 3 ~1×10 5 pcs / cm 2 .

[0138] In one embodiment, step S5 specifically involves culturing the cells from the 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, culture plate, or culture flask.

[0139] In this invention, in step S5, the culture medium is changed every 24 to 60 hours.

[0140] In one embodiment, step S6 specifically involves culturing the cells cultured in step S5 in a fifth culture medium for 1 to 3 days, and 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.

[0141] In this invention, the continued culture time is at least 10 days to obtain inner ear organoids that meet the requirements; in a preferred example, the continued culture time is at least 50 days; in a specific and preferred example, the continued culture time is about 150 days to still obtain inner ear organoids that meet the requirements.

[0142] In one embodiment, the continued cultivation time is 10 to 30 days, such as 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.

[0143] The method provided by this invention can self-assemble and differentiate into inner ear organoids from cryopreserved inner ear progenitor cells. It can self-assemble and induce differentiation into inner ear organoids from long-term cryopreserved (e.g., cryopreserved for 1 year, 2 years, 3 years, 4 years, 5 years or more).

[0144] On the one hand, the present invention provides an inner ear organoid obtained by the method according to any one of the preceding claims.

[0145] On the one hand, the present invention provides the use of inner ear organoids obtained according to any one of the above methods or as described above in the preparation of products related to the treatment of deafness.

[0146] The present invention will be further described below through specific embodiments. Unless otherwise specified, "%" represents volume percentage. The materials and reagents used in the following embodiments are all commonly used materials or reagents in the art, and can be obtained commercially or synthesized by known methods. Experimental methods in the following embodiments without specified conditions are generally performed according to conventional experimental conditions or the conditions recommended by the manufacturer of the relevant reagent (kit).

[0147] Example 1: Preparation of Inner Ear Progenitor Cells

[0148] A large number of inner ear progenitor cells were obtained through differentiation using human induced pluripotent stem cells (hPSCs). StemPro was used. TM Accutase TM hPSCs were single-celled and suspended in E8 medium supplemented with 10 μM Y27632 and 100 μg / ml Normocin. TM The cell suspension was then seeded into 6-well plates 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 day 0 of monolayer differentiation, and the medium was completely replaced on day 2 of differentiation. Subsequently, small molecules and growth factors were added at appropriate time points to induce hPSC differentiation toward the inner ear. Specifically, on day 4 of differentiation, cells were seeded in NB-CDM medium (DMEM-F12 medium, 1% N2, 2% B27, 0.1 mM β-mercaptoethanol, 50 µg / ml Normocin). TM Add 10 ng / ml BMP4, 10 ng / ml bFGF, and 1 μM SB431542 to the NB-CDM medium, changing the medium daily. On day 6 of monolayer differentiation, add 10 ng / ml BMP4, 10 ng / ml bFGF, 0.1 μM LDN193189, 2 μM IWP2, and 1 μM SB431542 to the NB-CDM medium, changing the medium daily until day 11 of monolayer differentiation to obtain inner ear progenitor cells (e.g., ...). Figure 2 (As shown).

[0149] Example 2: Cryopreservation of Inner Ear Progenitor Cells

[0150] The inner ear progenitor cells prepared according to Example 1 were cryopreserved. The method was as follows: Accutase was used... TM Cells were discretized into single cells, centrifuged, and then resuspended in cryopreservation solution. After counting, the cells were centrifuged at a concentration of 0.5–1.0 × 10⁻⁶ cells / cell. 7 Cells were aliquoted into cryovials. The cryopreservation solution consisted of 90% NB-CDM and 10% DMSO. After loading the cells to be cryopreserved into a gradient cryopreservation box, the cells were placed at -80°C for 48 hours, and then transferred to a liquid nitrogen tank for long-term storage.

[0151] Example 3: Preparation of Cochlear Neuron Organoids

[0152] According to experimental requirements, a corresponding number of inner ear progenitor cells that had been cryopreserved for 1096 days were removed from the liquid nitrogen tank and rapidly thawed in a 37°C water bath. After centrifugation at 1200 rpm for 3 minutes, the cryopreservation solution was discarded, and the cell pellet was resuspended 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-viscosity 96-well V plates, 10,000 cells per well, and this day was marked as day 0 of 3D differentiation. On day 6 of 3D differentiation, the medium was completely replaced, and the medium composition in the well plates was changed to NB-CDM medium supplemented with 1 μM ATRA, 500 ng / ml sonicedgehog (SHH), 10 ng / ml bFGF, 50 ng / ml IGF-1, and 20 ng / ml EGF. After 24 hours of culture, the pellets were transferred from the well plates to low-viscosity 10 cm culture dishes, and the dishes were placed in a 3D shaker for 6 more days of culture. The medium composition was changed every other day, with the same composition as on day 6. On day 13 of 3D differentiation, the medium composition was NIM medium (48% DMEM-F12 medium, 48% Neurobasal medium, 1% N2, 2% B27, and 50 µg / ml Normocin). TM The culture medium was 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 concentration of Y27632 in the above culture medium was changed to 1 μM. Inner ear neuron organoids were obtained 25 days after 3D differentiation.

[0153] Meanwhile, the inner ear progenitor cells obtained according to the preparation method in Example 1 were not cryopreserved, but directly subjected to the above-mentioned differentiation culture to obtain inner ear neuron organoids. The cell morphology images and morphological comparison statistical analysis results on day 4 and day 15 of 3D differentiation are shown below. Figure 3 ,Depend on Figure 3 It can be seen that cryopreservation of inner ear progenitor cells does not affect the 3D differentiation morphology of inner ear neuronal organoids. Furthermore, there was no significant difference in the gene expression levels of early markers of the inner ear neuronal lineage on day 0 and day 15 of 3D differentiation in different thawed batches of inner ear progenitor cells (hPSCs1 and hPSCs2 differentiated into inner ear progenitor cells, respectively) of the organoids (e.g., Figure 4 (As shown).

[0154] On day 25 after self-assembly of cryopreserved inner ear progenitor cells, organoids expressed the inner ear neuronal maturation markers MAP2, vGLUT1, and CALB2. Morphological analysis after single-cell culture of the organoids and seeding them on plates for 7 days revealed bipolar characteristics in surviving neurons. These findings confirm that the organoids at this stage are morphologically mature inner ear neuronal organoids. Immunofluorescence images are shown below. Figure 5 .Depend on Figure 6 To date, calcium imaging and analysis of calcium activity in organoids incubated with Fluo 4-AM calcium dye on day 25 of 3D differentiation and thereafter have revealed significant calcium release throughout the entire sphere. Figure 6 (China A), and a number of GDP-like events occurred ( Figure 6 Glutamate neuron B (a type of neuronal neuron) is an electrophysiological marker of neuronal functional maturity and exhibits specific responses to both activators and inhibitors of glutamatergic neurons. Figure 6 The middle C indicates that the inner ear organoids were functionally mature during this period.

[0155] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A method for discontinuous differentiation of pluripotent stem cells into inner ear organoids, characterized in that, The method includes the following steps: S1: Under the condition of the first culture medium, human pluripotent stem cells are cultured in contact with the extracellular matrix for 1-3 days. The first culture medium is a basal culture medium containing bone morphogenetic factor BMP4, fibroblast growth factor bFGF and SB-431542. The working concentration of BMP4 is 10 ng / mL, the working concentration of bFGF is 8-12 ng / mL, and the working concentration of SB-431542 is 0.8-1.5 μM. S2: The cells obtained in S1 were cultured in a first culture medium containing LDN-193189 and IWP-2 for 4 to 8 days to differentiate into inner ear progenitor cells; the inner ear progenitor cells were cryopreserved, with the working concentration of LDN-193189 being 0.1 μM and the working concentration of IWP-2 being 1 to 5 μM. S3: Revive the frozen inner ear progenitor cells; S4: The inner ear progenitor cells resuscitated in step S3 are suspended in the third culture medium and cultured for 4-8 days. The third culture medium is a basal culture medium containing CHIR99021, FGF2, IGF-1, and Y-27632. The working concentration of CHIR99021 is 2-4 μM, the working concentration of FGF2 is 8-12 ng / mL, the working concentration of IGF-1 is 45-55 ng / mL, and the working concentration of Y-27632 is 8-12 μM. S5: The cells after suspension culture in step S4 are cultured in the fourth culture medium for 5-9 days. The fourth culture medium is a basal culture medium containing ATRA, SHH, bFGF, IGF-1, and EGF. The working concentration of ATRA is 0.5-1.5 μM, the working concentration of SHH is 480-520 ng / mL, the working concentration of bFGF is 8-12 ng / mL, the working concentration of IGF-1 is 45-55 ng / mL, and the working concentration of EGF is 15-25 ng / mL. S6: The cells cultured in step S5 are cultured in the fifth culture medium to obtain the inner ear organoid. The fifth culture medium is a basal culture medium containing db-cAMP, BDNF, NT3, IGF-1, and Y-27632. The working concentration of db-cAMP is 90~110μM, the working concentration of BDNF is 15~25ng / mL, the working concentration of NT3 is 15~25ng / mL, the working concentration of IGF-1 is 20ng / mL, and the working concentration of Y-27632 is 8~12μM.

2. The method according to claim 1, characterized in that, The S6 step specifically involves culturing the cells cultured in the S5 step 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.

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